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  • 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
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  • G01N2333/475—Assays involving growth factors
  • G01N2333/4753—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II

Definitions

• transgenic mice which overexpress HGF become tumor-laden at many loci
• HGF/c-met in human cancer although the accumulated weight of the correlative data are convincing.
• a causal connection was established between germ-line c-met mutations, which constitutively activate its tyrosine kinase domain, and the occurrence of human papillary renal carcinoma (Schmidt, Duh et al. (1997) Nat Genet. 16:68-73 .
• Recent work on the relationship between inhibition of angiogenesis and the suppression or reversion of tumor progression shows great promise in the treatment of cancer (Boehm, Folkman et al (1997) Nature. 390:404-7).
• Angiogenesis is markedly stimulated by HGF, as well as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) (Rosen, Lamszus et al (1997) Ciba Found Symp.
• VEGF vascular endothelial growth factor
• bFGF basic fibroblast growth factor
• HGF and VEGF were recently reported to have an additive or synergistic effect on mitogenesis of human umbilical vein endothelial cells (HUVECs) (Van Belle,
• the amino-terminal 31 residue signal sequence of HGF is cleaved upon export, followed by proteolytic cleavage by uPA and/or other proteases.
• the mature protein is a heterodimer consisting of a 463 residue ⁇ -subunit and a 234 residue ⁇ -subunit, linked via a single disulfide bond.
• Human c-met protein is exported to the cell surface via a 23 amino acid signal sequence (reviewed in Comoglio (1993) Exs. 65:131-65; Rubin (1993) Biochim Biophys Acta. 1155:357-71: Jiang (1999) Crit Rev Oncol Hematol. 29:209-48).
• the exported form of c-met is initially a pro-peptide which is proteolytically cleaved.
• the mature protein is a heterodimer consisting of an extracellular 50 kDa ⁇ -subunit bound by disulfide bonds to a 140 kDa ⁇ -subunit.
• 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 subunits and 8 homologous ⁇ 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, partly 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 ⁇ ,,,, ( ⁇ ⁇ b ⁇ 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).
• ⁇ 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.
• 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).
• blockade of ⁇ v ⁇ 3 with a heterodimer-specific antibody prevents new vessel formation without affecting the pre-existing vasculature (Brooks et al. (1994) Science 264:569-71).
• ⁇ v ⁇ 3 expression on endothelial cells 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.
• Circulation 96:1809-18 ⁇ 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. (1994) J. Vase. Surg. 19:125-34; Srivatsa et ⁇ /. (1997) Cardiovasc. Res. 36:408-28; Slepian et ⁇ /. (1998) Circulation 97:1818-27; Coleman et al. (1999) Circ. Res. 84: 1268-76).
• ⁇ 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
• ⁇ ⁇ b ⁇ 3 (also referred to as GPIIbllla) 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.
• Inhibitors of ⁇ ub ⁇ 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 ⁇ ⁇ ⁇ inhibitors, Reopro, Integrilin and Aggrastat 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 ⁇ ⁇ b ⁇ 3 .
• Integrilin (COR Therapeutics) is a cyclic heptapeptide based on the integrin binding site of barbourin, an ⁇ ⁇ b ⁇ 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 that 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 ⁇ Ub ⁇ 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 Serial No. 07/536,428, filed June 11, 1990, entitled “Systematic Evolution of Ligands by Exponential Enrichment," now abandoned, U.S. Patent No. 5,475,096, entitled “Nucleic Acid Ligands," and U.S. Patent No.
• 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.
• 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.
• 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. Patent 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.
• nucleic acid ligands that act as inhibitors of HGF and c-met. It is a further object of the invention to provide therapeutic and diagnostic agents for tumorigenic conditions in which HGF and c-met are implicated.
• nucleic acid ligands to HGF and c- met to diagnose and treat hypertension, arteriosclerosis, myocardial infarction, and rheumatoid arthritis. It is an even further object of the invention to use nucleic acid ligands to HGF singly or in combination with other nucleic acid ligands that inhibit VEGF and/or bFGF, and/or possibly other angiogenesis factors.
• 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.
• nucleic acid ligands to HGF and c-met are provided.
• the methods use the SELEX process for ligand generation.
• the nucleic acid ligands to HGF and c-met provided by the invention are useful as therapeutic and diagnostic agents for a number of diseases.
• Figure 1 illustrates the template and primer oligonucleotides used in the 2'-F- pyrimidine RNA SELEX experiments.
• the 5' fixed region of the template and primers contains a T7 promoter to facilitate transcription of RNA by T7 RNA polymerase.
• Figure 5A shows that tRNA competes with RNA pools for binding to 2.7 nM HGF and Figure 5B shows conventional pool binding.
• Figure 6 illustrates inhibition of 10 ng/mL HGF stimulation of starved HUVECs by aptamers.
• Figure 6A shows a 1 st set of aptamers and
• Figure 6B illustrates a 2 nd set of aptamers.
• Figure 7 illustrates truncates of aptamer 8-102 (SEQ ID NO: 12).
• Figure 7A shows predicted two-dimensional structures of full-length and truncated sequences and
• Figure 7B shows binding of full-length and truncated aptamers to HGF.
• Figure 8 illustrates truncates of aptamer 8-17 (SEQ ID NO:68).
• Figure 8A shows a predicted two-dimensional structures of full-length and truncated sequences and
• Figure 8B shows binding of full-length and truncated aptamers to HGF.
• Figure 9 illustrates binding of HGF truncate SELEX pools.
• Figure 9A shows the
• HGF SELEX 4 30N7 series and Figure 9B shows the HGF SELEX 5 30N7 series.
• Figure 11 illustrates aptamer inhibition of 50 ng/mL HGF stimulation of 4MBr5 cells.
• Figure 11 A shows the effect of PEGylation of 36mer and
• Figure 1 IB shows a comparison of PEGylated 36mer to best full-length inhibitor 8-17.
• Figure 12 shows aptamer inhibition of 50 ng/mL HGF stimulation of 4MBr-5 cells.
• Figure 13 shows HUVEC mitogenesis by 10 ng/mL HGF, 10 ng/mL VEGF, or both
• Figure 14 illustrates aptamer-mediated inhibition of HUVEC mitogenesis.
• FIG. 14A shows stimulation by both HGF and VEGF inhibited by either HGF or VEGF aptamers or both.
• Figure 14B illustrates stimulation by HGF alone inhibited by either
• Figure 14C illustrates stimulation by VEGF alone inhibited by either HGF or VEGF aptamer or both.
• Figure 15 depicts ratios of selected to unselected partially 2'-OMe substituted purines in aptamer NX22354.
• FIG. 16 illustrates 2'-OMe substituted derivatives of NX22354 binding to HGF
• Figure 17 illustrates binding of SELEX pools to c-met.
• Figure 17A shows c-Met SELEX 40N7;
• Figure 17B shows c-Met SELEX 30N8; and
• Figure 17C shows both
• SELEXes a, c pools, 40N7; b, d pools, 30N8.
• Figure 18 illustrates binding of c-met SELEX pools to c-met and KDR Ig fusion proteins.
• Figure 19 shows binding of c-met 40N7 cloned aptamers to c-met and KDR Ig fusion proteins.
• Figure 19A shows clone 7c- 1 and Figure 19B shows clone7c-3.
• Figure 20 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.
• Figure 21 illustrates cross-reactivity of aptamer 17.16 (SEQ ID NO:249) to purified integrin ⁇ ⁇ b ⁇ 3 .
• Figure 22 illustrates cross-reactivity of aptamer 17.16 (SEQ ID NO:249) 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.
• Figure 23 illustrates ⁇ 3 aptamer inhibition of integrin ligand binding.
• Biotinylated fibrinogen or vitronectin were incubated in microtiter wells coated with either integrin ⁇ v ⁇ 3 or ⁇ ⁇ b ⁇ 3 in the presence or absence of varying concentrations of ligand binding competitors.
• Competitors included aptamer 17.16 (SEQ ID NO:249), 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.
• Figure 24 illustrates binding of aptamer 17.16 (SEQ ID NO:249) 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 /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.
• FIG. 25 illustrates biodistribution of [ 99m Tc] -aptamer 17.16 (SEQ ID NO:249) 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
• the method comprises: 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.
• 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 are 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.
• 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 that 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.
• the SELEX methodology is employed to obtain nucleic acid ligands to HGF, c-met and integrins or portions thereof. The SELEX methodology is described in the SELEX Patent Applications.
• 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.
• HGF refers to hepatocyte growth factor/scatter factor. This includes purified hepatocyte growth factor/scatter factor, fragments of hepatocyte growth factor/scatter factor, chemically synthesized fragments of hepatocyte growth factor/scatter factor, derivatives or mutated versions of hepatocyte growth factor/scatter factor, and fusion proteins comprising hepatocyte growth factor/scatter factor and another protein. "HGF” as used herein also includes hepatocyte growth factor/scatter factor isolated from species other than humans.
• 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.
• 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. Patent 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. Patent No. 5,637,459, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX,” and U.S. Patent 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 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 Serial
• 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.
• 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 become covalently attached to their targets upon irradiation of the nucleic acid ligand with light having a selected wavelength.
• 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 HGF or c-met, or fragments thereof as a target. Alternatively, full-length HGF or c-met, or discrete domains of HGF or c-met, 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 HGF or c-met. Determination of the precise number of amino acids needed for the optimal nucleic acid ligand is routine experimentation for skilled artisans. In preferred embodiments, the SELEX process is carried out using HGF or c-met attached to a solid support. A candidate mixture of single stranded RNA molecules is then contacted with the solid support.
• the single stranded RNA molecules have a 2'-fluoro modification on C and U residues, rather than a 2'-OH group.
• the solid support is washed to remove unbound candidate nucleic acid ligand.
• the nucleic acid ligands that bind to the HGF or c-met protein are 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.
• the solid support can be a nitrocellulose filter.
• Nucleic acids in the candidate mixture that do not interact with the immobilized HGF or c-met can be removed from this nitrocellulose filter by application of a vacuum.
• the HGF or c-met target is adsorbed on a dry nitrocellulose filter, and nucleic acids in the candidate mixture that do not bind to the HGF or c-met are removed by washing in buffer.
• the solid support is a microtiter plate comprised of, for example, polystyrene.
• the HGF or c-met protein is used as a target for
• nucleic acid ligands that inhibit the HGF/c-met interaction are used to inhibit tumorigenesis, by inhibiting, for example, angiogenesis and motogenesis.
• a nucleic acid ligand to HGF is used in combination with nucleic acid ligands to VEGF (vascular endothelial growth factor) and or bFGF (basic fibroblast growth factor) to inhibit tumor metastasis and angiogenesis.
• VEGF vascular endothelial growth factor
• bFGF basic fibroblast growth factor
• nucleic acid ligands that inhibit VEGF are described in U.S. Patent No. 5,849, 479, U.S. Patent No. 5,811,533 and U.S. Patent Application Serial No. 09/156,824, filed September 18, 1998, each of which is entitled "High Affinity Oligonucleotide Ligands to Vascular Endothelial Growth Factor," and each of which is specifically incorporated herein by reference in its entirety.
• Nucleic acid ligands to VEGF are also described in U.S. Patent No. 5,859,228, U.S. Patent No. 6,051,698, U.S. Patent Application Serial No.
• Nucleic acid ligands to integrins 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 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.
• 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 ligands.
• the nucleic acid ligands that bind to the integrin are then released into solution, reverse transcribed by reverse transcriptase and amplified using the Polymerase Chain Reaction (PCR).
• PCR Polymerase Chain Reaction
• the amplified candidate mixture is then used to begin the next round of the SELEX process.
• Example 5 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 accomplished 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 are analyzed using binding to human platelets as an assay. This can be done, for example, by fluorescently labeling the nucleic acid ligand by any of the numerous techniques known in the art. The fluorescent nucleic acid ligand is then contacted with platelets, and the amount of nucleic acid ligand is 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 labeled 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 radiolabelled 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.
• Radiolabelled nucleic acid ligand Blood flow through the vein is then re-established, and the radiolabelled nucleic acid ligand is introduced into the animals blood supply. The distribution of the radiolabelled 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.
• Anti-HGF monoclonal antibody MAB294 was purchased from R&D Systems, Inc. Human IgG j was produced in-house by stable expression from Chinese hamster ovary cells.
• the filter is washed in buffer, followed by vacuum release and RNA extraction.
• spot filter SELEX the protein is applied to a dry nitrocellulose 13 mm filter, allowed to adsorb for several minutes, then pre-incubated in Buffer S (HBSMC buffer plus 0.02% each of ficoll, polyvinylpyrrolidone, and human serum albumin) for 10 minutes at 37°C to remove unbound protein.
• Buffer S HBSMC buffer plus 0.02% each of ficoll, polyvinylpyrrolidone, and human serum albumin
• the wash buffer is removed, and then the RNA library is added in the same buffer, and incubated with the protein-bound filter.
• the filters are washed by repeated incubations in fresh buffer, followed by RNA extraction.
• RNA sequence libraries containing either a 30 or 40 nucleotide randomized region sequence ( Figure 1).
• the RNA libraries were transcribed from the corresponding synthetic DNA templates that were generated by Klenow extension (Sambrook, Fritsch et al. (1989)
• the DNA templates were transcribed in 1 mL reactions, each containing 0.25 nM template, 0.58 ⁇ M T7 RNA polymerase, 1 mM each of ATP and GTP, 3 mM each of 2'-F- CTP and 2'-F-UTP, 40 mM Tris-HCl (pH 8.0), 12 mM MgCl2, 1 mM spermidine, 5 mM DTT, 0.002% Triton X-100 and 4% polyethylene glycol (w/v) for at least 4 hours at 37°C.
• the full-length transcription products were purified by denaturing polyacrylamide gel electrophoresis.
• Radiolabeled RNA was obtained from transcription reactions as described above, but containing 0.2 nM ATP and 100 ⁇ Ci of ⁇ - 2 P-ATP.
• radiolabeled RNA was obtained by labeling the 5'-end of RNA with ⁇ - 32 P -ATP (NEN-DuPont), catalyzed by T4 polynucleotide kinase (New England Biolabs).
• T4 polynucleotide kinase New England Biolabs.
• transcription reactions included 5 mM guanosine.
• radiolabeled RNA pools were suspended in HBSMC buffer to which HGF protein was added, and incubated at 37°C for 30 minutes to 3 hours depending on the round.
• Binding reactions were then filtered under suction through 0.45 ⁇ m nitrocellulose filters (Millipore), pre-wet with binding buffer. The filters were immediately washed with at least 5 mL of HBSMC buffer. For each binding reaction, a protein-minus control reaction was done in parallel in order to determine the amount of background binding to the filters. The amount of RNA retained on the filters was quantified by Cherenkov counting, and compared with the amount input into the reactions.
• the bound DNA in the captured RNA/DNA complexes was then eluted by heat denaturation and amplified using conventional SELEX PCR primers. To complete the cycle, the resulting DNA was then used as a transcription template for generating RNA to be cleaved by RNaseH, and used in the next round of truncate SELEX.
• C-met protein was diluted in HBSMCK (50 mM HEPES, pH 7.4, 140 mM NaCl, 3 mM KC1, 1 mM CaCl 2 , 1 mM MgCl 2 ), and was adsorbed to polystyrene wells by incubating 100 ⁇ L of diluted protein per well for 60 minutes at 37°C.
• the wells were each washed with three 400 ⁇ L aliquots of HIT buffer (HBSMCK, 0.1% I-block, 0.05 % Tween 20), and then blocked in 400 ⁇ L of blocking agent for 60 minutes at 37°C.
• SELEX was initiated by incubating 100 ⁇ L of RNA in the protein-bound well for 60 minutes at 37°C.
• Competitor titration curves were generated essentially as a standard binding curve, except that the protein and RNA concentrations were kept constant, and the competitor concentration was varied. Competitors were also added at a fixed concentration in binding experiments to increase stringency for purposes of comparing pool binding affinities. In these experiments, the competitor concentration was chosen based on the results from the competitor titration curves.
• RNA oligomers were synthesized with an amino- linker at the 5'-position. This was subsequently reacted with NHS-ester 40K-PEG manufactured by Shearwater Polymers, Inc. (HuntsviUe, AL), and purified by HPLC on a reverse-phase preparative column.
• Boundaries and truncation Boundary determinations were done for a subset of aptamers that demonstrated in vitro inhibition of HGF activity. Using a standard alkaline hydrolysis procedure with 5'-end-labeled RNA, the 3'-boundaries of aptamers 8-17, 8-102, 8-104, 8-126, 10-1 and 10-2 were examined. Additionally, 3 '-end-labeled RNA was used for 5'-boundary experiments with aptamers 8-17 and 8-102. These experiments were mostly uninformative, probably because the high degree of non-specific binding of RNA fragments, regardless of size, obscured the binding of truncated high-affinity aptamers to HGF.
• Three rounds of hybridization truncate SELEX were done in parallel, using as starting pools HGF SELEX 1 round 8 and HGF SELEX 3 round 11.
• the truncate SELEX rounds were done at equi-molar RNA and protein, starting at 1 nM and decreasing to 0.5 and 0.1 nM. Signal-to-noise ratios were very high during selection. Subsequent manipulations were satisfactory even though the amount of recovered RNA was sub- picomolar.
• binding affinities of truncate rounds two and three were determined and compared to those of the RNaseH-cleaved starting pools (Figure 9). For both SELEXes, the third round pools bound with improved affinity for HGF compared with the earlier rounds.
• the dissociation constants for the third round truncate SELEX pools are 1-2 nM, representing a 2-3 fold improvement. While the magnitude of this improvement is not large, it is probably significant since HGF as a target did not easily yield affinity enrichment, probably because of its intrinsically high affinity for RNA.
• the two pools were cloned and sequenced, and binding affinities were determined (Table 5).
• the truncated aptamer with the best binding affinity, Tr51 is among several sequences which are novel, that is, they were not found in the clones sequenced from the full-length SELEX pools.
• the emergence of novel sequences suggests that the truncate SELEX succeeded in amplifying aptamers that were relatively rare in the full-length pools.
• Aptamer Tr51 appeared more frequently than any other sequence, consistent with the observation that it has better binding affinity than any other truncate.
• Other sequences that appeared multiple times also tend to be those with binding affinities near or better than the pool K ⁇ of 1-2 nM.
• the modified aptamer designated NX22354, was tested for inhibition of HGF-mediated proliferation 4MBr-5 cells ( Figure 11 A).
• the data indicate that the 36mer-PEG aptamer inhibits HGF, and that it performs at least as well as the full- length aptamer 8-17, which had previously exhibited the strongest inhibition of all aptamers tested.
• the non-PEGylated 36mer did not inhibit HGF, suggesting that the addition of PEG and/or the 3'-cap contribute to the aptamer's bioactivity.
• HGF-mediated stimulation of cell migration HGF readily stimulates cell movement, hence the name, scatter factor.
• the inhibitory effect of HGF aptamers was assayed by measuring their effect on A549 cell migration across a Matrigel coated membrane with 8.0 micron pores as described in Materials and Methods (Table 6).
• the NX22354 aptamer fully inhibited HGF-mediated migration at both 1 and 0.2 ⁇ M concentrations, but at 0.04 ⁇ M, the effect was negligible.
• the monoclonal antibody control (sample 3) was moderately effective at the 1 ⁇ g/mL dose, which is above its published EC 50 value of 0.1 -0.3 ⁇ g/mL for inhibition of 4MBr-5 cell proliferation.
• VEGF and HGF were added, singly and in combination, to HUVECs, and incorporation of H-thymidine was measured (Figure 13).
• stimulation by HGF was relatively weak compared with that of VEGF and together, the stimulatory effect was greater than that elicited by VEGF alone.
• each cytokine was added at 10 ng/mL for optimal stimulation in the aptamer inhibition experiments.
• the effect of adding one or both aptamers to the doubly-stimulated cells in the presence of both growth factors was then tested (Figure 14A). It was observed that each aptamer partially inhibits the stimulation and that both aptamers result in complete inhibition. Interestingly, the magnitude of the inhibitory effect of each aptamer roughly corresponds with the magnitude of the stimulation conferred by each cytokine. This observation suggests that the stimulatory effect of each cytokine can be inhibited independently, and that the two cytokines stimulate HUVECs independently.
• FIGs 14B and C depict controls in which each cytokine was administered separately, demonstrating that the HGF and VEGF aptamers do not cross-react, that is, each aptamer affects only the cytokine against which it was selected.
• HGF stimulated cells inhibition by the HGF aptamer ⁇ X22354 was observed, but not by the VEGF aptamer NXl 838 ( Figure 14B).
• stimulation by VEGF was inhibited by the VEGF aptamer NXl 838, but was unaffected by the HGF aptamer NX22354 ( Figure 14C).
• HGF like VEGF
• HGF aptamers which inhibit other growth factors suggest further combinations of the VEGF or the HGF aptamer in combination with other aptamers, for example, aptamers that inhibit bFGF, platelet-derived growth factor (PDGF), transforming growth factor beta (TGF), keratinocyte growth factor (KGF), and/or their receptors allowing for the possibility that any combination of these inhibitors may be relevant.
• PDGF platelet-derived growth factor
• TGF transforming growth factor beta
• KGF keratinocyte growth factor
• the goal is to have an array of aptamer-inhibitors of cytokines and their receptors and to be able to tailor combination treatments for specific disease states.
• the four partially-substituted oligonucleotides were synthesized with a 1 :1 ratio of 2'-OMe amidite:2'-OH amidite (Table 7).
• Table 7 The data analysis measures the ratios of the selected to unselected RNA at each substituted purine position, based on quantitation of bands from the gel.
• the data are summarized by position ( Figure 15).
• the three unsubstituted aptamers provide an important comparison, which is expressed as an average of the three unsubstituted aptamers with standard deviation represented by the error bars. Points that occur at ratios higher than that of the nearby positions are likely to require 2'-OH for binding.
• the data strongly indicate that two positions, G5 and A25, do not tolerate 2'-OMe substitution. Two other positions, A3 and G10, show a slight preference above the standard deviation of the unselected RNA.
• the set of OMe aptamers were also examined for binding to HGF (data not shown).
• the binding data indicate that the OMel and OMe3 bind as well as the parent unsubstituted 36mer, whereas OMe2 and OMe4 bind do not bind as well. This suggests that the substitutions in OMe2 and OMe4 are less well tolerated with respect to HGF binding in solution, consistent with the fact that OMe2 and OMe4 are substituted at A25 and G5, respectively.
• NX22354 tolerates 2'-OMe substitution at all purines except G5 and A25 (aptamer 2x Sub 2'-OH) with minimal loss of binding affinity.
• the other two positions in question apparently are not required to be 2'-OH since aptamer 4x Sub 2'-OH binds no better than aptamer 2x Sub 2'-OH.
• ML-124 5'-ACGAGTTTATCGAAAAAGAACGATGGTTCCAATGGAGCA-3' (SEQ ID NO: 188), was used that is complementary to the most prevalent N7-series human IgG, aptamer sequence, and differs by only a few bases from most other IgG, aptamers.
• This PCR primer is the same length as the selected sequence of the major IgG, so that it can tolerate mismatches and hybridize to similar sequences.
• the ML- 124 3'-primer ML-34; 5'- CGCAGGATCCTAATACGACTCACTATA-3' (SEQ ID NO: 189), was used with a 5'-primer containing the T7-promoter sequence present in all cloned aptamers to amplify 40N7 series nucleic acids pools: random, la, 2a, 3a and 4a (data not shown). Since IgG, aptamers have not been isolated from an N8 type library, this analysis was not done for the 30N8 SELEX. PCR of random and c-met SELEX round la pools yielded no signal after 20 cycles.
• a DNA template library of sequence: S'-ttat ⁇ cg ctc ⁇ ctat ⁇ gggagacaagaataaacgctcaarinnmm ⁇ riJ rmnnnttcgacaggaggctcacaacaggc-3' (SEQ ID NO: 190) was prepared by chemical synthesis. The italicized nucleotides correspond to a T7 RNA polymerase promoter. There are 40N residues (a, g, t or c).
• 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 ⁇ M cyclic RGD peptide ("cRGD”) (GPenGRGDSPCA, Life Technologies, Gibco BRL, Gaithersburg, MD). Eluted RNAs were extracted with phenol, then chloroform ⁇ soamyl alcohol (24: 1), and ethanol precipitated.
• cRGD cyclic RGD peptide
• Figure 24 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 ⁇ 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 ⁇ ub ⁇ 3 .
• ⁇ Clone series 8 is from HGF SELEX 1; series 11 is from HGF SELEX 3. Numbers in parentheses refer to repeat occurrences of the same exact sequence. For the series 8 clones, a second number refers to an exact match which was isolated in series 11. *N7 fixed sequences are not shown. (5'-GGGAGGACGAUGCGG-N- CAGACGACUCGCCCGA-3') °NO, not determined.
• *10-2 contains N8 fixed sequences; all others are N7.
• Tr70 UGGGAACCUAUUUAUGUCAUCGUCUGUGCC New 2.4 134
• Tr42 UGAUAACCUAUUUAUGACGUCGUGGCUCCC 11-162 6.1 138
• Tr4 (4,2) d GUGACUCAAAAUGGUGAUCCUCGUUUCCGC 8-126 1.4 148
• Trl5 GUUUGAGUGACGAUCGUCUUGUCCCAUGUG 11-208 8.8 150
• Trl -36 and Tr37-72 clones are from series which were carried through 8 and 11 conventional rounds, respectively.
• NX22354 is a synthetic truncate based on boundary experiments, derived from sequence

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