WO2008125116A2

WO2008125116A2 - Upar inhibition

Info

Publication number: WO2008125116A2
Application number: PCT/DK2008/050075
Authority: WO
Inventors: Jørgen Kjems; Daniel Miotto Dupont; Peter A. Andreasen
Original assignee: Aarhus Universitet
Priority date: 2007-03-31
Filing date: 2008-03-28
Publication date: 2008-10-23
Also published as: WO2008125116A3

Abstract

The present invention relates to aptamers capable of binding to a receptor-binding form of urokinase-type plasminogen activator protein, u-PA. Furthermore, the present invention relates to use of the aptamers for prevention of u-PA-mediated proteolytic activity at the cell surface for the preparation of a composition for preventing or counteracting proteolytic activity on the surface of a cell. The aptamers of the present invention may additionally be used as imaging agents or used for the treatment or prevention of cancer and/or metastatic spread. Within the scope of the present invention is also pharmaceutical compositions and diagnostic kits comprising aptamers.

Description

uPAR inhibition

All patent and non-patent references cited in the application, or in the present application, are also hereby incorporated by reference in their entirety.

Field of invention

The present invention relates to aptamers capable of binding to uPA and uses thereof in treatment and diagnostics.

Background of invention

In cancer patients, it is usually not the primary tumour, but its metastases at distant sites that are the main cause of death. However, it is today a tremendously difficult task to detect these metastases and to predict which patients who are at risk of experiencing recurrence after surgery. This means that many patients that would be cured by local treatment alone are over-treated and suffer the toxic side effects of chemotherapy needlessly (1 ).

Cancer metastasis is a very complex process, involving important steps such as detachment of tumor cells from the primary tumor, active migration and invasion into the surrounding host tissue, entrance into circulatory systems, extravasation, and formation of new solid tumors in a distant tissue. That non-malignant stromal host cells are stimulated to contribute to cancer dissemination, only adds to the complexity of the process. A major challenge in present day cancer medicine is to predict which patients are in need of further treatment after surgical removal of the primary tumour and to design treatment with optimal efficacy and minimal side effects. Usually, predictions are made based on standard clinical and biochemical characterisations of primary tumours (1 )-

Positron emission tomography (PET) in oncology

The increased application of PET and PET combined with computer tomography (PET/CT) in oncology during the last 5 years has been so remarkable, that the technology has been called "the fastest growing medical technology ever"(4). Today the cancer imaging ability of PET and PET/CT helps to determine primary diagnosis, staging of the disease, effects of treatment, recurrence after therapy and to plan radiation therapy.

¹⁸Fluorodeoxyglucose (¹⁸F-FDG) is a radiolabeled glucose molecule and the most used PET-imaging agent in oncology. This molecule is a marker of malignancy as viable tumour cells often have an upregulated glucose metabolism. However, this agent has its limitations, as it cannot be used for detecting all tumours, for instance not prostate and brain tumours. Therefore, to extend the power of PET and PET/CT in cancer diagnostics, a panel of PET-imaging agents have to be developed that with high sensitive and specificity target other traits of malignant cancer cells. Recent findings suggest that RNA aptamers may be of great interest in this respect and that targeting causally involved proteins in cancer metastasis can establish an important link between detection and therapy.

RNA aptamers are promising therapeutic and diagnostic agents Systematic evolution of ligands by exponential enrichment (SELEX) is a relatively new approach for generating potential therapeutic and diagnostic agents (3,5,7). The technique combines the ability of RNA or DNA oligonucleotides to fold into a variety of three-dimensional structures depending on their nucleic acid sequence, with the possibility of selecting from very large pools of random sequences (~10¹⁵), the ones capable of binding to a target of interest, including purified proteins and proteins on the surface of whole cells. Oligonucleotides selected in this way are called aptamers and can often not only bind the target but also inhibit its functionality. They are remarkable in terms of affinity and specificity, being comparable to antibodies with dissociation constants (K₀) in the low picomolar to low nanomolar range and ability to discriminate between closely related proteins that share common sets of structural domains. Though SELEX is a relatively new technology, the first aptamer drug has already received U.S. marketing approval in 2005 and several others are in pre-clinical and clinical trials (5,7).

Metastasis and the role of proteolytic enzyme systems - the target

The great majority (>80%) of life-threatening cancers are derived from epithelial tissues (Weinberg, 2007). The invasion-metastasis cascade for cancer cells of epithelial origin consists of several steps, detachment from the primary tumor, localised invasion into neighbouring tissue, intravasation, transport through the circulation, arrest in microvessels of various tissues, extravasation, and formation of micrometastases, which can eventually develop into macrometastases (colonisation). That non-malignant stromal cells are stimulated to contribute to cancer dissemination, only adds to the complexity of the process. For many steps in the invasion-metastasis cascade, cancer cells need to recruit molecular machinery associated with proteolytic degradation of the extracellular matrix (ECM). Such degradation is necessary in order for cancer cells to be able to detach from the primary tumor, breakdown basement membranes, invade and migrate into normal tissue, but it is also important in the cancer cell-directed tissue remodelling processes (like angiogenesis and desmoplasia), which serve to create an infrastructure in the tumor-stroma supporting tumor growth and metastasis. Proteolytic enzyme systems that have been found to be involved in cancer dissemination include the plasminogen activation system and the metalloproteinases (MMPs), two systems that in some degree have been found to have overlapping functions (Andreasen et al., 1997; Lund et al., 1999; Andreasen et al., 2000; Durand et al., 2004; Weinberg, 2007). It is becoming increasingly evident, however, that the components of such systems, as exemplified by the plasminogen activation system, apart from playing a role in proteolysis, can also contribute to both physiological and patho-physiological processes through proteolysis-independent activities.

In order to metastasise, tumor cells have to acquire characteristics of the invasive and migratory cell, which among other things means obtaining the molecular machinery associated with degradation of the extracellular matrix. This degradation is accomplished by several proteolytic enzyme systems, including the matrix metalloproteinases (MMPs) and the urokinase-type plasminogen activator (uPA) system (reviewed in 8,9,10). The uPA-system consists of the serine proteinase uPA, its glycolipid-anchored receptor uPAR, accumulating uPA at the cell surface, and the primary inhibitor of uPA, the serpin plasminogen activator inhibitor-1 (PAI-1 ). On the cell surface, uPA converts plasminogen to plasmin by proteolytic cleavage, which in turn degrades matrix components and activates latent metalloproteinases and latent growth factors. Since the late 1980^'s, numerous independent studies have demonstrated that cancer patients with low levels of uPA, uPAR and PAI-1 in their primary tumour tissue have a significantly better survival than patients with high levels of either factor. In breast cancer f.ex., high levels of uPA and PAI-1 are predictors for distant metastasis and when combining the two the prognostic value is superior to other tumour biological factors such as cathepsins B, D, L, p53, S-phase, MIB1 or DNA ploidy as well as to established prognostic factors such as tumor size, grade, hormone receptor or menopausal status (1 1 ). It now also seems beyond reasonable doubt that uPA-catalysed plasminogen activation plays not only a prognostic role but also a causal role in tumor growth, invasion and metastasis (10). Efforts to inhibit either the proteolytic activity of uPA directly or uPA^'s association to uPAR reduces the invasiveness of tumour cells in murine models of cancer in vivo (2). It therefore initially came as a surprise that uPA^'s inhibitor PAI-1 is a prognostic marker of tumour invasion and angiogenesis as well (12). It is today, however, realised that the uPA-system has additional non-proteolytic related activities which play important roles in cancer dissemination. Apart from inhibiting uPA, PAI-1 also interacts with the ECM protein vitronectin (10) and with endocytosis receptors of the low density lipoprotein receptor family (14), but the exact tumour biological function of PAI-1 is still not fully understood. Recent studies have also found that uPAR not only localises the proteinase to the surface, but that the mere binding also affects migration and proliferation by inducing intracellular signalling and interactions with ECM proteins and integrins. Though many aspects about the exact mechanism of the uPA-system, its components are highly attractive targets for anticancer therapy. Additionally, uPA and PAI-1 are often produced by the non-malignant stromal cells arguing for that therapeutics directed towards these proteins may not be as prone to development of resistance (17). uPAR, uPA and PAI-1 are also candidates for directing imaging agents to tumour cells. Upon inhibition of uPAR-bound uPA by PAI-1 , the three component complex is rapidly endocytosed, uPA and PAI-1 degraded and uPAR recycled to the cell surface, lnternalisation of membrane-proteins has previously been exploited to deliver siRNA (15) and toxins (16) to cancer cells using aptamers, suggesting that targeting uPA, PAI- 1 and uPAR with radiolabeled aptamers could potentially lead to uptake of radioactivity by cancer cells efficiently retaining signals in tumour tissue compared to normal tissue.

Summary of invention

The present invention relates to aptamer and use thereof for thereaputic and diagnostic use.

In one aspect the present invention relates to an aptamer capable of binding a receptor-binding form of urokinase-type plasminogen activator protein u-PA,

The aptamer of the present invention is a ribonucleic acid aptamer that may comprise at least one chamical modification which can take a number of forms such as agents for labelling, drugs, cytotoxic and toxic agents among others. The aptamers of the present invention prevents the binding of a receptor binding form of u-PA to a u-PA receptor (u-PAR), whereby u-PA-mediated cell-associated plasminogen activation is prevented.

Thus, in second aspect the present invention pertains to a use of the prevention of u- PA-mediated proteolytic activity at the cell surface for preparing a composition for preventing or counteracting proteolytic activity at the cell surface of a mammal.

However, in a third aspect, the invention relates to use of the aptamer for preventing the binding of a receptor-binding form of u-PA to a u-PAR in a mammal for the preparation of a composition for preventing the binding of u-PA to a uPAR.

Upon formation of a uPA,-uPAR -PAI-1 complex the complex is often internalised and the u PAR receptor recycled. Thus, a fourth aspect relates to the use of the aptamer for preventing a the binding of a receptor-binding form of uPA to a uPAR in a mammal for the preparation of a composition for preventing the internalisation of a uPA- PAI-1 - uPAR complex.

A fifth aspect relates to the use of the aptamer as an agent for imaging, for example for locating uPA. The imaging may be performed by a number of techniques as described herein.

A sixth aspect pertains to a use of the aptamer for the treatment of cancer and/or metastatic spread of the cancer in a mammal in need thereof. Furthermore, the invention relates in a seventh aspect to a use of the aptamer as a medicament for a cancer.

Another aspect relates to the use of the aptamer for characterising a tumor.

In another aspect the invention relates to a pharmaceutical composition comprising an aptamer as defined in the present invention or a pharmaceutically acceptable salt thereof, carrier, diluent or adjuvant. In yet another aspect the invention relates to a pharmaceutical composition for treating cancer and/or metastatic activity comprising an aptamer.

A further aspect relates to a compositition comprising an aptamer.

In yet another aspect the aptamer of the present invention is used as a medicament.

A further aspect pertains to diagnostic kit comprising an aptamer as defined in the present invention.

Yet a further aspect related to a method of diagnosing a disease comprising applying the aptamer of the present invention and detecting the presence or absence of said aptamer.

Description of Drawings

Figure 1 shows the serine protease domain of uPA. A cartoon diagram of the SPD and the catalytic residues, His204(57), Asp255(102) and Ser356(195). See the text for details (PDB: 1 LMW (Spraggon et al., 1995)).

Figure 2 is a schematic drawing of the serine protease proteolytic mechanism. A-C. The acylation step. D-F. The deacylation step. See the text for details. The figure is a modified version of the one found in Stryer, 1995

Figure 3 shows the complex between the amino terminal fragment of uPA and the uPA receptor, uPAR. uPAR consists of three domains (surface presentation; domain I in green, residues 1 -92; domain Il in yellow, residues 93-191 ; and domain III in blue, residues 192-283), which together form the binding pocket for uPA. The amino terminal fragment is illustrated in cartoons with the kringle domain in magenta and the growth factor domain in red. The red arrow indicates the position of the Ω-loop, which is buried in the uPAR structure (PDB: 2FD6 (Huai et al., 2006)).

Figure 4 shows a schematic overview of the plasminogen activation system. uPA and tPA catalyse the conversion of the inactive zymogen plasminogen to plasmin, and are inhibited by PAI-1 , PAI-2 and PN-1 . Once generated, plasmin is able to degrade ECM proteins and activate MMPs. The activity of plasmin is directly inhibited by Q₂-AP. The figure also illustrates the domain composition of the different proteins (see the text for details).

Figure 5 shows a representation of the RNA aptamer selection procedure. By transcription of a DNA library a large RNA library is generated. The RNA library is applied in a selection experiment towards a target, in this case uPA. RNA sequences with affinity towards the target are harvested and subjected to reverse transcription followed by PCR to generate dsDNA products. The dsDNA products are transcribed to generate a pool of RNA enriched in sequences with affinity towards the target and applied in a new round of selection. When sufficient enrichment has occurred, the sequences of the dsDNA products can be obtained to reveal the sequence of the RNA aptamers.

Figure 6 shows the RNA library preparation and amplification of recovered RNA after a selection round. For preparation of the initial library, equimolar amounts of the "N35 library" and "Forward N35" oligonucleotides are annealed and extended using the Klenow enzyme. A double-stranded DNA product is formed that serves as a transcription template in the presence of T7 RNA polymerase resulting in the generation of the RNA library. After a selection round, recovered RNA is reverse transcribed using the "Reverse N35" oligonucleotide and dsDNA products obtained by PCR using both "Forward N35" and "Reverse N35" oligonucleotides. By BamHI- digestion of the dsDNA products the correct 3^'- terminus of the dsDNA templates are formed and the templates can be used to generate an amplified pool of the recovered RNA. The figure is adapted from Kenan and Keene, 1999.

Figure 7 shows enrichment of RNA binding to uPA-Ab-beads. RNA pools applied in selection round 2-8 were internally labelled with [α-³²P]-dATP allowing detection using scintillation counting. The percentage of [α-³²P]-dATP-labelled RNA eluted from the uPA-Ab-beads is shown compared to the amount of [α-³²P]-dATP-labelled RNA that was applied in the different selection rounds. In round 2 to 5, 500 nM of RNA was applied in the selection procedure and 100 nM in round 6 to 8. Also, there was a difference in the mode of RNA purification for round 2-5 and 6-8 as described concerning the selection of 2^'-F-pyrimidine RNA aptamers towards PAI-1 above.

Figure 8 shows the screening the 29 individual RNA clones for binding to uPA by SPR analysis. A. Rabbit polyclonal anti-PAI-1 antibody (pAb) was immobilised on a SPR- sensor chip and uPA captured on the surface. Passing over RNA allowed identification of RNA clones capable of associating to the uPA-containing sensor surface. B. Injection of aptamer koloi 2 over the sensor surface gave a detectable response whereas a non-relevant clone did not.

Figure 9 shows no binding of RNA clones to uPAR-bound uPA as indicated by SPR analysis. A. An illustration of the SPR experiment. Human uPA receptor was immobilised covalently on a CM5 chip and uPA captured on the sensor surface. RNA binding to uPAR-bound uPA was subsequently studied by injecting RNA samples over the sensor surface. B. The sensorgram obtained when studying the interaction of RNA kolo12 with uPAR-bound uPA. No detectable association was seen for this clone and the same result was obtained when passing the remaining 28 RNA clones over the sensor surface.

Figure 10 shows that the RNA clone koloi 2 inhibits the uPA -uPAR interaction. A. An illustration of the SPR experiment. Human uPA receptor was immobilised covalently on a CM5 chip. The capture of uPA was investigated in the presence of RNA clones. B. The sensorgram when capturing uPA on the sensor surface in the presence of increasing concentrations of RNA clone kolo12. C. The experiment was repeated six times with koloi 2 and a non-relevant RNA clone and the relative uPA capture level plotted as a function of the RNA concentration.

Figure 1 1 shows the binding of RNA kolo12 to different forms of uPA. The different forms were captured on a CM5 sensor chip coupled with rabbit polyclonal anti-uPA antibody and RNA kolo12 subsequently passed over the sensor surface. The relative responses obtained for kolo12 per mole uPA variant captured. The figure shows the mean and standard deviations of three independent experiments.

Figure 12 shows the effect of kolo12 on uPA-mediated peptide hydrolysis. uPA (2 nM) was pre-incubated with PAI-1 (40 nM), kolo12 (100 nM) or the upain-1 peptide (50 μM) and the ability of uPA to subsequently hydrolyse the small peptidolytic substrate, S- 2444, measured for 50 minutes at 37 ^eC. The figure shows the result of one of three independent experiments with identical results. Figure 13 shows the effect of kolo12 on uPA-mediated plasminogen activation in solution. uPA (0.5 nM) was pre-incubated with or without PAI-1 (40 nM), kolo12 (250 nM), upain-1 (50 μM), or suPAR (250 nM) for 10 minutes at 37 ^eC before the addition of plasminogen (200 nM) and a plasmin substrate. The figure shows the result of one of three independent experiments with identical results.

Figure 14 shows KoIoI 2 and truncated versions. Shown are the discussed asymmetrical internal loop and hairpin loop (green circles). When truncating from the 5^'- and 3^'-end kolo12.49 (red rectangle) and kolo12.33 (blue rectangle) are generated. The secondary structures have been obtained using the MFOLD program (Mathews et al., 1999; Zuker, 2003).

Figure 15 shows KoIoI 2, Kolo21 , Kolo79, KoIoI 2trunc2, KoIoI 2trunc1. The secondary structures have been obtained using the MFOLD program (Mathews et al., 1999; Zuker, 2003).

Figure 16 shows truncated RNA variants based on kolo12.33 and kolo21.35. KoIoI 2.33ntGG contains a GG-overhang compared to the original kolo12.33 (figure 34). Kolo12.33ntEND and kolo21.35ntEND differ from the original truncated variants by having GG substituted into the 5^'-END concomitantly with a CC substitution in the 3^'- END to retain the duplexes. The modifications have been made to increase transcription efficiency.

Figure 17. KoIoI 2 prevents the association of ¹²⁵l-labelled human ATF to U937 cells. A. An illustration of the experiment. RNA and ATF was mixed and incubated with U937 cells overnight at 4 ^eC. B. Bound and free ¹²⁵l-labelled human ATF was then estimated by scintillation counting and bound over free plotted as a function of the concentration of RNA. The figure shows the mean and standard deviations of three independent experiments.

Figure 18 shows that Kolo12.49ntEND prevents the association of pro-uPA to U937 cells. Pro-uPA (0.5 nM) with or without RNA or soluble uPAR as indicated in the figure was incubated with U937 cells for 30 minutes at room temperature. After washing the cells, bound pro-uPA was detected by adding plasminogen and a plasmin substrate. Substrate cleavage was observed for 60 minutes at 37 degrees. The signal obtained with cells without addition of pro-uPA has been subtracted. The figure shows the result of one of three independent experiments with identical results.

Figure 19 shows that Kolo12.49ntEND prevents uPA-mediated cell-surface associated plasminogen activation. Cells, plasminogen and α₂-antiplasmin were mixed and pre- incubated for 15 minutes at 37 ^eC before the addition of uPA and a plasmin fluorogenic substrate in the presence or not of kolo12.49ntEND or suPAR. Substrate cleavage was observed for 60 minutes at 37 degrees. The figure shows the result of one of three independent experiments with identical results.

Figure 20 shows that KoIoI 2.49ntEND binds to uPA in complex with PAI-1 and inhibits cell-mediated endocytosis and degradation of the uPA-PAI-1 complex. A. A sensor surface was coupled with polyclonal anti-PAI-1 antibody. The uPA-PAI-1 complex was captured by running first PAI-1 (HT-1080) and then uPA over the sensor surface. Association of koloi 2 to the sensor surface could subsequently be detected. The sensorgram shown is one of two experiments with identical results. B. The ability of U937 cells to endocytose and degrade ¹²⁵l-labelled uPA-PAI-1 complex was investigated in the presence of kolo12.49ntEND, control RNA or uPA. The figure shows the mean and standard deviations of three independent experiments.

Figure 21 shows the stability of unmodified and 2^'-F-pyrimidine kolo12 variants in 80% serum. Unmodified or 2^'-F-pyrimidine containing kolo12 and kolo12.49ntEND was incubated in 80% serum as indicated in the figure. The concentration of RNA in the incubations was 80 μg/mL for the koloi 2 clones and 100 μg/mL for the kolo12.49ntEND clones.

Figure 22 shows the stability of 2^'-F-pyrimidine kolo12 and kolo12.33ntEND in 10% serum. KoIoI 2 (lane 1 , 3, 5, 7) and kolo12.33ntEND (lane 2, 4, 6, 8) were incubated in 10% serum for 14 hours at 37 ^eC (lane 1 , 2), 25 ^eC (lane 3, 4) and 4 ^eC (lane 5, 6). As a control RNA clones incubated for 14 hours at 4 ^eC without addition of serum was included (lane 7, 8).

Figure 23 shows secondary structure predictions and sequences of the aptamers of the present invention. Secondary structure predictions for RNA clones obtained from the RNA aptamer selection experiment towards human uPA. Detailed description of the invention Aptamers

The present invention relates to aptamers capable of binding a receptor-binding form of urokinase-type plasminogen activator protein u-PA and uses thereof.

Aptamers are nucleic acid molecules having specific binding affinity to molecules through interactions other than classic Watson-Crick base pairing. Aptamers are chemically synthesized short strands of nucleic acid that adopt specific three- dimensional conformations and are selected for their affinity to a particular target through a process of in vitro selection referred to as systematic evolution of ligands by exponential enrichment (SELEX). SELEX is a combinatorial chemistry methodology in which vast numbers of oligonucleotides are screened rapidly for specific sequences that have appropriate binding affinities and specificities toward any target. Using this process, novel aptamer nucleic acid ligands that are specific for a particular target may be created.

In some embodiments, the capped aptamers are RNA aptamers, DNA aptamers, or aptamers having a mixed (i. e. both RNA and DNA) composition or derivatives of these. The aptamers of the present invention are aptamers directed against a receptor- binding form of u-PA. The aptamer is a ribonucleic acid, and the aptamer is a single stranded ribonucleic acid, although segments of it forms internal double stranded segments.

Aptamers, like peptides generated by phage display or monoclonal antibodies ("mAbs"), are capable of specifically binding to selected targets and modulating the target's activity, e. g., through binding aptamers may block their target's ability to function. Created by an in vitro selection process from pools of random sequence oligonucleotides, aptamers have been generated for over 100 proteins including growth factors, transcription factors, enzymes, immunoglobulins, and receptors. A typical aptamer according to the literature is 10-15 kDa in size (30-45 nucleotides or longer), binds its target with 0.1 to 10 nM or sub-nanomolar affinity, and discriminates against closely related targets (e. g., aptamers will typically not bind other proteins from the same gene family). A series of structural studies have shown that aptamers are capable of using the same types of binding interactions (e.g., hydrogen bonding, electrostatic complementarities, hydrophobic contacts, steric exclusion) that drive affinity and specificity in antibody-antigen complexes.

A ribonucleic acid or RNA is a nucleic acid polymer consisting of nucleotide monomers. The monomers are ribonucleotides, a nucleotide in which a purine or pyrimidine base is linked to a ribose molecule. The base may be adenine (A), guanine (G), cytosine (C), or uracil (U). Thymine (T), which is found in deoxyribonucleotides, is not found as a ribonucleotides in living beings. Ribonucleotides have one, two, or three phosphate groups attached to the ribose sugar. The ribonucleotides can be modified by including chemical structures on the bases, sugars and/or phosphate backbone found in the ribonucleotides. For example, Gm represents 2'- methoxyguanylic acid, Am represents 2'-methoxyadenylic acid, Cf represents 2'-fluorocytidylic acid, Uf represents 2'-fluorouridylic acid, Ar, represents riboadenylic acid. The aptamer includes cytosine or any cytosine-related base including 5-methylcytosine, 4-acetylcytosine, 3-methylcytosine, 5- hydroxymethyl cytosine, 2- thiocytosine, 5-halocytosine (e. g. , 5-fluorocytosine, 5-bromocytosine, 5- chlorocytosine, and 5-iodocytosine), 5-propynyl cytosine, 6-azocytosine, 5- trifluoromethylcytosine, N4, N4-ethanocytosine, phenoxazine cytidine, phenothiazine cytidine, carbazole cytidine or pyridoindole cytidine. The aptamer further includes guanine or any guanine-related base including 6- methylguanine, 1 -methylguanine, 2,2-dimethylguanine, 2-methylguanine, 7-methylguanine, 2- propylguanine, 6- propylguanine, 8-haloguanine (e. g. , 8-fluoroguanine, 8-bromoguanine, 8- chloroguanine, and 8-iodoguanine), 8-aminoguanine, 8-sulfhydrylguanine, 8- thioalkylguanine, 8- hydroxylguanine, 7-methylguanine, 8-azaguanine, 7-deazaguanine or 3-deazaguanine. The aptamer further includes adenine or any adenine-related base including 6-methyladenine, N6- isopentenyladenine, N6-methyladenine, 1 - methyladenine, 2-methyladenine, 2-methylthio-N6- isopentenyladenine, 8-haloadenine (e. g. , 8-fluoroadenine, 8-bromoadenine, 8-chloroadenine, and 8- iodoadenine), 8- aminoadenine, 8-sulfhydryladenine, 8-thioalkyladenine, 8-hydroxyladenine, 7- methyladenine, 2-haloadenine (e. g., 2-fluoroadenine, 2-bromoadenine, 2- chloroadenine, and 2- iodoadenine), 2-aminoadenine, 8-azaadenine, 7-deazaadenine or 3-deazaadenine. Also included is uracil or any uracil-related base including 5- halouracil (e. g., 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil), 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouracil, 5- carboxymethylaminomethyluracil, dihydrouracil, 1 -methylpseudouracil, 5- methoxyaminomethyl-2-thiouracil, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 5- methyl-2- thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5- oxyacetic acid, pseudouracil, 5-methyl-2-thiouracil, 2-thiouracil, 3- (3-amino-3-N-2- carboxypropyl) uracil, 5-methylaminomethyluracil, 5-propynyl uracil, 6- azouracil, or 4-thiouracil. Examples of other modified base variants known in the art include, without limitation, for example 4-acetylcytidine, 5- (carboxyhydroxylmethyl) uridine, 2'-methoxycytidine, 5-carboxymethylaminomethyl-2-thioridine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2'- O-methylpseudouridine, β-D-galactosylqueosine, inosine, N6- isopentenyladenosine, 1 - methyladenosine, 1 -methylpseudouridine, 1 - methylguanosine, 1 -methylinosine, 2,2- dimethylguanosine, 2-methyladenosine, 2- methylguanosine, 3-methylcytidine, 5-methylcytidine, N6- methyladenosine, 7- methylguanosine, 5-methylaminomethyluridine, 5-methoxyaminomethyl-2- thiouridine, P-D-mannosylqueosine, 5-methoxycarbonylmethyluridine, 5-methoxyuridine, 2- methylthio-N6-isopentenyladenosine, N-((9-β-D-ribofuranosyl-2-methylthiopurine-6- yl) carbamoyl) threonine, N- ( (9-p-D-ribofuranosylpurine-6-yl) N-methyl-carbamoyl) threonine, urdine-5- oxyacetic acid methylester, uridine-5-oxyacetic acid (v), wybutoxosine, pseudouridine, queosine, 2- thiocytidine, 5-methyl-2-thiouridine, 2- thiouridine, 4-thiouridine, 5-methyluridine, N- ( (9-p-D- ribofuranosylpurine-6-yl) carbamoyl) threonine, 2'-O-methyl-5-methyluridine, 2'-O-methyluridine, wybutosine, 3- (3-amino-3-carboxypropyl) uridine. Nucleotides also include any of the modified nucleobases described in U. S. Patent Nos. 3,687, 808,3, 687,808, 4,845, 205,5, 130,302, 5,134, 066, 5,175, 273,5, 367,066, 5,432, 272,5, 457,187, 5,459, 255,5, 484,908, 5,502, 177,5, 525,71 1 , 5,552, 540, 5,587, 469,5, 594,121 , 5,596, 091 ,5, 614,617, 5,645, 985,5, 830,653, 5,763, 588,6, 005,096, and 5,681 , 941. Examples of modified nucleoside and nucleotide sugar backbone variants known in the art include, without limitation, those having, e. g., 2'ribosyl substituents such as F, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SOz, CH3, ONO2, NO2, N3, NH2 OCH2CH2OCH3, O (CH2) 2ON (CH3) 2, OCH2OCH2N (CH3) 2, O (C1 -10 alkyl), O(C2-10 alkenyl), O (C2 IO alkynyl), S (CM O alkyl), S(C2-10 alkenyl), S (C2-lo alkynyl), NH (CI-10 alkyl), NH (C2 IO alkenyl), NH (C2 IO alkynyl), and O-alkyl-0-alkyl.

In one embodiment of the present invention the aptamers comprise at least one chemical modification in the form of locked nucleic acids (LNA) which are known to a person skilled in the art. In one embodiment,the aptamers comprise a nucleotide analogue. The use of nucleotide analogues can be used to create a molecule which forms an RNA like structure and/or functions like an RNA molecule within the context of the invention whilst comprising fewer RNA molecules than the passenger strand of an equivalent unmodified siRNA, including few or even no actual RNA monomers. Thus, the aptamers of the present invention may comprise in the range of 1 -150 LNA monomers, such as 1 to 100 monomers, 20-80, 10 to 70, 10 to 50, or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43,44, 45, 46, 47, 48, 49 or 50 LNA monomers. It is also within the scope that the aptamers may be a combination of 2'F- pyrimidine ribonucleotides and LNA monomers .In one embodiment the passenger strand comprises nucleobase units which are only LNA and RNA, such as alternating LNA and RNA units.

Combination of LNA and other nucleotide analogues such as 2'OMethyl (2¹OMe) and 2'fluor[omicron] (2¹F) are also considered as possible designs for the aptamers of the present invention. For example alternating LNA and 2'OMethyl (2¹OMe), alternating

LNA and 2'fluoro, alternating 2'OMethyl (2¹OMe) and 2'fluoro. Strands rich in analogues are considered especially useful when considering RNA complexes which otherwise have a low Tm. An additional advantage of such analogue rich "RNA¹ molecules and strands is that they can be used to provide a highly stable and nuclease resistant molecule in vivo, even when the internucleoside linkages are or include linkages which otherwise would be nuclease sensitive, such as phosphodiester or phosphate linkages. Aptamers which comprise primarily or completely phosphodiester linkages may be preferable as they typically show a lower toxicity at high dosages as compared to equivalent molecules with phosphorothioate linkages.

However, in some embodiments, such as when using RNA molecules or strands which have a high RNA content it may be preferable to use nuclease resistant linkages such as phosphorothioate linkages. The internucleoside linkage may be selected form the group consisting of: -0-P(O)2-O-, -O-P(O,S)-O-, -0-P(S)2-O-, -S-P(O)2-O-, -S-P(O7S)- O-, -S-P(S)2-O-, -0-P(O)2-S-, -OP(OI S)- S-, -S-P(O)2-S-, -O-PO(R<H>)-O-, 0-

PO(0CH3)-O-, -O-PO(NR<H>)-O-, -0-PO(OCH2CH2S-R)-O-, -0-PO(BHs)-O-, -O- P0(NHR<H>)-0-, -O-P(0)2-NR<H>-, -NR<H>-P(O)2-O-, -NR<H>-CO-O-, -NR<H>-CO- NR<H>-, and/or the internucleoside linkage may be selected form the group consisting of: -0-C0-0-, -0~CO-NR<H>-, -NR<H>-CO-CH2-, -O-CH2-CO-NR<H>-, -O-CH2-CH2- NR<H>-, -CO-NR<H>-CH2-, -CH2-NR<H>- CO-, -0-CH2-CH2-S-, -S-CH2-CH2-O-, -S- CH2-CH2-S-, -CH2-SO2-CH2-, -CH2-CO-NR<8>-, -O- CH2-CH2-NR<H>-CO -, -CH2- NCH3-O-CH2-, where R<H> is selected from hydrogen and C1 -4-alkyl, Suitably, in some embodiments, sulphur (S) containing internucleoside linkages as provided above may be preferred

The aptamers of the present invention are made using double stranded DNA transcription templates for the synthesis of RNA oligonucleotides by use of the T7 RNA polymerase. Aptamers produced synthetically are also within the scope of the present invention.

When referring to the length of a an aptamer as referred to herein, the length corresponds to the number of monomer units, i.e. nucleobases, irrespective as to whether those monomer units are nucleotides or nucleotide analogues The aptamers of the present invention are ribonucleic acids containing in the range of 10 to 150 ribonucleotides, such as in the range of 50 to 100 nucleotides, for example in the range of 10 to 50 nucleotides, or such as in the range of 25 to 50 nucleotides. The aptamers of the present invention comprises 150, 149, 148, 147, 146, 145, 144, 143, 142, 141 , 140, 139, 138, 137, 136, 135, 134, 133, 132, 131 , 130, 129, 128, 127, 126, 125, 124, 123, 122, 121 , 120, 1 19, 1 18, 1 17, 1 16, 1 15, 1 14, 1 13, 1 12, 1 1 1 , 1 10, 109, 108, 107, 106, 105, 104, 103, 102, 101 , 100, 99, 98, 97, 96, 95, 94, 93, 92, 91 , 90, 89, 88, 87, 86,

85, 84, 83, 82, 81 , 80, 79, 78, 77, 76, 75, 74, 73, 72, 71 , 70, 69, 68, 67, 66, 65, 64, 63, 62, 61 , 60, 59, 58, 57, 56, 55, 54, 53, 52, 51 , 50, 49, 48, 47, 46, 45, 44, 43, 42, 41 , 40, 39, 38, 37, 36, 35, 34, 33, 32, 31 , 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , or 10 ribonucleotides.

The aptamer has in the absence of a receptor-binding form of u-PA intramolecularly, mutually complementary sequences of four or more consecutive nucleotides and has a stem-loop structure. The aptamer structure is dependent on the ribonucleotides of the aptamer and the complementarity of the ribonucleotides upon folding into a secondary/tertiary structure.

In the context of the present invention "complementary" refers to the capacity for precise pairing between two nucleotides within an RNA aptamer. Further, in the present invention, "complementary" and "complementary base pair formation" refers to pairing between nucleobases: adenine and uracil, and guanine and cytosine by a hydrogen bond. Examples of nucleobases capable of complementary base pair formation include the combinations of adenine and uracil, and guanine and cytosine. For example, if a nucleotide at a certain position of a ribonucleotide is capable of hydrogen bonding with a ribonucleotide within the RNA molecule, then the ribonucleotides are considered to be complementary to each other at that position. The ribonucleotides or regions of the aptamer need not to be 10O percent complementary to other regions. In regions where complementariy exist between ribonucleotides the possibility of the formation of stem structures exists. If no complementarity exists between regions within the aptamer then the possibility of the formation of bulge regions or loops exists. The complementary sequences may be over a stretch of 5 consecutive ribonucleotides, 6 consecutive ribonucleotides, 7 consecutive ribonucleotides, 8 consecutive ribonucleotides, 9 consecutive ribonucleotides, 10 consecutive ribonucleotides, 1 1 consecutive ribonucleotides, 12 consecutive ribonucleotides, 13 consecutive ribonucleotides, 14 consecutive ribonucleotides, 15 consecutive ribonucleotides, 16 consecutive ribonucleotides, 17 consecutive ribonucleotides, 18 consecutive ribonucleotides, 19 consecutive ribonucleotides or 20 consecutive ribonucleotides, respectively.

In one embodiment of the invention, the aptamer has a structure comprising at least one loop. At least one bulge region is also present. The number of bulge regions can vary such that two bulge regions, three bulge regions or for example four bulge regions are found in the aptamer. The structure of the aptamer also comprises at least one stem structure, such as two stem structures, for example three stem structures, such as five stem structures, for example six stem structures, such as seven stem structures.

In one embodiment of the present invention the aptamer comprises the general formula 5'-A-B-C-D-E-C'-A'-3', where A and A' interact to form a stem structure, C and C interact to form a stem structure, B and E interact to form a bulge region and D is a loop. However, the aptamer also comprises the general formula 5'-A-B-C-D-E-F-E'-G- C'-H-A'-3', where A and A' interact to form a stem structure, C and C interact to form a stem structure, E and E' interact to form a stem structure, B and H form a bulge region, and D and G form a bulge region, and F forms a loop.

The aptamer comprises at least one of the following sequences as shown below in a variable region or a variant thereof being at least 70 % identical to one of said sequences, such as at least 75 % identical to one of said sequences, such as at least 80 % identical to one of said sequences, such as at least 85 % identical to one of said sequences, such as at least 90 % identical to one of said sequences, such as at least 91 % identical to one of said sequences, such as at least 92 % identical to one of said sequences such as at least 93 % identical to one of said sequences such as at least 94 % identical to one of said sequences such as at least 95 % identical to one of said sequences, such as at least 96 % identical to one of said sequences, such as at least 97 % identical to one of said sequences, such as at least 98 % identical to one of said sequences, such as at least 99 % identical to one of said sequences: kolo31 ACTCCTCGGCGCAAGGATGTGGG-ATCGATGCAATC 35 kolo32 AACTAGAACCATACTCGCCGGCGCCAATGTCGTAG 35 kolol2 —TGCGACTGTTATAACCTAACAGCGACGTAAAG-ATA 35xx kolol4 —TGCGACTGTTATAACCTAACAGCGACGTAAAG-ATA 35 kolo66 —TGCGACTGTTATAACCTAACAGCGACGTAAAG-ATA 35xx kolo87 —TGCGACTGTTATAACCTAACAGCGACGTAAAG-ATA 35 kolo79 GAAACGACTCG-ACAACCT-CGAGCGACGTGAATCAT 35xx kolo84 -TACCGACTAGCAAAACCTGCTGGCGACGTTTAG-AT 35 x kolo21 AATAACCTTAATGCGACGTTGGTTTGTCAACAACG 35xx kolo22 AATAACCTTAATGCGACGTTGGTTTGTCAACAACG 35 kolo29 AATAACCTTAATGCGACGTTGGTTTGTCAACAACG 35 kolo76 AATAACCTTAATGCGACGTTGGTTTGTCAACAACG 35 kolo77 AATAACCTTAATGCGACGTTGGTTTGTCAACAACG 35 kolo74 AATAACCTTAATGCGACGTTGGTTTGTCAACAACG 35xx kolo71 AATAACCTTAATGCGACGTTGGTTCGTCAACATGG 35xx kolo28 ACAACCTAAATGCGACGTTGGGTCAAAAACGTGAA 35xx kolo25 GATAACCTCGATGCGACGTTCGGCC-TCAAAATCAA 35xx kolo78 ACAAC-TTAATGCGACGTTGGTAAAGCATATCAAAC 35 x kolo26 CTCCGCTATCTAACGTATGATA-GAATGGATGACTA 35xx kolo83 TGCGCATGAAATGACTGCATGTCTCCGGATTGATC— 35 kolo68 ATGACAGGATGCAGAGCTCCACTGTCTAGTGTTTA 35 x kolo24 CCGCGACAGTCGTAAGTTTTGACTGACTGAACGTT 35 x kolo88 TCGCTAATTATAGGCGGAGTGCGACGTTATAAATA 35 x kolo9 AGTGATTCGCCATAACC-TGGCTGTTTCAGGCTGTT 35 kolo69 AGTGATTCGCCATAACC-TGGCTGTTTCAGGCTGTT 35 kolo85 AGTGATTCGCCATAACC-TGGCTGTTTCAGGCTGTT 35 kolo75 TCGCATTCGTTATAACC-TAACAGTTTGCGGA-GTTA 35 kolol7 TAGTACGACC-TACTTATTTGAC 22 kolo73 TTGCACAACC-TGCATATTTGTCGTCGACGTACGAA 35 kolo82 CGATACAACC-TATCTGTTTGTCGTGGACATCAAAT 35 kolo67 TGCTATGACC-TAGACATTTGACG 23 kolo86 TACCATAACC-TGGATATCTGTCGTTATCATGGGAC 35 kolo20 TTGCGTAACCCGCAATTTCCGTTGGTATATAC 32 kolo70 ACCTGACTTGCGATAACC-TCGACATTTACGGTATT 35 kololO TAATGGCCGGTTTAACCGTT-TGCGCATTAAGTCGA 35 kolo89 TAATGGCCGGTTTAACCGTT-TGCGCATTAAGTCGA 35 kolol5 TACCATTTACCACAACC-TGGTTATCTGGTTTAAAC 35 kolo27 TTGTCGTCTTCTCCAGT—TAGTCAACGAACTTGTGT 35xx kolol9 TCCGTTTGCAGGTTTTGACCTGCCGGAGTTGTTTG 35 kolo81 GGCAGAAGTTGATTCCAACTTCATTTGCGTTTAAT 35 kolo80 ATATTTGTCTCATCCCACGACAATTATGATGCGAC 35 kololl ATCTTAGCGACGTGACACACGACTAGGGATTAATC 35

However, the aptamer comprises at least one of the following sequences as shown below in a variable region or a variant thereof being at least 70 % identical to one of said sequences, such as at least 75 % identical to one of said sequences, such as at least 80 % identical to one of said sequences, such as at least 85 % identical to one of said sequences, such as at least 90 % identical to one of said sequences, such as at least 91 % identical to one of said sequences, such as at least 92 % identical to one of said sequences such as at least 93 % identical to one of said sequences such as at least 94 % identical to one of said sequences such as at least 95 % identical to one of said sequences, such as at least 96 % identical to one of said sequences, such as at least 97 % identical to one of said sequences, such as at least 98 % identical to one of said sequences, such as at least 99 % identical to one of said sequences: kolol2 (4) —UGCGACUGUUAUAACCUAACAGCGACGUAAAG-AUA 35 kolo79 GAAACGACUCG-ACAACCU-CGAGCGACGUGAAUCAU 35 kolo84 -UACCGACUAGCAAAACCUGCUGGCGACGUUUAG-AU 35 kolo21(6) AAUAACCUUAAUGCGACGUUGGUUUGUCAACAACG 35 kolo71 AAUAACCUUAAUGCGACGUUGGUUCGUCAACAUGG 35 kolo28 ACAACCUAAAUGCGACGUUGGGUCAAAAACGUGAA 35 kolo25 GAUAACCUCGAUGCGACGUUCGGCC-UCAAAAUCAA 35 kolo78 ACAAC-UUAAUGCGACGUUGGUAAAGCAUAUCAAAC 35 kolo26 CUCCGCUAUCUAACGUAUGAUA-GAAUGGAUGACUA 35 kolo83 UGCGCAUGAAAUGACUGCAUGUCUCCGGAUUGAUC— 35 kolo68 AUGACAGGAUGCAGAGCUCCACUGUCUAGUGUUUA 35 kolo24 CCGCGACAGUCGUAAGUUUUGACUGACUGAACGUU 35 kolo88 UCGCUAAUUAUAGGCGGAGUGCGACGUUAUAAAUA 35 kolo9 (3) AGUGAUUCGCCAUAACC-UGGCUGUUUCAGGCUGUU 35 kolo75 UCGCAUUCGUUAUAACC-UAACAGUUUGCGGA-GUUA 35 kolol7 UAGUACGACC-UACUUAUUUGAC 22 kolo73 UUGCACAACC-UGCAUAUUUGUCGUCGACGUACGAA 35 kolo82 CGAUACAACC-UAUCUGUUUGUCGUGGACAUCAAAU 35 kolo67 UGCUAUGACC-UAGACAUUUGACG 23 kolo86 UACCAUAACC-UGGAUAUCUGUCGUUAUCAUGGGAC 35 kolo20 UUGCGUAACCCGCAAUUUCCGUUGGUAUAUAC 32 kolo70 ACCUGACUUGCGAUAACC-UCGACAUUUACGGUAUU 35 kololO (2) UAAUGGCCGGUUUAACCGUU-UGCGCAUUAAGUCGA 35 kolol5 UACCAUUUACCACAACC-UGGUUAUCUGGUUUAAAC 35 kolo27 UUGUCGUCUUCUCCAGU—UAGUCAACGAACUUGUGU 35 kolol9 UCCGUUUGCAGGUUUUGACCUGCCGGAGUUGUUUG 35 kolo81 GGCAGAAGUUGAUUCCAACUUCAUUUGCGUUUAAU 35 kolo80 AUAUUUGUCUCAUCCCACGACAAUUAUGAUGCGAC 35 kololl AUCUUAGCGACGUGACACACGACUAGGGAUUAAUC 35

The terms "sequence identity" in the context of two or more nucleic acid or protein sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981 ), by the homology alignment algorithm of Needleman & Wunsch, J. MoI. Biol. 48: 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Ausubel et al., infra).

One example of an algorithm that is suitable for determining percent sequence identity is the algorithm used in the basic local alignment search tool (hereinafter "BLAST"), see, e.g. Altschul et al., J. MoI. Biol. 215: 403-410 (1990) and Altschul et al., Nucleic Acids Res., 15: 3389-3402 (1997). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (hereinafter "NCBI"). The default parameters used in determining sequence identity using the software available from NCBI, e.g., BLASTN (for nucleotide sequences) and BLASTP (for amino acid sequences) are described in McGinnis et al., Nucleic Acids Res., 32: W20-W25 (2004).

However, in another embodiment the aptamer is selected from the group consisting of KoIoI 2, Kolo21 , Kolo24, Kolo25, Kolo26, Kolo27, Kolo28, K0I068, Kolo71 , Kolo78, Kolo79, Kolo84, K0I088, KoIoI 2.33ntEND, KoIoI 2.33ntGG, Kolo21 .35ntEND, KoIoI 2.49ntEND, Kolo79.44nt, Kolo79.44ntEND1 , Kolo79.44ntEND2, Kolo79.34nt and Kolo79.48ntfunny. In a further embodiment the aptamer is selected from the group consisting of KoIoI 2, Kolo21 , Kolo25, Kolo26, Kolo27, Kolo28, Kolo71 , Kolo79, KoIoI 2.33ntEND, KoIoI 2.33ntGG, Kolo21.35ntEND, KoIoI 2.49ntEND, Kolo79.44nt, Kolo79.44ntEND1 , Kolo79.44ntEND2, kolo79.34nt and Kolo79.48ntfunny.

In another embodiment the aptamer is selected from the group consisting of KoIoI 2, Kolo21 , Kolo25, Kolo26, Kolo71 , Kolo79, KoIoI 2.49ntEND, Kolo79.44nt and Kolo79.44ntEND2. In another embodiment the aptamer is selected from the group consisting of KoIoI 2, KoIoI 2.33ntEND, KoIoI 2.33ntGG and KoIoI 2.49ntEND. In yet another embodiment the aptamer is selected from the group consisting of Kolo79, Kolo79.44nt, Kolo79.44ntEND1 , Kolo79.44ntEND2 and Kolo79.48ntfunny.

Any of the aptamers Kolo31 , Kolo32, KoIoI 2, KoIoI 4, KoIo 66, Kolo87, Kolo79, Kolo84, Kolo21 , kolo22, Kolo29, Kolo76, Kolo77, Kolo74, Kolo71 , Kolo28, Kolo25, Kolo78, Kolo26, Kolo83, K0I068, Kolo24, K0I088, Kolo9, Kolo25, Kolo69, Kolo85, Kolo75, KoIoI 7, Kolo73, Kolo82, Kolo67, K0I086, Kolo20, Kolo70, KoIoI O, Kolo89, KoIoI 5, Kolo27, KoIoI 9, Kolo81 , Kolo80, KoIo1 1 , KoIoI 2.33ntEND, KoIoI 2.33ntGG, Kolo21 .35ntEND, KoIoI 2.49ntEND, Kolo79.44nt, Kolo79.44ntEND1 ,

Kolo79.44ntEND2, Kolo79.34nt, Kolo79.48ntfunny is a separate embodiment of the present invention.

Preferred separate embodiments are the following aptamers: KoIoI 2, Kolo21 , kolo25, Kolo26, Kolo79, Kolo79.44nt, Kolo79.49ntEND1 , Kolo79.44ntEND2, Kolo79.48ntfunny.

In a preferred embodiment the aptamer is selected from the group consisting of KoIoI 2, Kolo25 and KoIoI 2.49.In a further preferred embodiment the aptamer is selected from the group consisting of Kolo25 and KoIoI 2.49. In another preferred embodiment the aptamer is selected individually from KoIoI 2, Kolo25 and KoIoI 2.49. It is appreciated that shorter variants of the listed aptamers comprising the sequences at least one of the following sequences as shown below in a variable region or a variant thereof being at least 70 % identical to one of said sequences, such as at least 75 % identical to one of said sequences, such as at least 80 % identical to one of said sequences, such as at least 85 % identical to one of said sequences, such as at least 90 % identical to one of said sequences, such as at least 91 % identical to one of said sequences, such as at least 92 % identical to one of said sequences such as at least 93 % identical to one of said sequences such as at least 94 % identical to one of said sequences such as at least 95 % identical to one of said sequences, such as at least 96 % identical to one of said sequences, such as at least 97 % identical to one of said sequences, such as at least 98 % identical to one of said sequences, such as at least 99 % identical to one of said sequences: kolo31 ACTCCTCGGCGCAAGGATGTGGG-ATCGATGCAATC 35 kolo32 AACTAGAACCATACTCGCCGGCGCCAATGTCGTAG 35 kolol2 —TGCGACTGTTATAACCTAACAGCGACGTAAAG-ATA 35xx kolol4 —TGCGACTGTTATAACCTAACAGCGACGTAAAG-ATA 35 kolo66 —TGCGACTGTTATAACCTAACAGCGACGTAAAG-ATA 35xx kolo87 —TGCGACTGTTATAACCTAACAGCGACGTAAAG-ATA 35 kolo79 GAAACGACTCG-ACAACCT-CGAGCGACGTGAATCAT 35xx kolo84 -TACCGACTAGCAAAACCTGCTGGCGACGTTTAG-AT 35 x kolo21 AATAACCTTAATGCGACGTTGGTTTGTCAACAACG 35xx kolo22 AATAACCTTAATGCGACGTTGGTTTGTCAACAACG 35 kolo29 AATAACCTTAATGCGACGTTGGTTTGTCAACAACG 35 kolo76 AATAACCTTAATGCGACGTTGGTTTGTCAACAACG 35 kolo77 AATAACCTTAATGCGACGTTGGTTTGTCAACAACG 35 kolo74 AATAACCTTAATGCGACGTTGGTTTGTCAACAACG 35xx kolo71 AATAACCTTAATGCGACGTTGGTTCGTCAACATGG 35xx kolo28 ACAACCTAAATGCGACGTTGGGTCAAAAACGTGAA 35xx kolo25 GATAACCTCGATGCGACGTTCGGCC-TCAAAATCAA 35xx kolo78 ACAAC-TTAATGCGACGTTGGTAAAGCATATCAAAC 35 x kolo26 CTCCGCTATCTAACGTATGATA-GAATGGATGACTA 35xx kolo83 TGCGCATGAAATGACTGCATGTCTCCGGATTGATC— 35 kolo68 ATGACAGGATGCAGAGCTCCACTGTCTAGTGTTTA 35 x kolo24 CCGCGACAGTCGTAAGTTTTGACTGACTGAACGTT 35 x kolo88 TCGCTAATTATAGGCGGAGTGCGACGTTATAAATA 35 x kolo9 AGTGATTCGCCATAACC-TGGCTGTTTCAGGCTGTT 35 kolo69 AGTGATTCGCCATAACC-TGGCTGTTTCAGGCTGTT 35 kolo85 AGTGATTCGCCATAACC-TGGCTGTTTCAGGCTGTT 35 kolo75 TCGCATTCGTTATAACC-TAACAGTTTGCGGA-GTTA 35 kolol7 TAGTACGACC-TACTTATTTGAC 22 kolo73 TTGCACAACC-TGCATATTTGTCGTCGACGTACGAA 35 kolo82 CGATACAACC-TATCTGTTTGTCGTGGACATCAAAT 35 kolo67 TGCTATGACC-TAGACATTTGACG 23 kolo86 TACCATAACC-TGGATATCTGTCGTTATCATGGGAC 35 kolo20 TTGCGTAACCCGCAATTTCCGTTGGTATATAC 32 kolo70 ACCTGACTTGCGATAACC-TCGACATTTACGGTATT 35 kololO TAATGGCCGGTTTAACCGTT-TGCGCATTAAGTCGA 35 kolo89 TAATGGCCGGTTTAACCGTT-TGCGCATTAAGTCGA 35 kolol5 TACCATTTACCACAACC-TGGTTATCTGGTTTAAAC 35 kolo27 TTGTCGTCTTCTCCAGT—TAGTCAACGAACTTGTGT 35xx kolol9 TCCGTTTGCAGGTTTTGACCTGCCGGAGTTGTTTG 35 kolo81 GGCAGAAGTTGATTCCAACTTCATTTGCGTTTAAT 35 kolo80 ATATTTGTCTCATCCCACGACAATTATGATGCGAC 35 kololl ATCTTAGCGACGTGACACACGACTAGGGATTAATC 35

are within the scope of the present invention. Shorter variants are aptamers that comprise 50, 49, 48, 47, 46, 45, 44, 43, 42, 41 , 40, 39, 38, 37, 36, 35, 34, 33, 32, 31 , 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , or 10 ribonucleotides.

In a preferred embodiment the shorter variants of KoIoI 2.49 are aptamers that comprise 50, 49, 48, 47, 46, 45, 44, 43, 42, 41 , 40, 39, 38, 37, 36, 35, 34, 33, 32, 31 , 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , or 10 ribonucleotides, and in a further preferred embodiment the shorter variants of KoIoI 2.49 are aptamers that comprise 48, 47, 46, 45, 44, 43, 42, 41 , 40, 39, 38 or 37 ribonucleotides.

Chemical modification of the aptamer

The aptamer of the present invention may comprise at least one chemical modification. The at least one chemical modification is a chemical modification which selected from the group consisting of a chemical substitution at a sugar position, a chemical substitution at a phosphate position and a chemical substitution at a base position, of the ribonucleic acid.lt is appreciated that the at least one chemical is either a substitution at a sugar position, a substitution at the phosphate position or at a base position of the ribonucleic acid forming the aptamer, however, the at least one chemical modification may also be any combination of postions on the ribonucleic acid which can be chemically modified. By the term 'sugar position' is meant a ribose molecule, and by the term 'base position' is meant a purine or pyrimidine base, such as adenine, guanine or cytosine and uracil. The term 'phosphate position' refers to the phosphate group linked to the ribose sugar.

It is within the scope of the present invention that at least one chemical modification of the aptamer is present, such as two, for example three, such as four, for example five, such as six, for example seven, such as eight, for example nine, such as ten, for example 1 1 , such as 12, for example 13, such as 14, for example 15 chemical modifications are present. The at least one chemical modification is selected from the group consisting of a modified ribonucleotide, 3' capping, a high molecular weight non- immunogenic compound, a lipophilic compound, drug, a cytotoxic moiety, a labelling agent and phosphate bone modification. In another embodiment the at least one chemical modification is selected from the group consisting of a modified ribonucleotide, 3' capping, a high molecular weight non-immunogenic compound, a lipophilic compound, drug, a cytotoxic moiety and a labelling agent.

The types of modification of a ribonucleotide is described elsewhere herein It is appreciated that each of the ribonucleotides of the aptamer may be chemically modified in order to avoid degradation in for example serum or blood, this is described elsewhere herein, In embodiments of the present invention the chemical modification of the aptamer is 3'capping and/or 5' capping. High molecular weight non-immunogenic compounds such as polyethylene glycol, dextran, albumin, polyethylene glycol, proteins and magnetite may be used to chemically modify the aptamers of the present invention.

In one embodiment the at least one chemical modification of the aptamer is a drug. One example of a drug is a protein toxin. The protein toxin is selected from the group consisting of diphtheria toxin, ricin, abrin, gelonin and Pseudomonas exotoxin A. However, any of diphtheria toxin, ricin, abrin, gelonin and Pseudomonas exotoxin A constitutes a separate embodiment.

Lipophilic compounds such as for example cholesterol, dialkyl glycerol, oleic acid, choleic acid or diacyl glycerol are compounds which are within the scope of the at least one chemical modification of the aptamers of the present invention.

In one embodiment the aptamer of the present invention comprises at least one chemical modification in the form of a cytotoxic moiety. The cytotoxic moiety is preferably a small molecule cytotoxic agent. The small moiety cytotoxic agent is selected from cancer drugs for example said cancer drug is preferably selected from the group consisting of adrenocorticosteroids, such as prednisone, dexamethasone or decadron; altretamine (hexalen, hexamethylmelamine (HMM)); amifostine (ethyol); aminoglutethimide (cytadren); amsacrine (M-AMSA); anastrozole (arimidex); androgens, such as testosterone; asparaginase (elspar); bacillus calmette-gurin; bicalutamide (casodex); bleomycin (blenoxane); busulfan (myleran); carboplatin (paraplatin); carmustine (BCNU, BiCNU); chlorambucil (leukeran); chlorodeoxyadenosine (2- CDA, cladribine, leustatin); cisplatin (platinol); cytosine arabinoside (cytarabine); dacarbazine (DTIC); dactinomycin (actinomycin-D, cosmegen); daunorubicin (cerubidine); docetaxel (taxotere); doxorubicin (adriomycin); epirubicin; estramustine (emcyt); estrogens, such as diethylstilbestrol (DES); etopside (VP-16, VePesid, etopophos); fludarabine (fludara); flutamide (eulexin); 5-FUDR (floxuridine); 5-fluorouracil (5-FU); gemcitabine (gemzar); goserelin (zodalex); herceptin (trastuzumab); hydroxyurea (hydrea); idarubicin (idamycin); ifosfamide; IL-2 (proleukin, aldesleukin); interferon alpha (intron A, roferon A); irinotecan (camptosar); leuprolide (lupron); levamisole (ergamisole); lomustine (CCNU); mechlorathamine (mustargen, nitrogen mustard); melphalan (alkeran); mercaptopurine (purinethol, 6-MP); methotrexate (mexate); mitomycin-C (mutamucin); mitoxantrone (novantrone); octreotide (sandostatin); pentostatin (2-deoxycoformycin, nipent); plicamycin (mithramycin, mithracin); prorocarbazine (matulane); streptozocin; tamoxifin (nolvadex); taxol (paclitaxel); teniposide (vumon, VM-26); thiotepa; topotecan (hycamtin); tretinoin (vesanoid, all-trans retinoic acid); vinblastine (valban); vinblastine hydrazide; vincristine (Oncovin); calicheamicin; vinca alkaloid; cryptophycin; , a tubulysin, dolastatin-10, dolastatin-15, auristatin E, rhizoxin, epothilone B, epithilone D, taxoid, maytansinoid, and vinorelbine (navelbine), and any variants and derivatives thereof. In one preferred embodiment vinblastine is conjugated to the 3' end of the aptamer.

The aptamer of the present invention may be chemically modified to comprise at least one modification for example in the form of a labelling agent.

The labelling agent of the present invention may be selected from the group consisting of radioisotopes, non-radioactive agents, contrast agents for positron emission tomography (PET) imaging, contrast agents for x-ray or computated tomography (CT) x-ray imaging and contrast agents for magnetic resonance (MR) imaging. Radionuclides used in PET scanning decay by emitting a positron, which also has been chemically incorporated into a metabolically active molecule, is injected into the living subject (usually into blood circulation). Examples of radionuclides are isotopes with short half lives such as carbon-1 1 (-20 min), nitrogen-13 (~10 min), oxygen-15 (~2 min), and Fluorine-18 (-1 10 min). Due to their short half lives, the radionuclides must be produced in a cyclotron which is not too far away in delivery-time to the PET scanner. These radionuclides are incorporated into compounds normally used by the body such as glucose, water or ammonia and then injected into the body to trace where they become distributed. Such labelled compounds are known as radiotracers. The aptamers of the present invention comprise in one embodiment of the present invention radionucleotides. In particular fluorine-18 may be incorporated into the aptamers as at the 2' position of pyrimidines as described elsewhere herein.

For x-ray or computed tomography (CT) imaging, the labelling agent is in the form of an agent which provides a contrast to the surrounding tissue. Such an agent has a different electron density than the surrounding tissues (either more or less electron density than compared to the surrounding tissue) to make it visible. For CT imaging agents that will increase electron density in desired body parts, known as positive contrast agents. The positive contrast agents are selected from the group consisting of bromine moieties, fluorine moieties, iodine moieties and materials that comprise radioopaque metal atoms. It is understood, that bromine, fluorine or iodine moieties may be used separately or in combination. However, agents known as negative contrast agents are also within the scope of the present invention. Non-limiting examples of labelling agents for x-ray imaging or CT imaging is barium sulphate and/or iodine.

In one embodiment the aptamers of the present invention comprise at least one chemical modification in the form of a radioisotope. "Radioactive labeling" or "Radioactively label" refers to labeling using a substance including radioactive isotopes. Radioisotopes are selected from the group consisting of yttrium-90, indium- 1 1 1 , iodine-131 , lutetium-177, copper-67, rhenium-186, rhenium-188, bismuth-212, bismuth-213, astatine-21 1 , and actinium-225. However, the use of radioisotopes such as carbon-13 (¹³C), tritium (³H), carbon-14, S-35 or ³²P, is also within the scope of the present application. Short lived isotopes is in one embodiment coupled through reactive groups, for example thio groups,, wherease longer lived isotopes are incorporated by the the use of radiolabeled nucleotides during or after synthesis of the aptamer.

The labelling agent of the present invention may be at least one contrast agents for MR imaging. MRI is a a diagnostic imaging technique involving the use of a magnetic field, field gradients and radiofrequency energy to excite protons, resulting in the manufacture of an image of mobile protons in water or fats of the body being subjected to MRI. Examples of contrast agents for MRI are an imageable nucleus (such as 19Fe) radioisotopes, diamagnetic, paramagnetic, ferromagnetic or superparamagnetic substances. In one preferred embodiment the contrast agents for MRI are those which have paramagnetic properties such as for example gadolinium or manganese- pramagnetic substances. In a preferred embodiment of the present invention gadolinium-based contrast agents are used. Other examples are contrast agents , such as iron oxides, ferric ion, ferric ammonium citrate for example diethylenetriaminepentaacetic (gadolinium-DTPA) and the like.

In one preferred embodiment, superparamagnetic contrast agents such as iron oxide nanoparticles are used for the aptamers of the present invention.

"Non-radioactive labeling" or "non-radioactively label" refers to labeling not using radioactive substances. In non-radioactive labeling, labeling substances specifically include, but are not particular limited to luminescent molecules, fluorescent molecules such as fluorescein, enzymes such as peroxidase and alkali phosphatase, antibodies, and other molecules having binding specificity to specific molecules such as a biotin that are used in the art. By a fluorescent label as a chemical modification of an aptamer of the present invention is meant a fluorescent chemical group (fluorophore) with which the aptamer is modified, and the fluorescence of this label enables sensitive and quantitative detection of the aptamer. Fluorescein (and derivatives therof) is an example of a fluorophore chemically attached to the aptamer of the present invention. Other examples of flourescent dyes suitable for the present invention are derivatives of rhodamine, coumarin and cyanine. Derivatives of cyanine are for example Cy3 and Cy5. Non- limiting examples of rhodamine dyes are Rhodamine 6G, Rhodamine B, TRITC, TAMRA, sulforhodamine 101 (and its sulfonyl chloride form Texas Red) and Rhodamine Red. Phosphate bone modification such as phosphotioates as mentioned elsewhere herein are within the scope of the present invention.

In another embodiment of the invention the aptamer comprises at least one chemical modification to produce a nanoparticle. A nanoparticle also known as nanopowder, nanocluster or nanocrystal is a is a microscopic particle with at least one dimension less than 600 nm, such as less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm, or less than 100 nm. The size of the nanoparticles of the present invention ranges between 4 nm to 600 nm. In a preferred embodiment the nanoparticles of the present invention has a size in the range of 50 to 300 nm.The at least one chemical modification is chitosan, which is known to be a very good agent which binds nucleic acids including ribonucleic acids, with low toxicity. Chitosan has the advantage that it is well tolerated in the body. Without being bound by theory it is believed that chitosan minimises the charge of the nucleotide thereby allowing better uptake. Aptamers of the present invention form nanoparticles through electrostatic bridges between chitosan polymeric chains. An agent for chemical modification of the aptamers of the present invention which may be used, optionally with other agents, such as chitosan, include polyethyleneimine. The at least one chemical modification may in combination or on its own be the incorporation of a 3' terminal cholesterol conjugated nucleobase during oligonucleotide synthesis.

In further embodiments the at least one chemical modification may be selected individually from polyethyleneimine (PEI), poly (lysine) (PLL), poly(2-dimethyl- amino)ethyl, methacrylate (pDMAEMA), chitosan, histidine-based polypeptides, 5 poly(lactic acid) (PLA), polylactide/glycolide acid co-polymers (PLGA), poly(lacticglycolide) acid, polyethylene glycol (PEG), and poly[N-(2- hydroxpropyl)methacrylamide] (PHPMA).

In the present invention, an "aptamer" refers to a nucleic acid ligand artificially engineered to bind strongly and specifically with a particular target protein. A "modulate aptamer" refers to an aptamer wherein the core portion of the aptamer is designed such that it has a low Tm value and divided in two short oligonucleotide chains which bind to form a double strand only in the presence of a target protein. A "molecular beacon aptamer" refers to an aptamer which comprises two structural components, a loop sequence which becomes a complementary probe to a target sequence, and a stem structure which is formed by annealing of two complementary arm sequences present on both ends of the probe sequence.

A "conjugate" refers to a formation in a stabilized state between a single stranded or double stranded modulate aptamer according to the present invention and a target protein via hydrogen bonds and/or non-covalent bonds such as hydrophobic interactions. Here, "stabilized" refers to formation of a conjugate in which the binding- dissociation equilibrium is biased toward a bound state, and dissociation is difficult.

Stability of aptamers

RNA is relatively labile and can be degraded by a number of ribonucleases. This degradation can be greatly reduced by the introduction of modifications and substitutions at the 2'-prime position of the ribonucleotide and by modifications and substitutions along the phosphate backbone of the RNA. In addition a variety of modifications can be made on the nucleobases themselves which both inhibit degradation and which can increase desired nucleotide interactions or decrease undesired nucleotide interactions. Once the sequence of an aptamer is known modifications or substitutions can be made by the synthetic procedures described below or by procedures known to those of skill in the art.

The disclosed aptamers can be synthesized using any suitable method. Many synthesis methods are known. The following techniques are preferred for synthesis of for example, 2'-0-AIIyI modified oligomers that contain residual purine ribonucleotides, and bearing a suitable 3'-terminus such as an inverted thymidine residue (Ortigao et al., Antisense Research and Development 2:129-146 (1992)) or two phosphorothioate linkages at the 3'-terminus to prevent eventual degradation by 3'-exonucleases, can be synthesized by solid phase [beta]-cyanoethyl phosphoramidite chemistry (Sinha et al., Nucleic Acids Res. 12:4539-4557 (1984)) on any commercially available DNA/RNA synthesizer. A preferred method is the 2'-0-tert-butyldimethylsilyl (TBDMS) protection strategy for the ribonucleotides (Usman et al., J. Am. Chem. Soc. 109:7845-7854 (1987)), and all the required 3'-0-phosphoramidites are commercially available. In addition, the use of aminomethylpolystyrene is preferred as the support material due to its advantageous properties (McCollum and Andrus Tetrahedron Letters 32:4069-4072 (1991 )). Fluorescein can be added to the 5'-end of a substrate RNA during the synthesis by using commercially available fluorescein phosphoramidites. In general, a desired oligomer can be synthesized using a standard RNA cycle. Upon completion of the assembly, all base labile protecting groups are removed by an 8 hour treatment at 55°C. with concentrated aqueous ammonia/ethanol (3:1 v/v) in a sealed vial. The ethanol suppresses premature removal of the 2'-0-TBDMS groups which would otherwise lead to appreciable strand cleavage at the resulting ribonucleotide positions under the basic conditions of the deprotection (Usman et al., J. Am. Chem. Soc. 109:7845-7854 (1987)). After lyophilization the TBDMS protected oligomer is treated with a mixture of triethylamine trihydrofluoride/triethylamine/N-methylpyrrolidinone for 2 hours at 60⁰C. to afford fast and efficient removal of the silyl protecting groups under neutral conditions (Wincott et al., Nucleic Acids Res. 23:2677-2684 (1995)). The fully deprotected oligomer can then be precipitated with butanol according to the procedure of Cathala and Brunei (Nucleic Acids Res. 18:201 (1990)). Purification can be performed either by denaturing polyacrylamide gel electrophoresis or by a combination of ion-exchange HPLC (Sproat et al., Nucleosides and Nucleotides 14:255-273 (1995)) and reversed phase HPLC. For use in cells, it is preferred that synthesized oligomers be converted to their sodium salts by precipitation with sodium perchlorate in acetone. Traces of residual salts are then preferably removed using small disposable gel filtration columns that are commercially available. As a final step it is preferred that the authenticity of the isolated oligomers is checked by matrix assisted laser desorption mass spectrometry (Pieles et al., Nucleic Acids Res. 21 :3191 -3196 (1993)) and by nucleoside base composition analysis. In addition, a functional cleavage test with the oligomer on the corresponding chemically synthesized short oligoribonucleotide substrate is also preferred. [0058] The disclosed oligomers can also be produced through enzymatic methods, when the nucleotide subunits are available for enzymatic manipulation. For example, the RNA molecules can be made through in vitro RNA polymerase T7 reactions. They can also be made by strains of bacteria or cell lines expressing T7, and then subsequently isolated from these cells. As discussed below, the disclosed aptamers can also be expressed in cells directly using vectors and promoters.

In order for an aptamer to be suitable for use as a therapeutic, it is preferably inexpensive to synthesize, safe and stable in vivo. Wild-type RNA and DNA aptamers are typically not stable in vivo because of their susceptibility to degradation by nucleases. Resistance to nuclease degradation can be greatly increased by the incorporation of modifying groups at the 2'-position. Fluoro and amino groups have been successfully incorporated into oligonucleotide pools from which aptamers have been subsequently selected. In one embodiment, the present invention provides aptamers comprising combinations of 2'-OH, 2'-F, 2'-deoxy, and 2'-OMe modifications of the ATP, GTP, CTP, TTP, and UTP nucleotides. In another embodiment, the present invention provides aptamers comprising combinations of 2'-OH, 2'-F, 2'-deoxy, 2'-OMe, 2'-NH2, and 2'-methoxyethyl modifications of the ATP, GTP, CTP, TTP, and UTP nucleotides. In another embodiment, the present invention provides aptamers comprising combinations of 2'- OH, 2'-F, 2'-deoxy, 2'-0Me, 2'-NH2, and 2'-methoxyethyl modifications of the ATP, GTP, CTP, TTP, and UTP nucleotides.

For example, chemically modified aptamers include those containing one or more modified bases. For example, the modified pyrimidine bases of the present invention may have substitutions of the general formula 5'-X and/or 2'-Y, and the modified purine bases may have modifications of the general formula 8-X and/or 2-Y. The group X includes the halogens I, Br, Cl, or an azide or amino group. The group Y includes an amino group, fluorine, or a methoxy group. Other functional substitutions that would serve the same function may also be included. The aptamers of the present invention may have one or more X-modified bases, or one or more Y-modified bases, or a combination of X-and Y-modified bases. The present invention encompasses derivatives of these substituted pyrimidines and purines such as 5'-triphosphates, and 5'-dimethoxytrityl, 3'-beta-cyanoethyl, N, N- diisopropyl phosphoramidites with isobutyryl protected bases in the case of adenosine and guanosine, or acyl protection in the case of cytosine. Within the scope of the present invention is also aptamers bearing any of the nucleotide analogs herein disclosed. The present invention encompasses specific nucleotide analogs modified at the 5 and 2'positions, including 5- (3-aminoallyl) uridine triphosphate (5-AA-UTP), 5- (3-aminoallyl) deoxyuridine triphosphate (5-AA-dUTP), 5-fluorescein-12-uridine triphosphate (5-F-12-UTP), 5- digoxygenin-1 1 -uridine triphosphate (5-Dig-1 1 -UTP), 5-bromouridine triphosphate (5- Br-UTP), 2'-amino-uridine triphosphate (2'-NH₂-UTP) and 2'-amino-cytidine triphosphate (2'-NH₂-CTP), 2'-fluoro-cytidine triphosphate (2'-F-CTP), and 2'-fluoro- uridine triphosphate (2'-F-UTP).

Thus, in one embodiment of the present invention the aptamers are stable in serum. In one preferred embodiment the aptamers of the invention comprises at least one chemical modification in the form of 2'-Flouro (F) of the ribonucleotides, ATP, GTP, CTP and UTP nucleotides, such as particularly CTP, UTP and TTP. In particular a flour atom is present in the 2' position of the ribose-ring instead of the hydroxyl group of all pyrimidines of the aptamers according to the present invention. The pyrimidines are the ribonucleotides cytosine and uracil. It is appreciated by a person skilled in the art that the aptamers of the present invention comprise at least one chemical modification, at least 2 chemical modifications, at least 3 chemical modifications, at least 4 chemical modifications, at least 5 chemical modifications, at least 6 chemical modifications, at least 7 chemical modifications, at least 8 chemical modifications, at least 9 chemical modifications, at least 10 chemical modifications, at least 15 chemical modifications, at least 20 chemical modifications, at least 25 chemical modifications, at least 30 chemical modifications, at least 45 chemical modifications of the ribonucleotides in any combination to improve stability of the aptamers. The chemical modifications of the ribonucleotides of the aptamers may be combined with any other type of modification of the aptamer as described herein. From a comparison of the stability of modified versus non-modified aptamers of the present invention exemplified by KoIoI 2 and KoIoI 2.49ntEND it is evident that the non- modified versions of the aptamers are degraded so that no visible trace of the aptamers can be seen 6 and 24 hrs after the addition of serum. Presumably the degradation occurs rapidly after the addition of serum. In contrast, the modified aptamers can still be seen after 6 and 24 hrs, even though some degradation has occurred upon addition of serum.

The stability of the modified aptamers of the present invention enhances the applicability of the aptamers of the present invention as diagnostic tool for for example imaging, and as a therapeutic tool for treatment of the conditions as described elsewhere herein in addition to the ability of the aptamers as delivery vehicles for for eample cytotoxic agents and the like.

Dissociation constants

According to the present invention a complex is formed between the aptamers of the present invention and a receptor-binding form of u-PA. The dissociation constant, usually denoted K^ for a given complex provides a measure of how willing a complex between in this case the aptamer and u-PA is to dissociate into the aptamer on one hand and the u-PA on the other hand

Thus, a dissociation constant is a specific type of equilibrium constant that measures the propensity of a larger object to separate (dissociate) reversibly into smaller components, as when a complex falls apart into its component molecules. The dissociation constant is and is the inverse of the affinity constant.

For a general reaction

in which a complex A_xB_y breaks down into x A subunits and y B subunits, the dissociation constant is defined

where [A], [B], and [A_xB_y] are the concentrations of A, B, and the complex A_xB_y, respectively.

The smaller the value of the dissociation constant the less dissociation of the species of the complex in solution is observed. The dissociation constant is influenced by temperature, ionic strength and also the type of solvent.

In one embodiment of the present invention the complex between the aptamer and u- PA has a dissociation constant of about 100 nM or below, such as a dissociation constant of about 50 nM or below, or for example a dissociation constant in the range of 1 nM to 10 nM. In a specific embodiment the dissociation constant is 3.2 nM +/- 0.5 nM.

In some embodiments, the therapeutic aptamer-drug conjugates of the invention have the following general formula: (aptamer)n-linker-(drug)m, where n is between 1 and 10 and m is between 0 and 20, particularly where n is between 1 and 10 and m is between 1 and 20. In some embodiments, the linker is a polyalkylene glycol, particularly a polyethylene glycol. In some embodiments, the drug is encapsulated, e.g. in a nanoparticle. In some embodiments, the linker is a liposome, dendrimer or comb polymer. In some embodiments, the drug is a cytotoxin. A plurality of aptamer species and drug species may be combined to yield a therapeutic composition. In one embodiment, the therapeutic aptamer-drug conjugates of the invention are used in the targeted killing of tumor cells through aptamer- mediated delivery of cytotoxins. The efficiency of cell killing is improved if the target tumor marker is a marker that readily internalizes or recycles into the tumor cell. The present invention relates to aptamers capable of binding a receptor-binding form of urokinase-type plasminogen activator protein, u-PA.

By the term 'receptor-binding form of uPA' is meant is meant any form of uPA possessing a site that binds to a site at a uPAR. For example the uPA contains the uPAR binding site. The receptor binding form of uPA can thus be pro-uPA, uPA an amino terminal fragment of uPA, or a uPA irreversible inhibited by for example diisopropyl fluorophosphates, or any other inhibitor or modification of uPA that can bind to uPAR. uPA is found in all mammalian species. Thus, the aptamers of the present invention are capable of binding to uPA of any mammalian species, however, in a preferred embodiment the aptamer is capable of binding to human uPA. The receptor binding form of u-PA which is the target for the aptamers of the present invention, like plasmin, is produced and secreted as a pro-enzyme or zymogen, termed pro-uPA, having a catalytic activity towards plasminogen several hundred-fold lower than that of uPA (Andreasen et al., 1997). Moreover, the reaction between uPA and PAI-1 is more than a 1000-fold faster than that of pro-uPA with PAI-1 (Behrendt et al., 2003), and not dramatically affected by binding of uPA to the uPA receptor (Ellis et al., 1990). The plasma concentration of uPA is -20 pM, uPA being present in both the active form, the pro-form and in complex with PAI-1 (Andreasen et al., 1997). uPA has a molecular weight (M_r) of -50 kDa and consists of an N-terminal growth factor-like domain (GFD; amino acids 1 -49 according to uPA numbering), a kringle domain (KD; amino acids 50-131 ), an interdomain linker region also termed the connecting peptide (CP; 132-158), and a C-terminal serine protease domain (SPD, amino acids 159-41 1 ) (Spraggon et al., 1995; Andreasen et al., 2000). It undergoes several posttranslational modifications such as glycosylation of Asn302, phosphorylation on Ser138 and 303, and fucosylation of Thr18 (Alfano et al., 2005). Proteolytic cleavage of the peptide bond Lys158-lle159 between the KD and the GFD converts single-chain pro-uPA into the active two-chain uPA form with the two chains being held together by a single disulphide-bridge (Cys148-Cys279). Cleavage of the Lys158-lle159 brings about structural rearrangements in the SPD domain important for its catalytic capacity. Further proteolytic cleavage of uPA at K135-K136 in the interdomain linker region results in generation of the so-called amino terminal fragment (ATF) consisting of the GFD and the KD, and a fragment termed low molecular weight uPA (LMW-uPA). LMW- uPA contains the SPD and most of the interdomain linker region between SPD and the KD, and retains full catalytic activity compared to the two-chain version, however, it does not bind to uPAR as the part of uPA responsible for binding to uPAR is the GFD.

There are no structures of full-length uPA but several structures of the active serine protease domain of uPA in complex with active site inhibitors have been determined by X-ray crystallography and reveals that the catalytically active domain shares the overall common fold found among chymotrypsin-like serine proteases (figure 1 ) (see for example (Spraggon et al., 1995; Zhao et al., 2007)). The SPD is a globular domain consisting of two sub-domains both containing six-stranded β-barrels together forming a cleft central in the catalytic mechanism. This cleft is the active site containing the so- called catalytic triad consisting of His204(57), Asp255(102), and Ser356(195), the numbers in parenthesis being the numbering in chymotrypsin, the standard representative of the serine protease family. The hydrolysis of target peptide bonds (denoted P1 -P1 ^') occurs by a two-step enzymatic reaction, comprising an acylation step and a deacylation step (figure 2). In the acylation step (figure 2A-C); following the initial formation of a non-covalent Michaelis complex between the substrate and the SPD, the oxygen atom of Ser356(195) carries out a nucleophilic attack on the carbonyl carbon atom of the P1 residue. His204(57) accepts the proton of the hydroxyl group of Ser356(195) during the attack, while Asp255(102) keeps His204(57) in the right orientation compared to

Ser356(195) and stabilises the proton transfer. Usually, the -CH₂OH group of a serine is quite unreactive at physiological conditions, however in the aforementioned context highly reactive. As the result, a transient tetrahedral intermediate is formed with the carbonyl oxygen atom of P1 carrying an additional electron (figure 2B). This so-called carbonyl oxyanion is stabilised by interaction with the main chain protons of

Gly354(193) and Ser356(195) in what is termed the oxyanion hole. The protonated histidine then delivers its proton to the nitrogen atom of the peptide bond releasing the C-terminal part of the substrate, while the N-terminal part is bound to the protease by an esterbond (figure 2C). In the deacylation step (figure 2D-F), the oxygen atom of a water molecule makes a nucleophilic attack on the P1 carbonyl atom while delivering a proton to His204. A new tetrahedral oxyanion intermediate is formed this time leading to the release of the N-terminal part of the substrate and restoration of the active site for a new round of substrate hydrolysis (Stryer, 1995; Hedstrom, 2002). Plasmin is a broad-specific serine protease which directly or indirectly by activation of matrix metalloproteinases (MMPs) promotes the degradation of ECM components including fibrin, fibronectin, tenascin, proteoglycans, vitronectin and, as the main molecules of the basement membranes laminin and collagen IV (figure 4) (Andreasen et al., 1997; Andreasen et al., 2000; Durand et al., 2004; Castellino and Ploplis, 2005; Weinberg, 2007). Furthermore, the degradation of ECM leads to release and activation of latent sequestered growth factors.

Plasmin is secreted as a 791 amino acid zymogen, plasminogen, which is present in plasma at a concentration of 2 μM and in the extravasculature probably at the same concentration (Andreasen et al., 1997; Castellino and Ploplis, 2005). The catalytic activity of plasminogen is at least several hundred-fold lower than that of plasmin. Plasminogen has a molecular weight (M_r) of -90 kDa and consists of an N-terminal peptide sequence, Glu1 -Lys77, followed by five homologous kringle domains (KDs) and a C-terminal serine protease domain (SPD) responsible for its catalytic activity. Conversion of plasminogen to the active enzyme plasmin occurs by proteolytic cleavage between the SPD and the KDs which results in a two-chain form held together by two disulphide-bridges. This processing is primarily mediated by either of the two serine proteases urokinase-type plasminogen activator (uPA) or tissue-type plasminogen activator (tPA), but recent studies comparing mammary gland adipogenesis and wound healing in mice deficient in plasminogen with mice double deficient in uPA and tPA suggest the existence of additional in vivo relevant plasminogen activators (Selvarajan etal., 2001 ; Dano et al., 2005). uPA is mainly responsible for plasminogen activation in the extravasculature during invasion, migration and tissue remodelling processes, and tPA during fibrinolysis, however gene deficient mice have demonstrated that they in some degree are able to substitute for each other (Carmeliet et al., 1994; Bugge et al., 1996a).

uPA binds to cell surfaces via the uPA receptor (uPAR; K₀ 0.1 -1 nM), fibrin and cell surfaces are the sites of plasminogen activation by tPA and uPA, respectively. In contrast to plasmin the plasminogen activators when fibrin- or cell-associated are not protected from their primary inhibitor plasminogen activator inhibitor-1 (PAI-1 ), or the two other inhibitors, plasminogen activator inhibitor-2 (PAI-2) and protease nexin-1 (PN-1 ). Plasmin activity at fibrin clots or on cell surfaces is therefore a tightly controlled event determined by the rate of plasminogen activation, as a consequence of the presence of plasminogen activators and their inhibitors, relative to the rate of dissociation of plasmin from the fibrin clot or the cell surface.

The 3D-structure of the amino terminal fragment of u-PA has been obtained by nuclear magnetic resonance (Hansen et al., 1994) and recently by X-ray crystallography alone and in complex with uPAR (Barinka etal., 2006; Huai et al., 2006). The crystal structure of ATF-uPAR from Huai et al., 2006 is shown in figure 3. Apart from this, the structure of the kringle domain alone has also been determined by NMR (Li et al., 1994). The kringle domain has high homology with other kringle domains like the ones in for example plasminogen and especially those from tPA, all containing the characteristic three-disulphide triple-loop arrangement. While some of the plasminogen kringles bind to fibrin, the kringle of uPA does not, but it has been found to interact with heparin and related anionic polysaccharides (Stephens et al., 1992).

According to the NMR structure of the ATF, the two domains do not form interdomain interactions, but in the crystal structures of the ATF-uPAR complex and ATF alone the two domains are constrained by hydrophobic interactions with each other. The results seem to favour a locked relative orientation. The small GFD has sequence similarities with the epidermal growth factor (EGF) and is therefore sometimes also known as the EGF-like domain (Appella et al., 1987). The central feature in the GFD is a so-called Ω- loop (amino acids 22-28) connecting the strands of a two-strand β-sheet. According to alanine scanning mutagenesis on residues 20-30 of the GFD, this Ω-loop (amino acids 22-28) contains the majority of the functional epitope (Lys23, Tyr24, Phe25, He28 and Trp30) important for the high affinity interaction with uPAR (Magdolen et al., 1996). The structural epitope was revealed by the crystal structures of ATF-uPAR and is in agreement with the functional epitope. They show that the entire Ω-loop is deeply buried in the uPAR structure and that other parts of the GFD as well as parts of the kringle domain are in contact with the receptor (Barinka et al., 2006; Huai et al., 2006).

The uPA - uPAR interaction is characterised by pronounced species-specificity between the murine and human components, and it has been shown that human uPA can be "murinised" by swapping only four positions in the GFD (Asn22Tyr, Asn27Arg, His29Arg and Trp30Arg) (Quax et al., 1998). The aptamers of the present invention binds to the amino terminal part of the u-PA. The amino terminal part of the u-PA is also known as the N-terminal growth factor-like domain (GFD; amino acids 1 -49 according to uPA numbering).

Mature human uPAR on the cell surface is a glycosyl-phosphatidylinositol (GPI) - anchored 283-284 amino acid protein carrying a high level of heterogeneous, N-linked carbohydrates resulting in a molecular weight (M_r) between 50 and 60 kDa (Ploug, 2003). GPI-anchoring can occur at either position Ser282 or Gly283 and is dependent on a C-terminal moderately hydrophobic peptide sequence in nascent uPAR, which is excised during posttranslational processing. The protein is organised into three similar domains (from the N-terminus termed D1 , D2 and D3), so-called LU-domains, which have typical three-finger folds and are present in all members of the Ly6/uPAR/α- neurotoxin protein domain family. All three domains are necessary to form the binding pocket for uPA (figure 3) (Ploug, 2003; Llinas et al., 2005; Huai et al., 2006), and the functional epitope on uPAR has been mapped by comprehensive site-directed mutagenesis analyses revealing that high-affinity interaction depends on structural elements in domain I and Il while domain III plays an auxiliary role (Gardsvoll et al., 1999; Gardsvoll et al., 2006). Soluble forms of uPAR (suPAR) can be generated by proteolytic cleavage of uPAR close to the GPI-anchor or by hydrolysis of the GPI- anchor by phopholipases and have been found to exist in vivo (Sidenius and Blasi,

2003; Montuori et al., 2005). Though it has been suggested that GPI-anchored uPAR and soluble uPAR have different conformations, this so-called shedding does not alter its affinity for its two major ligands, uPA and VN (Ploug et al., 1994; Sidenius and Blasi, 2003).

The role of the uPA-uPAR interaction is not only to recruit cell-surface associated plasminogen activation. uPAR has been shown to interact directly with the ECM protein vitronectin and the interaction is stimulated when uPA is associated with uPAR (Waltz and Chapman, 1994; Wei et al., 1994; Kanse et al., 1996). Thus this interaction leads to cell-matrix attachment in contrast to proteolysis which favours cell detachment. The ECM protein vitronectin is -78 kDa glycoprotein containing from the N-terminus: a somatomedin B domain (SMB), an integrin binding-binding RGD sequence, a collagen- binding region and 2 hemopexin-like domains (Hp1 and Hp2) (for a review see (Andreasen et al., 1997)). The 3D structure of the SMB domain has been revealed both using NMR and X-ray crystallography (Zhou etal., 2003; Kamikubo et al., 2004; Mayasundari et al., 2004; Kjaergaard et al., 2007). Some discrepancies have however been obtained about the disulphide-bridge pattern of vitronectin with the one in the X- ray crystal structure proposed by Zhou et al., 2003 to be correct (Li et al., 2007; Zhou, 2007). Vitronectin seems to exist in two conformation: the "closed" conformation, which is the one present in blood plasma, or the "extended" multimeric conformation, which is deposited in the ECM (see review by (Hess et al., 1995)). The uPAR - VN interaction requires the intact three dimensional structure of uPAR (Hoyer-Hansen etal., 1997a; Sidenius and Blasi, 2000) and is thought to be mediated in part by binding of the SMB domain of vitronectin to a region in domain I and the linker region between domain I and Il (Deng et al., 1996; Gardsvoll and Ploug, 2007). This interaction alone, however, cannot account for the high affinity, which has been suggested to rely on an additional low affinity interaction site between uPA and vitronectin (Moser et al., 1995; Sidenius et al., 2002; Gardsvoll and Ploug, 2007). uPA binding to uPAR has also been suggested to infer susceptibility of the linker region between domain I and Il of uPAR to proteolytic attack by various proteases, including uPA it self, plasmin and matrix metalloproteases (Hoyer-Hansen et al., 1992; Fazioli et al., 1997; Hoyer-Hansen et al., 1997b). This leads to abolition of uPA binding and concomitantly loss of uPAR affinity for vitronectin (Hoyer-Hansen et al., 1997a). However, apart from these "loss-of-function" consequences, cleavage in this region exposes a chemotactic pentapeptide sequence (Ser88-Tyr92), which allows D2-D3 of uPAR to induce cytoskeletal changes and intracellular signal transduction via G-protein coupled receptors (GPCRs) in favour of cell motility (Fazioli et al., 1997; Degryse et al., 1999; Nguyen et al., 2000). The chemotactic sequence, however, has been suggested to be exposed by uPA binding alone as well (Resnati et al., 1996). uPA has also been shown to induce interactions of uPAR with integrins (Ploug, 2003) although these interactions can be both uPA-independent and uPA-dependent. Most reports describe uPAR-integrin interactions occuring in the cis-form, but the trans-form has also been described thereby inducing cell-cell interaction (Tarui et al., 2001 ). The result of the uPA-uPAR-integrin interaction is co-localisation of the uPA-uPAR complex at cell-ECM or cell-cell contact sites and modulation of integrin function in terms of cell adhesion and migration (Ploug, 2003).

USE Therapeutic use Aptamers have a number of desirable characteristics for use as therapeutics and diagnostics including high specificity and affinity, biological efficacy, and excellent pharmacokinetic properties. In addition, they offer specific competitive advantages over antibodies and other protein biologies, for example: 1 ) Speed and control. Aptamers are produced by an entirely in vitro process, allowing for the rapid generation of initial leads, including therapeutic leads. In vitro selection allows the specificity and affinity of the aptamer to be tightly controlled and allows the generation of leads, including leads against both toxic and non-immunogenic targets.

Aptamers as a class have demonstrated little or no toxicity or immunogenicity. In chronic dosing of rats or woodchucks with high levels of aptamer (10 mg/kg daily for 90 days), no toxicity is observed by any clinical, cellular, or biochemical measure. Whereas the efficacy of many monoclonal antibodies can be severely limited by immune response to antibodies themselves, it is extremely difficult to elicit antibodies to aptamers most likely because aptamers cannot be presented by T-cells via the MHC and the immune response is generally trained not to recognize nucleic acid fragments.

Therapeutic aptamers are chemically synthesized and consequently can be readily scaled as needed to meet production demand. Whereas difficulties in scaling production are currently limiting the availability of some biologies and the capital cost of a large-scale protein production plant is enormous, a single large-scale oligonucleotide synthesizer can produce upwards of 100 kg/year and requires a relatively modest initial investment. The current cost of goods for aptamer synthesis at the kilogram scale is estimated at $500/g, comparable to that for highly optimized antibodies. Continuing

Therapeutic aptamers are chemically robust. They are intrinsically adapted to regain activity following exposure to factors such as heat and denaturants and can be stored for extended periods (>1 yr) at room temperature as lyophilized powders.

There are several potential uses of an inhibitor of the uPA-uPAR interaction in terms of prevention of cancer progression. Pretreatment before surgery may prevent metastasis consequent to surgical manipulation of the tumor and long-term treatment may inhibit establishment and outgrowth of micrometastasis after primary tumor removal (Ignar et al., 1998). It has been demonstrated that a factor named angiostatin is secreted by primary tumors and thought to suppress growth of micrometastases. Levels of such an inhibitory factor would be diminished upon primary tumor ablation allowing the establishment and growth of micrometastasis. Use of an uPA-uPAR inhibitor as an adjunct to cytokic chemotherapy may extend the time periods between regimens of chemotherapy as increases in tumor latence has been observed with co-administration of antiangiogenic agents. The role of the uPA-uPAR interaction in relation to cell proliferation and apoptosis, however, also suggests that an inhibitor not only could have a preventive effect but also regress cancer development.

Cell surface associated plasminogen activation by uPA can be inhibited in two ways: either by inhibiting uPA^'s proteolytic activity or by blocking the association of uPA with uPAR. Several strategies have been employed in the attempt to generate highly specific, high-affinity inhibitors of the catalytic activity of uPA, including the use of antibodies, peptides, small organochemical compounds and natural inhibitors. The aptamers of the present invention are capable of preventing the binding of uPA and its receptor uPAR, i.e inhibits the uPA-uPAR interaction on the cell surface- Most inhibitors of the uPA-uPAR interaction are constructs based on the two proteins themselves, such as inhibitors based on full-length uPA or variants thereof, soluble uPAR, small peptides, or antibodies. An example is inhibitors based on full-length uPA include mutants with decreased or no catalytic activity as well as active-site-inhibited uPA thus functioning as uPAR antagonists have been described in the literature to have effect on cell surface- associated uPA activity by more than 95% (Cohen et al., 1991 ). In a following study (Crowley et al., 1993), the same result was obtained when using human PC3 prostate cancer cells, which subsequently exhibited a reduced metastatic capacity when subcutaneously inoculated into nude mice.

Thus, the many studies on inhibitors of the uPA-uPAR interaction have validated the uPA-uPAR interaction as a target for anti-cancer therapy.

The uPA-uPAR interaction supports the malignant phenotype of cancer cells in several ways: Firstly, by breaking down ECM-barriers during invasion; secondly, by mediating the cyclic cell attachment and detachment during migration; and thirdly, by stimulating cell motility through the control of cytoskeletal dynamics and chemotaxis. An increasing number of reports, reviewed extensively in (Alfano et al., 2005; Mazzieri and Blasi, 2005), suggest that the interaction between uPA and uPAR, in addition, may also promote cell proliferation and prevent apoptosis, independent of uPA catalytic activity, by mechanisms still unclear. The aptamers of the present invention upon binding to uPA prevents the binding of uPA to the uPA receptor. The prevention occurs upon binding of the aptamer to its target uPA which inhibit or reduce the ability of uPA to bind to its receptor uPAR. The present invention provides aptamers that upon binding to uPA prevents uPA-mediated cell-associated plasminogen activation.Thus, the aptamers of the present invention can be used for prevention of uPA-mediated proteolytic activity at the cell surface for preparing a composition for preventing or counteracting proteolytic activity at the surface of a mammal.

In the context of the present invention the term ' preventing or counteracting' refers to the situation in which the binding of uPA to uPAR is inhibited entirely, or a situation wherein the binding is inhibited in a sufficient manner so as to inhibit the undesired effect of the plasminogen activation.

By the term 'proteolytic activity' is meant a proteolytic activity which is local ie. located at one or more distinct regions in a human body, or specific cells. This is in contrast to proteolytic activity all over the body.

In particular, said mammal is a human being. In one preferred embodiment the proteolytic acitivity which is prevented is the conversion of plasminogen to plasmin, also referred to elsewhere herein as plasminogen activation. It is appreciated that this prevention of the conversion of plasminogen to plasmin is the conversion which takes place at the cell-surface. uPA is produced by many cultured cells of neoplastic origin. It has been found that explants of tumor tissue released more uPA than the corresponding normal tissue.

Furthermore, uPA has been identified in extracts of tumor tissue such as human lung, colon, endometrial, breast, prostate and renal carcinomas, human melanomas, and for example murine mammary tumor. Degradation of surrounding normal tissue is a prerequisite for invasiveness of malignant tumors. The fact that uPA is constantly found in malignant tumors and the findings that uPA is involved in the tissue degradation in normal physiological events suggests that uPA is involved in tissue destruction and cancer development or metastatic activity of cancer. Thus, one aspect of the invention is therefore the use of the aptamer for the treatment of cancer in a mammal in need thereof. In one embodiment the treatment of cancer is the treatment of metastatic activity of the cancer in a mammal in need thereof. It is envisaged that the treatment should include a therapeutically efficient amount of the aptamer.

Another aspect relates to the use of the aptamer according to the present invention for characterising a tumour. Such a characterisation of the uPA content of the tumor is useful for deciding on a treatment regime employing for example protease inhibitors in the therapeutic intervention against cancer. A high content of uPA would indicate that protease inhibitors against uPA should be employed.

Another aspect relates to the use of the aptamers of the present invention capable of preventing the binding of a receptor-binding form of uPA to a uPAR in a mammal for the preparation of a composition for preventing the binding of uPA to a uPAR.

Yet another aspect of the present invention is the use of the aptamers of the present invention capable of preventing the binding of a receptor-binding form of uPA to a uPAR in a mammal for the preparation of a composition for preventing the internalisation of a uPA- PAI-1 -uPAR complex. Upon inhibition of uPAR-bound uPA by PAI-1 , the uPA-PAI-1 -uPAR complex can be rapidly endocytosed through clathrin- coated pits via several transmembrane receptors of the low-density lipoprotein receptor (LDLR) family, including low-density lipoprotein receptor-related protein-1A (LRP1 A; (Nykjaer et al., 1992)) and -1 B (LRP-1 B; (Li et al., 2002)), megalin (Willnow et al., 1992; Moestrup et al., 1993), very-low-density lipoprotein receptor (VLDLR; (Argraves et al., 1995; Heegaard et al., 1995)), as well as via the related receptor sorting protein- related receptor (sorLA; (Gliemann et al., 2004)). Following internalisation, the complex is disrupted by the low pH in the early endosomes, the uPA-PAI-1 complex degraded in the lysosomes, while the intact receptors are recycled to the cell surface. The LDLR family of endocytosis receptors has been implicated in binding and endocytosis of a large number of structurally unrelated proteins such as apolipoproteins, protease- inhibitor complexes, extracellular matrix proteins and hormone carriers. Generally, the receptors are believed to mediate ligand binding via their so-called complement-type repeats (CTRs) and endocytosis via a cytoplasmic C-terminal domain. Though both free uPA and PAI-1 have affinity for the endocytosis receptors, K_D ^'s estimated to 7-200 nM and -30 nM, respectively (Nykjaer et al., 1994; Horn et al., 1998; Gliemann et al., 2004; Croucher et al., 2006), only the uPA-PAI-1 complex is endocytosed efficiently requiring association with uPAR (Nykjaer et al., 1992). uPA-PAI-1 recognition {K_d ~λ nM) relies on interactions of the endocytosis receptors with both uPA and PAI-1 involving at least both the SPD and GFD of uPA and the flexible-joint region of PAI-1 (Nykjaer et al., 1994; Horn et al., 1998; Rodenburg et al., 1998; Stefansson et al., 1998; Skeldal et al., 2006). Considering the result of the endocytosis of uPA-PAI-1 , i.e. degradation, the role of this process is probably to free the receptor and allow it to be redistributed at sites of necessity ones more (Nykjaer et al., 1997). A further aspect of the present invention relates to the aptamers for use as a medicament.

Yet another aspect of the invention therefore relates to the use of the aptamer as as a medicament for a cancer, wherein in one embodiment the medicament is for the prevention of metastatic activity of a cancer.

A further aspect of the present invention relates to a pharmaceutical composition comprising an aptamer or a pharmaceutically acceptable salt thereof, carrier, diluent or adjuvant.

The disclosed oligomers i.e. aptamers can be used in pharmaceutical mixtures that contain one or several aptamers as the active substance, and, optionally, pharmaceutically acceptable auxiliary substances, additives and carriers e g saline or dsstiϋed water Optionally, the formulations described herein also comprise excipients that stabilize the aptamer, whereby the therapeutic activity is mamtaiπrg. Excperts such as salts, sugars and alcohols may facilitate diffusior of the aptamer therapeutic Non limiting examples of excports that can bo used in combination with the presort invention include saccharides, such as sucrose, trehalose lactose, fructose galactose, mannitol, dexlrar and glucose Other lypes of excipients are poly alcohols, such as glycerol or sorbitol or proteins, such as albumin, hydrophobic molecules, such as oils, and hydrophilic polymers, such as polyethylene glycol, amonα others

Pharmaceutical formulations ot compounds of the irvention described herein comprise isomers such as diastereomers and enaπtiomers, mixtures of isomers, including racornic mixtures salts solvates, and polymorphs thereof Formulations may be in the form of tablets or capsules for oral administration, Intranasal formulations may be in the form of powders, nasal drops, or aerosols. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycolate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel. Therapeutic formulations may be in the form of liquid solutions or suspensions. Methods well known in the art for making formulations are found, for example, in Remington : The Science and Practice of Pharmacy (20th ed. , ed. A. R. Gennaro AR. ), Lippincott Williams & Wilkins, 2000.

The aptamers of the present invention may be encapsulated within or administered with a biocompatible polymer to provide controlled release of the aptamer. The biocompatible polymer can be either a biodegradable polymer or a biocompatible non- degradable polymer which releases over time the incorporated aptamer by diffusion. The aptamer can be homogeneously or heterogeneously distributed within the biocompatible polymer. A variety of biocompatible polymers are useful in the practice of the invention, the choice of the polymer depending on the rate of drug release required in a particular treatment regimen. The aptamers can be provided in a polymeric sustained release formulation in which the amount of aptamer in the composition varies from about 0.1% to about 30%, from about 0.1 % to about 10%, or from about 0. 5% to about 5% (w/w). Non-limiting examples of synthetic, biodegradable polymers include: polyamides such as poly (amino acids) and poly (peptides); polyesters such as poly (lactic acid), poly

(glycolic acid), poly (lactic-co-glycolic acid), and poly (caprolactone); poly (anhydrides); polyorthoesters ; polycarbonates; and chemical derivatives thereof (substitutions, additions of chemical groups (e. g., alkyl, alkylene), hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), copolymers and mixtures thereof.

The aptamer can also be encapsulated within a biocompatible non-degradable polymer. Non- limiting examples of non-degradable polymers include polysaccharides; polyethers, such as poly (ethylene oxide), poly (ethylene glycol), and poly (tetramethylene oxide); vinyl polymers, such as polyacrylates, acrylic acids, poly (vinyl alcohol), poly (vinyl pyrolidone), and poly (vinyl acetate); polyurethanes; cellulose- based polymers, such as cellulose, alkyl cellulose, hydroxyalkyl cellulose, cellulose ethers, cellulose esters, nitrocellulose, and cellulose acetates; polysiloxanes and other silicone derivatives. Alternatively, the aptamers can be encapsulated within liposomal formulations. Within the scope of pharmaceutical compositions of the present invention are also solutions, emulsions, and liposome-containing formulations. It is appreciated that the examples are non-limiting examples. The compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self- emulsifying solids and self-emulsifying semisolids. The delivery of drug to tumour tissue may be enhanced by carrier-mediated delivery including, but not limited to, cationic liposomes, cyclodextrins, porphyrin derivatives, branched chain dendrimers, polyethylenimine polymers, nanoparticles and microspheres (Dass CR. J Pharm Pharmacol 2002; 54(l):3-27).

The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Compositions of the invention are useful for administration to a subject suffering from, or predisposed to, a disease or disorder which is related to or derived from a target to which the aptamers of the invention specifically bind. Compositions of the invention can be used in a method for treating a patient or subject having a pathology, such as cancer. The method involves administering to the patient or subject a composition comprising aptamers, and/or aptamer-drug conjugates that bind to a specific cell surface component (e.g., an integral membrane protein) associated with the pathology, so that upon binding of the aptamer or aptamer-drug conjugate to the cell surface component (and delivery of a toxic payload to the cells on which the component is expressed occurs), treatment of the pathology is achieved.

The patient or subject having a pathology, i.e., the patient or subject treated by the methods of this invention, can be a mammal, or more particularly a human. In practice, the aptamers and/or the aptamer-drug conjugates or their pharmaceutically acceptable salts, are administered in amounts which will be sufficient to exert their desired biological activity.

One aspect of the invention comprises an aptamer composition of the invention in combination with other treatments for cancer related disorders. The aptamer composition of the invention may contain, for example, more than one aptamer,for example 2 aptamers, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 aptamers. In some examples, an aptamer composition of the invention, containing one or more compounds of the invention, is administered in combination with another useful composition such as a cytotoxic, cytostatic, or chemotherapeutic agent such as an alkylating agent, antimetabolite, mitotic inhibitor or cytotoxic antibiotic. In general, the currently available dosage forms of the known therapeutic agents for use in such combinations will be suitable.

"Combination therapy" (or "co-therapy") includes the administration of an aptamer composition of the invention and at least a second agent as part of a specific treatment regimen intended to provide the beneficial effect from the co-action of these therapeutic agents. The beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents. Administration of these therapeutic agents in combination typically is carried out over a defined time period (usually minutes, hours, days or weeks depending upon the combination selected).

"Combination therapy" may, but generally is not, intended to encompass the administration of two or more of these therapeutic agents as part of separate monotherapy regimens that incidentally and arbitrarily result in the combinations of the present invention. "Combination therapy" is intended to embrace administration of these therapeutic agents in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneous manner. Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single capsule having a fixed ratio of each therapeutic agent or in multiple, single capsules for each of the therapeutic agents.

Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, topical routes, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues. The therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the combination selected may be administered by injection while the other therapeutic agents of the combination may be administered topically.

Alternatively, for example, all therapeutic agents may be administered topically or all therapeutic agents may be administered by injection. The sequence in which the therapeutic agents are administered is not narrowly critical unless noted otherwise. "Combination therapy" also can embrace the administration of the therapeutic agents as described above in further combination with other biologically active ingredients. Where the combination therapy further comprises a non-drug treatment, the non-drug treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and non-drug treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the non-drug treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.

Therapeutic or pharmacological compositions of the present invention will generally comprise an effective amount of the active component(s) of the therapy, dissolved or dispersed in a pharmaceutically acceptable medium. Pharmaceutically acceptable media or carriers include any and all solvents, dispersion media, diluents, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the therapeutic compositions of the present invention.

The effectiveness of a composition according to the present invention is determined by the ability to migrate through the body and reach the target site(s) at therapeutically effective amounts. Whereas most currently approved antibody therapeutics are administered by intravenous infusion (typically over 2-4 hours), aptamers can be administered by subcutaneous injection (aptamer bioavailability via subcutaneous administration is >80% in monkey studies (Tucker et al., J. Chromatography B. 732: 203- 212,1999)). This difference is primarily due to the comparatively low solubility and thus large volumes necessary for most therapeutic mAbs. With good solubility (>150 mg/ml_) and comparatively low molecular weight (aptamer: 10-50 kDa; antibody: 150 kDa), a weekly dose of aptamer may be delivered by injection in a volume of less than 0.5 ml_. In addition, the small size of aptamers allows them to penetrate into areasof conformational constrictions that do not allow for antibodies or antibody fragments to penetrate, presenting yet another advantage of aptamer-based therapeutics or prophylaxis.

The compounds of the present invention can be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions.

Parenteral injectable administration is generally used for subcutaneous, intramuscular or intravenous injections and infusions. Additionally, one approach for parenteral administration employs the implantation of a slow-release or sustained-released systems, which assures that a constant level of dosage is maintained.

The pharmaceutical compositionof the present invention is preferably administered by exploiting mucosal routes, for example nasal, in order to avoid the first-pass hepatic clearance mechanism which is associated with intravenous administration.

Furthermore, preferred compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, inhalants, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen. Other preferred topical preparations include creams, ointments, lotions, aerosol sprays and gels, wherein the concentration of active ingredient would typically range from 0.01 % to 15%, w/w or w/v. The dosage regimen utilizing the aptamers is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular aptamer or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.

In a preferred embodiment of the present invention the pharmaceutical composition comprises one or more aptamers chemically modified with chitosan. Chitosan has mucoadhesive and mucosa permeation properties which makes the mucosal administration route for pharmaceutical compositions of the present invention comprising chitosan, the preferred route for administration.

Imaging and diagnostics

One aspect of the invention pertains to a diagnostic kit comprising an aptamer as described elsewhere herein. The diagnostic kit can be employed in detecting/locating the aptamer, or quantifying the amount of aptamer present for example in different types of tissues. The diagnostic kit may thus be used in methods for imaging. The invention therefore in another aspect also relates to a method for diagnosing a disease, such as cancer ie. a tumor, or metastatic activity related to said cancer, the method comprising applying the aptamer of the present invention, and detecting the presence or absence of said aptamer.

Another one aspect of the invention relates to the use of the aptamer as an agent for imaging which can be used to locate u-PA. A sample can be analysed by imaging for the presence or absence of uPA in various parts of the sample to be analysed. The amount of uPA can be detected and quantified.As described elsewhere herein uPA is found to be present in higher amounts in tumor tissue compared to the corresponding normal tissue. Thus, it is believed that the amount of uPA is higher in tumor tissue and a quantitative measure of the amount of uPA in the sample in question is indicative of the presence of a tumor. Sample in this context refers to for example a tissue sample of a mammal or a whole body or body part subjected to imaging. The types of imaging technology which is useful in the context of the present invention is selected from the group consisting of positron emission tomography (PET), magnetic resonance (MR) imaging, X-ray, X-ray computed tomography (CT), CT angiography (CTA) imaging, magnetic resonance angiography (NIA), nuclear imaging, ultrasound (US) imaging, optical imaging, infrared imaging and microwave imaging. In one embodiment the imaging is selected from the group consisting of PET, MRI, x-ray CT and x-ray. In another embodiment the imaging is selected form the group consisting of PET, MRI and x-ray CT. Any of positron emission tomography (PET), magnetic resonance (MR) imaging, X-ray, X-ray computed tomography (CT), CT angiography (CTA) imaging, magnetic resonance angiography (NIA), nuclear imaging, ultrasound (US) imaging, optical imaging, infrared imaging or microwave imaging represents separate embodiments of the present invention. One preferred embodiment is PET scanning. Another preferred embodiment is MRI.

The route of delivery of the aptamers into a mammal, particularly a human, for imaging purposes are as described elsewhere herein.

Examples

Human uPA was from Wakamoto Pharmaceutical Company (Japan) and pro-uPA from

Molecular Innovations (USA). Low molecular weight uPA (LMW-uPA), i.e., uPA N- terminally truncated at Lys136, and the amino terminal fragment (ATF) was prepared as described previously (Egelund et al., 2001 b). Murine uPA was from Molecular

Innovations (USA).

The monoclonal antibodies to human uPA have been described previously, mAb anti- uPA clone 2 and mAb anti-uPA clone 6 (Grondahl-Hansen et al., 1987; Petersen et al., 2001 ). Rabbit polyclonal anti-human uPA antibody has been described before (Knoop et al., 1998). Monoclonal antibodies towards murine uPA, mAb-H77B6 and mAb- H77A10 were obtained from Molecular Innovations, USA.

RNA aptamer selection Nucleic acid aptamers are oligonucleotides (DNA or RNA) identified by an in vitro selection process called SELEX (systematic evolution of ligands by exponential enrichment) to bind a given target (for a review see (Ellington and Szostak, 1990; Tuerk and Gold, 1990; Wilson and Szostak, 1999)). The technique takes advantage of the ability of RNA and DNA, like a peptide, to fold into a three-dimensional structure and the possibility of screening up to 10¹⁵ different nucleic acid sequences or structures for the ones capable of recognising for example a protein of interest. Numerous selection experiments have resulted in identification of protein binding aptamers with affinities and specificities comparable to antibodies even when binding of nucleic acids is not a normal function of the protein target. A typical nucleic acid library applied in the SELEX procedure contains oligonucleotides composed of a central region of random sequence, flanked by defined sequences in the 5^' and 3^' end, important during the steps of the selection procedure. Figure 5 illustrates the RNA aptamer selection procedure. The RNA library is generated from a double-stranded DNA (dsDNA) transcription template, incubated with the target of interest and the binding fraction partitioned usually by filtration through nitrocellulose or by affinity chromatography after extensive washing. Bound RNA sequences are then harvested and can be converted into new dsDNA transcription templates by reverse transcription and PCR using primers targeting the defined flanking sequences of the RNA. The dsDNA templates can then be inserted into vectors, which are electroporated into E.coli and used to identify RNA sequences by DNA sequencing of vectors from individual clones. Usually, as was the case also with phage-displayed peptide selection, several rounds of selection are necessary to enrich target specific RNA aptamers and dsDNA templates therefore used to produce the RNA pool for the subsequent selection round. Aptamers represent a promising new class of targeting agents with potential use in vivo (Hicke and Stephens, 2000; James, 2000). The susceptibility to nuclease degradation can be overcome by using modified nucleotides in the selection experiment, and when applied in vivo, aptamers have not shown toxicity or immunogenicity following testing in several mammalian species. The size of aptamers, 5 to 40 kDa, is intermediate between peptides and antibodies and has been shown to result in good tissue penetration. Additionally, the ease at which the aptamers are synthesised and modified allows for easy tailoring of aptamers for a wide range of applications, such as therapeutics, diagnostics and research. The in vivo potential of aptamers is illustrated by the FDA approval of the first therapeutic agent based on an aptamer in December 2004 (Ng et al., 2006) The guide lines presented in Kenan and Keene, 1999, were used as a starting point for generating RNA libraries and in vitro selecting RNA aptamers (Kenan and Keene, 1999).

Example 1 Generation of a RNA library with unmodified ribonucleotides For the first RNA aptamer selection experiment conducted, a RNA library was created consisting of unmodified normal ribonucleotides. The procedure for generating RNA libraries is illustrated in figure 6.

As shown here, a double-stranded DNA (dsDNA) template library was prepared in order to produce a library of 79mer RNA sequences having a variable 35 nucleotide central region flanked by two primer binding sites. 60 μg of dsDNA was used in the transcription reaction to generate the RNA library, which corresponds to a maximum theoretic library diversity of ~5^»10¹⁴ (the molecular weight of one dsDNA template is -69 kDa), when assuming that the degenerate "N35 library" primer supplied from a commercial source was completely degenerate.

Following oligonucleotides were obtained from a commercial source (DNA Technology A/S, Denmark): The "Forward N35" primer (5^'-CGCGGATCCTAATACGACTCACTATAGGGGCCACCAACGACATT-S^') provides a T7 promotor sequence and the "N35 library" primer (5^'-

GATCCATGGGCACTATTTATATCAAC(N₃₅)AATGTCGTTGGTGGCCC-S^') includes the degenerate sequence. To generate a library of dsDNA transcription templates, "Forward N35" primer and "N35 library" primer were mixed and annealed by heating to 75 ^eC for 15 minutes and slowly cooling to room temperature. Annealed primers were then extended by Klenow enzyme (3^'=>5^' exo^'; New England Biolabs, USA) for 1 hour at 37 ^eC and dsDNA products purified by electrophoresis in a non-denaturing 6% polyacrylamide gel (National Diagnostics, UK) using IxTBE (Invitrogen, Denmark) as running buffer. Gel pieces containing dsDNA were cut out, and DNA eluted overnight at 4 ^eC in sodium acetate (NaAc; pH 5.2). Eluted DNA was then phenol/chloroform (pH 7.9) extracted, precipitated with ethanol and resuspended in ddH₂O. The concentration of dsDNA was estimated by measuring the absorbance at 260 nm and using that one Abs_260nm-unit = 50 μg /ml_ double-stranded DNA.

For generating the RNA library with unmodified ribonucleotides, 60 μg of purified degenerate dsDNA template was transcribed in 5 ml_ 80 mM HEPES-NaOH (pH 7.5), 30 mM DTT, 25 mM MgCI₂, 2 mM spermidine-HCI containing 2.5 mM of each NTP (GE Healthcare/Amersham Biosciences, Denmark), 100 μg/mL acetylated BSA (Ambion/Applied Biosystems, Denmark) and 5 U/mL inorganic pyrophosphatase (Sigma-Aldrich, Denmark) using 100 μg/mL T7 RNA polymerase (kindly provided by Ray Brown, Department of Molecular Biology, Aarhus University, Denmark) for 4 hours at 37 ^eC. The reaction was then subjected to phenol/chloroform extraction (pH 6.6), and RNA ethanol precipitated and resuspended in 8 M urea. RNA was run on a BIO- RAD 491 prep column (Bio-Rad, Denmark) containing 12.5% denaturing polyacrylamide gel material (National Diagnostics, UK) and eluted from the column by electrophoresis using ¹/2xTBE as running buffer. Eluted fractions were analysed by denaturing polyacrylamide gel electrophoresis and fractions containing RNA of the expected size pooled, RNA ethanol precipitated and pellets resuspended in 10 mM Tris-HCI (pH 8), 1 mM EDTA, also known as TE8 buffer. The concentration of RNA was estimated by measuring the absorbance at 260 nm and using that one A₂₆o_nm-unit = 40 μg /ml_ single-stranded RNA.

Example 2

Generating the RNA library containing 2^'-F-pyrimidines

To select serum-stable aptamers, a new RNA library was generated where all pyrimidines in the RNA sequences were modified versions, having a fluor atom at the 2^' position of the ribose-ring instead of the hydroxyl-group. The dsDNA transcription template library used to generate the library had the same construction as mentioned for the unmodified RNA library (see figure 6). In the transcription reaction this time however, a mutant T7 polymerase had to be used, as the wildtype T7 polymerase does not efficiently incorporate 2^'-F-analogues into RNA transcripts (Sousa and Padilla, 1995; Huang et al., 1997). The amount of dsDNA template used for generating the RNA library was 1 17 μg, corresponding to a maximum theoretic diversity for the 2^'-F- pyrimidine RNA library of ~10¹⁵.

Example 3

Selecting 2^'-F-pyrimidine aptamers to human uPA

A selection experiment was set up aimed at selecting 2^'-F-pyrimidine RNA aptamers to human uPA. The isoelectric point of uPA is well above 7 (pl~9) indicating that it is an overall positively charged protein. It has been suggested that aptamers might have a major tendency to target areas containing positively charged residues while being repelled by surfaces containing negative side chains because of the phosphate containing backbone. This would mean that only a small part of a target protein might actually be accessible to the aptamers and suggests that affinities for favourable and unfavourable binding sites could differ by many orders of magnitude. This might also explain why aptamer binding sites often overlap with the binding area of naturally binding polyanions, why aptamers are biased to target certain regions and why aptamers binding to heparin and nucleic acid binding proteins usually bind with better affinities than aptamers targeting proteins that do not bind such polyanions (James, 2000). The high pi of uPA and the ability to bind heparin suggests that uPA is a good target for RNA aptamers. As found in the selection of 2^'-F-pyrimidine RNA aptamers to human PAI-1 , enrichment of target specific RNA was detectable after 6 rounds of selection, but this time it continued through round 7 and 8 as well (figure 7). Human uPA was captured on protein A beads using a mixture of the monoclonal anti- uPA antibody clone 6 and a rabbit polyclonal anti-uPA antibody. In the first selection round an amount of the 2^'-F RNA library was again applied corresponding to 5 copies of each of the 10¹⁵ different RNA species. A total of 8 rounds of selecting and amplifying 2^'-F-py RNA aptamers towards human uPA were completed, and in round 2 to 8 RNA transcripts were randomly labelled with trace amounts of [α-³²P]-dATP in order to follow the enrichment of RNA species binding to the uPA-Ab-beads (figure 7).

Example 4

The sequences of individual 2^'-F-py RNA clones from the human uPA selection experiment

After selection round 8, the sequences of 40 individual RNA clones were analysed and

29 different RNA clones identified. A sequence alignment using ClustalW is shown in table 1 . The alignment reveals that several clones have sequence similarities.

Clone# (frequency) Sequence of variable region length (nt )

kolol2 ( 4 ) —UGCGACUGUUAUAACCUAACAGCGACGUAAAG-AUA 35 kolo79 GAAACGACUCG-ACAACCU-CGAGCGACGUGAAUCAU 35 kolo84 -UACCGACUAGCAAAACCUGCUGGCGACGUUUAG-AU 35 kolo21 (6) AAUAACCUUAAUGCGACGUUGGUUUGUCAACAACG 35 kolo71 AAUAACCUUAAUGCGACGUUGGUUCGUCAACAUGG 35 kolo28 ACAACCUAAAUGCGACGUUGGGUCAAAAACGUGAA 35 kolo25 GAUAACCUCGAUGCGACGUUCGGCC-UCAAAAUCAA 35 kolo78 ACAAC-UUAAUGCGACGUUGGUAAAGCAUAUCAAAC 35 kolo26 CUCCGCUAUCUAACGUAUGAUA-GAAUGGAUGACUA 35 kolo83 UGCGCAUGAAAUGACUGCAUGUCUCCGGAUUGAUC- 35 kolo68 AUGACAGGAUGCAGAGCUCCACUGUCUAGUGUUUA 35 kolo24 CCGCGACAGUCGUAAGUUUUGACUGACUGAACGUU 35 kolo88 UCGCUAAUUAUAGGCGGAGUGCGACGUUAUAAAUA 35 kolo9 (3) AGUGAUUCGCCAUAACC-UGGCUGUUUCAGGCUGUU 35 kolo75 UCGCAUUCGUUAUAACC-UAACAGUUUGCGGA-GUUA 35 kolol7 UAGUACGACC-UACUUAUUUGAC 22 kolo73 UUGCACAACC-UGCAUAUUUGUCGUCGACGUACGAA 35 kolo82 CGAUACAACC-UAUCUGUUUGUCGUGGACAUCAAAU 35 kolo67 UGCUAUGACC-UAGACAUUUGACG 23 kolo86 UACCAUAACC-UGGAUAUCUGUCGUUAUCAUGGGAC 35 kolo20 UUGCGUAACCCGCAAUUUCCGUUGGUAUAUAC 32 kolo70 ACCUGACUUGCGAUAACC-UCGACAUUUACGGUAUU 35 kololO (2) UAAUGGCCGGUUUAACCGUU-UGCGCAUUAAGUCGA 35 kolol5 UACCAUUUACCACAACC-UGGUUAUCUGGUUUAAAC 35 kolo27 UUGUCGUCUUCUCCAGU—UAGUCAACGAACUUGUGU 35 kolol9 UCCGUUUGCAGGUUUUGACCUGCCGGAGUUGUUUG 35 kolo81 GGCAGAAGUUGAUUCCAACUUCAUUUGCGUUUAAU 35 kolo80 AUAUUUGUCUCAUCCCACGACAAUUAUGAUGCGAC 35 kololl AUCUUAGCGACGUGACACACGACUAGGGAUUAAUC 35

Table 1. Alignment of the variable regions of RNA sequences obtained in the selection experiment aimed at identifying 2 -F-pyrimidine RNA aptamers to human uPA. The clone name is found to the left with the number of identical clones obtained in parenthesis. In the middle, the random region of the RNA sequences is shown and at the right, the length of the random region.

Example 5

Screening the 29 individual RNA clones for binding to uPA using SPR To determine whether the obtained individual RNA clones had been selected in the selection experiment because of affinity to human uPA, the clones were analysed in terms of binding to human uPA using surface plasmon resonance. uPA was captured on the sensor surface using either immobilised monoclonal anti-uPA antibody clone 2, clone 6 or polyclonal anti-uPA antibody. The monoclonal anti-uPA antibody clone 2 binds to the serine protease domain of uPA, whereas clone 6 binds to the kringle domain. Reference surfaces always contained the respective antibodies alone to ensure that surface association was not due to binding of RNA clones to the antibodies. When 250-850 RU of uPA was captured in the different setups and 100-200 nM of the 29 individual RNA clones were passed over the sensor surface, though some of the responses were very small (2-3 RU), all clones were found to associate with the surface in at least one of the three setups, whereas non-relevant clones did not (data not shown). Figure 8 shows the result of passing RNA aptamer kolo12 and a non- relevant clone over a sensor surface with uPA captured using the immobilised polyclonal anti-uPA antibody. Attempts were made to do kinetic analysis on the interaction of several aptamer clones with uPA in the different setups but the data could not be fitted to simple 1 :1 binding models, maybe because of heterogeneity in the presentation of uPA or heterogeneity in the RNA samples (data not shown).

To test for cross-specificity the RNA clones were also analysed in terms of binding to the murine form of uPA using SPR. In this setup murine uPA was captured to a level of 250 RU on the active surface of a CM5 chip coupled with the monoclonal anti-murine uPA antibody mAb-H77A10. This setup has previously been used to study a peptide binding to the active site of murine uPA (Andersen et al., unpublished). None of the RNA clones from the human uPA selection experiment were found to associate detectably with murine uPA when passed over the surface at a concentration of 200 nM (data not shown).

All 29 clones were found to associate with uPA as determined using SPR analysis.

Example 6

Analysis of the inhibitory effects of RNA aptamers on the interaction between uPAR and uPA using SPR

An active sensor surface was also coupled with the receptor for uPA (uPAR) while the reference was blocked with ethanolamine. The chip was initially used to study whether aptamer clones to human uPA were able to associate to uPA when bound to uPAR. In figure 9, the result of passing 100 nM of kolo12 over a surface containing -900 RU uPAR-bound uPA is shown.

No measurable binding of kolo12 to uPA was detected in this setup and the same result was obtained for the remaining 28 individual clones from the selection experiment (data not shown). This finding suggested that the association of uPA with its receptor sterically hinders aptamer binding to uPA. None of the 29 clones were found to associate with uPA when uPA was bound to uPAR (figure 9).

The association of a fixed amount of uPA (2 nM) to the uPAR sensor surface was therefore studied in the presence of increasing concentrations of RNA aptamer clones. The results with kolo12 and a non-relevant aptamer clone is shown in figure 10. It is seen that kolo12 is able to dose-dependently prevent the association of uPA to uPAR, whereas the non-relevant clone is not. All clones were tested at concentrations up to 200 nM and IC₅₀ values estimated for the ones having a half-maximal inhibition at concentrations below 200 nM (see table 2). The ability of of RNA clones to inhibit the uPA-uPAR interaction was therefore investigated and several clones found to be low nanomolar inhibitors of the interaction (figure 10 and table 2) The specificity of the aptamers was analysed by investigating if they could inhibit the murine uPA - murine uPAR interaction. When a fixed concentration of murine uPA (3 nM) was passed over a sensor surface with murine uPAR in the presence of 250 nM of the different RNA aptamers, none were found to inhibit this interaction detectably (data not shown).

Clone name IC₅₀ (huPA-huPAR) IC₅₀ (huPA-muPAR)

κoioi2 5. 0 +/- 3.5 (n=6) 7.1 +/- 4.5 (n=3)

K0I021 4. 6 +/- 1.1 (n=6) 5.3 +/- 1.4 (n=3)

Kolo24 51. 2 +/- 6.6 (n=3)

Kolo25 5. 2 +/- 0.9 (n=3) 6.9 +/- 0.1 (n=3)

Kolo26 8. 0 +/- 0.5 (n=3) 11.5 +/- 1.6 (n=3)

Kolo27 11. 4 +/- 4.2 (n=3) 14.2 +/- 0.8 (n=3)

Kolo28 10. 1 +/- 1.1 (n=3) 10.2 +/- 1.7 (n=3)

K0I068 58. 3

Kolo71 6. 0 +/- 0.6 (n=3)

Kolo78 31. 0

Kolo79 7. 1 +/- 1.2 (n=3) 7.4 +/- 1.6 (n=3)

Kolo84 64. 8

K0I088 140. 0

Kolol2 .33ntEND 11. 6 +/- 4.5 (n=3) 11.1 +/- 1.6 (n=3)

Kolol2 .33ntGG 11. 2 +/- 4.8 (n=3)

Kolo21 .35ntEND 13. 6 +/- 4.6 (n=3) 10.3 +/- 1.4 (n=3)

Kolol2 .49ntEND 5. 6 5.2 +/- 0.6 (n=3)

Kolo79 .44nt 8. 0 7.8 +/- 1.5 (n=3)

Kolo79 .44ntENDl 6. 2

Kolo79 .44ntEND2 8. 6 4.0 +/- 0.3 (n=3)

Kolo79 .34nt 178. 0 232.0

Kolo79 .48ntf unny 13. 2 11.7

Table 2. The inhibitory constants (IC₅₀) for RNA clones with respect to inhibition of the human uPA - human uPAR interaction (huPA-huPAR) and the human uPA - murine uPAR interaction (huPA-muPAR) as determined by SPR analysis

The left column contains the names of the RNA clones found to have IC₅₀ values below 200 nM. The columns in the middle and at the right contain the IC₅₀ values determined for the human uPA-human uPAR and human uPA-murine uPAR interaction, respectively, with the number of repeats of the experiment in parenthesis. The IC₅₀-values have been obtained from plots like the one shown in figure 8C by nonlinear regression, fitting the data to a Standard equation for one site competition.

Example 7

Determining the affinity of kolo12 to human uPA using the SPR "Affinity in solution" procedure From SPR analysis of the direct binding of aptamers to uPA captured on the sensor surface using monoclonal or polyclonal anti-uPA antibodies it was not possible to determine the affinity constants (K₀) from the association and dissociation curves as the data could not be fitted using simple 1 :1 binding models. An alternative indirect SPR strategy called "Affinity in solution" was therefore applied in order to estimate an affinity constant for aptamer koloi 2 (the theoretical background can be found in appendix A). The approach takes advantage of the fact that the uPAR chip does not detect uPA in complex with kolo12. This means that when running a sample containing uPA and kolo12 over the sensor surface, the response reflects the amount of free uPA in the sample, which can be quantified by comparing it to the response obtained from a standard curve of uPA alone. By running a fixed concentration of uPA in the presence of increasing concentrations of kolo12, as done when estimating the IC₅₀ values, the K₀ can then be calculated from a plot of free uPA as a function of aptamer concentration. A requirement is that the measurement itself does not significantly disturb the equilibrium in the sample. The interactants in "affinity in solution" determination are determined A and B:

A + B = AB

Experiments are set up so that a fixed concentration of B is mixed with variable concentrations of A and allowed to reach equilibrium. The free concentration of B is then determined by injecting the sample over a ligand that binds B but not the complex AB (the interactant A or one with an overlapping binding epitope is usually suitable as ligand). It is assumed that the measurement itself does not significantly disturb the equilibrium in the sample.

The experimental setup requires a calibration curve with known concentrations of B determined over the same sensor surface, in order to calculate the free B concentration in the samples. The equilibrium constant for a 1 :1 interaction is given by:

^Afree « * free

K_D = ^■ or

AB

(A_tot - AB) . (B_tot - AB)

K_D =

AB Rearranging gives:

K_D • AB = A_m • B_m - AB • (A_tot + B_m ) + AB or AB² - AB • (A_tot + B_m + K_D ) + A_m • B_m = 0

Solving for AB:

_AB JΛ_{m +} B_{m +} K_D) (Λ_{m + Bm + K}J- _ tot tot

Substituting in the relationship: B_free = B_tot - AB gives

(B_tot - A_tot - K_D)

Bfree = ^" ±

This equation can be fitted to a plot of B_free against A_tot to calculate a value for K₀.

To test this, the ability of aptamers to inhibit the interaction between human uPA and murine uPAR was tested by SPR. As murine uPAR has a much lower affinity for human uPA compared with human uPAR, one would expect the IC₅₀ values to be different if this interaction affects the equilibrium. From table 2 it is seen that the IC₅₀ values were unchanged, suggesting that the equilibrium between human uPA and aptamers is not affected by the measurement itself. The interaction of human uPA with kolo12 was analysed three times using the "Affinity in solution" procedure of the Biacore T100, resulting in a K₀ of 3.2 nM +/- 0.5 nM.

Example 8

KoIoI 2 binds to the growth factor domain of uPA Using the SPR sensor surface coupled with polyclonal anti-uPA antibody the interaction of kolo12 with different forms of uPA was analysed (see figure 1 1 ).

When the same amount of the different forms of uPA were captured on the sensor surface, taking the differences in molecular mass into account, it was found that 50 nM kolo12 bound equally well to uPA and pro-uPA but not at all to uPA variants lacking the growth factor domain (ΔGFD) or the entire amino terminal fragment (LMW-UPA). The results suggest that kolo12 binds to the growth factor domain and it was therefore unexpected that the binding of kolo12 to the amino terminal fragment alone was reduced compared to uPA and pro-uPA. The explanation must be that when presenting ATF (-17 kDa) there is a much higher probability that the antibodies doing this will interfere with aptamer binding compared with when presenting full-length uPA. SDS- page analysis of the amino terminal fragment preparation did not suggest that the binding observed could be due to the presence of small amounts of full length uPA (data not shown). An identical binding profile was observed for clones kolo21 , -25, -26, -27, -28, -71 , and -79. Attempts were made to couple the amino terminal fragment directly on an SPR sensor surface (500 RU) and analyse the interaction with 200 nM kolo12, but no measurable binding was detected in this setup (data not shown). Interestingly, binding of soluble uPAR was detected in this setup, when applied at a concentration of 200 nM. When ATF was captured using the monoclonal anti-uPA antibody clone 6, which binds to the kringle of uPA, kolo12 was found to bind ATF (data not shown).

Example 9 Human uPA 2^'-F-py aptamers do not affect the plasminogen activation activity of human uPA

All 29 individual clones were tested for effects on the peptidolytic activity of human u PA. As shown in figure 12, hydrolysis of the chromogenic substrate S-2444 was completely inhibited upon pre-incubation of uPA with PAI-1 , partially by the upain-1 peptide, and was unaffected by the presence of 100 nM of aptamer clone koloi 2. The same result was obtained for all of the 29 individual clones (data not shown). In the experiment 1 mM of S-2444 substrate was used. Lowering the concentration of substrate to 50 μM made the assay more sensitive to inhibition by upain-1 but the RNA clones still did not show any sign of inhibitory activity (data not shown). Antibodies to the serine protease domain of uPA have previously been shown to inhibit plasminogen activation without having an effect on S-2444 hydrolysis (Petersen et al., 2001 ). Aptamers were therefore also tested for inhibitory activity towards uPA-mediated plasminogen activation in solution. The result with kolo12 is shown in figure 13 and represents the result obtained with all of the clones, i.e. no effect. There was a small inhibitory effect of the purified uPA receptor on the plasminogen activating activity of uPA but this has previously been reported (Ellis et al., 1991 ; Behrendt and Dano, 1996). A strong stimulation of plasminogen activation was seen with 250 nM auPA clone 6 (data not shown), which stimulates by a template mechanism involving plasminogen binding to a C-terminal lysine residue on the antibody (Ellis and Dano, 1993).

Example 10

Prediction of secondary structures and truncation of aptamers

5 of the 6 clones having IC₅₀ values below 10 nM have similarities in terms of sequence and predicted secondary structure. These five clones are koloi 2, 21 , 25, 71 and 79.

They are all characterised by having a conserved asymmetrical internal loop, CGA, and a conserved hairpin loop, (U/C)AACC, suggesting that these motifs are the important parts in the aptamers necessary for target recognition (see figure 14 and figure 15).

When predicting the secondary structures using the standard conditions of the MFOLD program only one structure was obtained for kolo12, 21 and 25. Kolo71 has two solutions but they only differ in how the 3^' and 5^' ends are paired. Kolo79 differs a bit from the four others, as two solutions are predicted where the second is completely different. Also, kolo79 is the only one of the five having a C at the first position in the hairpin loop instead of a U, and the duplex region between the hairpin loop and the asymmetrical internal loop is only 5 basepairs long compared to 6 for the others. For the clones having a duplex of 6 basepairs, the four central ones were not conserved. The similarities suggest that the important determinants for uPA binding and inhibitory activity towards the uPA-uPAR interaction perhaps could be retained in truncated versions like kolo12.33 (blue rectangle, figure 14). The truncated RNA variants koloi 2.33ntGG, koloi 2.33ntEND, and kolo21.35ntEND were generated and found to fold into the expected secondary structures (figure 16). Compared with KoIoI 2.33, kolo12.33ntGG has a 5^'-GG extension, which increases the efficiency of the transcription reaction (Milligan and Uhlenbeck, 1989). In kolo12.33ntEND and kolo21 .35ntEND, GG has been substituted into the 5^'-END concomitantly with a CC substitution in the 3^'-END to retain the duplexes (the END-modification).

By SPR analysis the truncated forms were shown to be only 2-3 fold poorer in terms of inhibitory activity towards the uPA - uPAR interaction compared to the full length versions (see figure 15, table 2). Attemps to truncate kolo79 according to the alternative predicted structure (Kolo79(1 ) in figure 15) into kolo79.34nt (figure 15) resulted in a ~25-fold reduction of the inhibitory activity, indicating that truncation steps should probably not be based on this predicted structure. Another truncated form of koloi 2, koloi 2.49ntEND (kolol 2.49 in figure 14 with the END-modification) containing an additional predicted internal loop of the aptamer compared to kolol 2.33ntGG and kolo12.33ntEND was comparable to full-length kolol 2 in terms of inhibitory activity, suggesting that the lack of this internal loop in kolol 2.33ntGG and kolol 2.33ntEND is the reason for the 2-3 fold loss in inhibitory activity. The region included in kolol 2.49ntEND compared to kolol 2.33ntEND or kolol 2.33ntGG is very different in the five otherwise similar clones. Kolol 2 and 79 have different internal loops, kolo25 has an asymmetrical internal loop, kolo21 has a three stem junction, and kolo71 either an asymmetrical internal loop or a three stem junction depending on the two predicted secondary structures for this clone. Truncated aptamers kolo79.44nt, kolo79.44END1 and kolo79.44ntEND2, similar to kolo12.49ntEND, were found to also retain full inhibitory activity towards the uPA - uPAR interaction compared to full length kolo79, whereas another truncated form of kolo79, kolo79.48ntfunny, was 2-3 fold poorer (figure 15, table 2). Kolo79.44nt, kolo79.44ntEND1 , kolo79.44ntEND2, and kolo79.48ntfunny illustrate different attempts to truncate kolo79 resulting in variants with different propensity to fold into the alternative secondary structure of kolo79 according to the MFOLD program.

If the extra region is necessary in all clones to retain maximum activity the shortest construct with full inhibitory activity might be generated from kolo25, resulting in a 41 mer (when including a 4 basepair duplex region between the 5^'- and 3^'-END).

Example 11

Kolol 2 inhibits the association of uPA variants to the cell surface of U937 cells The ability of RNA aptamers to inhibit the uPA - uPAR interaction was studied in cell culture assays. In one assay, the binding of ¹²⁵l-labelled human ATF to the uPAR expressing monocytic cell line U937 was investigated in the presence of increasing concentrations of full length kolo12 aptamer (see figure 17).

U937 cells were maintained in following growth medium; RPMI 1640 medium containing L-Glutamin (Cambrex, Denmark) supplemented with 10% FCS (Cambrex, Denmark), and 100 U/mL penicillin + 100 μg/m L streptomycin (Cambrex, Denmark). HT-1080 cells were maintained in following growth medium; DMEM medium with 4.5 g/L glucose and without L-Glutamin (Cambrex, Denmark) supplemented with 10% FCS (Cambrex), 2 mM L-Glutamine (Cambrex, Denmark) and 100 U/mL penicillin + 100 μg/mL streptomycin.

When the reactions had reached equilibrium it was found that association of ¹²⁵I-ATF to U937 cells was prevented in a dose-dependent manner, while a non-relevant RNA clone had no effect. As a positive control, it was shown that 100 nM of non-radioactive labelled uPA also inhibited the association of ¹²⁵I-ATF to U937 cells (data not shown). In another assay, U937 cells were loaded with pro-uPA in the presence of the truncated kolo12 variant, kolo12.49ntEND, soluble human uPAR or a non-relevant RNA clone. After a 30 minut incubation at room temperature, unbound pro-uPA was washed away and bound pro-uPA detected by adding plasminogen and measuring plasminogen activation using a plasmin sensitive fluorogenic substrate. In this assay, plasmin activity was decreased when cells had been loaded with pro-uPA in the presence of kolo12.49ntEND or soluble human uPAR, whereas the non-relevant RNA clone had no effect (see figure 18).

Example 12

KoIoI 2.49ntEND prevents cell surface associated plasminogen activation To investigate whether the uPA - uPAR inhibitory activity of aptamer kolo12 could ultimately lead to prevention of uPA-dependent cell-associated plasminogen activation, the aptamer was tested in a more complex in wVo-like cell culture assay. In plasma free plasmin only has a brief existence due to rapid inactivation by α₂-antiplasmin, whereas cell associated plasmin is protected from inhibition by α₂-antiplasmin. To monitor uPA- dependent cell associated plasminogen activation exclusively, pro-uPA was therefore mixed with U937 cells, plasminogen and the plasmin substrate 1-1390 in the presence of α₂-antiplasmin. It was verified that the plasmin activity was both cell-dependent (data not shown) and pro-uPA-dependent (see figure 19). In this assay the addition of soluble human uPAR and kolo12.49ntEND was found to prevent plasmin activity.

Example 13 Effect of kolo12.49ntEND on receptor mediated endocytosis of ¹²⁵I-UPA-PAI-1

By SPR analysis it was shown that kolol 2 also binds uPA when in complex with PAI-1 (see figure 20A). The uPA-PAI-1 complex was presented to the aptamer by first capturing active PAI-1 (HT-1080) on a sensor surface coupled with rabbit polyclonal anti-PAI-1 antibody followed by injection of uPA. Binding of aptamer kolol 2 to the uPA in complex with PAI-1 was expected, as the ATF of uPA is not affected by the inhibition of uPA by PAI-1. We subsequently investigated whether kolol 2.49ntEND is able to prevent cell-mediated endocytosis and degradation of ¹²⁵l-labelled uPA-PAI-1 complex (figure 20B). To show that the degradation was dependent on binding of the uPA-PAI-1 complex to uPAR on the cell surface we measured the degradation in the presence of excess uPA alone. No degradation was detected in this assay and we found that kolol 2.49ntEND dose-dependently inhibited ¹²⁵l-labelled uPA-PAI-1 degradation, whereas a non-relevant RNA clone did not.

Example 14 Serum-stability of kolol 2 and kolol 2.49ntEND

In order to assess the serum stability of kolol 2 and kolo12.49ntEND, samples of the two aptamers were incubated in 80% serum for 6 and 24 hours at 37 ^eC and subjected to agarose gel electrophoresis (see figure 21 ). To compare, the respective unmodified RNA clones were produced and included in the same analysis. Both unmodified clones were completely degraded after 6 and 24 hours of incubation in 80% serum, whereas the 2^'-F-pyrimidine modified RNA clones could still be detected. The extent of degradation was difficult to determine as the clones were found to run partially as a smear under the conditions of the experiment.

When incubating the two 2^'-pyrimidine modified kolol 2 versions in RPMI media containing 10% serum no pronounced smear or degradation was observed (figure 22).

Example 15

Assay for determining anticancer therapy and imaging of tumour tissue in vivo As model system for studying the aptamers in vivo, immunodeficient mice xenografted with human cancer cell lines expressing green fluorescent protein (GFP), will be used. Cell lines such as MDA-MB-231 -GFP (breast cancer) and PC-3-GFP (prostate cancer) are used for studying aptamers targeting uPA as they express the three proteins uPA, uPAR and PAI-1 in high amounts. The association of GFP with tumour cells will allow easy determination of cancer progression and metastasis using whole-body fluorescence imaging. Initial experiments will address whether fluorescent labelled aptamers co-localise with the GFP-expressing cancer cells in mice. For a more detailed analysis of biodistribution and pharmacokinetics, aptamers labelled with radioisotopes will be studied by PET and quantitative data acquired by radioactivity counts directly on blood, tumours and the different organs after surgery. Effects of aptamers on tumour dissemination will be assessed by daily injecting aptamers while monitoring the progression of cancer in treated and untreated mice.

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Claims

1. An aptamer capable of binding a receptor-binding form of urokinase-type plasminogen activator protein u-PA.

2. The aptamer according to claim 1 , wherein said aptamer is a ribonucleic acid.

3. The aptamer according to claim 2, wherein said aptamer is a single stranded ribonucleic acid.

4. The aptamer according to claim 1 , wherein the aptamer comprises at least one chemical modification.

5. The aptamer according to claim 4, wherein said at least one chemical modification is selected from the group consisting of a chemical substitution at a sugar position, a chemical substitution at a phosphate position and a chemical substitution at a base position, of the ribonucleic acid.

6. The aptamer according to claim 4, wherein said at least one chemical modification results in the production of a nanoparticle.

7. The aptamer according to claim 6, wherein said at least one chemical modification is chitosan, polyethyleneimine and/or cholesterol.

8. The aptamer according to claim 7, wherein said at least one chemical modification is chitosan.

9. The aptamer according to claim 4, wherein said at least one modification is selected from the group consisting of a modified ribonucleotide, 3' capping, a high molecular weight non-immunogenic compound, a lipophilic compound, drug, a cytotoxic moiety, a labelling agent and phosphate bone modification.

10. The aptamer according to claim 9, wherein the cytotoxic moiety is a small molecule cytotoxic agent.

1 1 . The aptamer according to claim 10, wherein said small molecule cytotoxic agent is selected from cancer drugs.

12. The aptamer according to claim 1 1 , wherein said cancer drug is selected from the group consisting of vinblastine hydrazide, calicheamicin, vinca alkaloid, a cryptophycin, a tubulysin, dolastatin-10, dolastatin-15, auristatin E, rhizoxin, epothilon B, epothilon D, taxoid, myatansinoid and any variants and derivatives thereof.

13. The aptamer according to claim 12, wherein said cancer drug is vinblastine.

14. The aptamer according to claim 9, wherein said at least one chemical modification is a protein toxin.

15. The aptamer according to claim 14, wherein said toxic protein is selected from the group consisting of diphtheria toxin, ricin, abrin, gelonin and Pseudomonas exotoxin A.

16. The aptamer according to claim 13, wherein vinblastine is conjugated to the 3' end of the aptamer

17. The aptamer according to claim 9, wherein the at least one chemical modification is a 2' Flouro substitution.

18. The aptamer according to claim 1 , wherein the aptamer is stable in serum.

19. The aptamer according to claim 9, wherein the labelling agent is selected from the group consisting of radioisotopes, non-radioactive agents, contrast agents for PET imaging, contrast agents for x-ray or CT x-ray imaging and contrast agents for MR imaging.

20. The aptamer according to claim 19, wherein said contrast agent for PET scanning is carbon-1 1 , nitrogen-13, oxygen-15, or Fluorine-18.

21 . The aptamer according to claim 19, wherein said contrast agent for MRI is an imageable nucleus (such as 19Fe) radioisotopes, diamagnetic, paramagnetic, ferromagnetic or superparamagnetic substances.

22. The aptamer according to claim 19, wherein said contrast agent for x-ray and CT imaging is barium sulphate and/or iodine.

23. The aptamer according to claim 19, wherein said non-radioactive agent is a derivative of fluorescein, rhodamine, coumarin and/or cyanine.

24. The aptamer according to claim 19, wherein the radioisotopes is selected from the group consisting of yttrium-90, indium-1 1 1 , iodine-131 , lutetium-177, copper-67, rhenium-186, rhenium-188, bismuth-212, bismuth-213, astatine-21 1 , and actinium- 225.

25. The aptamer according to claim 4, wherein the aptamer is modified by 2' F- pyrimidine.

26. The aptamer according to any of the preceding claims, wherein the complex between said aptamer and u-PA has a dissociation constant of about 100 nM or below.

27. The aptamer according to claim 26, wherein the complex between said aptamer and u-PA has a dissociation constant of about 50 nM or below.

28. The aptamer according to claim 26, wherein the complex between said aptamer and u-PA has a dissociation constant in the range of 1 nM to 10 nM

29. The aptamer according to claim 26, wherein the complex between said aptamer and u-PA has a dissociation constant of 3.2 nM +/- 0.5 nM.

30. The aptamer according to any of the preceding claims, wherein aptamer is a ribonucleic acid containing in the range of 10 and 150 nucleotides.

31 . The aptamer according to any of the preceding claims,, wherein said aptamer is a ribonucleic acid containing in the range of 50 and 100 nucleotides

32. The aptamer according to any of the preceding claims, wherein said aptamer is a ribonucleic acid containing in the range of 10 and 50 nucleotides.

33. the aptamer according to any of the preceding claims, wherein said aptamer is a ribonucleic acid containing in the range of 25 and 50 nucleotides.

34. The aptamer according to any of the preceding claims, wherein the aptamer has intramolecularly, mutually complementary sequences of four or more consecutive nucleotides and has a stem-loop structure in the absence of u-PA.

35. The aptamer according to claim 34, wherein the aptamer comprises the general formula 5'-A-B-C-D-E-C'-A'-3', where A and A' interact to form a stem structure, C and C interact to form a stem structure, B and E interact to form a bulge region and D is a loop.

36. The aptamer according to any of the preceding claims, wherein the aptamer comprises the general formula 5'-A-B-C-D-E-F-E'-G-C'-H-A'-3\ where A and A' interact to form a stem structure, C and C interact to form a stem structure, E and E' interact to form a stem structure, B and H form a bulge region, and D and G form a bulge region, and F forms a loop.

37. The aptamer according to any of the preceding claims, wherein the aptamer comprises at least one of the following sequences in a variable region or a variant thereof being at least 70 % identical to one of said sequences, such as at least 75 % identical to one of said sequences, such as at least 80 % identical to one of said sequences, such as at least 85 % identical to one of said sequences, such as at least 90 % identical to one of said sequencessuch as at least 91 % identical to one of said sequences, such as at least 92 % identical to one of said sequences such as at least 93 % identical to one of said sequences such as at least 94 % identical to one of said sequences such as at least 95 % identical to one of said sequences, such as at least 96 % identical to one of said sequences, such as at least 97 % identical to one of said sequences, such as at least 98 % identical to one of said sequences, such as at least 99 % identical to one of said sequences kolo31 ACTCCTCGGCGCAAGGATGTGGG-ATCGATGCAATC 35 kolo32 AACTAGAACCATACTCGCCGGCGCCAATGTCGTAG 35 kolol2 —TGCGACTGTTATAACCTAACAGCGACGTAAAG-ATA 35xx kolol4 —TGCGACTGTTATAACCTAACAGCGACGTAAAG-ATA 35 koloδδ —TGCGACTGTTATAACCTAACAGCGACGTAAAG-ATA 35xx kolo87 —TGCGACTGTTATAACCTAACAGCGACGTAAAG-ATA 35 kolo79 GAAACGACTCG-ACAACCT-CGAGCGACGTGAATCAT 35xx kolo84 -TACCGACTAGCAAAACCTGCTGGCGACGTTTAG-AT 35 X kolo21 AATAACCTTAATGCGACGTTGGTTTGTCAACAACG 35xx kolo22 AATAACCTTAATGCGACGTTGGTTTGTCAACAACG 35 kolo29 AATAACCTTAATGCGACGTTGGTTTGTCAACAACG 35 kolo76 AATAACCTTAATGCGACGTTGGTTTGTCAACAACG 35 kolo77 AATAACCTTAATGCGACGTTGGTTTGTCAACAACG 35 kolo74 AATAACCTTAATGCGACGTTGGTTTGTCAACAACG 35xx kolo71 AATAACCTTAATGCGACGTTGGTTCGTCAACATGG 35xx kolo28 ACAACCTAAATGCGACGTTGGGTCAAAAACGTGAA 35xx kolo25 GATAACCTCGATGCGACGTTCGGCC-TCAAAATCAA 35xx kolo78 ACAAC-TTAATGCGACGTTGGTAAAGCATATCAAAC 35 X kolo26 CTCCGCTATCTAACGTATGATA-GAATGGATGACTA 35xx kolo83 TGCGCATGAAATGACTGCATGTCTCCGGATTGATC— 35 kolo68 ATGACAGGATGCAGAGCTCCACTGTCTAGTGTTTA 35 X kolo24 CCGCGACAGTCGTAAGTTTTGACTGACTGAACGTT 35 X kolo88 TCGCTAATTATAGGCGGAGTGCGACGTTATAAATA 35 X kolo9 AGTGATTCGCCATAACC-TGGCTGTTTCAGGCTGTT 35 kolo69 AGTGATTCGCCATAACC-TGGCTGTTTCAGGCTGTT 35 kolo85 AGTGATTCGCCATAACC-TGGCTGTTTCAGGCTGTT 35 kolo75 TCGCATTCGTTATAACC-TAACAGTTTGCGGA-GTTA 35 kolol7 TAGTACGACC-TACTTATTTGAC 22 kolo73 TTGCACAACC-TGCATATTTGTCGTCGACGTACGAA 35 kolo82 CGATACAACC-TATCTGTTTGTCGTGGACATCAAAT 35 kolo67 TGCTATGACC-TAGACATTTGACG 23 kolo86 TACCATAACC-TGGATATCTGTCGTTATCATGGGAC 35 kolo20 TTGCGTAACCCGCAATTTCCGTTGGTATATAC 32 kolo70 ACCTGACTTGCGATAACC-TCGACATTTACGGTATT 35 kololO TAATGGCCGGTTTAACCGTT-TGCGCATTAAGTCGA 35 kolo89 TAATGGCCGGTTTAACCGTT-TGCGCATTAAGTCGA 35 kololδ TACCATTTACCACAACC-TGGTTATCTGGTTTAAAC 35 kolo27 TTGTCGTCTTCTCCAGT—TAGTCAACGAACTTGTGT 35xx kolol9 TCCGTTTGCAGGTTTTGACCTGCCGGAGTTGTTTG 35 koloδl GGCAGAAGTTGATTCCAACTTCATTTGCGTTTAAT 35 kolo80 ATATTTGTCTCATCCCACGACAATTATGATGCGAC 35 kolol l -ATCTTAGCGACGTGACACACGACTAGGGATTAATC-

38. The aptamer according to any of the preceding claims, wherein the aptamer comprises at least one of the following sequences in a variable region or a variant being at least 70 % identical to one of said sequences, such as at least 75 % identical to one of said sequences, such as at least 80 % identical to one of said sequences, such as at least 85 % identical to one of said sequences, such as at least 90 % identical to one of said sequences, such as at least 91 % identical to one of said sequences, such as at least 92 % identical to one of said sequences such as at least 93 % identical to one of said sequences such as at least 94 % identical to one of said sequences such as at least 95 % identical to one of said sequences, such as at least 96 % identical to one of said sequences, such as at least 97 % identical to one of said sequences, such as at least 98 % identical to one of said sequences, such as at least 99 % identical to one of said sequences:

kolol2 (4) —UGCGACUGUUAUAACCUAACAGCGACGUAAAG-AUA 35 kolo79 GAAACGACUCG-ACAACCU-CGAGCGACGUGAAUCAU 35 kolo84 -UACCGACUAGCAAAACCUGCUGGCGACGUUUAG-AU 35 kolo21 (6) AAUAACCUUAAUGCGACGUUGGUUUGUCAACAACG 35 kolo71 AAUAACCUUAAUGCGACGUUGGUUCGUCAACAUGG 35 kolo28 ACAACCUAAAUGCGACGUUGGGUCAAAAACGUGAA 35 kolo25 GAUAACCUCGAUGCGACGUUCGGCC-UCAAAAUCAA 35 kolo78 ACAAC-UUAAUGCGACGUUGGUAAAGCAUAUCAAAC 35 kolo26 CUCCGCUAUCUAACGUAUGAUA-GAAUGGAUGACUA 35 kolo83 UGCGCAUGAAAUGACUGCAUGUCUCCGGAUUGAUC— 35 kolo68 AUGACAGGAUGCAGAGCUCCACUGUCUAGUGUUUA 35 kolo24 CCGCGACAGUCGUAAGUUUUGACUGACUGAACGUU 35 kolo88 UCGCUAAUUAUAGGCGGAGUGCGACGUUAUAAAUA 35 kolo9 (3) AGUGAUUCGCCAUAACC-UGGCUGUUUCAGGCUGUU 35 kolo75 UCGCAUUCGUUAUAACC-UAACAGUUUGCGGA-GUUA 35 kolol7 UAGUACGACC-UACUUAUUUGAC 22 kolo73 UUGCACAACC-UGCAUAUUUGUCGUCGACGUACGAA 35 kolo82 CGAUACAACC-UAUCUGUUUGUCGUGGACAUCAAAU 35 kolo67 UGCUAUGACC-UAGACAUUUGACG 23 kolo86 UACCAUAACC-UGGAUAUCUGUCGUUAUCAUGGGAC 35 kolo20 UUGCGUAACCCGCAAUUUCCGUUGGUAUAUAC 32 kolo70 ACCUGACUUGCGAUAACC-UCGACAUUUACGGUAUU 35 kololO(2) UAAUGGCCGGUUUAACCGUU-UGCGCAUUAAGUCGA 35 kololδ UACCAUUUACCACAACC-UGGUUAUCUGGUUUAAAC 35 kolo27 UUGUCGUCUUCUCCAGU—UAGUCAACGAACUUGUGU 35 kolol9 UCCGUUUGCAGGUUUUGACCUGCCGGAGUUGUUUG 35 koloδl GGCAGAAGUUGAUUCCAACUUCAUUUGCGUUUAAU 35 kolo80 AUAUUUGUCUCAUCCCACGACAAUUAUGAUGCGAC 35 kolol 1 AUCUUAGCGACGUGACACACGACUAGGGAUUAAUC 35

39. The aptamer according to any of the preceding claims, wherein the aptamer is selected from the group consisting of KoIoI 2, Kolo21 , Kolo24, Kolo25, Kolo26, Kolo27, Kolo28, Kolo68, Kolo71 , Kolo78, Kolo79, Kolo84, Kolo88, KoIoI 2.33ntEND, KoIoI 2.33ntGG, Kolo21.35ntEND, KoIoI 2.49ntEND,

Kolo79.44nt, Kolo79.44ntEND1 , Kolo79.44ntEND2, Kolo79.34nt and Kolo79.48ntfunny.

40. The aptamer according to any of the preceding claims, wherein the aptamer is selected from the group consisting of KoIoI 2, Kolo21 , Kolo25, Kolo26, Kolo27, Kolo28, Kolo71 , Kolo79, KoIoI 2.33ntEND, KoIoI 2.33ntGG, Kolo21 .35ntEND,

KoIoI 2.49ntEND, Kolo79.44nt, Kolo79.44ntEND1 , kolo79.34nt and Kolo79.48ntfunny.

41. The aptamer according to claim 1 , wherein said aptamer is Kolo12.49ntEND.

42. The aptamer according to any of the preceding claims, wherein the aptamer comprises at least one of the following sequences in a variable region or a variant being at least 70 % identical to one of said sequences, such as at least 75 % identical to one of said sequences, such as at least 80 % identical to one of said sequences, such as at least 85 % identical to one of said sequences, such as at least 90 % identical to one of said sequences, such as at least 95 % identical to one of said sequences, such as at least 98 % identical to one of said sequences, such as at least 99 % identical to one of said sequences:

>kolo9 GGGGCCACCAACGACATTAGTGATTCGCCATAACCTGGCTGTTTCAGGCTGTTGTTGATATAAATAGTGCCCATGGATC

>kololO

GGGGCCACCAACGACATTTAATGGCCGGTTTAACCGTTTGCGCATTAAGTCGAGTTGATATAAATAGTGCCCATGGATC

>kololl

GGGGCCACCAACGACATTATCTTAGCGACGTGACACACGACTAGGGATTAATCGTTGATATAAATAGTGCCCATGGATC >kolo!2

GGGGCCACCAACGACATTTGCGACTGTTATAACCTAACAGCGACGTAAAGATAGTTGATATAAATAGTGCCCATGGATC

>kolo!4

GGGGCCACCAACGACATTTGCGACTGTTATAACCTAACAGCGACGTAAAGATAGTTGATATAAATAGTGCCCATGGATC

>kolo!5 GGGGCCACCAACGACATTTACCATTTACCACAACCTGGTTATCTGGTTTAAACGTTGATATAAATAGTGCCCATGGATC

>kolo!7 GGGGCCACCAACGACATTTAGTACGACCTACTTATTTGACGTTGATATAAATAGTGCCCATGGATC

>kolol9

GGGGCCACCAACGACATTTCCGTTTGCAGGTTTTGACCTGCCGGAGTTGTTTGGTTGATATAAATAGTGCCCATGGATC

>kolo20 GGGGCCACCAACGACATTTTGCGTAACCCGCAATTTCCGTTGGTATATACGTTGATATAAATAGTGCCCATGGATC

>kolo21

GGGGCCACCAACGACATTAATAACCTTAATGCGACGTTGGTTTGTCAACAACGGTTGATATAAATAGTGCCCATGGATC

>kolo22

GGGGCCACCAACGACATTAATAACCTTAATGCGACGTTGGTTTGTCAACAACGGTTGATATAAATAGTGCCCATGGATC >kolo24

GGGGCCACCAACGACATTCCGCGACAGTCGTAAGTTTTGACTGACTGAACGTTGTTGATATAAATAGTGCCCATGGATC

>kolo25

GGGGCCACCAACGACATTGATAACCTCGATGCGACGTTCGGCCTCAAAATCAAGTTGATATAAATAGTGCCCATGGATC

>kolo26 GGGGCCACCAACGACATTCTCCGCTATCTAACGTATGATAGAATGGATGACTAGTTGATATAAATAGTGCCCATGGATC

>kolo27

GGGGCCACCAACGACATTTTGTCGTCTTCTCCAGTTAGTCAACGAACTTGTGTGTTGATATAAATAGTGCCCATGGATC

>kolo28

GGGGCCACCAACGACATTACAACCTAAATGCGACGTTGGGTCAAAAACGTGAAGTTGATATAAATAGTGCCCATGGATC >kolo29

GGGGCCACCAACGACATTAATAACCTTAATGCGACGTTGGTTTGTCAACAACGGTTGATATAAATAGTGCCCATGGATC

>kolo31

GGGGCCACCAACGACATTACTCCTCGGCGCAAGGATGTGGGATCGATGCAATCGTTGATATAAATAGTGCCCATGGATC

>kolo32 GGGGCCACCAACGACATTAACTAGAACCATACTCGCCGGCGCCAATGTCGTAGGTTGATATAAATAGTGCCCATGGATC

>kolo66

GGGGCCACCAACGACATTTGCGACTGTTATAACCTAACAGCGACGTAAAGATAGTTGATATAAATAGTGCCCATGGATC

>kolo67

GGGGCCACCAACGACATTTGCTATGACCTAGACATTTGACGGTTGATATAAATAGTGCCCATGGATC >kolo68

GGGGCCACCAACGACATTATGACAGGATGCAGAGCTCCACTGTCTAGTGTTTAGTTGATATAAATAGTGCCCATGGATC

>kolo69

GGGGCCACCAACGACATTAGTGATTCGCCATAACCTGGCTGTTTCAGGCTGTTGTTGATATAAATAGTGCCCATGGATC

>kolo70 GGGGCCACCAACGACATTACCTGACTTGCGATAACCTCGACATTTACGGTATTGTTGATATAAATAGTGCCCATGGATC

>kolo71

GGGGCCACCAACGACATTAATAACCTTAATGCGACGTTGGTTCGTCAACATGGGTTGATATAAATAGTGCCCATGGATC

>kolo73

GGGGCCACCAACGACATTTTGCACAACCTGCATATTTGTCGTCGACGTACGAAGTTGATATAAATAGTGCCCATGGATC >kolo74

GGGGCCACCAACGACATTAATAACCTTAATGCGACGTTGGTTTGTCAACAACGGTTGATATAAATAGTGCCCATGGATC

>kolo75

GGGGCCACCAACGACATTTCGCATTCGTTATAACCTAACAGTTTGCGGAGTTAGTTGATATAAATAGTGCCCATGGATC

>kolo76 GGGGCCACCAACGACATTAATAACCTTAATGCGACGTTGGTTTGTCAACAACGGTTGATATAAATAGTGCCCATGGATC

>kolo77

GGGGCCACCAACGACATTAATAACCTTAATGCGACGTTGGTTTGTCAACAACGGTTGATATAAATAGTGCCCATGGATC

>kolo78

GGGGCCACCAACGACATTACAACTTAATGCGACGTTGGTAAAGCATATCAAACGTTGATATAAATAGTGCCCATGGATC >kolo 79

GGGGCCACCAACGACATTGAAACGACTCGACAACCTCGAGCGACGTGAATCATGTTGATATAAATAGTGCCCATGGATC

>kolo80

GGGGCCACCAACGACATTATATTTGTCTCATCCCACGACAATTATGATGCGACGTTGATATAAATAGTGCCCATGGATC >kolo81

GGGGCCACCAACGACATTGGCAGAAGTTGATTCCAACTTCATTTGCGTTTAATGTTGATATAAATAGTGCCCATGGATC

>kolo82

GGGGCCACCAACGACATTCGATACAACCTATCTGTTTGTCGTGGACATCAAATGTTGATATAAATAGTGCCCATGGATC

>kolo83 GGGGCCACCAACGACATTTGCGCATGAAATGACTGCATGTCTCCGGATTGATCGTTGATATAAATAGTGCCCATGGATC

>kolo84

GGGGCCACCAACGACATTTACCGACTAGCAAAACCTGCTGGCGACGTTTAGATGTTGATATAAATAGTGCCCATGGATC

>kolo85

GGGGCCACCAACGACATTAGTGATTCGCCATAACCTGGCTGTTTCAGGCTGTTGTTGATATAAATAGTGCCCATGGATC >kolo86

GGGGCCACCAACGACATTTACCATAACCTGGATATCTGTCGTTATCATGGGACGTTGATATAAATAGTGCCCATGGATC

>kolo87

GGGGCCACCAACGACATTTGCGACTGTTATAACCTAACAGCGACGTAAAGATAGTTGATATAAATAGTGCCCATGGATC

>kolo88 GGGGCCACCAACGACATTTCGCTAATTATAGGCGGAGTGCGACGTTATAAATAGTTGATATAAATAGTGCCCATGGATC

>kolo89

GGGGCCACCAACGACATTTAATGGCCGGTTTAACCGTTTGCGCATTAAGTCGAGTTGATATAAATAGTGCCCATGGATC

43. The aptamer according to any of the preceding claims, wherein the aptamer prevents the binding of a receptor binding form of u-PA to a u-PA receptor (u-PAR).

44. The aptamer of claim 43, wherein said binding of the aptamer to said receptor- binding form of u-PA prevents u-PA-mediated cell-associated plasminogen activation.

45. The aptamer according to claim 44, wherein the activation of plasminogen is the conversion of plasminogen to plasmin.

46. The aptamer according to any of the preceding claims, wherein the aptamer binds to the growth factor domain of u-PA.

47. Use of the aptamer as defined in any of the preceding claims 1 to 46 for prevention of u-PA-mediated proteolytic activity at the cell surface for preparing a composition for preventing or counteracting proteolytic activity at the cell surface of a mammal.

48. The use according to claim 47, wherein said proteolytic activity is the conversion of plasminogen to plasmin.

49. Use of the aptamer as defined in any of the preceding claims 1 -46 capable of preventing the binding of a receptor-binding form of u-PA to a u-PAR in a mammal for the preparation of a composition for preventing the binding of u-PA to a uPAR.

50. Use of the aptamer as defined in any of the preceding claims 1 -46 capable of preventing a the binding of a receptor-binding form of uPA to a uPAR in a mammal for the preparation of a composition for preventing the internalisation of a uPA- PAI-

1 -uPAR complex.

51 . Use of the aptamer as defined in any of claims 1 to 46 as an agent for imaging.

52. The use according to claim 51 , wherein the imaging is used to locate u-PA

53. The use according to claim 52, wherein the location of u-PA is indicative of the presence of a tumor.

54. The use according to claim 53, wherein said imaging is selected from the group consisting of positron emission tomography (PET), magnetic resonance (MR) imaging, X-ray, X-ray computed tomography (CT), CT angiography (CTA) imaging, magnetic resonance angiography (NIA), nuclear imaging, ultrasound (US) imaging, optical imaging, infrared imaging and microwave imaging.

55. Use of the aptamer as defined in any of claims 1 to 46 for the treatment of cancer and/or metastatic spread of the cancer in a mammal in need therof.

56. Use of the aptamer as defined in any of claims 1 to 46 as a medicament for a cancer.

57. Use of the aptamer as defined in any of claims 1 to 46 for characterising a tumour.

58. Use of the aptamer according to claims 56 as a medicament for prevention of metastatic activity of a cancer.

59. A pharmaceutical composition comprising an aptamer as defined in any of claim 1 to 46 or a pharmaceutically acceptable salt thereof, carrier, diluent or adjuvant.

60. A pharmaceutical composition for treating cancer and/or metastatic activity comprising the aptamer as defined in claims 1 to 46.

61. A composition comprising an aptamer as defined in any of claim 1 to 46.

62. The composition according to claim 60 for treatment of cancer and/or metastatic activity of a cancer.

63. An aptamer as defined in any of claims 1 to 46 for use as a medicament.

64. A diagnostic kit comprising an aptamer as defined in any of claims 1 to 46

65. A method of diagnosing a disease comprising applying the aptamer as defined in any of claims 1 to 46 and detecting the presence or absence of said aptamer.