Use of monoclonal antibodies to distinguish protein conformational isoforms

• US 20070281318A1
• Filed: 08/18/2003
• Published: 12/06/2007
• Est. Priority Date: 10/10/2002
• Status: Active Grant

• Current Assignee: Heinrich-Heine-Universität Düsseldorf, Regents of the University of California (University of California)
• Original Assignee: Heinrich-Heine-Universität Düsseldorf
• Inventors: Korth, Carsten, Lingappa, Vishwanath
• Granted Patent: US 7,932,043 B2
• Time in Patent Office: Days
• US Class Current: 435/7.100
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 16/2872 against prion molecules, e.g. CD230
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Abstract

Methods of preparing monoclonal antibodies that differentially bind to a single conformer of a protein of interest are described. Passive immunization using these antibodies as well as use of conformer-specific antibodies as diagnostic reagents for the purpose of stratification of patient populations with regards to disease outcome, drug efficacy or drug sensitivity is also disclosed as well as active immunization with the protein conformer. In the screening techniques, detection can be for example by tissue immunostaining, western blotting or solution IP. A specific mab termed 7VC which shows conformation specificity to CtmPrP, a prion protein conformer that triggers neurodegeneration under specific assay conditions of pH and copper concentration, is described. A second specific antibody termed 19B10 shows conformation specificity for NtmPrP, a prion protein conformer that downregulates total PrP expression and effects cell differentiation.

Claims

1. A method of detecting prion disease in a sample, said method comprising:

contacting said sample with a diagnostically effective amount of an antibody characterized by its ability to bind to PrP^CTM in situ; and

determining whether said antibody binds specifically to any material in said sample under conditions whereby differential binding of said antibody to PrP isoforms associated with prion disease occurs, wherein antibody binding is indicative of prion disease.

2. The method according to claim 1, wherein said sample is a tissue or a bodily fluid.

3. The method according to claim 1, wherein said conditions comprise a local concentration of Cu²⁺ ions in an amount that promotes said differential binding.

4. The method according to claim 3, wherein said local concentration is about 1 μ

M to about 50 μ

M.

5. The method according to claim 1, wherein said conditions comprise a pH of about 7.8.

29. The method according to claim 2, wherein said antibody is a monoclonal antibody.

30. The method according to claim 29, wherein said monoclonal antibody is a monoclonal antibody designated 7VC.

6. A composition comprising:

a hybridoma cell culture producing monoclonal antibodies which bind specifically to one conformer of PrP selected from the group consisting of PrpCTM and PrPNTM and have low or no cross reactivity with a second conformer of PrP.

7. The composition according to claim 6, wherein said monoclonal antibodies are designated 7VC or 19B10.

8. A composition comprising:

a monoclonal antibody which binds to an antigenic determinant of a PrP conformer on the surface of a neuroblastoma cell and has low or no cross reactivity with non cell surface antigenic determinants of PrP conformers.

9. A hybridoma which expresses monoclonal antibodies having the same antigenic specificity as that of monoclonal antibodies 7VC or 19B10.

10. Monoclonal antibodies derived from a hybridoma according to claim 9.

11. A monoclonal antibody fragment derived from a monoclonal antibody having the binding specificity of a monoclonal antibody according to claim 10.

12. Monoclonal antibodies according to claim 10, labelled with an agent capable of providing a detectable signal.

24. The monoclonal antibody according to claim 9, wherein said monoclonal antibody recognizes PrP^NTM.

28. The monoclonal antibody according to claim 9, wherein said monoclonal antibody recognizes PrP^CTM.

13. A method for promoting cell differentiation, said method comprising:

providing a cell with a sufficient amount of a monoclonal antibody that binds to an antigenic determinant on PrPNTM but not other conformers of mature PrP, whereby said cell undergoes differentiation.

14. The method according to claim 13, wherein said cell is a stem cell or a cancer cell.

15. A method of inhibiting PrP expression in a cell, said method comprising:

providing a cell with a sufficient amount of a monoclonal antibody that binds to an antigenic determinant on PrPNTM but not other conformers of mature PrP, whereby PrP expression in said cell is inhibited.

16. The method according to claim 15, wherein said PrP is PrPSc.

17. A method for obtaining prion protein conformer-specific monoclonal antibodies, said method comprising:

contacting prion protein conformers each prepared using a cell-free translation system, with a composition comprising one or more monoclonal antibodies that bind to a prion protein conformer;

detecting binding of said monoclonal antibodies to one or more of said prion protein conformers by solution immunoprecipitation; and

isolating monoclonal antibodies that bind substantially specifically to a single prion protein conformer;

whereby conformer-specific monoclonal antibodies are obtained.

18. A method for screening hybridomas for those that express conformer-specific monoclonal antibodies said method comprising:

combining a panel of radiolabelled conformer-enriched translation products obtained using a cell-free translation system comprising microsomal membranes derived from the endoplasmic reticulum with individual samples of monoclonal antibodies expressed by said hybridomas; and

immunopreciptating any antibody-conformer complexes, whereby detection of complexes with a single conformer from said panel of radiolabelled conformer-enriched translation products is indicative of a hybridoma that expresses conformer-specific monoclonal antibodies.

19. The method according to claim 18, wherein conformer enrichment of said radiolabelled translation products is obtained by signal sequence swapping.

20. The method according to claim 18, wherein conformer enrichment of said radiolabelled translation products is obtained by alteration of the ribosome-membrane junction.

21. The method according to claim 18, wherein conformer enrichment of said radiolabelled translation products is obtained by translation with glycoprotein-depleted microsomal membranes.

22. The method according to claim 18, wherein conformer enrichment is obtained by expressing signal sequence swapped cDNAs in transfected mammalian cells.

23. A specific antibody (7VC) that recognizes prion protein in a copper-dependent fashion.

25. A specific antibody (SS) that recognizes the signal sequence of PrP

26. A monoclonal antibody designated 19B10.

27. A monoclonal antibody designated 7VC.

31. An epitope recognized by monoclonal antibody 7VC, wherein said epitope occurs in an amino acid sequence selected from the group consisting of an octarepeat sequence of a prion protein, WGQPHGGGWGQPHGG, GWGQPHGGGWGQPH, and GGGEGQGGGTHNQWN.

32. A kit for detecting prion disease in a sample, said kit comprising:

an antibody that exhibits differential binding to PrP^CTM in situ as compared to other PrP conformers under conditions that promote said differential binding.

33. The kit according to claim 32, wherein said sample is a tissue or a bodily fluid.

34. The kit according to claim 32, wherein said conditions comprise a local concentration of Cu²⁺ ions in an amount that promotes said differential binding.

35. The kit according to claim 34, wherein said local concentration is about 1 μ

M to about 50 μ

M.

36. The kit according to claim 32, wherein said conditions comprise a pH of about 7.8.

39. The kit according to claim 33, wherein said antibody is a monoclonal antibody.

40. The kit according to claim 39, wherein said monoclonal antibody is a monoclonal antibody designated 7VC.

37. Prion protein conformer-specific monoclonal antibodies, obtainable by a method comprising:

contacting a panel of prion protein conformers, wherein each member of said panel is prepared using a cell-free translation system, with a composition comprising one or more monoclonal antibodies that bind to a prion protein conformer;

detecting binding of said monoclonal antibodies to one or more of members of said panel of prion protein conformers by solution immunoprecipitation; and

isolating monoclonal antibodies that bind substantially specifically to a single prion protein conformer;

whereby conformer-specific monoclonal antibodies are obtained.

38. Hybridomas that express prion protein conformer-specific monoclonal antibodies according to claim 37.