Novel method for down-regulation of amyloid

Abstract

Disclosed are novel methods for combating diseases characterized by deposition of amyloid. The methods generally rely on immunization against amyloidogenic proteins (proteins contributing to formation of amyloid) such as beta amyloid (Aβ). Immunization is preferably effected by administration of analogues of autologous amyloidogenic polypeptides, said analogues being capable of inducing antibody production against the autologous amyloidogenic polypeptides. Especially preferred as an immunogen is autologous Aβ which has been modified by introduction of one single or a few foreign, immunodominant and promiscuous T-cell epitopes while substantially preserving the majority of Aβ's B-cell epitopes. Also disclosed are nucleic acid vaccination against amyloidogenic polypeptides and vaccination using live vaccines as well as methods and means useful for the vaccination. Such methods and means include methods for identification of useful immunogenic analogues of the amyloidogenic proteins, methods for the preparation of analogues and pharmaceutical formulations, as well as nucleic acid fragments, vectors, transformed cells, polypeptides and pharmaceutical formulations.

1 . A method for in vivo down-regulation of autologous beta amyloid (Aβ) protein or autologous amyloid precursor protein (APP) in an animal, including a human being, the method comprising effecting presentation to the animal's immune system of an immunogenically effective amount of at least one analogue of the animal's autologous Aβ or autologous APP wherein is introduced at least one isolated foreign T helper epitope (T H  epitope) by means of insertion, addition, deletion, or substitution, or by means of separate coupling to a polyhydroxypolymer carrier backbone of the T H  epitope and an Aβ or APP derived peptide sequence, so that immunization of the animal with the analogue induces production of antibodies against the autologous Aβ or autologous APP in the animal, wherein the foreign T H  epitope is introduced into Aβ or APP as schematically shown for the P2 and P30 epitopes in  FIG. 1 , or wherein the foreign T H  epitope is coupled to a polyhydroxypolymer carrier backbone that also carries an Aβ or APP derived peptide sequence. 
   
   
       2 . The method according to  claim 1 , wherein the introduction has as a result that a substantial fraction of B-cell epitopes of Aβ or APP are preserved and that
 at least one first moiety is introduced which effects targeting of the analogue to an antigen presenting cell (APC) or a B-lymphocyte, and/or   at least one second moiety is introduced which stimulates the immune system, and/or   at least one third moiety is introduced which optimizes presentation of the analogue to the immune system.   
   
   
       3 . The method according to  claim 2 , wherein the analogue is modified by introduction as side groups, by covalent or non-covalent binding to suitable chemical groups in the Aβ, APP, or a subsequence thereof, of the first and/or of the second and/or of the third moiety. 
   
   
       4 . The method according to  claim 1 , wherein introduction of the amino acid substitution and/or deletion and/or insertion and/or addition results in a substantial preservation of the overall tertiary structure of the autologous Aβ or autologous APP. 
   
   
       5 . The method according to  claim 1 , wherein the analogue includes a duplication of at least one B-cell epitope of the autologous Aβ or autologous APP and/or an introduction of a hapten. 
   
   
       6 . The method according to  claim 1 , wherein the foreign T-cell epitope is immunodominant in the animal. 
   
   
       7 . The method according to  claim 1 , wherein the foreign T-cell epitope is promiscuous. 
   
   
       8 . The method according to  claim 7 , wherein the T-cell epitope is selected from a Tetanus toxoid epitope, a diphtheria toxoid epitope, an influenza virus hemaglutinin epitope, and a  P. falciparum  CS epitope. 
   
   
       9 . The method according to  claim 2 , wherein the first moiety is a substantially specific binding partner for a B-lymphocyte specific surface antigen or for an APC specific surface antigen. 
   
   
       10 . The method according to  claim 2 , wherein the second moiety is selected from a cytokine a hormone; and a heat-shock protein. 
   
   
       11 . The method according to  claim 2 , wherein the third moiety is of lipid nature or a polyhydroxypolymer. 
   
   
       12 . The method according to  claim 11 , wherein the polysaccharide serves as a carrier backbone to which the Aβ or APP derived peptide and the foreign T cell epitope are separately bound. 
   
   
       13 . The method according to  claim 12 , wherein the Aβ or APP derived peptide and the foreign T cell epitope are bound via an amide bond to the polysaccharide. 
   
   
       14 . The method according to  claim 1 , wherein the autologous Aβ or APP has been modified so as to preserve B-cell epitopes which are not exposed to the extracellular phase when present in a cell-bound form of the autologous APP. 
   
   
       15 . The method according to  claim 14 , wherein the autologous Aβ or autologous APP has been modified so as to lack at least one B-cell epitope which is exposed to the extracellular phase when present in a cell-bound form of the autologous APP. 
   
   
       16 . The method according to  claim 1  which comprises a substitution of at least one amino acid sequence within autologous Aβ or APP with an amino acid sequence of equal or different length which gives rise to a foreign T H  epitope in the analogue. 
   
   
       17 . The method according to  claim 1 , wherein the analogue comprises the amino acid sequence corresponding to amino acids 672-714 in SEQ ID NO: 2, wherein is inserted an amino acid sequence which gives rise to a foreign T H  epitope in the analogue, or wherein the analogue comprises an amino acid sequence corresponding to amino acids 672-714 of SEQ ID NO: 2, wherein at least one amino acid sequence is substituted by an amino acid sequence of equal or different length so as to give rise to a foreign T H  epitope. 
   
   
       18 . The method according to  claim 1 , wherein presentation to the immune system is effected by having at least two copies of the analogue covalently of non-covalently linked to a carrier molecule capable of effecting presentation of multiple copies of antigenic determinants. 
   
   
       19 . The method according to  claim 1 , wherein the analogue has been formulated with an adjuvant which facilitates breaking of autotolerance to autoantigens. 
   
   
       20 . The method according to  claim 1 , wherein an effective amount of the analogue is administered to the animal via a route selected from the parenteral route, the subcutaneous, and the intramuscular routes; the peritoneal route; the oral route; the buccal route; the sublingual route; the epidural route; the spinal route; the anal route; and the intracranial route. 
   
   
       21 . The method according to  claim 20 , wherein the effective amount is between 0.5 μg and 2,000 μg of the analogue. 
   
   
       22 . The method according to  claim 20 , wherein the analogue is contained in a virtual lymph node (VLN) device. 
   
   
       23 . The method according to  claim 1 , wherein presentation of modified autologous Aβ or autologous APP to the immune system is effected by introducing nucleic acid(s) encoding the analogue into the animal's cells and thereby obtaining in vivo expression by the cells of the nucleic acid(s) introduced. 
   
   
       24 . The method according to  claim 23 , wherein the nucleic acid(s) introduced is/are selected from naked DNA, DNA formulated with charged or uncharged lipids, DNA formulated in liposomes, DNA included in a viral vector, DNA formulated with a transfection-facilitating protein or polypeptide, DNA formulated with a targeting protein or polypeptide, DNA formulated with Calcium precipitating agents, DNA coupled to an inert carrier molecule, DNA encapsulated in chitin or chitosan, and DNA formulated with an adjuvant. 
   
   
       25 . The method according to  claim 24 , wherein the nucleic acid(s) is/are contained in a VLN device. 
   
   
       26 . The method according to  claim 20  which includes at least one administration/introduction per year. 
   
   
       27 . A method for treating and/or preventing and/or ameliorating Alzheimer's disease or other diseases and conditions characterized by Aβ deposits, the method comprising down-regulating autologous Aβ or APP according to the method of  claim 1  to such an extent that the total amount of amyloid is decreased or that the rate of amyloid formation is reduced with clinical significance. 
   
   
       28 . An analogue of an autologous Aβ or autologous APP which is derived from an animal Aβ or APP wherein is introduced at least one isolated foreign T H  epitope as schematically shown for the P2 and P30 epitopes in  FIG. 1 , or wherein at least one foreign T H  epitope is coupled to a polyhydroxypolymer carrier backbone that also carries an Aβ or APP derived peptide sequence so that immunization of the animal with the analogue induces production of antibodies against the autologous Aβ or autologous APP. 
   
   
       29 . An analogue according to  claim 28 , wherein the modification is as defined in  claim 2 . 
   
   
       30 . An immunogenic composition comprising an immunogenically effective amount of an analogue according to  claim 28 , the composition further comprising a pharmaceutically and immunologically acceptable carrier and/or vehicle and optionally an adjuvant. 
   
   
       31 . A nucleic acid fragment which encodes an analogue according to  claim 28 . 
   
   
       32 . A vector carrying the nucleic acid fragment according to  claim 31 . 
   
   
       33 . The vector according to  claim 32  which is selected from the group consisting of a plasmid, a phage, a cosmid, a mini-chromosome, and a virus. 
   
   
       34 . The vector according to  claim 32  comprising, in the 5′→3′ direction and in operable linkage, a promoter for driving expression of the nucleic acid fragment, optionally a nucleic acid sequence encoding a leader peptide enabling secretion of or integration into the membrane of the polypeptide fragment, the nucleic acid fragment according to  claim 31 , and optionally a terminator. 
   
   
       35 . The vector according to  claim 32  which, when introduced into a host cell, is capable or incapable of being integrated in the host cell genome. 
   
   
       36 . The vector according to  claim 34 , wherein a promoter drives expression in a eukaryotic cell and/or in a prokaryotic cell. 
   
   
       37 . A transformed cell carrying the vector of  claim 32 , such as a transformed cell which is capable of replicating the nucleic acid fragment. 
   
   
       38 . The transformed cell according to  claim 37 , which is a microorganism selected from a bacterium, a yeast, a protozoan, or a cell derived from a multicellular organism selected from a fungus, an insect cell, a plant cell, and a mammalian cell. 
   
   
       39 . The transformed cell according to  claim 37 , which expresses the nucleic acid fragment according to  claim 31 . 
   
   
       40 . The method according to  claim 1 , wherein presentation to the immune system is effected by administering a non-pathogenic microorganism or virus which is carrying a nucleic acid fragment which encodes and expresses the analogue. 
   
   
       41 . A composition for inducing production of antibodies against Aβ or APP, the composition comprising
 a nucleic acid fragment according to  claim 31  or a vector according to  claim 32 , and   a pharmaceutically and immunologically acceptable carrier and/or vehicle and/or adjuvant.   
   
   
       42 . A stable cell line which carries the vector according to  claim 32  and which expresses the nucleic acid fragment, and which optionally secretes or carries the analogue according to  claim 28  on its surface. 
   
   
       43 . A method for the preparation of the cell according to  claim 37 , the method comprising transforming a host cell with the nucleic acid fragment according to  claim 31  or with the vector according to  claim 32 . 
   
   
       44 . A method for treating an animal by down-regulating amyloid which comprises administering an effective amount of an analogue according to  claim 28 . 
   
   
       45 . A method for the treatment, prophylaxis or amelioration of Alzheimer's disease or other conditions characterized by amyloid deposits which comprises administering to a patient an effective amount of an analogue according to  claim 28 . 
   
   
       46 . The method according to  claim 7 , wherein said T-cell epitope is a foreign T-cell epitope which is selected from a natural promiscuous T-cell epitope and an artificial MHC-II binding peptide sequence. 
   
   
       47 . The method according to  claim 28 , wherein said Tetanus toxiod epitope is P 2  or P 30 . 
   
   
       48 . The method according to  claim 9 , wherein the first moiety is a hapten or a carbohydrate for which there is a receptor on the B-lymphocyte or the APC. 
   
   
       49 . The method according to  claim 1 , wherein said cytokine is a member selected from the groups consisting of interferon γ (IFN-γ) or an effective part thereof, Flt3L or an effective part thereof, interleukin 1 (IL-1) or an effective part thereof, interleukin 2 (IL-2) or an effective part thereof, interleukin 4 (IL-4) or an effective part thereof, interleukin 6 (IL-6) or an effective part thereof, interleukin 12 (IL-12) or an effective part thereof, interleukin 13 (IL-13) or an effective part thereof, interleukin 15 (IL-15) or an effective part thereof, and granulocyte-macrophage colony stimulating factor (GM-CSF) or an effective part thereof; and granulocyte-macrophage colony stimulating factor (GM-CSF) or an effective part thereof; and said heat stock protein is a member selected from the group consisting of HSP70 or an effective part thereof such as HSP70 or an effective part thereof, HSP90 or an effective part thereof, HSC70 or an effective part thereof, GRP94 or an effective part thereof, and calreticulin (CRT) or an effective part thereof. 
   
   
       50 . The method according to  claim 11 , wherein said third moiety of a liquid nature is a member selected from the group consisting of a palmitoyl group, a myristyl group, a farnesyl group, a geranyl-geranyl group, a GPI-anchor, and an N-acyl diglyceride group and said polyhydroxy polymer is a polysaccharide. 
   
   
       51 . The method according to  claims 20 , wherein the parenteral route is intracutaneous. 
   
   
       52 . The method according to  claim 26 , which comprises at least 2, 3, 4, 6 or 12 administrations/introduction per year. 
   
   
       53 . The method according to  claim 32 , which is capable of autonomous replication. 
   
   
       54 . The transformed cell according to  claim 37 , which is a transformed cell which is capable of replicating the nucleic acid fragment. 
   
   
       55 . The transformed cell according to  claim 38 , wherein said insect cell is an S 2  or an SF cell. 
   
   
       56 . The transformed cell according to  claim 37 , wherein the transformed cell secretes or carries on its surface the analogue according to  claim 28 . 
   
   
       57 . The analogue according to  claim 28 , wherein said T H  epitope is at least one member selected from the group consisting of tetanus toxoid P20, tetanus toxoid P30, diphtheria toxoid, hemaglutinin,  P. falciparum  CS antigen, artificial MHC-II binding peptide sequence and PADRE. 
   
   
       58 . The analogue according to  claim 29 , wherein said first molecule is at least one member selected from the group consisting of a carbohydrate for which there is a receptor on a B-lymphocyte, a binding partner for a B-lymphocyte specific surface antigen, an antibody that specifically binds an APC surface molecule and an antibody that specifically binds a lymphocyte surface molecule. 
   
   
       59 . The analogue according to  claim 58 , wherein said carbohydrate is mannan, mannose or both mannan and mannose. 
   
   
       60 . The analogue according to  claim 58 , wherein said APC surface molecule is at least one member selected from the group consisting of an FCγ receptor, CD40 and CTLA-4. 
   
   
       61 . The analogue according to  claim 58 , wherein said lymphocyte surface molecule is at least one member selected from the group consisting of an FCγ receptor, CD40 and CTLA-4. 
   
   
       62 . The analogue according to  claim 68 , wherein said second molecule is at least one member selected of the group consisting of haptens, cytokines, heat-shock proteins and toxins. 
   
   
       63 . The analogue according to  claim 62 , wherein said cytokine is at least one member selected from the group consisting of interferon γ, interleukin 1, interleukin 2, interleukin 4, interleukin 6, interleukin 12, interleukin 13, interleukin 15 and granulocyte-macrophage colony stimulating factor. 
   
   
       64 . The analogue according to  claim 62 , wherein said heat-shock protein is at least one member selected from the group consisting of HSP70, HSP90 and HSC70. 
   
   
       65 . The analogue according to  claim 62 , wherein said toxin is at least one member selected from the group consisting of listeriolycin, lipid A and heat-labile enterotoxin. 
   
   
       66 . The analogue according to  claim 69 , wherein said third molecule is at least one member selected of the group consisting of lipidation anchors, OspA, complement factor C3 and the C3d fragment of complement factor C3. 
   
   
       67 . The analogue according to  claim 66 , wherein said lipidation anchor is at least one member selected from the group consisting of a myristyl group, a farnesyl group, a geranyl-geranyl group, a GPI-anchor and an N-acyl diglyceride group). 
   
   
       68 . The analogue according to  claim 28 , wherein said analogue is further modified by including a second molecule which stimulates the immune system. 
   
   
       69 . The analogue according to any one of  claims 28 ,  29  and  68 , wherein said analogue is further modified by including a third molecule which improves presentation of the analogue to the immune system.