Ager-peptides and use thereof

Abstract

The present invention relates to the identification, functionality and use of domains from the N terminus of the receptor for Advanced Glycation End Products (AGER). These domains, called receptor mulitimerization epitope (RME), are highly conserved in all AGER protein sequences. They represent the mediators for AGER self-association and heteromerization with other proteins. The invention likewise relates to the identification, functionality and use of peptides derived from the C domain of AGER (AGER-CDP). The AGER RMEs and AGER-CDPs of the invention are suitable as target for identifying AGER ligands which modulate the natural ligand interaction; as immunogen for active or passive immunization of individuals, as diagnostic means for identifying immunogenic reactions, and as peptide ligands for modulating protein-protein interactions involving AGER.

1 . The use of the receptor multimerization epitope (RME) of the Advanced Glycation End Products Receptor (AGER), comprising a peptide fragment, capable of auto-multimerization, of the N-terminal AGER ectodomain, or of a peptide (AGER-CDP) derived from the AGER Ig-like C domain, or of a functional, immunogenic equivalent of AGER-RME or of AGER-CDP, as an immunogen for preparing a polyclonal antiserum or monoclonal antibodies against AGER-RME or AGER-CDP. 
     
     
         2 . The use according to  claim 1 , where AGER-RME with a length of about 8 to 50 amino acid residues which is derived from the human AGER ectodomain having an amino acid sequence as shown in Genbank Ref. Seq. sequence NM_001136 or a functionally equivalent ectodomain, in particular the V domain thereof, is employed. 
     
     
         3 . The use according to  claim 1 , where AGER-RME comprises the following sequence: 
       
         
           
                 
               
                   (SEQ ID NO: I) 
                 
                 
                 
               
                     
                   C(K/R)GAPKKP(P/T)Q(Q/R/K)LE 
                 
             
                
               
            
             
                
               
            
           
         
       
     
     
         4 . The use according to  claim 3 , where AGER-RME comprises a sequence which is 
       
         
           
                 
               
                   (SEQ ID NO: 2) 
                 
                 
                 
               
                     
                   CRGAPKKPPQQLE, 
                 
                     
                     
                 
                 
               
                   (SEQ ID NO: 3) 
                 
                 
                 
               
                     
                   CKGAPKKPPQRLE, 
                 
                     
                     
                 
                 
               
                   (SEQ ID NO: 4) 
                 
                 
                 
               
                     
                   CKGAPKKPTQKLE. 
                 
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
               
            
           
         
       
     
     
         5 . The use according to  claim 4 , where AGER-RME comprises a sequence which is selected from:
   X 1 —X 2 —X 3 —X 4  
   in which   X 1  is a hydrogen atom, or the amino acid Q or the dipeptide DQ;   X 2  is NITARIG(K/E)PL(V/M)L(N/S/K) (SEQ ID NO:5);   X 3  is a sequence according to  claim 4 ; and   X 4  is the peptide sequence WKLN.   
     
     
         6 . The use according to  claim 5 , where AGER-RME is selected from SEQ ID NO: 1, 2, 3 and 5, and sequences of the general formula
 X 1 —X 2 —X 3 —X 4  in which X 1  to X 4  are as defined according to  claim 5 .   
     
     
         7 . The use according to  claim 1 , where AGER-CDP with a length of about 5 to 50 amino acid residues which is derived from the human AGER ectodomain having an amino acid sequence as shown in Genbank Ref. Seq. sequence NM_00 1 136 or a functionally equivalent ectodomain, in particular the C-like domain thereof, is employed. 
     
     
         8 . The use according to  claim 7 , where the peptide comprises one of the following sequences: 
       
         
           
                 
                 
               
                     
                   a) 
                 
                 
               
                   (SEQ ID NO: 31) 
                 
                 
                 
               
                     
                   DGKPLVPNEKGVSVKEQTRRHPETGLFTLQ, 
                 
                     
                     
                 
                     
                   b) 
                 
                 
               
                   (SEQ ID NO: 32) 
                 
                 
                 
               
                     
                   TLQSELMVTPARGGDPRPTFSCSFSPGLPR, 
                 
                     
                   or and 
                 
                     
                   c) 
                 
                 
               
                   (SEQ ID NO: 33) 
                 
                 
                 
               
                     
                   LPRHRALRTAPIQPRVWEPVPLEEVQLVVE. 
                 
             
                
               
            
             
                
               
            
             
                
                
                
               
            
             
                
               
            
             
                
                
                
               
            
             
                
               
            
             
                
               
            
           
         
       
     
     
         9 . The use according to  claim 8 , where the AGER-CDP is selected from SEQ ID NO: 31, 32 and 33. 
     
     
         10 . The use according to  claim 1 , where the AGER-RME or AGER-CDP is in the form of a cyclic peptide. 
     
     
         11 . The use of an AGER-RME or of an AGER-CDP as defined in  claim 1  for the diagnosis of diseases or stages of diseases in which auto-antibodies against the AGER-RME or the AGER-CDP occur. 
     
     
         12 . The use of an AGER-RME or of an AGER-CDP as defined in  claim 1 , of the AGER ectodomain having an amino acid sequence as shown in Genbank Ref. Seq. sequence NM_00 1 136 and N-terminal subfragments thereof, and of muteins and derivatives of these AGER molecules, or of AGER-RME- or AGER-CDP binding ligands for producing a pharmaceutical composition for the diagnosis or therapy of AGER-mediated diseases or stages of diseases. 
     
     
         13 . The use according to  claim 12 , where an AGER-RME- or AGER-CDP-binding ligand is used. 
     
     
         14 . The use according to  claim 12 , where the diseases or stages of diseases are associated with an AGER/AGER, AGER/ligand, AGER/receptor, AGER/receptor/ligand, AGER/receptor/coreceptor and/or AGER/receptor/counter-receptor interaction. 
     
     
         15 . The use according to  claim 12 , where the diseases or stages of diseases are selected from
 a) mechanical injuries of the skull and spinal cord,   b) ischaemic damage,   c) chronic disorders selected from neurodegenerative, inflammatory and autoimmune disorders,   d) diabetic sequelae,   e) tumor progression and metastasis,   f) altered neurogenesis processes associated with psychotic disorders and chronic states of pain caused by excessive neurite sprouting and/or pathological synaptogenesis,   g) impairments of neuronal regeneration, of axonal sprouting, of neurite extension and of neuronal plasticity,   h) central/peripheral amyloid disorders; and   i) arteriosclerosis.   
     
     
         16 . The use according to  claim 12 , where the AGER-RME- or AGER-CDP-binding ligand is an anti-AGER-RME or anti-AGER-CDP antibody. 
     
     
         17 . The use of AGER-RME or of an AGER-CDP according to  claim 1  as target for the detection or identification of AGER-binding ligands. 
     
     
         18 . The use of AGER-RME or of an AGER-CDP according to  claim 1  as an immunogen for active or passive immunization. 
     
     
         19 . A polyclonal anti-AGER-RME or anti-AGER-CDP antiserum obtainable by immunization of a mammal with an antigenic amount of an AGER-RME peptide or of an AGER-CDP as defined in  claim 1 . 
     
     
         20 . A monoclonal anti-AGER-RME or anti-AGER-CDP antibody or an antigen-binding fragment thereof, if appropriate in humanized form. 
     
     
         21 . The antiserum according to  claim 19 , having at least one of the following properties:
 a) improved specificity for an AGER-RME or an AGER-CDP,   b) improved specificity for a new epitope formed with involvement of the AGER-RME or an AGER-CDP,   c) inhibition of the AGER-RME-mediated multimerization with sRAGE or anti-AGER-RME antibody;   d) specific recognition of an AGER ligand-induced receptor status of sRAGE;   e) induction of a receptor configuration of sRAGE which modulates the binding of the AGER ligand to sRAGE.   
     
     
         22 . A monoclonal bispecific antibody comprising
 a) first antigen-binding domain derived from a monoclonal antibody according to  claim 20 , and   b) a second antigen-binding domain with specificity for cell surface receptors which are able to interact with AGER-RME or AGER-CDP, or with specificity for a ligand, coreceptor or counter-receptor for one of these receptors, or an antigen-binding fragment thereof, if appropriate in humanized form.   
     
     
         23 . A hybrid protein comprising an AGER-RME or an AGER-CDP according to  claim 1 . 
     
     
         24 . The hybrid protein according to  claim 23 , additionally comprising a functional part of a protein selected from immunglobulins and fragments thereof. 
     
     
         25 . The hybrid protein according to  claim 23 , comprising an Ig Fc fragment functionally linked to AGER-RME or AGER-CDP. 
     
     
         26 . An AGER-RME derivative or AGER-CDP derivative comprising AGER-RME or AGER-CDP according to  claim 1  in PEGylated form or coupled to a marker. 
     
     
         27 . A pharmaceutical composition comprising in a pharmaceutically acceptable carrier the monoclonal anti-AGER-RME or anti-AGER-CDP antibody or an antigen-binding fragment thereof of  claim 20 . 
     
     
         28 . (canceled) 
     
     
         29 . The pharmaceutical composition according to  claim 27 , additionally comprising as active ingredient an active substance selected from
 f) neurotrophic factors, inosine, neuroimmunophilins, chondroitin sulfate proteoglycan-degrading enzymes;   g) antibodies against neurite growth inhibitors, Nogo-A, MAG, Omgp, and/or their receptors,   h) soluble NgR fragment, Nogo-A peptide fragments,   i) inhibitors of the p75-mediated signal cascade, and   j) cAMP and functional analogs, protein kinase A, arginase I, polyamines, ciliary neurotrophis factor.   
     
     
         30 . The pharmaceutical composition according to  claim 27 , for intrathecal, intravenous, subcutaneous, oral or parenteral, nasal and inhalational administration. 
     
     
         31 . An immunogen comprising AGER-RME or AGER-CDP according to  claim 1  in a pharmaceutically acceptable carrier and if appropriate with an adjuvant for active immunization. 
     
     
         32 . A method for detecting effectors of the AGER receptor, where a sample in which an effector is suspected is incubated with an AGER-RME polypeptide or an AGER-CDP according to  claim 1 , and the mixture is investigated for the formation of an effector-AGER-RME complex or an effector-AGER-CDP complex. 
     
     
         33 . An expression vector comprising at least one coding nucleic acid sequence for AGER-RME or AGER-CDP as defined according to  claim 1 , operatively linked to at least one regulatory nucleic acid sequence. 
     
     
         34 . A recombinant microorganism which harbors at least one vector according to  claim 33 . 
     
     
         35 . A hybridoma cell line which produces a monoclonal antibody according to  claim 20 . 
     
     
         36 . A method for preparing AGER-RME or AGER-CDP, where a recombinant microorganism according to  claim 34  is cultivated, and the produced protein product is isolated from the culture. 
     
     
         37 . A method for producing a monoclonal antibody, where a hybridoma cell line according to  claim 35  is cultivated, and the produced protein product is isolated from the culture. 
     
     
         38 . A functional equivalent of AGER-RME as defined in  claim 1 , which has a degree of homology of less than 100% to SEQ ID NO: 6. 
     
     
         39 . The functional equivalent of AGER-RME according to  claim 38 , where this has at least one of the following properties:
 a) inhibition of signal transduction in the actin cytoskeletal rearrangement (ACR) assay;   b) competition with sRAGE for binding with an AGER ligand;   c) auto-multimerization or multimerization with AGER-RME or s-RAGE.   
     
     
         40 . The functional equivalent of AGER-RME according to  claim 38 , which has a core sequence with high positive charge density of the following general formula:
   ZX 1 ZZX 2 Z   in which   the residues Z are independently of one another an amino acid residue having a positively charged side chain; and   the residues X 1  and X 2  are independently of one another any 1 to 5 identical or different amino acid which have no positively charged side chains.   
     
     
         41 . A combination of at least two monoclonal antibodies differing in antigenic specificity, where at least one first monoclonal antibody binds to an antigen which is formed wholly or partly from a sequence region of the Ig-like V domain of AGER, and at least one second monoclonal antibody binds to an antigen which is formed wholly or partly by a sequence region of an AGER domain different from the Ig-like V domain. 
     
     
         42 . The antibody combination as claimed in  claim 41 , where the AGER domain different from the Ig-like V domain is the Ig-like C domain of AGER. 
     
     
         43 . The antibody combination as claimed in  claim 41 , where at least one first monoclonal antibody and, if appropriate, at least one second monoclonal antibody competes with the binding of AGER or of a soluble equivalent thereof with a binding partner. 
     
     
         44 . The antibody combination as claimed in  claim 43 , where the binding partner is an Aβ globulomer. 
     
     
         45 . The antibody combination as claimed in  claim 41  for use as medicament. 
     
     
         46 . A pharmaceutical composition comprising an antibody combination as claimed  claim 41 . 
     
     
         47 . The use of an antibody combination as claimed in  claim 41  for producing a pharmaceutical composition for the therapy of diseases or pathological states.