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Anti-addl antibodies and uses thereof

A technology of antibodies and ligands, applied in the direction of antibodies, anti-animal/human immunoglobulins, instruments, etc., can solve the problem of low correlation between dementia and amyloid plaques

Inactive Publication Date: 2008-03-05
MERCK & CO INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Despite strong experimental support for fibrillar Aβ and memory loss, the correlation between dementia and the amount of amyloid plaques is low (Katzman (1988) Ann. Neurol. 23: 138-144)

Method used

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  • Anti-addl antibodies and uses thereof
  • Anti-addl antibodies and uses thereof
  • Anti-addl antibodies and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1: General Materials and Methods

[0089]Preparation of ADDL. ADDLs in F12 medium (Biosource, Camarillo, CA) were prepared from Aβ1-42 following established methods (Lambert et al. (2001) supra). Briefly, the Aβ1-42 peptide (American Peptide Co., Sunnyvale, CA or California Peptide Research, Inc., Napa, CA) was weighed and placed in a container capable of holding a sufficient amount of HFIP (1, 1, 1, 3, 3 , 3-hexafluoro-2-propanol) in a glass vial to bring the peptide concentration to 10 mg / mL. HFIP was added to the dried peptide, the glass vial was capped, vortexed gently to mix, and the peptide / HFIP solution was stored at room temperature for at least 1 hour. Aliquots (50 or 100 μL, containing 0.5 or 1.0 mg, respectively) of the peptide solution were dispensed into a series of 1.5 mL conical centrifuge tubes. The tubes were placed in a speedvac overnight to remove HFIP. Tubes containing dried peptide films were capped and stored at -70°C in a sealed contai...

Embodiment 2

[0111] Example 2: Development and characterization of anti-ADDL antibodies

[0112] Three mice were inoculated with ADDLs (194±25 μg protein per injection) every three weeks for a total of six inoculations. Hybridomas obtained from the fusion of spleen cells from these mice with SP2 cells were cultured in 96-well plates. Supernatants from these wells were screened in dot blots and synthetic ADDLs were used to identify positive clones that could be compared to dot blots of endogenous fibrils to discern differences. Look for hybridomas that bind only synthetic ADDLs and not endogenous fibrils. To learn more about what the products of the hybridomas bind to and under what conditions, 3 western blots were performed on each positive clone: ​​SDS-PAGE of ADDLs, native gel of ADDLs, and SDS-PAGE with endogenous fibrils . Approximately 40 clones were screened for further examination. Each clone was tested under different conditions for recognition of soluble Alzheimer's patient br...

Embodiment 3

[0116] Example 3: Immunohistochemical analysis of endogenous and synthetic ADDLs associated with cultured hippocampal cells

[0117] Cultured hippocampal cells were also analyzed to determine whether monoclonal antibodies capable of distinguishing Alzheimer's disease from control brain extracts were able to identify ADDLs (endogenous or synthetic) bound to cultured cells. Hippocampal cultures were prepared according to established protocols and allowed to grow for 3-4 weeks. Synthetic ADDLs are prepared according to standard procedures (eg, U.S. Patent 6,218,506). Endogenous ADDLs were extracted from the brains of Alzheimer's patients according to the method of Gong et al. ((2003) supra). ADDLs (100 nM in F12, or 2 mg total protein in F12) were incubated with cells for 1 hour, then washed and fixed according to standard methods. After washing, cells were incubated with 20C2, 3B7, M94, 2A10, 4E2, 2D6, 4C2, 2B4, 5F10, or 5G12 monoclonal antibody, followed by an anti-mouse seco...

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PUM

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Abstract

The present invention relates to antibodies capable of differentially recognizing the multidimensional conformation of A[beta]-derived diffusible ligands, also known as ADDLs. The antibody of the present invention can distinguish the brain extracts of Alzheimer's patients and control humans, and can be used in methods for detecting ADDLs and diagnosing Alzheimer's disease. The antibody of the present invention also blocks the binding of ADDLs to neurons, the aggregation of ADDLs, and the phosphorylation of tau protein, and thus can be used in methods for preventing and treating diseases related to soluble oligomers of amyloid β1-42.

Description

[0001] Introduction to the invention [0002] This application claims U.S. Provisional Patent Application Serial Nos. 60 / 621,776, filed October 25, 2004, 60 / 652,538, filed February 14, 2005, 60 / 695,526, filed June 30, 2005, and Priority to 60 / 695,528 filed on 30 October, the contents of these provisional applications are hereby incorporated by reference in their entirety. [0003] This invention was made during the course of research funded in part by the National Institutes of Health (grant numbers NIHRO1-AG18877 and NIH RO1-AG22547). The US Government may have certain rights in this invention. Background of the invention [0004] Alzheimer's Disease (Alzheimer's Disease) is a progressive degenerative dementia (Terry et al. (1991) Ann.Neurol.30: 572-580; Coyle (1987) In: Encyclopedia of Neuroscience , Adelman (ed.), Bifkh (usef, Boston-Basel-Stuttgart, pp29-31). In its early stages, Alzheimer's disease is primarily manifested by a profound inability to form new memories (S...

Claims

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Application Information

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IPC IPC(8): A61K39/395
CPCC07K16/18C07K2317/565C07K2317/92G01N2800/2821C07K2317/55C07K2317/24
Inventor 保罗·阿克东安志强安德鲁·J·贝特罗伯特·布里斯伊丽莎白·申·多德森吉恩·金尼威廉·克莱因玛丽·P·兰伯特梁小平保罗·舒格鲁威廉·R·斯特罗尔希尔斯滕·L·维奥拉常磊
Owner MERCK & CO INC
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