Monoclonal antibodies specific PHF-TAU, hybridomas secreting them, antigen recognition by these antibodies and their applications

Abstract

A peptide from 6 to 100 amino acids long, including an amino acid sequence depicted by one of a) Val-Arg-Thr-Pro-Pro (amino acid 229-233; human tau numbering, SEQ ID NO 2) wherein the peptide is able to form an immunological complex with the monoclonal antibody AT180 produced by the hybridoma deposited at the ECACC on Dec. 22, 1992 under No.92122204 and b) Pro-Lys-Thr-Pro-Pro (amino acid 179-183; human tau numbering, SEQ ID NO 3) wherein the peptide is able to form an immunological complex with the monoclonal antibody AT270 produced by the hybridoma deposited at the ECACC on Jul. 7,1993 under No.93070774, with Thr being phosphorylated. A method of detecting PHF-tau protein one of the peptides is also disclosed.

1 . A monoclonal antibody which forms an immunological complex with a phosphorylated epitope of an antigen belonging to abnormally phosphorylated tau protein (phf-tau) residing in the region spanning posotions 143-254 with the following amino acid sequence:  
       
         
           
                 
                 
                 
               
                     
                 
                           143                         150 
                   (SEQ ID NO 1) 
                     
                 
                      NH 2 - Lys Gly Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg 
                 
                     
                 
                                   160                                     170 
                 
                   Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg 
                 
                     
                 
                                                       180 
                 
                   Ile Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser 
                 
                     
                 
                                   190                                     200 
                 
                   Gly Glu Pro Pro Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro 
                 
                     
                 
                                                       210 
                 
                   Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu 
                 
                     
                 
                                   220                                     230 
                 
                   Pro Thr Pro Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg 
                 
                     
                 
                                                       240 
                 
                   Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr 
                 
                     
                 
                                   250 
                 
                   Ala Pro Val Pro Met Pro Asp Leu Lys COOH 
                 
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       with said monoclonal antibody being characterized by the the fact that it is capable of specifically detecting abnormally phosphorylated tau protein (PHF-tau) in cerebrospinal fluid (CSF).  
     
     
         2 . A monoclonal antibody according to  claim 1 , characterized by the fact it forms an immunological complex: 
 either with a phosphorylated epitope located within the sequence defined in  claim 1  (SEQ ID NO 1),    or with any other phosphorylated peptide capable of forming an immunological complex with a monoclonal antibody, which itself is capable of forming a complex with a phosphorylated epitope located within the tau protein region as defined in  claim 1  (SEQ ID NO 1).    
     
     
         3 . A monoclonal antibody AT180 according to any of claims  1  or  2  secreted by the hybridoma deposited at ECACC on Dec. 22, 1992 under No. 92122204.  
     
     
         4 . A monoclonal antibody AT270 according to any of claims  1  or  2  secreted by the hybridoma deposited at ECACC on Jul. 7, 1993 under No. 93070774.  
     
     
         5 . A hybridoma, which secretes a monoclonal antibody according to anyone of  claims 1  to  4 , more particularly the hybridoma deposited at ECACC on Dec. 22, 1992 under No. 92122204, or the monoclonal antibody deposited at ECACC on Jul. 7, 1993 under No. 93070774.  
     
     
         6 . A peptide containing from 6 to 100 amino acids, and with said peptide specifically forming an immunological complex with any of the monoclonal antibodies, according to anyone of  claims 1  to  4 , with said peptide being in the phosphorylated form, and 
 with the sequence of said peptide comprising, or consisting of phosphorylated parts of the sequence as shown in SEQ ID NO 1, or,  
 with the sequence of said peptide comprising or consisting of the sequence of the peptides capable of forming an immunological complex with a monoclonal antibody according to anyone of  claims 1  to  4 .  
 
     
     
         7 . A peptide according to  claim 6 , characterized in that its amino acid sequence comprises or consists of its amino acid sequence the following sequence: Val-Arg-Thr-Pro-Pro (SEQ ID NO 2), with said Thr being phosphorylated and with said peptide being characterized in that it specifically forms an immunological complex with the monoclonal antibody according to  claim 3 .  
     
     
         8 . A peptide according to  claim 6  consisting of or comprising in its amino acid sequence the following sequence: 
 Pro-Lys-Thr-Pro-Pro (SEQ ID NO 3), with said Thr being phosphorylated and with said eptide being characterized in that it specifically forms an immunological complex with the monoclonal antibody according to  claim 4 .  
 
     
     
         9 . A phosphorylated peptide according to any of  claims 6  to  8 , which is capable of generating a monoclonal antibody according to anyone of  claims 1  to  4  upon immunization.  
     
     
         10 . A process for obtaining and isolating a hybridoma according to  claim 5 , secreting a monoclonal antibody according to anyone of  claims 1  to  4 , characterized in that it involves: 
 starting from the spleen cells of an animal, e.g. mouse or rat, previously immunized in vivo, or from spleen cells of such cells previously immunized in vitro with an antigen, preferably abnormally phosphorylated tau (PHF-tau), or a phosphorylated peptide according to  claims 6  to  9 , recognized by a monoclonal antibody as defined in any of  claims 1  to  4 ;  
 fusing said immunized cells with myeloma cells under hybridoma-forming conditions; and,  
 selecting those hybridomas which secrete monoclonal antibodies which specifically recognize a phosphorylated epitope of abnormally phosphorylated tau (PHF-tau) in cerebrospinal fluid (CSF).  
 
     
     
         11 . Monoclonal antibodies according to any of  claims 1  to  4  such as obtained by a process according to  claim 10 , characterized in that it involves: 
 starting from the spleen cells of a mouse previously immunized in vitro with abnormally phosphorylated tau (PHF-tau), or a phosphorylated tau peptide according to any of  claims 6  to  9 , recognized by a monoclonal antibody according to any of  claims 1  to  4 ;  
 fusing said immunized spleen cells with myeloma cells under hybridoma-forming conditions, 
 selecting those hybridomas which secrete monoclonal antibodies which specifically recognize PHF-tau and which are capable of specfically detecting PHF-tau in CSF.  
 
 
     
     
         12 . A process for producing monoclonal antibodies according to anyone of  claims 1  to  4  which involves: 
 culturing the selected hybridomas according to  claim 5  or the hybridomas such as obtained through the process of  claim 9 , in an appropriate medium culture; and  
 recovering the monoclonal antibodies excreted by said selected hybridomas; or alternatively:  
 implanting the selected hybridomas of  claim 5 , or the hybridomas obtained through the process of  claim 9 , into the peritoneum of a mouse and, when ascites has been produced by the animal, recovering the monoclonal antibodies then formed from said ascites.  
 
     
     
         13 . Process for the post-mortem detection or diagnosis of brain/neurological disease involving PHF-tau protein, such as Alzheimer's disease, which comprises at least the following steps: 
 contacting a monoclonal antibody according to anyone of  claims 1  to  4 , with a preparation of NFT or a detergent-extracted brain homogenate isolated from a patient having had Alzheimer's disease or any other disease involving abnormally phosphorylated tau (PHF-tau) under conditions suitable for producing an antigen-antibody complex; and    detecting the immunological binding of said antibody to said brain homogenate, and possibly separating the antigen from said complex and recovering the antigen sought in a purified form.    
     
     
         14 . Process for the detection or diagnosis in vitro of brain disease involving abnormally phosphorylated tau protein, such as Alzheimer's disease, which includes: 
 bringing a sample of CSF, preferably unconcentrated CSF, or of serum from a patient suspected of suffering from a neurological disorder involving PHF-tau, more particularly Alzheimer's disease, or proteins or polypeptides extracted therefrom, into contact under in vitro conditions with a monoclonal antibody according to anyone of  claims 1  to  4 , with said conditions suitable for producing an antigen-antibody complex; and,    detecting the immunological binding of said antibody to said sample of cerebrospinal fluid, or of serum, or extract thereof.    
     
     
         15 . Kit for the diagnosis in vitro of one of the following diseases: Alzheimer's disease, Down's syndrome, Pick's disease and other neurological disorders in which abnormally phosphorylated tau protein or paired helical filaments are implicated, characterized in that the kit comprises: 
 at least one monoclonal antibody according to anyone of  claims 1  to  4 , deposited on a microplate;    a preparation containing the sample (CSF, serum, or proteins extracted therefrom) to be diagnosed in vitro,    a second antibody 
 which can be a monoclonal antibody recognizing an epitope of abnormally phosphorylated tau protein, or an epitope of any phosphorylated tau peptide carrying an epitope of PHF-tau, with said epitopes being different from the one of the invention, or  
 which can be a polyclonal antibody recognizing abnormally phosphorylated tau or a polyclonal antibody recognizing a peptide carrying an epitope of PHF-tau, with said polyclonal antibody being capable of forming an immunological complex with epitopes which are different from the epitope of the invention, with said polyclonal antibody being preferably purified by immunoaffinity chromatography using immobilized tau protein;  
   a marker either for specific tagging or coupling with said second antibody;    appropriate buffer solutions for carrying out the immunological reaction between the monoclonal antibody of the invention and a test sample on the one hand, and the bound second antibody and the marker on the other hand,    possibly a peptide carrying an epitope of PHF-tau comprised in the region as defined in  claim 1  (SEQ ID NO 1) for standard purposes, or for competition purposes with respect to the antigen which is sought.    
     
     
         16 . Method or kit according to any of  claims 13  to  15 , for the in vitro detection or diagnosis of brain/neurological disease involving abnormally phosphorylated tau protein, such as Alzheimer's disease comprising at least one of the following combinations of monoclonal antibodies: 
 a mixture comprising at least two monoclonal antibodies according to any of  claims 1  to  4 ,  
 a mixture comprising at least one monoclonal antibody according to any of  claims 1  to  4  as well as at least one other monoclonal antibody recognizing tau or PHF-tau.  
 
     
     
         17 . Method or kit according to  claim 16 , further characterized in that it involves a sandwich detection format comprising coating and detecting antibodies, with said coating antbodies consisting of at least one antibody according to any one of  claims 1  to  4 , and with said detecting antibodies consisting of at least one monoclonal antibody capable of detecting both normal and/or abnormally phosphorylated human tau of which the epitope is different from the epitope of any of the monoclonal antibodies according to any of  claims 1  to  4 .