US20070010473A1 - Nucleic acid ligands to integrins - Google Patents

Nucleic acid ligands to integrins Download PDF

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US20070010473A1
US20070010473A1 US11/508,024 US50802406A US2007010473A1 US 20070010473 A1 US20070010473 A1 US 20070010473A1 US 50802406 A US50802406 A US 50802406A US 2007010473 A1 US2007010473 A1 US 2007010473A1
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nucleic acid
binding
integrin
selex
ligands
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Judy Ruckman
Larry Gold
Andrew Stephens
Nebojsa Janjic
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Gilead Sciences Inc
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • G—PHYSICS
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Definitions

  • This invention is directed towards nucleic acid ligands of integrins isolated using the SELEX process.
  • SELEX is an acronym for Systematic Evolution of Ligands by EXponential Enrichment.
  • This invention relates to integrin proteins, and methods and compositions for treating and diagnosing diseases involving integrins.
  • the integrins are a class of heterodimeric integral membrane proteins, one or more of which are expressed by most cell types (Hynes (1992) Cell 69:11-25). Some 16 homologous alpha subunits and 8 homologous beta subunits associate in various combinations to yield an extensive family of receptors. Each integrin heterodimer has a large extracellular domain that mediates binding to specific ligands. These ligands may include plasma proteins, proteins expressed on the surface of adjacent cells, or components of the extracellular matrix. Several of the integrins show affinity for more than one ligand and many have overlapping specificities (Hynes (1992) Cell 69:11-25).
  • Integrins play an important role in cellular adhesion and migration, and these properties are controlled by the cell, in part, by modulation of integrin affinity for its ligands (so-called “inside-out” signaling). Conversely, the presence or absence of integrin ligation provides specific information about the cellular microenvironment, and in many instances integrins serve as a conduit for signal transduction.
  • Ligand binding by an integrin may promote its incorporation into focal adhesions, the assembly of cytoskeletal and intracellular signaling molecules into supra-molecular complexes, and the initiation of a cascade of downstream signaling events including protein phosphorylation, calcium release, and an increase in intracellular pH (reviewed by Schwartz et al. (1995) Ann. Rev. Cell Dev. Biol. 11:549-99).
  • Such “outside-in” signaling ties into pathways controlling cell proliferation, migration and apoptosis (Stromblad et al. (1996) J. Clin. Invest. 98:426-33; Eliceiri et al. (1998) J. Cell. Biol. 140:1255-63).
  • Integrins have been shown to play a role in such diverse physiological settings as embryonic development, wound healing, angiogenesis, clot formation, leukocyte extravasation, bone resorption and tumor metastasis.
  • the ⁇ 3 -containing integrins are among the best studied of the receptor superfamily.
  • the ⁇ 3 subunit forms heterodimers with either ⁇ v ( ⁇ v ⁇ 3 ) or ⁇ IIb ( ⁇ IIb ⁇ 3 ), While these integrins show substantial overlap in ligand specificity, they play very different roles in normal physiology and in disease.
  • ⁇ v ⁇ 3 is expressed by activated endothelial cells, smooth muscle cells, osteoclasts, and, at a very low level, by platelets. It is also expressed by a variety of tumor cell types.
  • the integrin binds to a number of plasma proteins or proteins of the extracellular matrix, many of which are associated with sites of inflammation or wound healing (Albelda (1991) Am. J. Resp. Cell Mol. Biol. 4:195-203). These include vitronectin, fibronectin, osteopontin, von Willebrand factor, thrombospondin, fibrinogen, and denatured collagen Type I (Hynes (1992) Cell 69:11-25). Each of these proteins share a common sequence motif, arginine-glycine-aspartic acid (RGD), that forms the core of the integrin binding site.
  • RGD arginine-glycine-aspartic acid
  • ⁇ v ⁇ 3 has been most intensely studied in the context of new blood vessel formation (angiogenesis) where it mediates the adhesion and migration of endothelial cells through the extracellular matrix.
  • Angiogenesis in adults is normally associated with the cyclical development of the corpus luteum and endometrium and with the formation of granulation tissue during wound repair. In the latter case, microvascular endothelial cells form vascular sprouts that penetrate into the temporary matrix within a wound. These cells transiently express ⁇ v ⁇ 3 and inhibition of the ligand binding function of the integrin temporarily inhibits the formation of granulation tissue (Clark et al. (1996) Am. J. Pathol. 148:1407-21).
  • Solid tumors are unable to grow to significant size without an independent blood supply. It is currently hypothesized that the acquisition of an angiogenic phenotype is one of the limiting steps in the growth of primary tumors and of tumors at secondary sites (Folkman (1995) Nat. Med. 1:27-31).
  • the vasculature that penetrates a tumor mass provides a source of oxygen and nutrients, it also serves as a conduit for metastatic cells to leave the primary tumor and migrate throughout the body.
  • inhibition of angiogenesis may limit both the growth and metastasis of cancerous lesions.
  • ⁇ v ⁇ 3 is not only expressed by the microvasculature within tumors, but in some cases, is also found on the surface of tumor cells themselves.
  • expression of ⁇ v ⁇ 3 integrin has been detected in tissue sections from tumors of melanocytic and astroglial origin (Albelda et al. (1990) Canc. Res. 50:6757-64; Gladson and Cheresh (1991) J. Clin. Invest. 88:1924-32), and the level of integrin expression has been correlated with the stage or metastatic potential of the tumor (Albelda et al. (1990) Canc. Res. 50:6757-64; Gladson et al. (1996) Am. J. Pathol.
  • melanoma cells grown in vitro in a three-dimensional matrix of denatured collagen undergo apoptosis upon ⁇ v ⁇ 3 blockade.
  • Vitaxin is a chimeric Fab fragment derived from the ⁇ v ⁇ 3 -specific monoclonal antibody, LM609 (Wu et al. (1998) Proc. Nat. Acad. Sci. 95:6037-42).
  • LM609 a chimeric Fab fragment derived from the ⁇ v ⁇ 3 -specific monoclonal antibody
  • EMD121974 is a cyclic pentapeptide inhibitor of ⁇ v ⁇ 3 .
  • a Phase I study of this compound in Kaposi's sarcoma, brain tumors and solid tumors is scheduled to begin in 1999.
  • Angiogenesis (and ⁇ v ⁇ 3 ) are implicated in the pathology of several other diseases, including psoriasis (Creamer et al. (1995) Am. J. Pathol. 147:1661-7), rheumatoid arthritis (Walsh et al. (1998) Am. J. Pathol. 152:691-702; Storgard et al. (1999) J. Clin. Invest. 103:47-54), endometriosis (Healy et al. (1998) Hum. Reprod. Update 4:736-40), and several proliferative diseases of the eye (Casaroli Marano et al. (1995) Exp. Eye Res.
  • Atheromatous plaque and restenosis following angioplasty are pathologies characterized by thickening of the intima, the innermost layer of the arterial wall.
  • the proliferation and/or migration of smooth muscle cells into the neointima with concomitant deposition of fibrous extracellular proteins contributes to vessel wall thickening and subsequent vessel occlusion.
  • Platelets may also contribute to the development of restenotic lesions through adhesion to endothelial cells and the release of growth factors and cytokines that stimulate the underlying smooth muscle cell layer (Le Breton et al. (1996) J. Am. Coll. Cardiol. 28:1643-51).
  • ⁇ v ⁇ 3 integrin is expressed on arterial smooth muscle cells (Hoshiga et al. (1995) Circ.
  • ⁇ v ⁇ 3 blockade with RGD-containing peptides or a monoclonal antibody was found to limit neointimal hyperplasia in several animal models of restenosis following arterial injury, (Choi et al.
  • ⁇ v ⁇ 3 mediates the attachment of osteoclasts to matrix proteins, particularly osteopontin, on the surface of bone. Osteoclasts are responsible for the resorption of bone in normal physiology as well as in pathological conditions such as osteoporosis.
  • a monoclonal antibody specific for ⁇ v ⁇ 3 inhibited the binding and resorption of bone particles by osteoclasts in vitro (Ross et al. (1993) J. Biol. Chem. 268:9901-7).
  • an RGD-containing protein, echistatin was shown to block parathyroid-stimulated bone resorption in an animal model, as monitored by serum calcium levels (Fisher et al. (1993) Endocrin. 132:1411-3).
  • Inhibitors of ⁇ v ⁇ 3 integrin are thus considered of potential utility in treating debilitating bone loss such as occurs in osteoporosis.
  • ⁇ IIb ⁇ 3 (also referred to as GPIIbIIIa) is the major integrin on the surface of platelets where it mediates the adhesion of activated platelets to the plasma protein fibrinogen (Nachman and Leung (1982) J. Clin. Invest. 69:263-9; Shattil et al. (1985) J. Biol. Chem. 260:11107-14). During clot formation, fibrinogen dimers cross-link platelets to one another through the integrin receptor.
  • ⁇ IIb ⁇ 3 also binds to several other plasma and cell matrix proteins, including von Willebrand factor, vitronectin, and fibronectin (Faull and Ginsberg (1996) J. Am. Soc. Nephrol. 7:1091-7).
  • Clot formation is a tightly regulated process that balances the need for rapid response to vascular injury with the risk of aberrant occlusion of critical vessels.
  • the ⁇ IIb ⁇ 3 heterodimer is constitutively expressed on the surface of resting platelets at approximately 80,000 copies per cell (Wagner et al. (1996) Blood 88:907-14); however, the affinity of the integrin for fibrinogen is very low on these cells.
  • Activation of platelets by ADP, epinephrine, collagen or thrombin leads to a dramatic enhancement in integrin ligand binding activity (Bennett and Vilaire (1979) J. Clin. Invest. 64:1393-401; Marguerie et al. (1979) J. Biol. Chem.
  • Inhibitors of ⁇ IIb ⁇ 3 ligand binding have been primarily explored in the context of cardiovascular disease (Chong (1998) Am. J. Health Syst. Pharm. 55:2363-86; Topol et al. (1999) Lancet 353:227-31), but may have application in any of a number of indications where thrombus formation is suspected or is likely.
  • Three ⁇ IIb ⁇ 3 inhibitors have been approved for use in patients experiencing acute coronary syndrome and/or in patients who are undergoing percutaneous coronary intervention.
  • Reopro (Centocor/Eli Lilly) is a humanized murine monoclonal antibody Fab fragment with specificity for the ⁇ 3 chain of ⁇ IIb ⁇ 3
  • Integrilin (COR Therapeutics) is a cyclic heptapeptide based on the integrin binding site of barbourin, an ⁇ IIb ⁇ 3 inhibitory protein derived from snake venom.
  • Aggrastat (Merck & Co.) is a non-peptide small molecule antagonist of the integrin.
  • Reopro cross-reacts with ⁇ v ⁇ 3 , a fact which may account for the greater reduction in long-term rates of death and non-fatal myocardial infarction associated with its use (see above).
  • a significant effort is underway to identify new inhibitors of the platelet integrin with characteristics not found in the cohort of approved drugs.
  • compounds with specificity for the active, ligand-binding conformation of ⁇ IIb ⁇ 3 may reduce the risk of bleeding complications associated with the existing anti-clotting therapies.
  • Orally available compounds would be particularly useful for longer term therapy of patients at risk for recurrent myocardial infarction or unstable angina.
  • SELEX process Systematic Evolution of Ligands by EXponential enrichment
  • nucleic acids have three dimensional structural diversity not unlike proteins.
  • the SELEX process is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules and is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution of Ligands by EXponential Enrichment,” now abandoned, U.S. Pat. No. 5,475,096, entitled “Nucleic Acid Ligands” and U.S. Pat. No.
  • the SELEX process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets.
  • the SELEX method applied to the application of high affinity binding involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity.
  • the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
  • nucleic acids as chemical compounds can form a wide array of shapes, sizes and configurations, and are capable of a far broader repertoire of binding and other functions than those displayed by nucleic acids in biological systems.
  • the SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions.
  • SELEX process-identified nucleic acid ligands containing modified nucleotides are described in U.S. Pat. No. 5,660,985, entitled “High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,” that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2′-positions of pyrimidines.
  • the SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. Pat. No. 5,637,459, entitled “Systematic Evolution of Ligands by EXponential Enrichment: Chimeric SELEX,” and U.S. Pat. No. 5,683,867, entitled “Systematic Evolution of Ligands by EXponential Enrichment: Blended SELEX,” respectively.
  • These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules.
  • the SELEX method further encompasses combining selected nucleic acid ligands with lipophilic compounds or non-immunogenic, high molecular weight compounds in a diagnostic or therapeutic complex as described in U.S. patent application Ser. No. 08/434,465, filed May 4, 1995, entitled “Nucleic Acid Ligand Complexes”.
  • nucleic acid ligands to integrins, particularly to the ⁇ 3 integrins.
  • the methods use the SELEX process for ligand generation.
  • Particular embodiments describe the isolation of nucleic acid ligand inhibitors of both ⁇ v ⁇ 3 and ⁇ IIb ⁇ 3 .
  • the nucleic acid ligand inhibitors are derived from a library of 2′-fluoro-pyrimidine RNA sequences and were selected for high affinity binding to ⁇ v ⁇ 3 .
  • One of the modified nucleic acid ligands is shown to inhibit the binding of either vitronectin or fibrinogen to both of the purified integrins in vitro. This nucleic acid ligand binds to the surface of both resting and activated platelets with equivalent affinity and accumulates at the site of a preformed clot in an animal model of venous thrombosis.
  • the nucleic acid ligands provided by the invention are useful as therapeutic agents for a number of diseases including thrombosis and cancer.
  • the nucleic acid ligands of the instant invention are also useful as diagnostic agents for thrombosis.
  • FIG. 1 illustrates the binding of affinity-enriched RNA pools to immobilized ⁇ v ⁇ 3 .
  • 5′-biotinylated RNA pools were incubated at varying concentrations in 96-well microtiter plates coated with integrin ⁇ v ⁇ 3 .
  • Bound RNAs were detected via the biotin moiety by a chromogenic assay. Data are expressed in absorbance units at 405 nm as a function of input RNA concentration.
  • FIG. 2 illustrates cross-reactivity of aptamer 17.16 (SEQ ID NO:60) to purified integrin ⁇ IIb ⁇ 3 .
  • 5′-biotinylated aptamer 17.16 was incubated at varying concentrations in microtiter wells coated with either integrin ⁇ v ⁇ 3 or ⁇ IIb ⁇ 3 .
  • Bound RNA was detected via the biotin moiety using a chromogenic assay. Data are expressed as the per cent of the maximum signal to normalize for differences in protein coating.
  • FIG. 3 illustrates cross-reactivity of aptamer 17.16 (SEQ ID NO:60) to purified integrin ⁇ v ⁇ 5 .
  • 5′-biotinylated aptamer 17.16 or a control RNA of similar length and base composition were incubated at varying concentrations in microtiter wells coated with either ⁇ v ⁇ 3 or ⁇ v ⁇ 5 .
  • Bound RNAs were detected via the biotin moiety by a chromogenic assay. Data are expressed in absorbance units at 405 nm as a function of input RNA concentration.
  • FIGS. 4 A-C illustrate ⁇ 3 aptamer inhibition of integrin ligand binding.
  • Biotinylated fibrinogen or vitronectin were incubated in microtiter wells coated with either integrin ⁇ v ⁇ 3 or ⁇ IIb ⁇ 3 in the presence or absence of varying concentrations of ligand binding competitors.
  • Competitors included aptamer 17.16 (SEQ ID NO:60), a control RNA of similar length and base composition, a cyclic RGD peptide (cRGD, see Materials and Methods), an ⁇ v ⁇ 3 -specific monoclonal antibody (LM609), or unmodified fibrinogen or vitronectin.
  • Bound ligands were detected via biotin using a chromogenic assay. Data are expressed in absorbance units at 405 nm as a function of input competitor concentration.
  • FIG. 4A shows competition of vitronectin binding to immobilized ⁇ v ⁇ 3 ;
  • FIG. 4B shows competition of fibrinogen binding to immobilized ⁇ v ⁇ 3 ;
  • FIG. 4C shows competition of fibrinogen binding to immobilized ⁇ IIb ⁇ 3 .
  • An estimate of the maximum absorbance value was determined for each ligand/integrin pair in the absence of competitor. The baseline absorbance value was determined by adding 5 mM EDTA to the incubation buffer. The maximum and minimum values so determined were FIG. 4A , 0.914/0.113; FIG. 4B , 1.042/0.122; FIG. 4C , 0.889/0.128.
  • FIG. 5 illustrates binding of aptamer 17.16 (SEQ ID NO:60) to activated or resting human platelets.
  • 5′-fluorescein-conjugated aptamer 17.16 or a control RNA of similar length and base composition were incubated at various concentrations with resting or thrombin-activated human platelets (10 6 I/mL). Incubations were at room temperature in buffered saline containing divalent cations, 0.1% BSA and 0.01% sodium azide.
  • Mean fluorescence intensity of the sample was determined by flow cytometry both before and after the addition of EDTA to 5 mM final concentration. The difference in fluorescence intensity between the two samples (the EDTA-sensitive signal) is shown as a function of the concentration of aptamer or control RNA.
  • FIG. 6 illustrates biodistribution of [ 99m Tc]-aptamer 17.16 (SEQ ID NO:60) or control RNA in a rabbit venous clot model.
  • a clot derived from human platelet-rich plasma was generated in situ by temporary isolation of the jugular vein of an anesthetized rabbit. After restoration of circulation over the clot, [ 99m Tc]-labeled aptamer or control RNA were injected into the bloodstream of the rabbit via the ipsilateral ear vein. After one hour, the animal was sacrificed and tissues were weighed and counted in a gamma counter. Accumulation of radioactivity in various tissues is reported as the percentage of the injected dose per gram wet weight of tissue.
  • the central method utilized herein for identifying nucleic acid ligands to Integrins is called the SELEX process, an acronym for Systematic Evolution of Ligands by Exponential enrichment and involves (a) contacting the candidate mixture of nucleic acids with integrins, or expressed domains or peptides corresponding to integrins, (b) partitioning between members of said candidate mixture on the basis of affinity to integrins, and c) amplifying the selected molecules to yield a mixture of nucleic acids enriched for nucleic acid sequences with a relatively higher affinity for binding to integrins.
  • SELEX process an acronym for Systematic Evolution of Ligands by Exponential enrichment and involves (a) contacting the candidate mixture of nucleic acids with integrins, or expressed domains or peptides corresponding to integrins, (b) partitioning between members of said candidate mixture on the basis of affinity to integrins, and c) amplifying the selected molecules to yield a mixture of nucleic acids enriched for nu
  • nucleic acid ligand is a non-naturally occurring nucleic acid having a desirable action on a target.
  • Nucleic acid ligands are often referred to as “aptamers”.
  • the term aptamer is used interchangeably with nucleic acid ligand throughout this application.
  • a desirable action includes, but is not limited to, binding of the target, catalytically changing the target, reacting with the target in a way which modifies/alters the target or the functional activity of the target, covalently attaching to the target as in a suicide inhibitor, facilitating the reaction between the target and another molecule.
  • the action is specific binding affinity for a target molecule, such target molecule being a three dimensional chemical structure other than a polynucleotide that binds to the nucleic acid ligand through a mechanism which predominantly depends on Watson/Crick base pairing or triple helix binding, wherein the nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target molecule.
  • the target is an integrin, or portions thereof.
  • Nucleic acid ligands include nucleic acids that are identified from a candidate mixture of nucleic acids, said nucleic acid ligand being a ligand of a given target, by the method comprising: a) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and c) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids.
  • candidate mixture is a mixture of nucleic acids of differing sequence from which to select a desired ligand.
  • the source of a candidate mixture can be from naturally-occurring nucleic acids or fragments thereof, chemically synthesized nucleic acids, enzymatically synthesized nucleic acids or nucleic acids made by a combination of the foregoing techniques.
  • each nucleic acid has fixed sequences surrounding a randomized region to facilitate the amplification process.
  • nucleic acid means either DNA, RNA, single-stranded or double-stranded, and any chemical modifications thereof. Modifications include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole.
  • modifications include, but are not limited to, 2′-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3′ and 5′ modifications such as capping.
  • SELEX methodology involves the combination of selection of nucleic acid ligands which interact with a target in a desirable manner, for example binding to a protein, with amplification of those selected nucleic acids. Optional iterative cycling of the selection/amplification steps allows selection of one or a small number of nucleic acids which interact most strongly with the target from a pool which contains a very large number of nucleic acids. Cycling of the selection/amplification procedure is continued until a selected goal is achieved. In the present invention, the SELEX methodology is employed to obtain nucleic acid ligands to integrins.
  • SELEX target or “target” means any compound or molecule of interest for which a ligand is desired.
  • a target can be a protein, peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye, nutrient, growth factor, etc. without limitation.
  • the SELEX targets are integrins.
  • solid support is defined as any surface to which molecules may be attached through either covalent or non-covalent bonds. This includes, but is not limited to, membranes, microtiter plates, magnetic beads, charged paper, nylon, Langmuir-Bodgett films, functionalized glass, germanium, silicon, PTFE, polystyrene, gallium arsenide, gold, and silver. Any other material known in the art that is capable of having functional groups such as amino, carboxyl, thiol or hydroxyl incorporated on its surface, is also contemplated. This includes surfaces with any topology, including, but not limited to, spherical surfaces and grooved surfaces.
  • the nucleic acid ligands of the present invention are derived from the SELEX methodology.
  • the SELEX process is described in U.S. patent application Ser. No. 07/536,428, entitled “Systematic Evolution of Ligands by Exponential Enrichment,” now abandoned, U.S. Pat. No. 5,475,096, entitled “Nucleic Acid Ligands,” and U.S. Pat. No. 5,270,163 (see also WO 91/19813) entitled “Methods for Identifying Nucleic Acid Ligands.”
  • These applications, each specifically incorporated herein by reference, are collectively called the SELEX Patent Applications.
  • the SELEX process provides a class of products which are nucleic acid molecules, each having a unique sequence, and each of which has the property of binding specifically to a desired target compound or molecule.
  • Target molecules are preferably proteins, but can also include among others carbohydrates, peptidoglycans and a variety of small molecules.
  • SELEX methodology can also be used to target biological structures, such as cell surfaces or viruses, through specific interaction with a molecule that is an integral part of that biological structure.
  • the SELEX process may be defined by the following series of steps:
  • a candidate mixture of nucleic acids of differing sequence is prepared.
  • the candidate mixture generally includes regions of fixed sequences (i.e., each of the members of the candidate mixture contains the same sequences in the same location) and regions of randomized sequences.
  • the fixed sequence regions are selected either: (a) to assist in the amplification steps described below, (b) to mimic a sequence known to bind to the target, or (c) to enhance the concentration of a given structural arrangement of the nucleic acids in the candidate mixture.
  • the randomized sequences can be totally randomized (i.e., the probability of finding a base at any position being one in four) or only partially randomized (e.g., the probability of finding a base at any location can be selected at any level between 0 and 100 percent).
  • the candidate mixture is contacted with the selected target under conditions favorable for binding between the target and members of the candidate mixture. Under these circumstances, the interaction between the target and the nucleic acids of the candidate mixture can be considered as forming nucleic acid-target pairs between the target and those nucleic acids having the strongest affinity for the target.
  • nucleic acids with the highest affinity for the target are partitioned from those nucleic acids with lesser affinity to the target. Because only an extremely small number of sequences (and possibly only one molecule of nucleic acid) corresponding to the highest affinity nucleic acids exist in the candidate mixture, it is generally desirable to set the partitioning criteria so that a significant amount of the nucleic acids in the candidate mixture (approximately 5-50%) are retained during partitioning.
  • nucleic acids selected during partitioning as having the relatively higher affinity for the target are then amplified to create a new candidate mixture that is enriched in nucleic acids having a relatively higher affinity for the target.
  • the newly formed candidate mixture contains fewer and fewer unique sequences, and the average degree of affinity of the nucleic acids to the target will generally increase.
  • the SELEX process will yield a candidate mixture containing one or a small number of unique nucleic acids representing those nucleic acids from the original candidate mixture having the highest affinity to the target molecule.
  • the SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions.
  • SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. Pat. No. 5,660,985, entitled “High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,” that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2′-positions of pyrimidines.
  • the SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. Pat. No. 5,637,459, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX,” and U.S. Pat. No. 5,683,867, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX,” respectively.
  • These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules.
  • nucleic acid ligand can be made to increase the in vivo stability of the nucleic acid ligand or to enhance or to mediate the delivery of the nucleic acid ligand. See, e.g., U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, now abandoned, and U.S. Pat. No.
  • nucleic acid ligands contemplated in this invention include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole.
  • Such modifications include, but are not limited to, 2′-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate or alkyl phosphate modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3′ and 5′ modifications such as capping.
  • the nucleic acid ligands are RNA molecules that are 2′-fluoro (2′-F) modified on the sugar moiety of pyrimidine residues.
  • the modifications can be pre- or post-SELEX process modifications.
  • Pre-SELEX process modifications yield nucleic acid ligands with both specificity for their SELEX target and improved in vivo stability.
  • Post-SELEX process modifications made to 2′-OH nucleic acid ligands can result in improved in vivo stability without adversely affecting the binding capacity of the nucleic acid ligand.
  • the nucleic acid ligands of the invention are prepared through the SELEX methodology that is outlined above and thoroughly enabled in the SELEX applications incorporated herein by reference in their entirety.
  • the SELEX process can be performed using purified integrins, or fragments thereof as a target.
  • full-length integrins, or discrete domains of integrins can be produced in a suitable expression system.
  • the SELEX process can be performed using as a target a synthetic peptide that includes sequences found in an integrin. Determination of the precise number of amino acids needed for the optimal nucleic acid ligand is routine experimentation for skilled artisans.
  • the nucleic acid ligands become covalently attached to their targets upon irradiation of the nucleic acid ligand with light having a selected wavelength.
  • Methods for obtaining such nucleic acid ligands are detailed in U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection of Nucleic Acid Ligands,” now abandoned, U.S. Pat. No. 5,763,177, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX” and U.S. patent application Ser. No. 09/093,293, filed Jun. 8 1998, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX,” each of which is specifically incorporated herein by reference in its entirety.
  • the SELEX process is carried out using integrins attached to polystyrene beads.
  • a candidate mixture of single stranded RNA molecules is then contacted with the beads.
  • the single stranded RNA molecules have a 2′-fluoro modification on C and U residues, rather than a 2′-OH group.
  • the beads are washed to remove unbound candidate nucleic acid ligand.
  • the nucleic acid ligand that binds to the integrin is then released into solution, then reverse transcribed by reverse transcriptase and amplified using the Polymerase Chain Reaction.
  • the amplified candidate mixture is then used to begin the next round of the SELEX process.
  • Example 2 illustrates-one possible way of performing the SELEX process using integrins as targets.
  • the nucleic acid ligands thus obtained are assayed for their ability to inhibit the interaction of the integrin with its cognate ligand. In one embodiment, this is performed by first coating microtiter plates with the appropriate integrin(s). A ligand for the integrin, such as vitronectin or fibrinogen, is then biotinylated and contacted with the coated integrin in the presence of the nucleic acid ligand to be assayed.
  • a ligand for the integrin such as vitronectin or fibrinogen
  • the microtiter plate After incubation for a suitable period of time, the microtiter plate is washed, and the amount of vitronectin or fibrinogen binding to integrin is quantitated by adding a streptavidin-alkaline phosphatase conjugate, followed by a colorimetric substrate for alkaline phosphatase, such as p-nitrophenyl phosphate.
  • the alkaline phosphatase signal in each well of the plate is thus inversely proportional to the effectiveness of the nucleic acid ligand as an inhibitor of the interaction between the bound integrin and its cognate ligand.
  • the nucleic acid ligands can be analyzed using binding to human platelets as an assay. This can be done, for example, by fluorescently labelling the nucleic acid ligand by any of the numerous techniques known in the art. The fluorescent nucleic acid ligand can then be contacted with platelets, and the amount of nucleic acid ligand can be quantitated using Fluorescence Activated Cell Sorting (FACS).
  • FACS Fluorescence Activated Cell Sorting
  • nucleic acid ligands of the instant invention can also be studied in vivo.
  • nucleic acid ligands are labelled with a radiolabel used in the art of radioimaging.
  • a nucleic acid ligand can be conjugated to the isotope 99m Tc using one of a number of techniques known in the art.
  • the radiolabeled nucleic acid can then be studied in an animal model of venous thrombosis.
  • a human blood clot can be generated in rabbit vein by first isolating the vein in situ by ligation, and then infusing the vein with human platelet-rich plasma and heparin to induce the formation of a blood clot.
  • Radiolabeled nucleic acid ligand Blood flow through the vein is then re-established, and the radiolabeled nucleic acid ligand is introduced into the animals blood supply. The distribution of the radiolabeled nucleic acid ligand can then be studied in the rabbit's tissues to determine whether the nucleic acid ligand has accumulated in the clot, rather than in other areas.
  • nucleic acid ligands provided by the instant invention have a number of potential uses as therapeutic and diagnostic agents.
  • nucleic acid ligands that inhibit the interaction between platelet-expressed integrins and their cognate ligands are administered, along with pharmaceutically accepted excipients, in order to prevent the formation of blood clots in patients susceptible to deep vein thrombosis.
  • the nucleic acid ligands are used to treat acute thrombosis formation during and following percutaneous coronary intervention.
  • the nucleic acid ligands of the invention are used to treat patients with acute coronary syndromes such as unstable angina or myocardial infarction.
  • radiolabeled nucleic acid ligands to platelet-expressed integrins are administered to individuals who are to undergo major surgery, or have suffered major trauma.
  • Such nucleic acid ligands can function as imaging agents for the detection of thrombi, by showing sites in the body where large aggregations of platelets are present. If a thrombosis is detected by radioimaging at a critical site in the body, then anticoagulant and thrombolytic treatment—including treatment with the inhibitory nucleic acid ligands of the instant invention—can be given locally.
  • nucleic acid ligand imaging agent provides a nucleic acid ligand imaging agent.
  • anticoagulant and thrombolytic treatments which can cause harm if administered prophylactically by allowing internal bleeding to continue without efficient clotting—can be given only to those individuals who definitely have a dangerous thrombosis.
  • these treatments can be specifically injected at the site where the thrombosis has been detected by the nucleic acid ligand, instead of injecting higher concentrations into the bloodstream in the hope that some active agent will be carried to all potential sites of thrombosis.
  • Nucleic acid ligands to ⁇ v ⁇ 3 integrin can be used to inhibit tumor growth and metastasis. They can also be used to treat ocular diseases including, but not limited to, diabetic retinopathy, retinopathy of prematurity, and macular degeneration. Other diseases for which ⁇ v ⁇ 3 nucleic acid ligands are useful therapeutic agents include, but are not limited to, endometriosis, psoriasis, rheumatoid arthritis, stroke, osteoporosis, and restenosis.
  • ⁇ v ⁇ 3 integrin was isolated from human placenta and purified by immunoaffinity chromatography essentially as described by (Smith and Cheresh (1988) J. Biol. Chem. 263:18726-31). In brief, human placentas were diced and the tissue fragments were extracted in a buffer containing 100 mM octyl- ⁇ -D-glucopyranoside detergent (Calbiochem, San Diego, Calif.). The extract was cleared by centrifugation and applied to an immunoaffinity column ⁇ v ⁇ 3 -specific monoclonal antibody LM609 affixed to Affi-Gel 10, (Chemicon International, Inc., Temecula, Calif.)).
  • Protein bound to the column was eluted with a low-pH buffer and fractions were immediately neutralized and analyzed for integrin content by SDS-polyacrylamide gel electrophoresis. Integrin-containing fractions were pooled and aliquots of the purified material were stored at ⁇ 80° C.
  • Purified human ⁇ v ⁇ 3 was also purchased from Chemicon International, Inc, as was human ⁇ v ⁇ 5 integrin.
  • ⁇ IIb ⁇ 3 and fibrinogen were purchased from Enzyme Research Laboratories, Inc. (South Bend, Ind.). Vitronectin was purified from outdated human plasma according to the procedure of (Yatohgo et al. (1988) Cell Struct. Func. 13:281-92), using heparin affinity chromatography.
  • a DNA template library of sequence: 5′- ttatacgactcactata gggagacaagaataaac (SEQ ID NO:1) gctcaannnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnttcgacaggaggctcacaacaggc-3′ was prepared by chemical synthesis.
  • the italicized nucleotides correspond to a T7 RNA polymerase promoter.
  • a short DNA primer “3N8”:5′-gcctgttgtgagcctcctgtcgaa-3′ was annealed to the template and extended using Klenow DNA polymerase (New England Biolabs, Beverly, Mass.).
  • the double-stranded DNA product served as a product for T7 RNA polymerase transcription (enzyme obtained from Enzyco, Inc., Denver, Colo.) to generate a library of random-sequence RNAs.
  • 2′-fluoro-CTP and -UTP were used in place of the 2′-OH-pyrimidines.
  • the purified protein was diluted 1000-fold from detergent-containing storage buffer into 50 mM MES (2-[N-morpholino]ethanesulfonic acid), pH 6.1, 150 mM NaCl, 2 mM CaCl 2 , to a final concentration of approximately 0.2 ⁇ g/mL.
  • MES 2-[N-morpholino]ethanesulfonic acid
  • pH 6.1 150 mM NaCl
  • 2 mM CaCl 2 a final concentration of approximately 0.2 ⁇ g/mL.
  • 3.2 ⁇ polystyrene particles IDEXX Laboratories, Inc., Westbrook, Me.
  • the beads were collected by centrifugation and blocked by incubation in 3% BSA in MES buffer (above) for one hour at room temperature.
  • Blocked beads were washed several times by resuspension in binding buffer (50 mM Tris.HCl, pH 7.4 (at 37° C.), 145 mM NaCl, 4 mM KCl, 1 mM MgCl 2 , 2 mM CaCl 2 , 0.1 mM MnCl 2 , 0.01% BSA).
  • binding buffer 50 mM Tris.HCl, pH 7.4 (at 37° C.), 145 mM NaCl, 4 mM KCl, 1 mM MgCl 2 , 2 mM CaCl 2 , 0.1 mM MnCl 2 , 0.01% BSA.
  • binding buffer 50 mM Tris.HCl, pH 7.4 (at 37° C.), 145 mM NaCl, 4 mM KCl, 1 mM MgCl 2 , 2 mM CaCl 2 , 0.1 mM MnCl 2 , 0.01% B
  • RNAs that remained bound to the beads were eluted overnight at 37° C. in binding buffer plus 100 ⁇ M cyclic RGD peptide (“cRGD”) (GPenGRGDSPCA, Life Technologies, Gibco BRL, Gaithersburg, Md). Eluted RNAs were extracted with phenol, then chloroform:isoamyl alcohol (24:1), and ethanol precipitated. The RNA pellet was resuspended and annealed to primer 3N8 for reverse transcription using avian myeloblastosis virus reverse transcriptase (Life Sciences, Inc., St. Moscow, Fla.).
  • cRGD cyclic RGD peptide
  • the cDNA pool was amplified by the polymerase chain reaction using the 3N8 primer and primer “5N8“:5′-taatacgactcactatagggagacaagaataaacgctcaa-3′ (SEQ ID NO:3) and T. aquaticus DNA polymerase (Perkin Elmer-Cetus, Foster City, Calif.). Transcription of the PCR product generated an RNA pool to initiate a new round of selection. For the first round of selection 1 nmol of RNA (approximately 6 ⁇ 10 14 sequences) was incubated at 2 ⁇ M concentration with a volume of bead suspension equivalent to 50 pmol of protein (assuming all the integrin had adsorbed to the beads). In subsequent rounds, the concentration of RNA and protein-coated beads were both reduced to demand higher affinity binding interactions.
  • RNA affinity was determined by titration of biotinylated RNA with a small quantity of immobilized integrin. Bound RNA was detected through the biotin moiety.
  • Biotinylated RNA was prepared according to standard transcription protocols, but including a 5-fold molar excess of a 5′-biotin-modified GMP over GTP in the reaction mixture. Methods for synthesizing 5′-biotin-modified guanosine nucleotides are described in WO 98/30720 entitled “Bioconjugation of Oligonucleotides,” specifically incorporated herein by reference in its entirety.
  • the modified nucleotide is incorporated at the 5′ end of the transcript in proportion to its representation in the guanosine pool.
  • 96-well microtiter plates (Immulon 2, Dynatech Laboratories, Inc., Chantilly, Va.) were coated overnight at 4° C. with 100 ⁇ L purified ⁇ v ⁇ 3 at a concentration of 0.25 ⁇ g/mL in 20 mM TrisHCl, pH 7.5, 150 mM NaCl, 1 mM MgCl 2 , 2 mM CaCl 2 , 0.1 mM MnCl 2 .
  • Coating concentrations were 0.8 ⁇ g/mL for ⁇ v ⁇ 3 and 0.3 ⁇ g/mL for ⁇ v ⁇ 5 .
  • Wells were blocked with 200 ⁇ L of a solution of 3% BSA in the same buffer (1 hour at room temperature) then rinsed 3 times with 200 ⁇ L binding buffer (50 mM TrisHCl, pH 7.5, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 2 mM CaCl 2 , 0.1 mM MnCl 2 , 0.1% BSA).
  • Individual RNAs or RNA pools were denatured briefly at 93° C. in binding buffer without divalent cations or BSA, then serially diluted in the same buffer.
  • RNAs were allowed to incubate in the integrin-coated wells at 37° C. for 30-60 minutes. Unbound RNAs were removed by 3 rapid washes in binding buffer.
  • 100 ⁇ L of a 1:2500 dilution in binding buffer of streptavidin-alkaline phosphatase conjugate (Calbiochem) were incubated in each well for 30 minutes at room temperature, followed by three rapid washes, as above.
  • 100 ⁇ L/well p-nitrophenyl phosphate Sigma Chemical Co., St.
  • Binding data were fit to an equation that describes the fraction of RNA or protein bound as a function of K D , and the total concentrations of RNA and protein in the binding reaction for both monophasic and biphasic binding behavior (Green et al. (1996) Biochem. 35:14413-24).
  • RNA specifically bound to the integrin-coated beads had increased substantially (data not shown).
  • immobilized ⁇ v ⁇ 3 showed no detectable affinity for random sequence RNA
  • the Round 7 RNA pool bound with an equilibrium dissociation constant (K D ) of approximately 4 ⁇ 10 ⁇ 7 M ( FIG. 1 ).
  • the Round 7 affinity-enriched pool was cloned and sequences were determined for individual molecules in the mixture. Of 92 sequences obtained, 35 (38%) were very highly related to one another, in many cases differing at no more than a single base position.
  • Table 1 shows the sequences of the major family of 2′-F-pyrimidine RNAs with high affinity for ⁇ v ⁇ 3 (Family 1).
  • Clone names indicate the selected RNA pool from which each sequence was derived (round 7, round 15 or round 17) followed by a unique clone number. Note that in several cases identical sequences were isolated from different RNA pools; in these cases, both clone names are given. (Clones 17.12A and B were isolated as end-to-end inserts in a single plasmid.) Numbers in parentheses indicate the frequency with which a particular sequence was isolated; if no number is given the clone was obtained only once from the selected RNA pool.
  • Family 2 sequences isolated from the ⁇ v ⁇ 3 SELEX are shown in Table 2. The short motif (CCUGCC) held in common among all the sequences is indicated in boldface letters. In Table 3, sequences with no obvious relationship to Families 1 or 2 are shown. Groups of similar sequences with only two (7.41 and 7.93) or three (7.11, 7.82 and 7.101) members are also included in Table 3.
  • aptamers selected for high-affinity binding to a particular target protein show relatively weak binding to other related proteins, except in cases where the degree of homology is very high (for example, see (Green et al. (1996) Biochem. 35:14413-24; Ruckman et al. (1998) J. Biol. Chem. 273:20556-67)).
  • Significant homology exists within the families of integrin alpha and beta sub-units, and both alpha and beta sub-units are shared among members of the integrin superfamily.
  • the affinities were determined using the methods described above.
  • the Family 1 aptamer 17.16 (SEQ ID NO:60) was chosen as a representative of the major sequence family.
  • FIG. 2 shows that aptamer 17.16 bound with identical affinity to purified, utilized ⁇ v ⁇ 3 and to the platelet integrin, ⁇ IIb ⁇ 3 in a 96-well plate binding assay. Although these two proteins share the ⁇ 3 sub-unit in common, an alignment of the ⁇ v and ⁇ IIb amino acid sequences shows only 36% overall sequence identity (Fitzgerald et al. (1987) Biochem. 26:8158-65). Short stretches of exact sequence identity, 5 to 9 amino acids in length, do occur, primarily within four putative calcium-binding domains of each a sub-unit.
  • Binding of aptamer 17.16 to integrin ⁇ v ⁇ 5 was also tested.
  • the ⁇ 5 sub-unit shares 56% sequence identity with ⁇ 3 and is more closely related to ⁇ 3 than other members of the beta sub-unit family (McLean et al. (1990) J. Biol. Chem. 265:17126-31; Suzuki et al. (1990) Proc. Nat. Acad. Sci. 87:5354-8).
  • No aptamer binding to immobilized integrin ⁇ v ⁇ 5 was observed ( FIG. 3 ), although an ⁇ v -specific antibody detected the presence of ⁇ v ⁇ 5 protein adsorbed to the surface of the well (data not shown).
  • aptamer 17.16 and by extension the other members of sequence Family 1, bind primarily to the 3 sub-unit of ⁇ v ⁇ 3
  • the high-affinity binding of the aptamer to the platelet integrin, ⁇ v ⁇ 3 extends its range of potential application to indications involving detection of platelets or inhibition of their function.
  • RNA sequences with high affinity for a particular target While the SELEX process identifies RNA sequences with high affinity for a particular target, the procedure used in this example was designed to bias for the recovery of ligand binding site inhibitors by the inclusion of a cRGD peptide competitor in the elution buffer.
  • aptamer 17.16 could block the ligand binding site of ⁇ v ⁇ 3 or ⁇ IIb ⁇ 3 .
  • purified vitronectin and fibrinogen were biotinylated and incubated with one or both of the immobilized integrins in the presence or absence of varying concentrations of the aptamer or a non-binding control RNA.
  • 96-well microtiter plates were coated as described above with either ⁇ v ⁇ 3 or ⁇ IIb ⁇ 3 and blocked with BSA.
  • a fixed concentration of biotinylated ligand (fibrinogen: 6 nM final; vitronectin: 10 nM final) was pre-mixed in binding buffer (see “Measurement of Aptamer Binding Affinities,” above) with varying concentrations of aptamer, control RNA, cyclic RGD peptide, antibody, or unmodified ligand.
  • the mixtures were incubated in the integrin-coated wells for 60 minutes at room temperature.
  • biotinylated ligand was detected by addition of 100 ⁇ L/well 1:500 dilution streptavidin-alkaline phosphatase conjugate (Calbiochem) (30 minutes at room temperature) followed by 100 ⁇ L/well p-nitrophenyl phosphate, as described above. Absorbance was read at 405 nm. The data were fit to an equation that describes mutually exclusive binding of two ligands to a single target species (Gill et al. (1991) J. Mol. Biol. 220:307-24). The concentration of competitor that inhibited 50% of the maximum signal above background (IC 50 ) was determined from the fitted curve.
  • FIG. 4A shows inhibition of biotinylated vitronectin binding to immobilized ⁇ v ⁇ 3 .
  • Aptamer 17.16 inhibited the binding interaction with an IC 50 of 4.7 nM while the control RNA showed no inhibition.
  • the IC 50 of RGD peptide inhibition was 1.4 nM and that of LM609 was 2.7 nM.
  • Unmodified vitronectin inhibited the binding of the biotinylated material with an IC 50 of 59 nM.
  • IC 50 values for the data in FIG. 4B were: 17.16, 9.5 nM; control RNA, not measurable; RGD peptide, 1.0 nM; LM609, 6.3 nM; unmodified fibrinogen, 43 nM.
  • IC 50 values for FIG. 4C were: 17.16, 6.5 nM; control RNA, not measurable; RGD peptide, 21 nM; unmodified fibrinogen, 15 nM.
  • aptamer 17.16 is an effective competitor of ⁇ 3 integrin ligand binding and, on a molar basis, has an inhibitory potency nearly equivalent to that of a bivalent antibody.
  • Aptamer 17.16 (SEQ ID NO:60) was selected for binding to purified human ⁇ v ⁇ 3 adsorbed to the surface of a polystyrene bead.
  • In vitro assays to measure the affinity of the aptamer for purified ⁇ 3 integrins were also done in the context of hydrophobically-adsorbed protein.
  • an important test of aptamer function was to determine its capacity to bind to native protein on the surface of cells. Human platelets were chosen for this purpose because of their ease of isolation and their high level of expression of integrin ⁇ IIb ⁇ 3 .
  • Platelet-rich plasma was prepared from freshly-drawn citrated human blood by centrifugation at 1000 rpm for 15 minutes in a table top centrifuge.
  • activated platelets cells were incubated for 15 minutes at room temperature at 2 ⁇ 10 7 /mL in calcium- and magnesium-free Dulbecco's PBS with 2.5 U/mL thrombin and 5 mM Gly-Pro-Arg-Pro (GPRP) to inhibit platelet aggregation.
  • Cells were diluted 1:10 into binding buffer (20 mM HEPES, pH 7.5, 111 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 0.1% BSA, 0.01% sodium azide).
  • Resting cells were diluted similarly, without exposure to thrombin or GPRP.
  • the activation state of resting and thrombin-treated cells was monitored by staining with fluorophore-conjugated antibodies to CD61 ( ⁇ 3 integrin subunit), which binds to all platelets, and to CD62 (P-selectin), a marker of platelet activation.
  • Antibodies were obtained from Becton-Dickinson Immunocytometry Systems, San Jose, Calif. Fluorescein-conjugated RNAs were diluted in water to 4 ⁇ M and denatured briefly at 93° C., then diluted to 2 ⁇ M with 2 ⁇ -concentrated binding buffer. RNAs were then serially diluted in binding buffer.
  • Non-specific RNA binding to platelets was measured using a control RNA of similar length and base composition to aptamer 17.16. Non-specific binding became significant at concentrations above approximately 100 nM. Specific binding of the aptamer was distinguished from non-specific binding by the addition of 5 mM EDTA to the sample: EDTA had no effect on the binding of the control RNA but reduced aptamer binding to the level of the control. Specific binding of the aptamer was thus defined as the difference between the fluorescence intensity of the sample before the addition of EDTA (specific+non-specific) and the fluorescence intensity after the addition of EDTA (non-specific only).
  • FIG. 5 shows representative data for the EDTA-sensitive component of aptamer binding to both resting and thrombin-activated human platelets.
  • the maximum binding signal is approximately 2-fold higher to activated platelets, consistent with the slightly higher level of ⁇ IIb ⁇ 3 on such cells (Wagner et al. (1996) Blood 88:907-14).
  • the estimated K D for aptamer binding to platelets was approximately 10 nM for both cell populations, equivalent to the value determined for binding in vitro to purified ⁇ IIb ⁇ 3 .
  • aptamer 17.16 binds to both resting and activated platelets with an affinity equivalent to that reported for Reopro (abciximab, chimeric 7E3 Fab), an approved ⁇ IIb ⁇ 3 antagonist (Mousa et al. (1998) J. Pharm. Exp. Ther. 286:1277-84).
  • aptamer 17.16 was labeled at the 5′ end with technitium-99m ( 99m Tc) and its biodistribution was monitored in a rabbit model of venous thrombosis.
  • a clot is generated in situ in the isolated jugular vein of a rabbit from human platelet-rich plasma. Blood flow across the clot is re-established and the radiolabeled aptamer (or a non-binding control RNA) are introduced into the bloodstream via the ipsilateral ear vein. The distribution of the radiolabel into various tissues is reported as the per cent of the injected dose per gram of tissue.
  • RNA was conjugated to Hi 15 at 50 mg/mL aptamer in 30% dimethylformamide with 5 molar equivalents of Hi 15 -NHS buffered in 100 mM NaBorate pH 9.3, for 30 minutes at room temperature.
  • the conjugation reactions were washed over a 30,000 molecular weight cut-off filter (Microcon 30, Amicon, Inc., Beverly, Mass.) to remove excess Hi 15 cage.
  • RNAs were then labeled with 99m Tc in the following manner: to 1 nmol Hi 15 -aptamer was added 200 ⁇ l of 100 mM NaPO 4 buffer, pH 8.5, 23 mg/mL NaTartrate, and 50 ⁇ L [ 99m Tc] pertechnetate (5.0 mCi) eluted from a 99 Mo column (Syncor, Denver) within 12 hours prior to use.
  • the labeling reaction was initiated by the addition of 10 ⁇ L 5 mg/mL SnCl 2 .
  • the reaction mixture was incubated for 15 minutes at 90° C.
  • rabbits were anesthetized with isofluorane.
  • a two centimeter section of the right jugular vein was isolated in situ and all the branches were ligated.
  • a catheter was inserted into the facial vein.
  • the isolated vein segment was temporarily ligated above and below the catheter.
  • the vein segment was flushed with saline.
  • 1000 USP units of heparin was administered intravenously.
  • 300-400 ⁇ L of fresh human platelet-rich plasma (citrate) activated with calcium and thrombin was instilled into the isolated vein segment and allowed to clot.
  • aptamer 17.16 radiolabel accumulated in the clot to a significant degree by one hour after injection, while similar accumulation was not observed with the control RNA ( FIG. 6 ). Blood clearance of the radiolabel was apparently rapid and mediated primarily by a renal mechanism as judged by moderate accumulation of radioactivity in the kidney for both the aptamer and control RNA.
  • aptamers specific for ⁇ IIb ⁇ 3 or for other proteins expressed at high levels on the surface of platelets or within the matrix of a clot will serve as useful agents for rapid imaging of thrombi. TABLE 1 ⁇ v ⁇ 3 Family 1 aptamer sequences.

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Abstract

Methods are described for the isolation of nucleic acid ligands to integrins using the SELEX process. SELEX is an acronym for Systematic Evolution of Ligands by EXponential enrichment. The nucleic acid ligands of the present invention are useful as therapeutic and diagnostic agents.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • This application is a divisional of U.S. patent application Ser. No. 10/024,997, filed Oct. 17, 2002, now U.S. Pat. No. 7,094,535, which is a divisional of U.S. patent application Ser. No. 09/364,543, filed Jul. 29, 1999, now U.S. Pat. No. 6,331,394, both of which are entitled “Nucleic Acid Ligands to Integrins”. U.S. patent application Ser. No. 09/364,543 is a continuation in part of U.S. patent application Ser. No. 09/606,477, filed Jun. 29, 2000, now U.S. Pat. No. 6,465,189, which is a continuation of U.S. patent application Ser. No. 08/956,699, filed Oct. 23, 1997, now U.S. Pat. No. 6,083,696, which is a continuation of U.S. patent application Ser. No. 08/234,997, filed Apr. 28, 1994, now U.S. Pat. No. 5,683,867, all entitled “Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX.” U.S. Pat. No. 5,683,867 is a continuation in part of U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled “Nucleic Acid Ligands,” now U.S. Pat. No. 5,475,096, each of which are incorporated herein by reference in their entirety.
    • FIELD OF THE INVENTION
  • This invention is directed towards nucleic acid ligands of integrins isolated using the SELEX process. SELEX is an acronym for Systematic Evolution of Ligands by EXponential Enrichment. This invention relates to integrin proteins, and methods and compositions for treating and diagnosing diseases involving integrins.
    • BACKGROUND OF THE INVENTION
  • The integrins are a class of heterodimeric integral membrane proteins, one or more of which are expressed by most cell types (Hynes (1992) Cell 69:11-25). Some 16 homologous alpha subunits and 8 homologous beta subunits associate in various combinations to yield an extensive family of receptors. Each integrin heterodimer has a large extracellular domain that mediates binding to specific ligands. These ligands may include plasma proteins, proteins expressed on the surface of adjacent cells, or components of the extracellular matrix. Several of the integrins show affinity for more than one ligand and many have overlapping specificities (Hynes (1992) Cell 69:11-25). Both the α and β subunits contribute to a small intracellular domain that contacts components of the actin cytoskeleton, thus forming a physical link between proteins outside and inside the cell. Integrins play an important role in cellular adhesion and migration, and these properties are controlled by the cell, in part, by modulation of integrin affinity for its ligands (so-called “inside-out” signaling). Conversely, the presence or absence of integrin ligation provides specific information about the cellular microenvironment, and in many instances integrins serve as a conduit for signal transduction. Ligand binding by an integrin may promote its incorporation into focal adhesions, the assembly of cytoskeletal and intracellular signaling molecules into supra-molecular complexes, and the initiation of a cascade of downstream signaling events including protein phosphorylation, calcium release, and an increase in intracellular pH (reviewed by Schwartz et al. (1995) Ann. Rev. Cell Dev. Biol. 11:549-99). Such “outside-in” signaling ties into pathways controlling cell proliferation, migration and apoptosis (Stromblad et al. (1996) J. Clin. Invest. 98:426-33; Eliceiri et al. (1998) J. Cell. Biol. 140:1255-63). Integrins have been shown to play a role in such diverse physiological settings as embryonic development, wound healing, angiogenesis, clot formation, leukocyte extravasation, bone resorption and tumor metastasis.
  • The β3-containing integrins are among the best studied of the receptor superfamily. The β3 subunit forms heterodimers with either αv (αvβ3) or αIIb (αIIbβ3), While these integrins show substantial overlap in ligand specificity, they play very different roles in normal physiology and in disease.
  • αvβ3 is expressed by activated endothelial cells, smooth muscle cells, osteoclasts, and, at a very low level, by platelets. It is also expressed by a variety of tumor cell types. The integrin binds to a number of plasma proteins or proteins of the extracellular matrix, many of which are associated with sites of inflammation or wound healing (Albelda (1991) Am. J. Resp. Cell Mol. Biol. 4:195-203). These include vitronectin, fibronectin, osteopontin, von Willebrand factor, thrombospondin, fibrinogen, and denatured collagen Type I (Hynes (1992) Cell 69:11-25). Each of these proteins share a common sequence motif, arginine-glycine-aspartic acid (RGD), that forms the core of the integrin binding site.
  • αvβ3 has been most intensely studied in the context of new blood vessel formation (angiogenesis) where it mediates the adhesion and migration of endothelial cells through the extracellular matrix. Angiogenesis in adults is normally associated with the cyclical development of the corpus luteum and endometrium and with the formation of granulation tissue during wound repair. In the latter case, microvascular endothelial cells form vascular sprouts that penetrate into the temporary matrix within a wound. These cells transiently express αvβ3 and inhibition of the ligand binding function of the integrin temporarily inhibits the formation of granulation tissue (Clark et al. (1996) Am. J. Pathol. 148:1407-21). In cytokine-stimulated or unstimulated angiogenesis on the chick chorioallantoic membrane, blockade of αvβ3 with a heterodimer-specific antibody prevents new vessel formation without affecting the pre-existing vasculature (Brooks et al. (1994) Science 264:569-71). Furthermore, the loss of adhesive contacts by endothelial cells activated for angiogenesis induces a phenotype characteristic of apoptotic cells (Brooks et al. (1994) Cell 79:1157-64); that is, ligand binding by αvβ3 appears to transmit a survival signal to the cell. Thus, adhesion and/or signaling mediated by αvβ3 is essential for the formation of new blood vessels.
  • Solid tumors are unable to grow to significant size without an independent blood supply. It is currently hypothesized that the acquisition of an angiogenic phenotype is one of the limiting steps in the growth of primary tumors and of tumors at secondary sites (Folkman (1995) Nat. Med. 1:27-31). In addition, while the vasculature that penetrates a tumor mass provides a source of oxygen and nutrients, it also serves as a conduit for metastatic cells to leave the primary tumor and migrate throughout the body. Thus, inhibition of angiogenesis may limit both the growth and metastasis of cancerous lesions. In experimental settings of tumor-induced angiogenesis, inhibition of ligand-binding by endothelial αvβ3 prevented the formation of new blood vessels (Brooks et al. (1994) Cell 79:1157-64; Brooks et al. (1995) J. Clin. Invest. 96:1815-22), and inhibitors of αvβ3 were shown to reduce the growth of experimental tumors in vivo (Brooks et al. (1995) J. Clin. Invest. 96:1815-22; Carron et al. (1998) Canc. Res. 58:1930-5).
  • αvβ3 is not only expressed by the microvasculature within tumors, but in some cases, is also found on the surface of tumor cells themselves. In particular, expression of αvβ3 integrin has been detected in tissue sections from tumors of melanocytic and astroglial origin (Albelda et al. (1990) Canc. Res. 50:6757-64; Gladson and Cheresh (1991) J. Clin. Invest. 88:1924-32), and the level of integrin expression has been correlated with the stage or metastatic potential of the tumor (Albelda et al. (1990) Canc. Res. 50:6757-64; Gladson et al. (1996) Am. J. Pathol. 148:1423-34; Hieken et al. (1996) J. Surg. Res. 63:169-73). Furthermore, melanoma cells grown in vitro in a three-dimensional matrix of denatured collagen undergo apoptosis upon αvβ3 blockade.
  • Data such as these have driven an interest in inhibitors of αvβ3 for the treatment of cancer. At present, two such inhibitors are in or near clinical trial: Vitaxin is a chimeric Fab fragment derived from the αvβ3-specific monoclonal antibody, LM609 (Wu et al. (1998) Proc. Nat. Acad. Sci. 95:6037-42). A phase I trial in late-stage cancer patients has been completed and no significant treatment-associated toxicities were observed (Gutheil et al. (1998) Am. Soc. Clin. Onc.). EMD121974 is a cyclic pentapeptide inhibitor of αvβ3. A Phase I study of this compound in Kaposi's sarcoma, brain tumors and solid tumors is scheduled to begin in 1999.
  • Angiogenesis (and αvβ3) are implicated in the pathology of several other diseases, including psoriasis (Creamer et al. (1995) Am. J. Pathol. 147:1661-7), rheumatoid arthritis (Walsh et al. (1998) Am. J. Pathol. 152:691-702; Storgard et al. (1999) J. Clin. Invest. 103:47-54), endometriosis (Healy et al. (1998) Hum. Reprod. Update 4:736-40), and several proliferative diseases of the eye (Casaroli Marano et al. (1995) Exp. Eye Res. 60:5-17; Friedlander et al. (1996) Proc. Nat. Acad. Sci. 93:9764-9; Hammes et al. (1996) Nat. Med. 2:529-33). Inhibition of integrin ligand binding in each of these contexts may provide significant therapeutic benefit.
  • Atheromatous plaque and restenosis following angioplasty are pathologies characterized by thickening of the intima, the innermost layer of the arterial wall. The proliferation and/or migration of smooth muscle cells into the neointima with concomitant deposition of fibrous extracellular proteins contributes to vessel wall thickening and subsequent vessel occlusion. Platelets may also contribute to the development of restenotic lesions through adhesion to endothelial cells and the release of growth factors and cytokines that stimulate the underlying smooth muscle cell layer (Le Breton et al. (1996) J. Am. Coll. Cardiol. 28:1643-51). αvβ3 integrin is expressed on arterial smooth muscle cells (Hoshiga et al. (1995) Circ. Res. 77:1129-35) and mediates their migration on vitronectin and osteopontin (Brown et al. (1994) Cardiovasc. Res. 28:1815-20; Jones et al. (1996) Proc. Nat. Acad. Sci. 93:2482-7; Liaw et al. (1995) J. Clin. Invest. 95:713-24; Panda et al. (1997) Proc. Nat. Acad. Sci. 94:9308-13), both matrix proteins that are associated with atheroschlerotic tissues in vivo (Brown et al. (1994) Cardiovasc. Res. 28:1815-20; Giachelli et al. (1995) Ann. N. Y. Acad. Sci. 760:109-26; Panda et al. (1997) Proc. Nat. Acad. Sci. 94:9308-13). In addition, αvβ3 expression on endothelial cells, and to a much lesser extent on platelets, is responsible for at least part of the adhesive interaction between these cell types (Le Breton et al. (1996) J. Am. Coll. Cardiol. 28:1643-51; Gawaz et al. (1997) Circulation 96:1809-18). αvβ3 blockade with RGD-containing peptides or a monoclonal antibody was found to limit neointimal hyperplasia in several animal models of restenosis following arterial injury,, (Choi et al. (1994) J. Vasc. Surg. 19:125-34; Srivatsa et al. (1997) Cardiovasc. Res. 36:408-28; Slepian et al. (1998) Circulation 97:1818-27; Coleman et al. (1999) Circ. Res. 84:1268-76). Furthermore, treatment of patients undergoing percutaneous coronary intervention with an anti-β3 antibody (Reopro/abciximab/c7E3), which blocks both the platelet fibrinogen receptor, αIIbβ3, and αvβ3, provided long term reduction in the rates of death or myocardial infarction and in the rate of reocclusion of the artery (Lefkovits et al. (1996) Am. J. Cardiol. 77:1045-51), an effect that may be mediated through inhibition of αvβ3 ligation. The observation that αvβ3 is expressed by microvascular smooth muscle cells after experimentally-induced focal cerebral ischemia (Okada et al. (1996) Am. J. Pathol. 149:37-44) suggests that this integrin may also play some role in the development of ischemia/reperfusion injury in stroke.
  • Finally, αvβ3 mediates the attachment of osteoclasts to matrix proteins, particularly osteopontin, on the surface of bone. Osteoclasts are responsible for the resorption of bone in normal physiology as well as in pathological conditions such as osteoporosis. A monoclonal antibody specific for αvβ3 inhibited the binding and resorption of bone particles by osteoclasts in vitro (Ross et al. (1993) J. Biol. Chem. 268:9901-7). Furthermore, an RGD-containing protein, echistatin, was shown to block parathyroid-stimulated bone resorption in an animal model, as monitored by serum calcium levels (Fisher et al. (1993) Endocrin. 132:1411-3). Inhibitors of αvβ3 integrin are thus considered of potential utility in treating debilitating bone loss such as occurs in osteoporosis.
  • αIIbβ3 (also referred to as GPIIbIIIa) is the major integrin on the surface of platelets where it mediates the adhesion of activated platelets to the plasma protein fibrinogen (Nachman and Leung (1982) J. Clin. Invest. 69:263-9; Shattil et al. (1985) J. Biol. Chem. 260:11107-14). During clot formation, fibrinogen dimers cross-link platelets to one another through the integrin receptor. αIIbβ3 also binds to several other plasma and cell matrix proteins, including von Willebrand factor, vitronectin, and fibronectin (Faull and Ginsberg (1996) J. Am. Soc. Nephrol. 7:1091-7).
  • Clot formation is a tightly regulated process that balances the need for rapid response to vascular injury with the risk of aberrant occlusion of critical vessels. The αIIbβ3 heterodimer is constitutively expressed on the surface of resting platelets at approximately 80,000 copies per cell (Wagner et al. (1996) Blood 88:907-14); however, the affinity of the integrin for fibrinogen is very low on these cells. Activation of platelets by ADP, epinephrine, collagen or thrombin leads to a dramatic enhancement in integrin ligand binding activity (Bennett and Vilaire (1979) J. Clin. Invest. 64:1393-401; Marguerie et al. (1979) J. Biol. Chem. 254:5357-63), probably accomplished through a conformational change in the receptor (Shattil et al. (1985) J. Biol. Chem. 260:11107-14; O'Toole et al. (1990) Cell Reg. 1:883-93; Du et al. (1993) J. Biol. Chem. 268:23087-92). In this prototypic example of “inside-out” control of integrin function, cross-linking of platelets through the αIIbβ3-fibrinogen interaction is confined to local sites of platelet activation.
  • Inhibitors of αIIbβ3 ligand binding have been primarily explored in the context of cardiovascular disease (Chong (1998) Am. J. Health Syst. Pharm. 55:2363-86; Topol et al. (1999) Lancet 353:227-31), but may have application in any of a number of indications where thrombus formation is suspected or is likely. Three αIIbβ3 inhibitors have been approved for use in patients experiencing acute coronary syndrome and/or in patients who are undergoing percutaneous coronary intervention. Reopro (Centocor/Eli Lilly) is a humanized murine monoclonal antibody Fab fragment with specificity for the β3 chain of αIIbβ3, Integrilin (COR Therapeutics) is a cyclic heptapeptide based on the integrin binding site of barbourin, an αIIbβ3 inhibitory protein derived from snake venom. Aggrastat (Merck & Co.) is a non-peptide small molecule antagonist of the integrin. Unlike the small molecule inhibitors, Reopro cross-reacts with αvβ3, a fact which may account for the greater reduction in long-term rates of death and non-fatal myocardial infarction associated with its use (see above). A significant effort is underway to identify new inhibitors of the platelet integrin with characteristics not found in the cohort of approved drugs. Specifically, compounds with specificity for the active, ligand-binding conformation of αIIbβ3 may reduce the risk of bleeding complications associated with the existing anti-clotting therapies. Orally available compounds would be particularly useful for longer term therapy of patients at risk for recurrent myocardial infarction or unstable angina.
  • Given the role of integrins in the various disease states described above, it would be desirable to have high specificity inhibitors of particular integrins. The present invention provides such agents.
  • The dogma for many years was that nucleic acids had primarily an informational role. Through a method known as Systematic Evolution of Ligands by EXponential enrichment, termed the SELEX process, it has become clear that nucleic acids have three dimensional structural diversity not unlike proteins. The SELEX process is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules and is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution of Ligands by EXponential Enrichment,” now abandoned, U.S. Pat. No. 5,475,096, entitled “Nucleic Acid Ligands” and U.S. Pat. No. 5,270,163 (see also WO 91/19813), entitled “Methods for Identifying Nucleic Acid Ligands,” each of which is specifically incorporated by reference herein in its entirety. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule. The SELEX process provides a class of products which are referred to as nucleic acid ligands or aptamers, each having a unique sequence, and which has the property of binding specifically to a desired target compound or molecule. Each SELEX-identified nucleic acid ligand is a specific ligand of a given target compound or molecule. The SELEX process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets. The SELEX method applied to the application of high affinity binding involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
  • It has been recognized by the present inventors that the SELEX method demonstrates that nucleic acids as chemical compounds can form a wide array of shapes, sizes and configurations, and are capable of a far broader repertoire of binding and other functions than those displayed by nucleic acids in biological systems.
  • The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat. No. 5,707,796, both entitled “Method for Selecting Nucleic Acids on the Basis of Structure,” describe the use of the SELEX process in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection of Nucleic Acid Ligands,”, now abandoned, U.S. Pat. No. 5,763,177, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX” and U.S. patent application Ser. No. 09/093,293, filed Jun. 8, 1998, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX,” describe a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. Pat. No. 5,580,737, entitled “High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine,” describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, which can be non-peptidic, termed Counter-SELEX. U.S. Pat. No. 5,567,588, entitled “Systematic Evolution of Ligands by EXponential Enrichment: Solution SELEX,” describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule.
  • The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX process-identified nucleic acid ligands containing modified nucleotides are described in U.S. Pat. No. 5,660,985, entitled “High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,” that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2′-positions of pyrimidines. U.S. Pat. No. 5,580,737, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2′-amino (2′-NH2), 2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method of Preparation of Known and Novel 2′ Modified Nucleosides by Intramolecular Nucleophilic Displacement,” now abandoned, describes oligonucleotides containing various 2′-modified pyrimidines.
  • The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. Pat. No. 5,637,459, entitled “Systematic Evolution of Ligands by EXponential Enrichment: Chimeric SELEX,” and U.S. Pat. No. 5,683,867, entitled “Systematic Evolution of Ligands by EXponential Enrichment: Blended SELEX,” respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules.
  • The SELEX method further encompasses combining selected nucleic acid ligands with lipophilic compounds or non-immunogenic, high molecular weight compounds in a diagnostic or therapeutic complex as described in U.S. patent application Ser. No. 08/434,465, filed May 4, 1995, entitled “Nucleic Acid Ligand Complexes”. Each of the above described patent applications which describe modifications of the basic SELEX procedure are specifically incorporated by reference herein in their entirety.
  • It is an object of the present invention to provide methods that can be used to identify nucleic acid ligands that bind with high specificity and affinity to particular integrins.
  • It is a further object of the present invention to obtain nucleic acid ligands to particular integrins that inhibit the ability of that integrin to bind its cognate ligand.
  • It is a further object of the present invention to obtain integrin inhibiting pharmaceutical compositions for controlling thrombosis, tumor angiogenesis, tumor cell migration, proliferative ocular diseases, rheumatoid arthritis, psoriasis, osteoporosis, and restenosis.
  • It is yet a further object of the invention to obtain imaging agents for the non-invasive detection of deep vein or arterial thrombi.
    • SUMMARY OF THE INVENTION
  • Methods are provided for generating nucleic acid ligands to integrins, particularly to the β3 integrins. The methods use the SELEX process for ligand generation. Particular embodiments describe the isolation of nucleic acid ligand inhibitors of both αvβ3 and αIIbβ3. The nucleic acid ligand inhibitors are derived from a library of 2′-fluoro-pyrimidine RNA sequences and were selected for high affinity binding to αvβ3. One of the modified nucleic acid ligands is shown to inhibit the binding of either vitronectin or fibrinogen to both of the purified integrins in vitro. This nucleic acid ligand binds to the surface of both resting and activated platelets with equivalent affinity and accumulates at the site of a preformed clot in an animal model of venous thrombosis.
  • The nucleic acid ligands provided by the invention are useful as therapeutic agents for a number of diseases including thrombosis and cancer. The nucleic acid ligands of the instant invention are also useful as diagnostic agents for thrombosis.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 illustrates the binding of affinity-enriched RNA pools to immobilized αvβ3. 5′-biotinylated RNA pools were incubated at varying concentrations in 96-well microtiter plates coated with integrin αvβ3. Bound RNAs were detected via the biotin moiety by a chromogenic assay. Data are expressed in absorbance units at 405 nm as a function of input RNA concentration.
  • FIG. 2 illustrates cross-reactivity of aptamer 17.16 (SEQ ID NO:60) to purified integrin αIIbβ3. 5′-biotinylated aptamer 17.16 was incubated at varying concentrations in microtiter wells coated with either integrin αvβ3 or αIIbβ3. Bound RNA was detected via the biotin moiety using a chromogenic assay. Data are expressed as the per cent of the maximum signal to normalize for differences in protein coating.
  • FIG. 3 illustrates cross-reactivity of aptamer 17.16 (SEQ ID NO:60) to purified integrin αvβ5. 5′-biotinylated aptamer 17.16 or a control RNA of similar length and base composition were incubated at varying concentrations in microtiter wells coated with either αvβ3 or αvβ5. Bound RNAs were detected via the biotin moiety by a chromogenic assay. Data are expressed in absorbance units at 405 nm as a function of input RNA concentration.
  • FIGS. 4A-C illustrate β3 aptamer inhibition of integrin ligand binding. Biotinylated fibrinogen or vitronectin were incubated in microtiter wells coated with either integrin αvβ3 or αIIbβ3 in the presence or absence of varying concentrations of ligand binding competitors. Competitors included aptamer 17.16 (SEQ ID NO:60), a control RNA of similar length and base composition, a cyclic RGD peptide (cRGD, see Materials and Methods), an αvβ3-specific monoclonal antibody (LM609), or unmodified fibrinogen or vitronectin. Bound ligands were detected via biotin using a chromogenic assay. Data are expressed in absorbance units at 405 nm as a function of input competitor concentration. FIG. 4A shows competition of vitronectin binding to immobilized αvβ3; FIG. 4B shows competition of fibrinogen binding to immobilized αvβ3; and FIG. 4C shows competition of fibrinogen binding to immobilized αIIbβ3. An estimate of the maximum absorbance value was determined for each ligand/integrin pair in the absence of competitor. The baseline absorbance value was determined by adding 5 mM EDTA to the incubation buffer. The maximum and minimum values so determined were FIG. 4A, 0.914/0.113; FIG. 4B, 1.042/0.122; FIG. 4C, 0.889/0.128.
  • FIG. 5 illustrates binding of aptamer 17.16 (SEQ ID NO:60) to activated or resting human platelets. 5′-fluorescein-conjugated aptamer 17.16 or a control RNA of similar length and base composition were incubated at various concentrations with resting or thrombin-activated human platelets (106I/mL). Incubations were at room temperature in buffered saline containing divalent cations, 0.1% BSA and 0.01% sodium azide. Mean fluorescence intensity of the sample was determined by flow cytometry both before and after the addition of EDTA to 5 mM final concentration. The difference in fluorescence intensity between the two samples (the EDTA-sensitive signal) is shown as a function of the concentration of aptamer or control RNA.
  • FIG. 6 illustrates biodistribution of [99mTc]-aptamer 17.16 (SEQ ID NO:60) or control RNA in a rabbit venous clot model. A clot derived from human platelet-rich plasma was generated in situ by temporary isolation of the jugular vein of an anesthetized rabbit. After restoration of circulation over the clot, [99mTc]-labeled aptamer or control RNA were injected into the bloodstream of the rabbit via the ipsilateral ear vein. After one hour, the animal was sacrificed and tissues were weighed and counted in a gamma counter. Accumulation of radioactivity in various tissues is reported as the percentage of the injected dose per gram wet weight of tissue.
    • DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • The central method utilized herein for identifying nucleic acid ligands to Integrins is called the SELEX process, an acronym for Systematic Evolution of Ligands by Exponential enrichment and involves (a) contacting the candidate mixture of nucleic acids with integrins, or expressed domains or peptides corresponding to integrins, (b) partitioning between members of said candidate mixture on the basis of affinity to integrins, and c) amplifying the selected molecules to yield a mixture of nucleic acids enriched for nucleic acid sequences with a relatively higher affinity for binding to integrins.
    • DEFINITIONS
  • Various terms are used herein to refer to aspects of the present invention. To aid in the clarification of the description of the components of this invention, the following definitions are provided:
  • As used herein, “nucleic acid ligand” is a non-naturally occurring nucleic acid having a desirable action on a target. Nucleic acid ligands are often referred to as “aptamers”. The term aptamer is used interchangeably with nucleic acid ligand throughout this application. A desirable action includes, but is not limited to, binding of the target, catalytically changing the target, reacting with the target in a way which modifies/alters the target or the functional activity of the target, covalently attaching to the target as in a suicide inhibitor, facilitating the reaction between the target and another molecule. In the preferred embodiment, the action is specific binding affinity for a target molecule, such target molecule being a three dimensional chemical structure other than a polynucleotide that binds to the nucleic acid ligand through a mechanism which predominantly depends on Watson/Crick base pairing or triple helix binding, wherein the nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target molecule. In the present invention, the target is an integrin, or portions thereof. Nucleic acid ligands include nucleic acids that are identified from a candidate mixture of nucleic acids, said nucleic acid ligand being a ligand of a given target, by the method comprising: a) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and c) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids.
  • As used herein, “candidate mixture” is a mixture of nucleic acids of differing sequence from which to select a desired ligand. The source of a candidate mixture can be from naturally-occurring nucleic acids or fragments thereof, chemically synthesized nucleic acids, enzymatically synthesized nucleic acids or nucleic acids made by a combination of the foregoing techniques. In a preferred embodiment, each nucleic acid has fixed sequences surrounding a randomized region to facilitate the amplification process.
  • As used herein, “nucleic acid” means either DNA, RNA, single-stranded or double-stranded, and any chemical modifications thereof. Modifications include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to, 2′-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3′ and 5′ modifications such as capping.
  • “SELEX” methodology involves the combination of selection of nucleic acid ligands which interact with a target in a desirable manner, for example binding to a protein, with amplification of those selected nucleic acids. Optional iterative cycling of the selection/amplification steps allows selection of one or a small number of nucleic acids which interact most strongly with the target from a pool which contains a very large number of nucleic acids. Cycling of the selection/amplification procedure is continued until a selected goal is achieved. In the present invention, the SELEX methodology is employed to obtain nucleic acid ligands to integrins.
  • The SELEX methodology is described in the SELEX Patent Applications.
  • “SELEX target” or “target” means any compound or molecule of interest for which a ligand is desired. A target can be a protein, peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye, nutrient, growth factor, etc. without limitation. In this application, the SELEX targets are integrins.
  • As used herein, “solid support” is defined as any surface to which molecules may be attached through either covalent or non-covalent bonds. This includes, but is not limited to, membranes, microtiter plates, magnetic beads, charged paper, nylon, Langmuir-Bodgett films, functionalized glass, germanium, silicon, PTFE, polystyrene, gallium arsenide, gold, and silver. Any other material known in the art that is capable of having functional groups such as amino, carboxyl, thiol or hydroxyl incorporated on its surface, is also contemplated. This includes surfaces with any topology, including, but not limited to, spherical surfaces and grooved surfaces.
  • Note that throughout this application, various references are cited. Every reference cited herein is specifically incorporated in its entirety.
  • A. Preparing Nucleic Acid Ligands to Integrins.
  • In the preferred embodiment, the nucleic acid ligands of the present invention are derived from the SELEX methodology. The SELEX process is described in U.S. patent application Ser. No. 07/536,428, entitled “Systematic Evolution of Ligands by Exponential Enrichment,” now abandoned, U.S. Pat. No. 5,475,096, entitled “Nucleic Acid Ligands,” and U.S. Pat. No. 5,270,163 (see also WO 91/19813) entitled “Methods for Identifying Nucleic Acid Ligands.” These applications, each specifically incorporated herein by reference, are collectively called the SELEX Patent Applications.
  • The SELEX process provides a class of products which are nucleic acid molecules, each having a unique sequence, and each of which has the property of binding specifically to a desired target compound or molecule. Target molecules are preferably proteins, but can also include among others carbohydrates, peptidoglycans and a variety of small molecules. SELEX methodology can also be used to target biological structures, such as cell surfaces or viruses, through specific interaction with a molecule that is an integral part of that biological structure.
  • In its most basic form, the SELEX process may be defined by the following series of steps:
  • 1) A candidate mixture of nucleic acids of differing sequence is prepared. The candidate mixture generally includes regions of fixed sequences (i.e., each of the members of the candidate mixture contains the same sequences in the same location) and regions of randomized sequences. The fixed sequence regions are selected either: (a) to assist in the amplification steps described below, (b) to mimic a sequence known to bind to the target, or (c) to enhance the concentration of a given structural arrangement of the nucleic acids in the candidate mixture. The randomized sequences can be totally randomized (i.e., the probability of finding a base at any position being one in four) or only partially randomized (e.g., the probability of finding a base at any location can be selected at any level between 0 and 100 percent).
  • 2) The candidate mixture is contacted with the selected target under conditions favorable for binding between the target and members of the candidate mixture. Under these circumstances, the interaction between the target and the nucleic acids of the candidate mixture can be considered as forming nucleic acid-target pairs between the target and those nucleic acids having the strongest affinity for the target.
  • 3) The nucleic acids with the highest affinity for the target are partitioned from those nucleic acids with lesser affinity to the target. Because only an extremely small number of sequences (and possibly only one molecule of nucleic acid) corresponding to the highest affinity nucleic acids exist in the candidate mixture, it is generally desirable to set the partitioning criteria so that a significant amount of the nucleic acids in the candidate mixture (approximately 5-50%) are retained during partitioning.
  • 4) Those nucleic acids selected during partitioning as having the relatively higher affinity for the target are then amplified to create a new candidate mixture that is enriched in nucleic acids having a relatively higher affinity for the target.
  • 5) By repeating the partitioning and amplifying steps above, the newly formed candidate mixture contains fewer and fewer unique sequences, and the average degree of affinity of the nucleic acids to the target will generally increase. Taken to its extreme, the SELEX process will yield a candidate mixture containing one or a small number of unique nucleic acids representing those nucleic acids from the original candidate mixture having the highest affinity to the target molecule.
  • The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat. No. 5,707,796 both entitled “Method for Selecting Nucleic Acids on the Basis of Structure,” describe the use of the SELEX process in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection of Nucleic Acid Ligands,” now abandoned, U.S. Pat. No. 5,763,177, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX” and U.S. patent application Ser. No. 09/093,293, filed Jun. 8, 1998, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX,” all describe a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. Pat. No. 5,580,737, entitled “High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine,” describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed Counter-SELEX. U.S. Pat. No. 5,567,588, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Solution SELEX,” describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. U.S. Pat. No. 5,496,938, entitled “Nucleic Acid Ligands to HIV-RT and HIV-1 Rev,” describes methods for obtaining improved nucleic acid ligands after SELEX has been performed. U.S. Pat. No. 5,705,337, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Chemi-SELEX,” describes methods for covalently linking a ligand to its target.
  • The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. Pat. No. 5,660,985, entitled “High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,” that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2′-positions of pyrimidines. U.S. Pat. No. 5,637,459, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2′-amino (2′-NH2), 2′-fluoro(2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method of Preparation of Known and Novel 2′ Modified Nucleosides by Intramolecular Nucleophilic Displacement,” now abandoned, describes oligonucleotides containing various 2′-modified pyrimidines.
  • The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. Pat. No. 5,637,459, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX,” and U.S. Pat. No. 5,683,867, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX,” respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules.
  • In U.S. Pat. No. 5,496,938 methods are described for obtaining improved nucleic acid ligands after the SELEX process has been performed. This patent, entitled Nucleic Acid Ligarids to HIV-RT and HIV-1 Rev, is specifically incorporated herein by reference.
  • One potential problem encountered in the diagnostic use of nucleic acids is that oligonucleotides in their phosphodiester form may be quickly degraded in body fluids by intracellular and extracellular enzymes such as endonucleases and exonucleases before the desired effect is manifest. Certain chemical modifications of the nucleic acid ligand can be made to increase the in vivo stability of the nucleic acid ligand or to enhance or to mediate the delivery of the nucleic acid ligand. See, e.g., U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, now abandoned, and U.S. Pat. No. 5,660,985, both entitled “High Affinity Nucleic Acid Ligands Containing Modified Nucleotides”, and the U.S. patent application entitled “Transcription-Free SELEX”, U.S. patent application Ser. No. 09/356,578, filed Jul. 28, 1999, each of which is specifically incorporated herein by reference. Modifications of the nucleic acid ligands contemplated in this invention include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to, 2′-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate or alkyl phosphate modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3′ and 5′ modifications such as capping. In preferred embodiments of the instant invention, the nucleic acid ligands are RNA molecules that are 2′-fluoro (2′-F) modified on the sugar moiety of pyrimidine residues.
  • The modifications can be pre- or post-SELEX process modifications. Pre-SELEX process modifications yield nucleic acid ligands with both specificity for their SELEX target and improved in vivo stability. Post-SELEX process modifications made to 2′-OH nucleic acid ligands can result in improved in vivo stability without adversely affecting the binding capacity of the nucleic acid ligand.
  • Other modifications are known to one of ordinary skill in the art. Such modifications may be made post-SELEX process (modification of previously identified unmodified ligands) or by incorporation into the SELEX process.
  • The nucleic acid ligands of the invention are prepared through the SELEX methodology that is outlined above and thoroughly enabled in the SELEX applications incorporated herein by reference in their entirety. The SELEX process can be performed using purified integrins, or fragments thereof as a target. Alternatively, full-length integrins, or discrete domains of integrins, can be produced in a suitable expression system. Alternatively, the SELEX process can be performed using as a target a synthetic peptide that includes sequences found in an integrin. Determination of the precise number of amino acids needed for the optimal nucleic acid ligand is routine experimentation for skilled artisans.
  • In some embodiments, the nucleic acid ligands become covalently attached to their targets upon irradiation of the nucleic acid ligand with light having a selected wavelength. Methods for obtaining such nucleic acid ligands are detailed in U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection of Nucleic Acid Ligands,” now abandoned, U.S. Pat. No. 5,763,177, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX” and U.S. patent application Ser. No. 09/093,293, filed Jun. 8 1998, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX,” each of which is specifically incorporated herein by reference in its entirety.
  • In preferred embodiments, the SELEX process is carried out using integrins attached to polystyrene beads. A candidate mixture of single stranded RNA molecules is then contacted with the beads. In especially preferred embodiments, the single stranded RNA molecules have a 2′-fluoro modification on C and U residues, rather than a 2′-OH group. After incubation for a predetermined time at a selected temperature, the beads are washed to remove unbound candidate nucleic acid ligand. The nucleic acid ligand that binds to the integrin is then released into solution, then reverse transcribed by reverse transcriptase and amplified using the Polymerase Chain Reaction. The amplified candidate mixture is then used to begin the next round of the SELEX process. Example 2 illustrates-one possible way of performing the SELEX process using integrins as targets.
  • In preferred embodiments, the nucleic acid ligands thus obtained are assayed for their ability to inhibit the interaction of the integrin with its cognate ligand. In one embodiment, this is performed by first coating microtiter plates with the appropriate integrin(s). A ligand for the integrin, such as vitronectin or fibrinogen, is then biotinylated and contacted with the coated integrin in the presence of the nucleic acid ligand to be assayed. After incubation for a suitable period of time, the microtiter plate is washed, and the amount of vitronectin or fibrinogen binding to integrin is quantitated by adding a streptavidin-alkaline phosphatase conjugate, followed by a colorimetric substrate for alkaline phosphatase, such as p-nitrophenyl phosphate. The alkaline phosphatase signal in each well of the plate is thus inversely proportional to the effectiveness of the nucleic acid ligand as an inhibitor of the interaction between the bound integrin and its cognate ligand.
  • In other embodiments, the nucleic acid ligands can be analyzed using binding to human platelets as an assay. This can be done, for example, by fluorescently labelling the nucleic acid ligand by any of the numerous techniques known in the art. The fluorescent nucleic acid ligand can then be contacted with platelets, and the amount of nucleic acid ligand can be quantitated using Fluorescence Activated Cell Sorting (FACS).
  • The distribution of the nucleic acid ligands of the instant invention can also be studied in vivo. In some embodiments, nucleic acid ligands are labelled with a radiolabel used in the art of radioimaging. For example, a nucleic acid ligand can be conjugated to the isotope 99mTc using one of a number of techniques known in the art. The radiolabeled nucleic acid can then be studied in an animal model of venous thrombosis. For example, a human blood clot can be generated in rabbit vein by first isolating the vein in situ by ligation, and then infusing the vein with human platelet-rich plasma and heparin to induce the formation of a blood clot. Blood flow through the vein is then re-established, and the radiolabeled nucleic acid ligand is introduced into the animals blood supply. The distribution of the radiolabeled nucleic acid ligand can then be studied in the rabbit's tissues to determine whether the nucleic acid ligand has accumulated in the clot, rather than in other areas.
  • The nucleic acid ligands provided by the instant invention have a number of potential uses as therapeutic and diagnostic agents. In some embodiments, nucleic acid ligands that inhibit the interaction between platelet-expressed integrins and their cognate ligands are administered, along with pharmaceutically accepted excipients, in order to prevent the formation of blood clots in patients susceptible to deep vein thrombosis. In other embodiments, the nucleic acid ligands are used to treat acute thrombosis formation during and following percutaneous coronary intervention. In still other embodiments, the nucleic acid ligands of the invention are used to treat patients with acute coronary syndromes such as unstable angina or myocardial infarction.
  • In other embodiments, radiolabeled nucleic acid ligands to platelet-expressed integrins are administered to individuals who are to undergo major surgery, or have suffered major trauma. Such nucleic acid ligands can function as imaging agents for the detection of thrombi, by showing sites in the body where large aggregations of platelets are present. If a thrombosis is detected by radioimaging at a critical site in the body, then anticoagulant and thrombolytic treatment—including treatment with the inhibitory nucleic acid ligands of the instant invention—can be given locally. The advantage of using such a nucleic acid ligand imaging agent is that the anticoagulant and thrombolytic treatments—which can cause harm if administered prophylactically by allowing internal bleeding to continue without efficient clotting—can be given only to those individuals who definitely have a dangerous thrombosis. Moreover, these treatments can be specifically injected at the site where the thrombosis has been detected by the nucleic acid ligand, instead of injecting higher concentrations into the bloodstream in the hope that some active agent will be carried to all potential sites of thrombosis.
  • Nucleic acid ligands to αvβ3 integrin can be used to inhibit tumor growth and metastasis. They can also be used to treat ocular diseases including, but not limited to, diabetic retinopathy, retinopathy of prematurity, and macular degeneration. Other diseases for which αvβ3 nucleic acid ligands are useful therapeutic agents include, but are not limited to, endometriosis, psoriasis, rheumatoid arthritis, stroke, osteoporosis, and restenosis.
    • EXAMPLES
  • The following examples are given for illustrative purposes only. They are not to be taken as limiting the scope of the invention in any way.
    • Example 1 Isolation of Integrins and Integrin Ligands
  • αvβ3 integrin was isolated from human placenta and purified by immunoaffinity chromatography essentially as described by (Smith and Cheresh (1988) J. Biol. Chem. 263:18726-31). In brief, human placentas were diced and the tissue fragments were extracted in a buffer containing 100 mM octyl-β-D-glucopyranoside detergent (Calbiochem, San Diego, Calif.). The extract was cleared by centrifugation and applied to an immunoaffinity column αvβ3-specific monoclonal antibody LM609 affixed to Affi-Gel 10, (Chemicon International, Inc., Temecula, Calif.)). Protein bound to the column was eluted with a low-pH buffer and fractions were immediately neutralized and analyzed for integrin content by SDS-polyacrylamide gel electrophoresis. Integrin-containing fractions were pooled and aliquots of the purified material were stored at −80° C. Purified human αvβ3 was also purchased from Chemicon International, Inc, as was human αvβ5 integrin. αIIbβ3 and fibrinogen were purchased from Enzyme Research Laboratories, Inc. (South Bend, Ind.). Vitronectin was purified from outdated human plasma according to the procedure of (Yatohgo et al. (1988) Cell Struct. Func. 13:281-92), using heparin affinity chromatography.
    • Example 2 Generating Nucleic Acid Ligands to Integrins Using the SELEX Method
  • A DNA template library of sequence:
    5′-ttatacgactcactatagggagacaagaataaac (SEQ ID NO:1)
    gctcaannnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
    nnnnnnnnnttcgacaggaggctcacaacaggc-3′

    was prepared by chemical synthesis. The italicized nucleotides correspond to a T7 RNA polymerase promoter. There are 40 n residues (a,g,t, or c). A short DNA primer “3N8”:5′-gcctgttgtgagcctcctgtcgaa-3′ (SEQ ID NO:2) was annealed to the template and extended using Klenow DNA polymerase (New England Biolabs, Beverly, Mass.). The double-stranded DNA product served as a product for T7 RNA polymerase transcription (enzyme obtained from Enzyco, Inc., Denver, Colo.) to generate a library of random-sequence RNAs. 2′-fluoro-CTP and -UTP were used in place of the 2′-OH-pyrimidines.
  • For application of the SELEX process to αvβ3 integrin, the purified protein was diluted 1000-fold from detergent-containing storage buffer into 50 mM MES (2-[N-morpholino]ethanesulfonic acid), pH 6.1, 150 mM NaCl, 2 mM CaCl2, to a final concentration of approximately 0.2 μg/mL. 3.2 μ polystyrene particles (IDEXX Laboratories, Inc., Westbrook, Me.) were added to the diluted protein and the mixture was rotated overnight at 4° C. The beads were collected by centrifugation and blocked by incubation in 3% BSA in MES buffer (above) for one hour at room temperature. Blocked beads were washed several times by resuspension in binding buffer (50 mM Tris.HCl, pH 7.4 (at 37° C.), 145 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 0.1 mM MnCl2, 0.01% BSA). For one round of selection, integrin-coated beads were mixed with RNA and rotated at 37° C. for 4 hours to allow equilibration of the RNA with the immobilized protein. The beads were then collected by centrifugation and washed at least 5 times in binding buffer by rapid resuspension and pelleting, without additional incubation. RNAs that remained bound to the beads were eluted overnight at 37° C. in binding buffer plus 100 μM cyclic RGD peptide (“cRGD”) (GPenGRGDSPCA, Life Technologies, Gibco BRL, Gaithersburg, Md). Eluted RNAs were extracted with phenol, then chloroform:isoamyl alcohol (24:1), and ethanol precipitated. The RNA pellet was resuspended and annealed to primer 3N8 for reverse transcription using avian myeloblastosis virus reverse transcriptase (Life Sciences, Inc., St. Petersburg, Fla.). The cDNA pool was amplified by the polymerase chain reaction using the 3N8 primer and primer “5N8“:5′-taatacgactcactatagggagacaagaataaacgctcaa-3′ (SEQ ID NO:3) and T. aquaticus DNA polymerase (Perkin Elmer-Cetus, Foster City, Calif.). Transcription of the PCR product generated an RNA pool to initiate a new round of selection. For the first round of selection 1 nmol of RNA (approximately 6×1014 sequences) was incubated at 2 μM concentration with a volume of bead suspension equivalent to 50 pmol of protein (assuming all the integrin had adsorbed to the beads). In subsequent rounds, the concentration of RNA and protein-coated beads were both reduced to demand higher affinity binding interactions.
  • The affinity of individual RNAs and RNA pools for αvβ3 was determined by titration of biotinylated RNA with a small quantity of immobilized integrin. Bound RNA was detected through the biotin moiety. Biotinylated RNA was prepared according to standard transcription protocols, but including a 5-fold molar excess of a 5′-biotin-modified GMP over GTP in the reaction mixture. Methods for synthesizing 5′-biotin-modified guanosine nucleotides are described in WO 98/30720 entitled “Bioconjugation of Oligonucleotides,” specifically incorporated herein by reference in its entirety. The modified nucleotide is incorporated at the 5′ end of the transcript in proportion to its representation in the guanosine pool. 96-well microtiter plates (Immulon 2, Dynatech Laboratories, Inc., Chantilly, Va.) were coated overnight at 4° C. with 100 μL purified αvβ3 at a concentration of 0.25 μg/mL in 20 mM TrisHCl, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 2 mM CaCl2, 0.1 mM MnCl2. Coating concentrations were 0.8 μg/mL for αvβ3 and 0.3 μg/mL for αvβ5. Wells were blocked with 200 μL of a solution of 3% BSA in the same buffer (1 hour at room temperature) then rinsed 3 times with 200 μL binding buffer (50 mM TrisHCl, pH 7.5, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 0.1 mM MnCl2, 0.1% BSA). Individual RNAs or RNA pools were denatured briefly at 93° C. in binding buffer without divalent cations or BSA, then serially diluted in the same buffer. 50 μL binding buffer containing 2X-concentrations of divalent cations and BSA were added to each well, followed by 50 μL RNA dilution. RNAs were allowed to incubate in the integrin-coated wells at 37° C. for 30-60 minutes. Unbound RNAs were removed by 3 rapid washes in binding buffer. To detect bound RNA, 100 μL of a 1:2500 dilution in binding buffer of streptavidin-alkaline phosphatase conjugate (Calbiochem) were incubated in each well for 30 minutes at room temperature, followed by three rapid washes, as above. 100 μL/well p-nitrophenyl phosphate (Sigma Chemical Co., St. Louis, Mo.) was added and incubated at room temperature for 30 minutes. Color development was monitored by absorbance at 405 nm. Binding data were fit to an equation that describes the fraction of RNA or protein bound as a function of KD, and the total concentrations of RNA and protein in the binding reaction for both monophasic and biphasic binding behavior (Green et al. (1996) Biochem. 35:14413-24). A control RNA corresponding to a sequence-scrambled version of aptamer 7.24:
    5′-gggagacaagaauaaucgcucaacguugaaugcu (SEQ ID NO:4)
    gcauuauggaguaauugaccgcuacaucccuuucgac
    aggaggcucacaacaggc-3′

    was used to monitor non-specific binding of RNA under the conditions of the assay.
  • After seven rounds of the SELEX process, the amount of RNA specifically bound to the integrin-coated beads had increased substantially (data not shown). Although immobilized αvβ3 showed no detectable affinity for random sequence RNA, the Round 7 RNA pool bound with an equilibrium dissociation constant (KD) of approximately 4×10−7 M (FIG. 1). The Round 7 affinity-enriched pool was cloned and sequences were determined for individual molecules in the mixture. Of 92 sequences obtained, 35 (38%) were very highly related to one another, in many cases differing at no more than a single base position. These sequences are collectively referred to as “Family 1.” It is likely that many if not most of these RNAs derived from a single precursor as a result of errors in replication during the RT and PCR steps. Another 25 sequences (27%) shared a short motif (CCUGCC) that defined a second sequence family (“Family 2”). The remaining 32 sequences (35%) were not obviously related to sequences in Families 1 or 2 and were thus termed “orphan” sequences. The large percentage of orphan sequences in the round 7 pool suggested that a great deal of sequence complexity remained in the population. Therefore, the SELEX process was continued in the hope of further enriching for high affinity sequences whose representation in the round 7 pool may have been low. Indeed, a substantial improvement in the affinity of the RNA pool was observed after 8 additional rounds of affinity selection (Round 15, FIG. 1). No further improvement was seen after two more rounds of selection (Round 17, FIG. 1), so clones were isolated from the Round 15 and Round 17 RNA pools and the sequences of individual isolates were compared to those obtained at Round 7. Twenty-seven of 39 sequences derived from the Round 15 pool (69%) were members of the highly conserved sequence family, Family 1. Three sequences (8%) could be grouped with Family 2 and 9 sequences (23%) were orphans. All of the 18 sequences isolated from the Round 17 pool were members of sequence Family 1. Thus, in this case, additional rounds of the SELEX process served to focus the RNA population on a single high-affinity sequence family that was already predominate at Round 7.
  • Table 1 shows the sequences of the major family of 2′-F-pyrimidine RNAs with high affinity for αvβ3 (Family 1). Clone names indicate the selected RNA pool from which each sequence was derived (round 7, round 15 or round 17) followed by a unique clone number. Note that in several cases identical sequences were isolated from different RNA pools; in these cases, both clone names are given. (Clones 17.12A and B were isolated as end-to-end inserts in a single plasmid.) Numbers in parentheses indicate the frequency with which a particular sequence was isolated; if no number is given the clone was obtained only once from the selected RNA pool. Sequences of the 5′ and 3′ fixed sequence regions common to all of the clones are shown at the top in lower case letters. Gaps have been inserted into many of the sequences to highlight the strong sequence conservation among most of the clones. The length of the random sequence region is shown for each RNA, as well as an estimate of the KD for binding to immobilized αvβ3, where it was determined (ND=not determined). The KD value provided is generally based on one or the average of two determinations. Family 2 sequences isolated from the αvβ3 SELEX are shown in Table 2. The short motif (CCUGCC) held in common among all the sequences is indicated in boldface letters. In Table 3, sequences with no obvious relationship to Families 1 or 2 are shown. Groups of similar sequences with only two (7.41 and 7.93) or three (7.11, 7.82 and 7.101) members are also included in Table 3.
  • The substantial affinity improvement between rounds 7 and 15 must be due in part to the loss of lower affinity species from the population; however, the introduction of and selection for higher affinity sequence variants of Family 1 may also have contributed to the overall affinity enrichment of the pool. While the affinity of relatively few sequences from the Round 7 pool were measured, their affinities for immobilized αvβ3 were generally less than that of RNAs derived from Rounds 15 and 17 (Tables 1-3).
    • Example 3 Specificity of the Nucleic Acid Ligands to Integrins
  • In general, aptamers selected for high-affinity binding to a particular target protein show relatively weak binding to other related proteins, except in cases where the degree of homology is very high (for example, see (Green et al. (1996) Biochem. 35:14413-24; Ruckman et al. (1998) J. Biol. Chem. 273:20556-67)). Significant homology exists within the families of integrin alpha and beta sub-units, and both alpha and beta sub-units are shared among members of the integrin superfamily. Thus, it was of interest to assess the relative affinity of the αvβ3 aptamers for closely related integrins. The affinities were determined using the methods described above. The Family 1 aptamer 17.16 (SEQ ID NO:60) was chosen as a representative of the major sequence family. FIG. 2 shows that aptamer 17.16 bound with identical affinity to purified, utilized αvβ3 and to the platelet integrin, αIIbβ3 in a 96-well plate binding assay. Although these two proteins share the β3 sub-unit in common, an alignment of the αv and αIIb amino acid sequences shows only 36% overall sequence identity (Fitzgerald et al. (1987) Biochem. 26:8158-65). Short stretches of exact sequence identity, 5 to 9 amino acids in length, do occur, primarily within four putative calcium-binding domains of each a sub-unit. Binding of aptamer 17.16 to integrin αvβ5 was also tested. The β5 sub-unit shares 56% sequence identity with β3 and is more closely related to β3 than other members of the beta sub-unit family (McLean et al. (1990) J. Biol. Chem. 265:17126-31; Suzuki et al. (1990) Proc. Nat. Acad. Sci. 87:5354-8). No aptamer binding to immobilized integrin αvβ5 was observed (FIG. 3), although an αv-specific antibody detected the presence of αvβ5 protein adsorbed to the surface of the well (data not shown). Together, these data strongly suggest that aptamer 17.16, and by extension the other members of sequence Family 1, bind primarily to the 3 sub-unit of αvβ3 Furthermore, the high-affinity binding of the aptamer to the platelet integrin, αvβ3 extends its range of potential application to indications involving detection of platelets or inhibition of their function.
    • Example 4 Aptamer Inhibition of Ligand Binding to Purified Integrins
  • While the SELEX process identifies RNA sequences with high affinity for a particular target, the procedure used in this example was designed to bias for the recovery of ligand binding site inhibitors by the inclusion of a cRGD peptide competitor in the elution buffer. To test whether aptamer 17.16 could block the ligand binding site of αvβ3 or αIIbβ3, purified vitronectin and fibrinogen were biotinylated and incubated with one or both of the immobilized integrins in the presence or absence of varying concentrations of the aptamer or a non-binding control RNA. This was done as follows: purified integrin ligands, vitronectin and fibrinogen, were biotinylated according to (Smith et al. (1990) J. Biol. Chem. 265:12267-71). Briefly, proteins were dialyzed into 0.1 M NaHCO3, 0.1 M NaCl. N-hydroxysuccinimido-LC-biotin (Pierce) was dissolved at 1 mg/mL in DMSO and added to the protein at a ratio of 0.1 mg biotin per 1 mg protein. The reaction was allowed to rotate at room temperature for 2 hours. Biotinylated proteins were dialyzed into phosphate-buffered saline and their concentrations determined by absorbance at 280 nm. 96-well microtiter plates were coated as described above with either αvβ3 or αIIbβ3 and blocked with BSA. A fixed concentration of biotinylated ligand (fibrinogen: 6 nM final; vitronectin: 10 nM final) was pre-mixed in binding buffer (see “Measurement of Aptamer Binding Affinities,” above) with varying concentrations of aptamer, control RNA, cyclic RGD peptide, antibody, or unmodified ligand. The mixtures were incubated in the integrin-coated wells for 60 minutes at room temperature. After washing, bound biotinylated ligand was detected by addition of 100 μL/well 1:500 dilution streptavidin-alkaline phosphatase conjugate (Calbiochem) (30 minutes at room temperature) followed by 100 μL/well p-nitrophenyl phosphate, as described above. Absorbance was read at 405 nm. The data were fit to an equation that describes mutually exclusive binding of two ligands to a single target species (Gill et al. (1991) J. Mol. Biol. 220:307-24). The concentration of competitor that inhibited 50% of the maximum signal above background (IC50) was determined from the fitted curve.
  • Known ligand binding inhibitors, including an RGD peptide and the αvβ3-specific antibody LM609, were included as positive controls for the assay. FIG. 4A shows inhibition of biotinylated vitronectin binding to immobilized αvβ3. Aptamer 17.16 inhibited the binding interaction with an IC50 of 4.7 nM while the control RNA showed no inhibition. By comparison, the IC50 of RGD peptide inhibition was 1.4 nM and that of LM609 was 2.7 nM. Unmodified vitronectin inhibited the binding of the biotinylated material with an IC50 of 59 nM. Similar data were obtained for aptamer inhibition of fibrinogen binding to αvβ3 (FIG. 4B) and for fibrinogen binding to αIIbβ3 (FIG. 4C). IC50 values for the data in FIG. 4B were: 17.16, 9.5 nM; control RNA, not measurable; RGD peptide, 1.0 nM; LM609, 6.3 nM; unmodified fibrinogen, 43 nM. IC50 values for FIG. 4C were: 17.16, 6.5 nM; control RNA, not measurable; RGD peptide, 21 nM; unmodified fibrinogen, 15 nM. Thus, aptamer 17.16 is an effective competitor of β3 integrin ligand binding and, on a molar basis, has an inhibitory potency nearly equivalent to that of a bivalent antibody.
    • Example 5 Nucleic Acid Ligand Binding to Human Platelets
  • Aptamer 17.16 (SEQ ID NO:60) was selected for binding to purified human αvβ3 adsorbed to the surface of a polystyrene bead. In vitro assays to measure the affinity of the aptamer for purified β3 integrins were also done in the context of hydrophobically-adsorbed protein. Thus, an important test of aptamer function was to determine its capacity to bind to native protein on the surface of cells. Human platelets were chosen for this purpose because of their ease of isolation and their high level of expression of integrin αIIbβ3. Because αIIbβ3 undergoes a conformational change upon platelet activation, binding of the aptamer to both resting and thrombin-activated platelets was tested. This was done as follow: fluorescein-conjugated RNA was prepared according to (Davis et al. (1998) Nuc. Acids Res. 26:3915-24). Briefly, RNA was transcribed in the presence of a 5-fold molar excess of the initiator nucleotide guanosine-5′-O-(2-thiodiphosphate) (Calciochem), followed by conjugation of the gel-purified RNA to 5-iodoacetamidofluorescein (Pierce, Rockford, Ill.). Platelet-rich plasma was prepared from freshly-drawn citrated human blood by centrifugation at 1000 rpm for 15 minutes in a table top centrifuge. For activated platelets, cells were incubated for 15 minutes at room temperature at 2×107/mL in calcium- and magnesium-free Dulbecco's PBS with 2.5 U/mL thrombin and 5 mM Gly-Pro-Arg-Pro (GPRP) to inhibit platelet aggregation. Cells were diluted 1:10 into binding buffer (20 mM HEPES, pH 7.5, 111 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 0.1% BSA, 0.01% sodium azide). Resting cells were diluted similarly, without exposure to thrombin or GPRP. The activation state of resting and thrombin-treated cells was monitored by staining with fluorophore-conjugated antibodies to CD61 (β3 integrin subunit), which binds to all platelets, and to CD62 (P-selectin), a marker of platelet activation. Antibodies were obtained from Becton-Dickinson Immunocytometry Systems, San Jose, Calif. Fluorescein-conjugated RNAs were diluted in water to 4 μM and denatured briefly at 93° C., then diluted to 2 μM with 2×-concentrated binding buffer. RNAs were then serially diluted in binding buffer. Each dilution was mixed 1:1 with resting or activated platelets and allowed to incubate in the dark at room temperature for 30 minutes. The incubation mixtures were applied directly to a Becton Dickinson FACSCalibur flow cytometer to determine the mean fluorescence intensity of the sample. Under such equilibrium binding conditions, an estimate of the KD for aptamer binding to the cell surface integrin could be obtained.
  • Non-specific RNA binding to platelets was measured using a control RNA of similar length and base composition to aptamer 17.16. Non-specific binding became significant at concentrations above approximately 100 nM. Specific binding of the aptamer was distinguished from non-specific binding by the addition of 5 mM EDTA to the sample: EDTA had no effect on the binding of the control RNA but reduced aptamer binding to the level of the control. Specific binding of the aptamer was thus defined as the difference between the fluorescence intensity of the sample before the addition of EDTA (specific+non-specific) and the fluorescence intensity after the addition of EDTA (non-specific only).
  • FIG. 5 shows representative data for the EDTA-sensitive component of aptamer binding to both resting and thrombin-activated human platelets. The maximum binding signal is approximately 2-fold higher to activated platelets, consistent with the slightly higher level of αIIbβ3 on such cells (Wagner et al. (1996) Blood 88:907-14). However, the estimated KD for aptamer binding to platelets was approximately 10 nM for both cell populations, equivalent to the value determined for binding in vitro to purified αIIbβ3. Furthermore, aptamer 17.16 binds to both resting and activated platelets with an affinity equivalent to that reported for Reopro (abciximab, chimeric 7E3 Fab), an approved αIIbβ3 antagonist (Mousa et al. (1998) J. Pharm. Exp. Ther. 286:1277-84).
    • Example 6 Nucleic Acid Ligand Biodistribution in Rabbit Venous Clot Model
  • To explore the application of a β3-specific aptamer in clot imaging, aptamer 17.16 was labeled at the 5′ end with technitium-99m (99mTc) and its biodistribution was monitored in a rabbit model of venous thrombosis. In this model, a clot is generated in situ in the isolated jugular vein of a rabbit from human platelet-rich plasma. Blood flow across the clot is re-established and the radiolabeled aptamer (or a non-binding control RNA) are introduced into the bloodstream via the ipsilateral ear vein. The distribution of the radiolabel into various tissues is reported as the per cent of the injected dose per gram of tissue.
  • The experiment was performed as follows: Aptamer 17.16 and a control RNA of similar length and base composition were transcribed using a 5-fold molar excess of 5′-(O-hexylamino) guanosine monophosphate. Each RNA was conjugated to Hi15 at 50 mg/mL aptamer in 30% dimethylformamide with 5 molar equivalents of Hi15-NHS buffered in 100 mM NaBorate pH 9.3, for 30 minutes at room temperature. The conjugation reactions were washed over a 30,000 molecular weight cut-off filter (Microcon 30, Amicon, Inc., Beverly, Mass.) to remove excess Hi15 cage. The RNAs were then labeled with 99mTc in the following manner: to 1 nmol Hi15-aptamer was added 200 μl of 100 mM NaPO4 buffer, pH 8.5, 23 mg/mL NaTartrate, and 50 μL [99mTc] pertechnetate (5.0 mCi) eluted from a 99Mo column (Syncor, Denver) within 12 hours prior to use. The labeling reaction was initiated by the addition of 10 μL 5 mg/mL SnCl2. The reaction mixture was incubated for 15 minutes at 90° C. Unreacted 99mTc was removed by spin dialysis through a 30,000 molecular weight cut-off membrane (Centrex, Schleicher & Schuell) with two 300 μL washes. This labeling protocol results in 30-50% of the added 99mTc being incorporated with a specific activity of 2-3 mCi/nmol RNA.
  • For biodistribution studies, rabbits were anesthetized with isofluorane. A two centimeter section of the right jugular vein was isolated in situ and all the branches were ligated. A catheter was inserted into the facial vein. The isolated vein segment was temporarily ligated above and below the catheter. The vein segment was flushed with saline. 1000 USP units of heparin was administered intravenously. 300-400 μL of fresh human platelet-rich plasma (citrate) activated with calcium and thrombin was instilled into the isolated vein segment and allowed to clot. After 30 minutes the ligatures were removed and blood flow over the thrombus was re-established (confirmed by the injection of 200 μl of air into the ipsilateral ear vein). [99mTc]-conjugated aptamer or control RNA was injected into the ipsilateral ear vein. At 1 hour the rabbit was exsanguinated and tissues were weighed and counted in a Wallac 1470 gamma counter. The aptamer and control RNA were tested at 1 nmol/kg (approximately 0.03 mg/kg).
  • For aptamer 17.16, radiolabel accumulated in the clot to a significant degree by one hour after injection, while similar accumulation was not observed with the control RNA (FIG. 6). Blood clearance of the radiolabel was apparently rapid and mediated primarily by a renal mechanism as judged by moderate accumulation of radioactivity in the kidney for both the aptamer and control RNA. Thus, aptamers specific for αIIbβ3 or for other proteins expressed at high levels on the surface of platelets or within the matrix of a clot will serve as useful agents for rapid imaging of thrombi.
    TABLE 1
    αvβ3 Family 1 aptamer sequences.
    Sequence of variable region SEQ
    Clone name 5′-gggagacaagaauaaacgcucaa [variable region] Sequence KD ID
    (# of isolates) uucgacaggaggcucacaacaggc-3′ length (nM) NO
    7.3 (2) uucuacgu uguuuaagggcuuauaugagcgcauuauaccc 40  22 5
    7.6; 17.12A uucaacgc uguuuaagggcuuauaugagcgcguuauaccc 40 ND 6
    7.12 uucaacgc uguuuaagggcuuauaugagcgcguuacaccc 40 ND 7
    7.24 (5) uucaacgc uguucaagggcuuauaugagcgcguuauaccc 40 170 8
    7.25 uucaacga uguuuaagggcuuauaugagcgcguuauaccc 40 ND 9
    7.34 uucau gaa guccaagggcuuauaugagcgcguuauaccc 39 ND 10
    7.36 (3) uucaacgc ugucaaagggcuuauaugagcgccguuauaccc 40 ND 11
    7.37 (2) uu aacgu uguucaaggguuuauaugagugcguuuauaccc 39 ND 12
    7.38 (2) uucaacgc uguccaagggcuuauaugagcgcguuauaccc 40  49 13
    7.49 uucaacggauguccaagggcuu uaugagcgcguuauaccc 40 ND 14
    7.53 uucgacgcuguucaagggcuuauaugagcgcauuauaucc 40 ND 15
    7.54 (2) uucgacgc uguucaagggcuuauaugagcgcguuauaccc 40 230 16
    7.57 (2) uucgacga uguccaagggcuuauaugagcgcauuauaccc 40 ND 17
    7.63 uucaacgc uguucaagggcuuauaugagcgcguuacaccc 40 ND 18
    7.64 uuc auga uguucaagggcuuauaugagcgcauuauaccc 39 ND 19
    7.77 (2) uucaacga uguugaggggcuuauaugagcgcauuauaccc 40 770 20
    7.80 uucaacga uguccaagggcuuauaugagcgcauuauaccc 40 ND 21
    7.86 uucaacgc uguucaagggcuuaugugagcgcguuauaccc 40 ND 22
    7.91 uucaacgu uguccaagggcuuauaugagcgcauuauaccc 40 ND 23
    7.115 uucaacgcuguucaagggcuuauaugagcgcauuauaccc 40 ND 24
    7.121 uucaacga uguccaagggcuuauaugagcggauua ccc 38 ND 25
    7.124 uucaacac ugu gaagggcuuauaugagcgcgucauaccc 39 ND 26
    7.127 uucaacguuguucaagggcuuauaugagcgcgcguauaccc 40 ND 27
    15.2 uucaacgu ugucaaagggcuuauaugagcggauua ccc 38  6 28
    15.3 (3); 17.17 uucaacgu uguccaagggcuuauaugagcggauua ccc 38  8 29
    15.7 uucuacga ugucaaagggcuuauaugagcggauua ccc 38  5 30
    15.8 uucgacgc uguugaagggcuuauacgagcggauua ccc 38  5 31
    15.10 uucaacgc uguucaagggcuuauaugagcggauua ccc 38  20 32
    15.14 uucaacau uguccaagggcuuauaugagcggauua ccc 38  6 33
    15.17 uucaacgu ugucaaagggcuuauacgggcggauua ccc 38  4 34
    15.18 (2) uucaacgc ugug aagggcuuauaugagcggauua ccc 37  2 35
    15.20 uucaacgc uguccaagggcuuauaugagcgcauuauaccc 40  20 36
    15.27 uucgacua uguccaagggcuuauaugagcggauua ccc 38 ND 37
    15.28 uucgacga ugucuaagggcuuauaugagcggauua ccc 38 ND 38
    15.40; 17.12B uucaacgc uguugaagggcuuauacgagcggauua ccc 38 ND 39
    15.41 uucaacgu uguccaagggcuuauacgagcggauua ccc 38 ND 40
    15.42; 17.14 (2) uucaacgc uguccaagggcuuauacgagcggauua ccc 38 ND 41
    15.46; 17.20 uucgacgc ugug aagggcuuauaugagcggauua ccc 37  40 42
    15.47 uucaacgu ugucaaagggcuuauacgagcggauua ccc 38 ND 43
    15.48 uucaacgc uguugaagggcuuauaugagcggauua ccc 38 ND 44
    15.49 uucuacgu ugucuaagggcuuauaugagcggauua ccc 38 ND 45
    15.50; 17.3 uucgacgc ugug aagggcuuauacgagcggauua ccc 37  30 46
    15.52 uucaacgc uguucaagggcuuauacgagcggauua ccc 38 ND 47
    15.53 uucaacgc uguccuagggcuuauaugagcgcaggauaccc 40  70 48
    15.55 uucuacgc uguuuaagggcuuauaugagcgaauua ccc 38 ND 49
    15.57 uucuacgu uguccaagggcuuauaugagcggauua ccc 38 ND 50
    15.58 uucgacgu uguugaagggcuuauaugagcggauua ccc 38 ND 51
    17.1 uucaacgc ugucaaagggcuuauauaagcggauua ccc 38 380 52
    17.2 (2) uucuacgc ugug aagggcuuauaugagcggauua ccc 37  2 53
    17.5 uucgacgc ugug aagggcuuauaugagcggau acaccc 38  5 54
    17.7 (2) uucuacgc ugug aagggcuuauacgagcggauua ccc 37  6 55
    17.8 uucaacgu ugucuaagggcuuauaugagcggauua ccc 38  18 56
    17.10 uucuacgu uguugaagggcuuauaugagcggauua ccc 38 ND 57
    17.11 uucuacgc ugug aagggcuuauaugagcgaauua ccc 37  4 58
    17.13 uucaacgc uguccaagggcuuauaugggcggauua ccc 38  10 59
    17.16 uucaacgc ugug aagggcuuauacgagcggauua ccc 37  8 60
  • TABLE 2
    αvβ3 Family 2 aptamer sequences
    Clone
    name Sequence of variable region SEQ
    (# of 5′-gggagacaagaauaaacgcucaa [variable region] Sequence KD ID
    isolates uucgacaggaggcucacaacaggc-3′ length (nM) NO:
    7.4 GUACCGGAUCGCCCUGCCACGGUAUUUGAGACAUUGAAA 39 ND 61
    7.5 (3) GGUAGUAAAUGGACUCCUGCCAUCCAAUACUAUCUCUGAG 40 >1000 62
    7.13 UGUAGUCGCAUGUCGAGCAGCAAUUCCUGCCAUUGUAGG 39 >1000 63
    7.14 (2) UGAAGAACUAGACCUGCCCAAGUCCUUCAUCGUGCUUGCU 40 ND 64
    7.27 (2) CGAUUAUACUAUCCCUGCCAGUAGUAAUCAGUGCUAUA 38 ND 65
    7.29 CGGUGAAGACCUCUAUUAACAACAUGACCUGCCUGCGUUG 40 ND 66
    7.32 CGCAAAUAUGUUCCUGCCAAAUACGGGCGUUGACGCUAGA 40 ND 67
    7.43 GGACCCUGCCGAGCACAUUUAUUCUGGUAACUGAGCCCCC 40 ND 68
    7.51 CGCUGAGAGAAAGCCCUGCCCUUUCAGCUCGAGAGUUAUA 40 ND 69
    7.58 UGAGAUGCAGUUCCUGCCUGCUGCAUUUCUUAGAGUGUGU 40 ND 70
    7.83 GAUUAACGGUUAUCCUGCCAACCGAUUAUAAGAGCAUGGA 40 ND 71
    7.89 UGAGAGACUACAAUAGAACUUAUGUAACCUGCCACAUAGG 40 ND 72
    7.97 UAGGAAGUGUAACCUGCCUCACGGUCCUAUCGAGUAGUUU 40 ND 73
    7.100 UGAAAACGCAACCUGCCGGCGUCGUCCUGGGUAAUUUA 40 ND 74
    7.104 AUAGGGGGUACCUGCCGACCCCAGAAAUAAGCGUGAUU 39 ND 75
    7.105 UCCUGCCAUAGCGUCUUCAUGUCUGACGUUUGAGUUUCCG 40 ND 76
    7.107 UCCUAGGUUGGUCCUGCCACAGCUCAAAGGUUUAGCUUCA 40 ND 77
    7.109 ACAUGCAGACAACCCUGCCUUCUGCGUGGUUUAGGAGUA 39 ND 78
    7.120 AACCUCAGGCGACCUGCCGCUGUCUGAAGUUCGAGCAUAA 40 ND 79
    7.122 ACUCAAGACCCUGCCACUAUGUGUUACUGAGUAGGAGCGU 40 ND 80
    7.125 AUUCGAAAUACGGGUUAAAUCCCUGCCUUUAACACGACA 39 ND 81
    15.19 UGUAGCCGCAUGUCGAGCAGCAAUUCCUGCCAUUGUAGG 39  770 82
    15.21 CGGUGAAGACCUCUAUUAACAACAUGACCUGCCUGCGUUG 40  200 83
    15.34 UCCCACCCUGCCUUGUCUGUUUGAUAGAGACACUGUCCUU 40  190 84
  • TABLE 3
    αvβ3 orphan aptamer sequences
    Sequence of variable region SEQ
    Clone name
    5′-gggagacaagaauaaacgcucaa [variable region] Sequence KD ID
    (# of isolates) uucgacaggaggcucacaacaggc-3′ length (nM) NO
    7.1 gguuugaaagauugccuguagcuccaaaucuuggugagcu 40 ND 85
    7.2 ucccgccgauagcuuccacgaagaguuaucuguaaaacaa 36 ND 86
    7.11 ugagcuccugauuccaaaccuauuccguuucuggu 40 ND 87
    7.30 acuggacaagucaaucucuccggcuugagacuugguuuac 40 ND 88
    7.33 (2) cgagcucuugcuuccaaaccuauuccagacguuu cuggg 40 ND 89
    7.41 (2) gcgagccuauugucuaagaugcaccaggccuguuaagcau 40 >1000 90
    7.42 gccuguacggcgauuaugucuuuaccuuaacuguucc 37 ND 91
    7.46 uaccaauggcacgaauaacugacuaccccccaaaauggaa 40 ND 92
    7.47 gcggggcuuugcucaaguguuugcaaacgguaaauuccac 40 ND 93
    7.61 ccuaccgacguccgccgcuggguuaaccuguaaagucacu 40 ND 94
    7.66 (2) gugaaccgauaagcgaaaguaguaccccugcuugacuacu 40 >1000 95
    7.67 ggagcuccuaguuccaaaccuauuccagaaguuuucugggu 41 ND 96
    7.75 uaguacgcagucauagcggggcagggacuuucuccgugca 40 ND 97
    7.76 uuauacugguaugccgccgaccagaauuaauccaaugcgu 40 ND 98
    7.82 ugagcuccugguuccaaaccuauuccagacguuucagggu 40 ND 99
    7.85 ucuggccugugacuguagucguuucuucgaguugugacgc 40 ND 100
    7.92 cucaacgauguccaagggcuuauaugagcgcguuacccc 39 ND 101
    7.93 gcgagccuauugucuaaqaugcgccaagccuguaaagcau 40 ND 102
    7.94 gacuagccggccugagauccuuguucgccacacaugcugg 40 ND 103
    7.96 cuucccccgcaaacacauguuuaguacugggagacuuggg 40 ND 104
    7.101 ugagcuccugauuccgaaccuauuccagacguuucugggu 40 ND 105
    7.102 cugauccucuugucauuguacaucucgcag 30 ND 106
    7.106 uacuaagccuaacaaaagagcggauauuggcgcggcacg 39 ND 107
    7.108 agucuuaguaguaccgccugcuucuaaccuugggcgcuuu 40 ND 108
    7.112 ugauuucaugacuuaugccgccggcaugacuucaaugacg 40 ND 109
    7.114 ucaaaggacggaagugccugugcccgacuaaagaguugag 40 ND 110
    7.118 cuaucgaucguuuuuucauuucccccugaccaucgccug 39 ND 111
    7.123 uugucccgcgcagaaacgugacaaaauuuaacacgcaccgu 40 ND 112
    7.128 uucaacguuguucaagggcuuauaugagcgcguuauaccc 40 ND 113
    15.4(4) ugauuucaugacuuaugccgccggcaugacuucaaugacg 40  2000 114
    15.5 gcauucaaaauuugcgagaacgaauagaaguccgagagcc 40  4000 115
    15.13 (2) gcgggauuuuccugaucaucccacugauucggggccuuac 40  790 116
    15.39 ucaaucucggacuagacuaacgaccuugguugacgcuca 39  410 117
    15.43 cgccguuaucacgacgugcguucugggcgguacucgcgca 40   45 118

Claims (5)

1. A method for the treatment of a disease resulting from platelet activation, the method comprising administering a biologically-effective amount of a nucleic acid ligand to a β3 integrin.
2. A method for treating deep vein thrombosis comprising administering a biologically-effective amount of a nucleic acid ligand to a β3 integrin.
3. A pharmaceutical composition for the treatment of deep vein thrombosis comprising a nucleic acid ligand to a β3 integrin and a pharmaceutically acceptable excipient.
4. A method for the treatment of a disease in which αvβ3 activation is a contributing pathogenic factor, the method comprising administering a biologically-effective dose of a nucleic acid ligand to αvβ3 and a pharmaceutically acceptable excipient.
5. The method of claim 6 wherein said disease is selected from the group consisting of cancer, diabetic retinopathy, retinopathy of prematurity, macular degeneration, endometriosis, psoriasis, rheumatoid arthritis, stroke, osteoporosis, and restenosis.
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