Amyloid beta(1-42) oligomers, derivatives thereof and antibodies thereto, methods of preparation thereof and use thereof

Abstract

The invention relates to neuromodulatory oligomers of the amyloid β(1-42) protein, a particular production method, by means of which the oligomer can be obtained in a reproducible manner at high yield, the use of the oligomers and diagnostic and therapeutic agents, for the generation of oligomer-specific antibodies and for the discovery of substances which can interact with the oligomers and in the formation thereof.

1 . An oligomer of the amyloid β(1-42) protein, having an apparent molecular weight of about 15 kDa in SDS gel electrophoresis, or a derivative thereof. 
     
     
         2 . An oligomer of the amyloid β(1-42) protein, having an apparent molecular weight of about 20 kDa in SDS gel electrophoresis, or a derivative thereof. 
     
     
         3 . An oligomer of the amyloid β(1-42) protein, having an apparent molecular weight of about 38 kDa in SDS gel electrophoresis, or a derivative thereof. 
     
     
         4 . An oligomer of the amyloid β(1-42) protein, having an apparent molecular weight of about 48 kDa in SDS gel electrophoresis, or a derivative thereof. 
     
     
         5 . A composition, comprising a mixture of an oligomer as claimed in  claim 1  and an oligomer as claimed in  claim 2 , or of derivatives thereof. 
     
     
         6 . A composition, comprising a mixture of an oligomer as claimed in  claim 3  and an oligomer as claimed in  claim 4 , or of derivatives thereof. 
     
     
         7 . The oligomer or composition as claimed in any of  claims 1  to  6 , characterized in that the derivatives have a detectable label. 
     
     
         8 . The oligomer or composition as claimed in  claim 7 , characterized in that the label is a fluorescent, luminescent, colorimetric, radioactive or magnetic label or is a label having affinity to complementary binding partners. 
     
     
         9 . The oligomer or composition as claimed in any of  claims 1  to  8 , characterized in that the oligomer or the derivative is crosslinked. 
     
     
         10 . The oligomer or composition as claimed in any of  claims 1  to  8 , characterized in that the derivative is obtainable by proteolytic cleavage of the oligomer. 
     
     
         11 . The oligomer or composition as claimed in  claim 10 , characterized in that the derivative is an oligomer of an Aβ(x-42) fragment, where x is 8 to 24, preferably from 10 to 22, and in particular from 12 to 20. 
     
     
         12 . The oligomer or composition as claimed in  claim 11 , characterized in that the derivative is an oligomer of the Aβ(12-42) fragment or of the Aβ(20-42) fragment. 
     
     
         13 . A method for preparing oligomers of a derivatized or nonderivatized amyloid β(1-42) protein, characterized in that monomeric amyloid β(1-42) protein or a derivative thereof is exposed to a detergent and, optionally, at least one oligomer or derivative thereof is obtained in a manner known per se. 
     
     
         14 . A method for preparing oligomers of a derivatized or nonderivatized amyloid β(1-42) protein, characterized in that monomeric amyloid β(1-42) protein or a derivative thereof is exposed to a detergent, the detergent action is then reduced and incubation is continued, and, optionally, at least one oligomer or derivative thereof is obtained in a manner known per se. 
     
     
         15 . The method as claimed in  claim 14 , characterized in that the detergent is ionic. 
     
     
         16 . The method as claimed in  claim 15 , characterized in that the detergent is sodium dodecyl sulfate. 
     
     
         17 . The method as claimed in any of  claims 13  to  16 , characterized in that the time of exposure to the detergent is from about 1 to 20 hours. 
     
     
         18 . The method as claimed in  claim 17 , characterized in that the temperature of exposure is from about 20 to 50° C. 
     
     
         19 . The method as claimed in any of  claims 14  to  16 , characterized in that the time of continued incubation is from 10 to 30 hours. 
     
     
         20 . The method as claimed in  claim 19 , characterized in that the temperature of exposure is from about 20 to 50° C. 
     
     
         21 . The method as claimed in any of  claims 13  to  20 , characterized in that the amyloid β(1-42) protein is unfolded at least partially beforehand. 
     
     
         22 . The method as claimed in  claim 21 , characterized in that for this purpose the protein is exposed to a hydrogen bond-breaking agent, preferably HFIP. 
     
     
         23 . The method as claimed in any of  claims 13  to  22 , characterized in that the oligomer is exposed to a crosslinker. 
     
     
         24 . The method as claimed in any of  claims 13  to  23 , characterized in that the oligomer is exposed to a protease. 
     
     
         25 . The use of an oligomer, of a derivative thereof or of a composition as claimed in any of  claims 1  to  12  in a diagnostic in vitro detection method. 
     
     
         26 . The use of an oligomer, of a derivative thereof or of a composition as claimed in any of  claims 1  to  12  in a diagnostic in vivo detection method. 
     
     
         27 . The use of an oligomer, of a derivative thereof or of a composition as claimed in any of  claims 1  to  12  for generating oligomer-specific antibodies. 
     
     
         28 . A method for characterizing a substance or a substance mixture, which comprises
 i) providing said substance or said substance mixture;   ii) exposing an oligomer, a derivative thereof or a composition as claimed in any of  claims 1  to  12  to said substance or said substance mixture; and   iii) determining, whether said substance or particular portions of said substance mixture bind to said oligomer, said derivative thereof or to at least one oligomer or derivative thereof present in said composition.   
     
     
         29 . A substance or a portion of a substance mixture, characterized in that said substance or said portion is identifiable by a method as claimed in  claim 28  as a ligand binding to the oligomer, the derivative thereof or to at least one oligomer or derivative thereof present in the composition. 
     
     
         30 . The use of a substance or of a portion of a substance mixture as claimed in  claim 29  for preparing a medicament for the treatment of amyloid β-associated, in particular dementing, disorders. 
     
     
         31 . The use of a substance or of a portion of a substance mixture as claimed in  claim 29  for preparing a composition for diagnosing amyloid β-associated, in particular dementing, disorders. 
     
     
         32 . A method for producing antibodies, which comprises
 i) immunizing a host with at least one oligomer, derivative thereof or composition as claimed in any of  claims 1  to  12 ; and   ii) obtaining an antibody-containing host serum produced as a response to said immunization.   
     
     
         33 . The method as claimed in  claim 32 , characterized in that at least one antibody of the serum is selected, which recognizes the oligomer or derivative thereof used as immunogen or at least one oligomer or derivative thereof present in the composition used as immunogen. 
     
     
         34 . A method for preparing antibodies with specificity for an oligomer or derivative thereof as claimed in any of  claims 1  to  12 , which comprises
 i) providing an antigen comprising said oligomer or derivative thereof;   ii) exposing an antibody repertoire to said antigen; and   iii) selecting from said repertoire an antibody which specifically binds to said oligomer or derivative thereof.   
     
     
         35 . The method as claimed in  claim 34 , wherein the antibody repertoire is exposed to the antigen in vivo by immunizing an animal with said antigen. 
     
     
         36 . The method as claimed in  claim 34 , wherein furthermore a number of hybridomas from lymphocytes of the animal are established and a hybridoma secreting an antibody which specifically binds to the oligomer or derivative thereof is selected. 
     
     
         37 . The method as claimed in  claim 34 , wherein the animal is selected from among mice, rats, chickens, camelids, cynomolus monkeys, rabbits and goats. 
     
     
         38 . The method as claimed in  claim 37 , wherein the animal is a knockout mouse with APP deficiency. 
     
     
         39 . The method as claimed in  claim 37 , wherein the animal is a transgenic mouse having human immunoglobulin genes, which generates human antibodies after an antigenic stimulus. 
     
     
         40 . The method as claimed in  claim 37 , wherein the animal is a mouse with severe combined immunodeficiency (SCID), which has been reconstituted with human peripheral mononuclear blood cells or lymphoid cells or precursors thereof. 
     
     
         41 . The method as claimed in  claim 37 , wherein the animal is a mouse which has been treated with lethal total body irradiation, then protected against radiation with bone marrow cells from a mouse with severe combined immunodeficiency (SCID) and into which subsequently functional human lymphocytes or precursors thereof have been transplanted. 
     
     
         42 . The method as claimed in  claim 34 , wherein the antibody repertoire is exposed to the antigen in vitro by screening a recombinant antibody library with said antigen. 
     
     
         43 . The method as claimed in  claim 42 , wherein the recombinant antibody library is expressed on the surface of a bacteriophage. 
     
     
         44 . The method as claimed in  claim 42 , wherein the recombinant antibody library is expressed on the surface of yeast cells. 
     
     
         45 . The method as claimed in  claim 42 , wherein the recombinant antibody library is expressed on the surface of bacterial cells. 
     
     
         46 . The method as claimed in  claim 42 , wherein the recombinant antibody library is expressed as RNA-protein fusions. 
     
     
         47 . The method as claimed in  claim 42 , wherein the recombinant antibody library is an scFv library or an Fab library. 
     
     
         48 . The method as claimed in  claim 42 , wherein the recombinant antibody library is a single domain library. 
     
     
         49 . The method as claimed in  claim 34 , wherein the antibody repertoire is exposed to the antigen by immunizing an animal in vivo with said antigen and a recombinant antibody library prepared from the lymphoid cells of said animal is then screened with said antigen. 
     
     
         50 . The method as claimed in  claim 34 , wherein the antibody repertoire is exposed to the antigen by immunizing an animal in vivo with said antigen and a recombinant antibody library prepared from the lymphoid cells of said animal is then subjected to affinity maturation in vitro. 
     
     
         51 . The method as claimed in  claim 34 , wherein the antibody repertoire is exposed to the antigen by immunizing an animal with said antigen and individual cells secreting the antigen-binding antibody are then selected and cDNAs for the variable regions of the heavy and light chains are obtained from the single cells. 
     
     
         52 . An antibody, obtainable by a method as claimed in any of  claims 32  to  51 . 
     
     
         53 . A protein, which binds specifically to an oligomer or derivative thereof as claimed in any of  claims 1  to  12 . 
     
     
         54 . The protein as claimed in  claim 53 , which binds the oligomer or derivative thereof with an affinity in the range of K D =10 −6 -10 −12  M. 
     
     
         55 . The protein as claimed in  claim 54 , which binds the oligomer or derivative thereof with an affinity greater than K d =10 −8  M. 
     
     
         56 . The protein as claimed in  claim 54 , which binds the oligomer or derivative thereof with an affinity greater than K d =10 −9  M. 
     
     
         57 . The protein as claimed in  claim 54 , which binds the oligomer or derivative thereof with an affinity greater than K d =10 −10  M. 
     
     
         58 . The protein as claimed in  claim 54 , which binds the oligomer or derivative thereof with an affinity greater than K d =10 −11  M. 
     
     
         59 . The protein as claimed in any of  claims 53  to  58 , which binds monomeric Aβ(1-42) protein and/or monomeric Aβ(1-40) protein with an affinity of less than K d =10 −8  M. 
     
     
         60 . The protein as claimed in any of  claims 53  to  59 , wherein the affinity of said protein to the oligomer or derivative thereof is at least 10 times greater than to monomeric Aβ(1-42) protein and/or monomeric Aβ(1-40) protein. 
     
     
         61 . The protein as claimed in any of  claims 53  to  59 , wherein the affinity of said protein to the oligomer or derivative thereof is at least 100 times greater than to monomeric Aβ(1-42) protein and/or monomeric Aβ(1-40) protein. 
     
     
         62 . The protein as claimed in any of  claims 53  to  59 , wherein the affinity of said protein to the oligomer or derivative thereof is at least 1000 times greater than to monomeric Aβ(1-42) protein and/or monomeric Aβ(1-40) protein. 
     
     
         63 . The protein as claimed in any of  claims 53  to  62 , which binds the oligomer or derivative thereof with a rate constant k off  of 0.1 s −1  or lower, as determined by means of surface plasmon resonance. 
     
     
         64 . The protein as claimed in any of  claims 53  to  63 , which inhibits the activity of the oligomer or derivative thereof with an inhibition constant IC 50  of 1×10 −5  or lower. 
     
     
         65 . The protein as claimed in any of  claims 53  to  64 , which is an antibody or an antigen-binding moiety thereof. 
     
     
         66 . The antibody as claimed in  claim 65 , which is a substantially human antibody or an antigen-binding moiety thereof. 
     
     
         67 . The antibody as claimed in  claim 65 , which is a chimeric antibody or an antigen-binding moiety thereof. 
     
     
         68 . The antibody as claimed in  claim 65 , which is a CDR graft antibody or an antigen-binding moiety thereof. 
     
     
         69 . The protein as claimed in any of  claims 53  to  64 , which is a molecule derived from the T-cell receptor or a receptor domain derived from the T-cell receptor or a fusion protein of said receptor domain with an Fc moiety of an immunoglobulin. 
     
     
         70 . A pharmaceutical agent, comprising a protein as claimed in any of  claims 53  to  69  and, optionally, a pharmaceutically suitable carrier. 
     
     
         71 . The use of a protein as claimed in any of  claims 53  to  69  for preparing a medicament for the treatment of amyloid β-associated, in particular dementing, disorders. 
     
     
         72 . The use of a protein as claimed in any of  claims 53  to  69  for preparing a composition for diagnosing amyloid β-associated, in particular dementing, disorders. 
     
     
         73 . A vaccine, comprising at least one oligomer or at least one derivative thereof or a composition as claimed in any of  claims 1  to  12 . 
     
     
         74 . The use of an oligomer or derivative thereof or of a composition as claimed in any of  claims 1  to  12  for preparing a vaccine for the treatment of amyloid β-associated, in particular dementing, disorders.