High affinity nucleic acid ligands of cytokines

• US 6,028,186 A
• Filed: 06/07/1995
• Issued: 02/22/2000
• Est. Priority Date: 06/10/1991
• Status: Expired due to Term

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: NeXstar Pharmaceuticals, Inc. (Gilead Sciences Inc.)
• Inventors: Gold, Larry, Pagratis, Nikos, Jayasena, Sumedha, Tasset, Diane
• Primary Examiner(s): ZITOMER, STEPHANIE W
• Application Number:
  US08/481,710
• Time in Patent Office: 1,721 Days
• Field of Search: 536/23.1, 536/24.31, 536/22.1, 536/24.3, 435/6, 435/91.2, 935/77, 935/78
• US Class Current: 536/24.31
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 14/001 by chemical synthesis
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: C12N 15/115 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
• CPC Class Codes: C12N 2310/13 Decoys
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: G01N 2333/16 HIV-1, HIV-2
• CPC Class Codes: G01N 2333/163 Regulatory proteins, e.g. tat, nef, rev, vif, vpu, vpr, vpt, vpx
• CPC Class Codes: G01N 2333/503 : basic FGF [bFGF]
• CPC Class Codes: G01N 2333/8125 : Alpha-1-antitrypsin
• CPC Class Codes: G01N 2333/96433 : Serine endopeptidases (3.4.21)
• CPC Class Codes: G01N 2333/96436 : Granzymes
• CPC Class Codes: G01N 2333/96486 : Metalloendopeptidases (3.4.24)
• CPC Class Codes: G01N 2333/966 : Elastase
• CPC Class Codes: G01N 2333/974 : Thrombin
• CPC Class Codes: G01N 33/532 : Production of labelled immunochemicals
• CPC Class Codes: G01N 33/535 : with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: G01N 33/56988 : HIV or HTLV
• CPC Class Codes: G01N 33/68 : involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/76 : Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors

Abstract

Methods are described for the identification and preparation of high-affinity nucleic acid ligands to cytokines. Included in the invention are specific nucleic acid ligands to IFN-gamma, IL-4, IL-10, TNF-alpha, and RANTES.

Claims

1. A purified and isolated non-naturally occurring nucleic acid ligand to a cytokine selected from the group consisting of interleukin-4 (IL4), interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-alpha) and Regulated on Activation, Normal T-cell Expressed and Secreted (RANTES) protein.

2. The purified and isolated non-naturally occurring nucleic acid ligand of claim 1 wherein said nucleic acid ligand is single-stranded.

3. The purified and isolated non-naturally occurring nucleic acid ligand of claim 2 wherein said nucleic acid ligand is RNA.

4. The purified and isolated non-naturally occurring nucleic acid ligand of claim 2 wherein said nucleic acid ligand is DNA.

7. The purified and isolated non-naturally occurring RNA ligand of claim 3 wherein said ligand is to IL-4.

8. The purified and isolated non-naturally occurring RNA ligand to IL-4 of claim 7 wherein said ligand is selected from the group consisting of the sequences set forth in Tables 7 and 8 (SEQ ID NOS:

79-185).

9. The purified and isolated non-naturally occurring RNA ligand to IL-4 of claim 7 wherein said ligand is substantially homologous to and has substantially the same ability to bind IL-4 as a ligand selected from the group consisting of the sequences set forth in Tables 7 and 8 (SEQ ID NOS:

79-185).

10. The purified and isolated non-naturally occurring RNA ligand to IL-4 of claim 7 wherein said ligand has substantially the same structure and substantially the same ability to bind IL-4 as a ligand selected from the group consisting of the sequences set forth in Tables 7 and 8 (SEQ ID NOS:

79-185).

11. The purified and isolated non-naturally occurring RNA ligand of claim 3 wherein said ligand is to IL-10.

12. The purified and isolated non-naturally occurring RNA ligand to IL-10 of claim 11 wherein said ligand is selected from the group consisting of the sequences set forth in Table 10 (SEQ ID NOS:

189-205).

13. The purified and isolated non-naturally occurring RNA ligand to IL-10 of claim 11 wherein said ligand is substantially homologous to and has substantially the same ability to bind IL-10 as a ligand selected from the group consisting of the sequences set forth in Table 10 (SEQ ID NOS:

189-205).

14. The purified and isolated non-naturally occurring RNA ligand to IL-10 of claim 11 wherein said ligand has substantially the same structure and substantially the same ability to bind IL-10 as a ligand selected from the group consisting of the sequences set forth in Table 10 (SEQ ID NOS:

189-205).

15. The purified and isolated non-naturally occuring RNA ligand of claim 3 wherein said ligand id to TNF-alpha.

16. The purified and isolated non-naturally occurring RNA ligand to TNF-alpha of claim 15 wherein said ligand is selected from the group consisting of the sequences set forth in Table 12 (SEQ ID NOS:

209-255).

17. The purified and isolated non-naturally occurring RNA ligand to TNF-alpha of claim 15 wherein said ligand is substantially homologous to and has substantially the same ability to bind TNF-alpha as a ligand selected from the group consisting of the sequences set forth in Table 12 (SEQ ID NOS:

209-255).

18. The purified and isolated non-naturally occurring RNA ligand to TNF-alpha of claim 15 wherein said ligand has substantially the same structure and substantially the same ability to bind TNF-alpha as a ligand selected from the group consisting of the sequences set forth in Table 12 (SEQ ID NOS:

189-205).

19. The purified and isolated non-naturally occurring nucleic acid ligand of claim 1 wherein said ligand is to RANTES.

5. A nucleic acid ligand to a cytokine selected from the group consisting of IL-4, IL-10, TNF-α

and RANTES, identified according to the method comprising;

a) preparing a candidate mixture of nucleic acids;

b) contacting the candidate mixture of nucleic acids with said cytokine, wherein nucleic acids having an increased affinity to said cytokine relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and

d) amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched for nucleic acid sequences with relatively higher affinity and specificity for binding to said cytokine, whereby a nucleic acid ligand of said cytokine may be identified.

6. The purified and isolated non-naturally occuring RNA ligand to IFN-gamma of claim 5 wherein said ligand is selected from the group consisting of the sequences set forth in Tables 3 and 4 (SEQ ID NOS:

7-73).