Methods and compositions for the generation and use of conformation-specific antibodies

Patent metadata

Field	Value
Publication number	US-9796784-B2
Application number	US-201414325013-A
Country	US
Kind code	B2
Filing date	Jul 7, 2014
Priority date	Oct 27, 2009
Publication date	Oct 24, 2017
Grant date	Oct 24, 2017

Abstract

The present invention features methods and compositions for the generation and use of conformation-specific antibodies or fragments thereof.

First claim

What is claimed is:

1. A method of generating a conformation-specific antibody or fragment thereof that specifically binds to a Xaa-Pro motif of the naturally occurring form of a polypeptide, where Xaa is any amino acid residue, said method comprising:
(i) administering a proline-analog-containing antigenic peptide to a non-human host animal, said antigenic peptide comprising a Xaa-Pro motif comprising said proline analog;
(ii) isolating antisera containing said antibody or fragment thereof produced in said animal; and
(iii) purifying said conformation-specific antibody or fragment thereof from said antisera; wherein said conformation-specific antibody or fragment thereof specifically binds to the Xaa-Pro motif of the naturally occurring form of said polypeptide;
wherein said proline analog is selected from the group consisting of homoproline, azetidine-2-carboxylic acid (Aze), tert-butyl-L-proline (TBP), trans-4-fluoro-L-proline (t-4F-Pro), and cis-4-fluoro-L-proline (c-4F-Pro), and wherein the peptidyl-prolyl bond of said Xaa-Pro motif of said antigenic peptide is in a cis conformation, or is in a trans conformation.

2. The method of claim 1 , wherein said Xaa is serine or threonine.

3. The method of claim 1 , wherein said Xaa is phosphorylated.

4. The method of claim 1 , wherein said Xaa is not phosphorylated.

5. The method of claim 1 , wherein the peptidyl-prolyl bond of said Xaa-Pro motif of said antigenic peptide is in a cis conformation.

6. The method of claim 5 , wherein said antibody or fragment thereof binds to the cis conformation of said Xaa-Pro motif of said polypeptide with at least 10- to 100-fold greater affinity than to the trans conformation of said Xaa-Pro motif of said polypeptide.

7. The method of claim 1 , wherein the peptidyl-prolyl bond of said Xaa-Pro motif of said antigenic peptide is in a trans conformation.

8. The method of claim 7 , wherein said antibody or fragment thereof binds to the trans conformation of said Xaa-Pro motif of said polypeptide with at least 10- to 100-fold greater affinity than to the cis conformation of said Xaa-Pro motif of said polypeptide.

9. The method of claim 1 , wherein said polypeptide is a peptidyl-prolyl cis/trans isomerase (PPlase) substrate.

10. The method of claim 9 , wherein said PPlase substrate is a Pin1 substrate.

11. The method of claim 10 , wherein said Pin1 substrate is NIMA, RAB4, CDC25, WEE1, PLK1, MYT1, CDC27, CENP-F, Incenp, RBP1, NHERF-1, KRMP1, CK2, Topollα, DAB2, p54nrb, Sil, EMI1, cyclin D1, Ki67, c-Myc, cyclin E, c-Jun, β-catenin, Cf-2, NF-κB, RAF1, C-Fos, RARα, AlB1/SRC-3, HBx, STAT3, p53, Bcl-2, p73, BimEL, p66 Shc , CHE1, tau, amyloid precursor protein (APP), APP fragment, synphilin-1, gephyrin, MCL1, NFAT, AUF1, IRF3, BTK, SIN3-RPD3, or hSpt5.

12. The method of claim 1 , wherein the peptidyl-prolyl bond of said Xaa-Pro motif of said antigenic peptide is fixed in the cis conformation or the trans conformation.

13. The method of claim 1 , wherein said antigenic peptide is at least 8 amino acid residues in length.

14. The method of claim 13 , wherein said antigenic peptide is between 8 and 20 amino acid residues in length.

15. The method of claim 1 , wherein said conformation-specific antibody is a monoclonal antibody.

16. The method of claim 1 , wherein said conformation-specific antibody is a polyclonal antibody.

17. The method of claim 1 , wherein said host animal is a rabbit.

18. The method of claim 1 , wherein said host animal is a bovine, equine, canine, ovine, feline, goat, sheep, or chicken.

19. The method of claim 1 , wherein the conformation-specific antibody specifically binds to the cis conformation of said Xaa-Pro motif of said polypeptide with a Kd of at least about 10 −4 M or greater, or wherein the conformation-specific antibody specifically binds to the trans conformation of said Xaa-Pro motif of said polypeptide with a Kd of at least about 10 −4 M or greater.

20. The method of claim 1 , wherein Xaa is serine or threonine and wherein said polypeptide is a peptidyl-prolyl cis/trans isomerase (PPlase) substrate.

Assignees

• Beth Israel Deaconess Medical Ct Inc

Inventors

• Lu Kun Ping
• Zhou Xiao Zhen

Classifications

• A61P37/04

  Immunostimulants · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• A61P25/28

  for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia · CPC title

• C07K14/4711 Primary

  Alzheimer's disease; Amyloid plaque core protein · CPC title

• G01N2333/99

  Isomerases (5.) · CPC title