Antibodies specific for the protofibril form of beta-amyloid protein

Abstract

Isolated antibodies have been characterized which show specific affinity to a repeating conformational epitope of a protofibril form of the human β-amyloid peptide as compare to low molecular weight forms of β-amyloid peptide. These isolated antibodies and related pharmaceutically effective compositions may be useful in the therapeutic and/or prophylactic treatment of Alzheimer's disease by effectively blocking the ability of the protofibril form of β-amyloid peptide to form fibril forms linked with complications associated with Alzheimer's disease. The isolated antibodies of the present invention are also useful in various diagnostic assays and associated kits.

1 . An isolated antibody, or an antigen binding fragment thereof, that specifically interacts and shows a measurable affinity to a conformational epitope of a protofibril form of Aβ peptide, whereby the protofibril epitope is represented by an exposed region of an Aβ-protofibril form comprising the amino acid sequence as set forth in SEQ ID NO:2, wherein said antibody shows minimal or no affinity for monomer or dimer forms of Aβ peptide. 
     
     
         2 . The isolated antibody or antigen binding fragment of  claim 1 , that specifically interacts and shows a measurable affinity to a conformational epitope of a protofibril form of Aβ peptide, whereby wherein the protofibril epitope is represented by an exposed region of an Aβ-protofibril form comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4, wherein said antibody shows minimal or no affinity for monomer or dimer forms of Aβ peptide. 
     
     
         3 . The antibody or antigen binding fragment of  claim 2 , wherein the antibody is a monoclonal antibody. 
     
     
         4 . The antibody or antigen binding fragment of  claim 3  wherein the antibody is designated 13C3. 
     
     
         5 . The antibody or antigen binding fragment of  claim 3  wherein the antibody is a humanized monoclonal antibody. 
     
     
         6 . The antibody or antigen binding fragment of  claim 3  wherein the antibody is a human monoclonal antibody. 
     
     
         7 . The antibody or antigen binding fragment of  claim 2  further comprising a variable light chain comprised of the amino acid sequence as set forth in SEQ ID NO:5 or a variable heavy chain comprised of the amino acid sequence as set forth in SEQ ID NO:7. 
     
     
         8 . The antibody or antigen binding fragment of  claim 2  further comprising a variable light chain comprising a CDR1 region as set forth in SEQ ID NO:13, a CDR2 region as set forth in SEQ ID NO:14, and a CDR3 as set forth in SEQ ID NO:15. 
     
     
         9 . The antibody or antigen binding fragment of  claim 2  further comprising a variable heavy chain comprised of a CDR1 region as set forth in SEQ ID NO:20, a CDR2 region as set forth in SEQ ID NO:21, and a CDR3 as set forth in SEQ ID NO:22. 
     
     
         10 . A method of producing a monoclonal antibody which specifically binds in vitro to a repeating conformational epitope of a protofibril form of β-amyloid peptide while showing minimal affinity to low molecular weight forms of β-amyloid peptide, comprising:
 (a) immunizing a mammal with the protofibril form of β-amyloid peptide; 
 (b) harvesting B-cells of said mammal; 
 (c) creating hybridomas from the harvested B-cells, wherein said hybridomas produce antibodies; and, 
 (d) selecting hybridomas which produce antibodies specifically binding to the protofibril form of β-amyloid peptide while showing minimal affinity to monomer or dimer forms of β-amyloid peptide. 
 
     
     
         11 . A method for quantifying the amount of a protofibril form of β-amyloid peptide in a tissue or fluid sample, comprising:
 (a) obtaining the tissue or fluid sample from a subject; 
 (b) contacting the tissue or fluid sample with the antibody of  claim 1  or fragment thereof that specifically binds to the protofibril form of β-amyloid peptide while showing minimal affinity to low molecular weight forms of β-amyloid peptide; and, 
 (c) quantifying the amount of protofibril form of β-amyloid peptide in the sample. 
 
     
     
         12 . A kit for detecting protofibril form of β-amyloid peptide while showing minimal affinity to low molecular weight forms of β-amyloid peptide, comprising:
 (a) the antibody of  claim 1  or a fragment thereof, capable of specifically binding in vitro to a repeating conformational epitope of a protofibril form of β-amyloid peptide while showing minimal affinity to low molecular weight forms of β-amyloid peptide; and, 
 (b) a reagent that binds, directly, or indirectly, to said antibody or the fragment thereof. 
 
     
     
         13 . A method of inhibiting deposition of β-amyloid fiber plaques within a mammal which comprises administering to the mammal pharmaceutically effective amount of the antibody or antigen binding fragment of  claim 1  which specifically interacts with the protofibril form of β-amyloid peptide so as to inhibit formation and deposition of β-amyloid fiber plaques. 
     
     
         14 . A hybridoma which secretes an antibody selected from the group consisting of 13C3, 19A6 and 1D1. 
     
     
         15 . An isolated nucleic acid molecule encoding a variable chain fragment of the antibody of  claim 1  wherein the variable chain fragment comprises the amino acid sequence as set forth in SEQ ID NO:7 or 5. 
     
     
         16 . The isolated nucleic acid molecule of  claim 15 , wherein the nucleic acid molecule comprises the nucleotide sequence as set for in SEQ ID NO:8 or 6. 
     
     
         17 . An expression vector comprising the nucleic acid molecule of  claim 16 . 
     
     
         18 . A host cell comprising the expression vector of  claim 17 . 
     
     
         19 . An isolated variable chain fragment of the antibody of  claim 1  comprising the amino acid sequence set forth in SEQ ID NO:7 or 5. 
     
     
         20 . A pharmaceutical composition comprising the antibody or antigen binding fragment of  claim 1 , which specifically interacts with the protofibrillar form of β-amyloid, wherein said specific interaction is characterized by a ratio of the affinity of said variable region fragment for the protofibrillar Aβ form to the affinity for other Aβ forms greater than about 2.