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Nucleic acid ligands to integrins

a technology of integrins and ligands, applied in the field of integrin proteins, can solve the problems of limiting the growth and metastasis of cancerous lesions, solid tumors cannot grow to significant size without an independent blood supply, and the affinity of integrins for fibrinogen is very low on these cells

Inactive Publication Date: 2007-01-11
GILEAD SCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0027] 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 vitro

Problems solved by technology

Solid tumors are unable to grow to significant size without an independent blood supply.
Thus, inhibition of angiogenesis may limit both the growth and metastasis of cancerous lesions.
(1996) Blood 88:907-14); however, the affinity of the integrin for fibrinogen is very low on these cells.

Method used

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  • Nucleic acid ligands to integrins
  • Nucleic acid ligands to integrins

Examples

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example 1

Isolation of Integrins and Integrin Ligands

[0070]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 fi...

example 2

Generating Nucleic Acid Ligands to Integrins Using the SELEX Method

[0071] A DNA template library of sequence:

5′-ttatacgactcactatagggagacaagaataaac(SEQ ID NO:1)gctcaannnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnttcgacaggaggctcacaacaggc-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.

[0072] 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]etha...

example 3

Specificity of the Nucleic Acid Ligands to Integrins

[0077] 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 t...

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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 [0001] 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 “Nucle...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K31/7088A61K31/7105G01N33/50A61K31/711A61K31/712A61K38/00A61P7/02A61P9/10A61P15/00A61P17/06A61P19/10A61P27/06A61P29/00A61P35/00C07H21/00C07H21/02C07H21/04C12N15/09C12P19/34C12Q1/68
CPCA61K38/00C12N2310/322C12N15/115C07H21/00C12N2310/16G01N33/5308G01N33/74G01N2333/4753A61P15/00A61P17/06A61P19/10A61P27/06A61P29/00A61P35/00A61P7/02A61P9/10C12N2310/3533C12N2310/321C12N2310/3521
Inventor RUCKMAN, JUDYGOLD, LARRYSTEPHENS, ANDREWJANJIC, NEBOJSA
Owner GILEAD SCI INC
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