Nucleic acids for high throughput screening of CpG-based immuno-agonist/antagonist

• US 6,943,240 B2
• Filed: 09/17/2001
• Issued: 09/13/2005
• Est. Priority Date: 09/15/2000
• Status: Expired due to Fees

• Current Assignee: Coley Pharmaceutical GmbH (Pfizer Inc.)
• Original Assignee: Coley Pharmaceutical GmbH (Pfizer Inc.)
• Inventors: Lipford, Grayson, Bauer, Stefan, Wagner, Hermann
• Primary Examiner(s): Marschel, Ardin H.
• Assistant Examiner(s): Smith, Carolyn L.
• Application Number:
  US09/954,987
• Publication Number:
  US 20030104523A1
• Time in Patent Office: 1,457 Days
• Field of Search: 536/23.1, 435/320.1, 435/325
• US Class Current: 536/23.1
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748

Abstract

The invention pertains to murine TLR9 and related TLR9s which include murine-specific amino acids, as well as nucleic acids which encode those polypeptides. The present invention also includes fragments and biologically functional variants of the murine TLR9. The invention further relates to methods of using such murine and non-murine TLR9 nucleic acids and polypeptides, especially in methods for screening for agonists and antagonists of immunostimulatory CpG nucleic acids. Also included are murine TLR9 inhibitors which inhibit murine TLR9 activity by inhibiting the expression or function of murine TLR9. In a further aspect the present invention pertains to murine TLR7 and murine TLR8, as well as related TLR7 and TLR8 molecules which include murine-specific amino acids, as well as nucleic acids which encode those polypeptides. The present invention also includes fragments and biologically functional variants of the murine TLR7 and TLR8. Methods are included for screening for ligands of TLR7 and TLR8, as well as for inhibitors and agonists and antagonists of signaling mediated by TLR7 and TLR8.

Claims

1. An isolated nucleic acid molecule which encodes a Toll-like receptor 9 (TLR9) polypeptide HTLR9-CXXCm, said polypeptide comprising an amino acid sequence of SEQ ID NO:

6 except for substitution of amino acids 269-274 of SEQ ID NO;

6 with amino acids 269-274 of SEQ ID NO;

3.

2. An expression vector comprising the isolated nucleic acid molecule of claim 1 operably linked to a promoter.

3. A host cell comprising the expression vector of claim 2.

4. The host cell of claim 3, further comprising at least one expression vector selected from the group consisting of:

(a) an expression vector comprising a nucleic acid molecule which encodes an isolated TLR7 polypeptide operably linked to a promoter, and

(b) an expression vector comprising a nucleic acid molecule which encodes an isolated TLR8 polypeptide operably linked to a promoter.

5. The host cell of claim 3, further comprising a reporter gene construct comprising a reporter gene operatively linked to a promoter sensitive to NF-κ

B.

6. The host cell of claim 3, wherein the host cell is a 293 fibroblast cell.

7. The host cell of claim 5, wherein the host cell is a 293 fibroblast cell.