Method for the identification of extracellular domains of the epstein barr virus (ebv), tumor-associated latent membrane proteins and for the selection of antibody reagents reactive therewith

Abstract

Described are a method for the identification of extracellular epitopes of Epstein Barr virus (EBV) encoded membrane proteins, expressed on the outer cell surface of EBV-transformed mammalian cells, methods for the selection and preparation of antibody reagents specific for the said epitopes, as well as peptides, including extracellular domains of Epstein Barr Virus encoded tumour cell associated membrane proteins, the use of said peptide for immunisation and therapeutic vaccination to induce antibodies and T-cells reactive with said domains, and the use of said antibody reagents for the production of targeting cells, tumour cell purging and as diagnostic for and medicament against EBV-mediated malignant cell growth.

1 - 30 . (canceled)  
     
     
         31 . A method for the preparation of monoclonal antibody reagents capable of binding with extracellular epitopes of Epstein Barr virus (EBV) encoded membrane proteins, expressed on the outer cell surface of EBV-transformed mammalian cells, comprising the steps of: 
 1) providing an expression library, comprising mammalian genetic material, the expression library being a high-diversity library containing at least 10 9  different clones;    2) contacting with the said library a peptide, comprising the extracellular epitope, the said peptide being identified by a method, comprising the steps of: 
 A) providing the amino acid sequence of at least one open reading frame (ORF) of the EBV genome;  
 B) determining at least one putative extracellular domain contained within the amino acid sequence of A);  
 C) preparation of a peptide comprising the putative extracellular domain of B) or a part thereof, the peptide not comprising the full-length ORF;  
 D) preparation of an antibody reagent capable of specifically binding with the peptide of C);  
 E) reacting the antibody reagent of D) with viable EBV transformed mammalian cells;  
 F) detecting specific binding of the antibody reagent to the cell surface of the EBV transformed mammalian cells;  
   3) allowing the peptide to specifically bind to organisms of the said library;    4) isolating the organisms whereto the peptide is specifically bound;    5) preparing single clones of the isolated organisms;    6) culturing a clone of step 5) under conditions that the mammalian genetic material is expressed; and    7) isolating the expression product of step 6).    
     
     
         32 . A method for the preparation of monoclonal antibody reagents capable of binding with extracellular epitopes of Epstein Barr virus (EBV) encoded membrane proteins, expressed on the outer cell surface of EBV-transformed mammalian cells, comprising the steps of: 
 1) providing an expression library, comprising mammalian genetic material, the expression library being a high-diversity library containing at least 10 9  different clones;    2) contacting with the said library viable EBV-transformed cells expressing on their surface one or more peptide epitopes, identified according to a method, comprising the steps of: 
 A) providing the amino acid sequence of at least one open reading frame (ORF) of the EBV genome;  
 B) determining at least one putative extracellular domain contained within the amino acid sequence of A);  
 C) preparation of a peptide comprising the extracellular domain of B) or a part thereof, the peptide not comprising the full-length ORF;  
 D) preparation of an antibody reagent capable of specifically binding with the peptide of C);  
 E) reacting the antibody reagent of D) with viable EBV transformed mammalian cells;  
 F) detecting specific binding of the antibody reagent to the cell surface of the EBV transformed mammalian cells;  
   3) allowing the cells to specifically bind to organisms of the said library;    4) isolating the organisms whereto the cells are specifically bound;    5) preparing single clones of the isolated organisms;    6) culturing a clone of step 5) under conditions that the mammalian genetic material is expressed; and    7) isolating the expression product of step 6).    
     
     
         33 . The method according to  claim 31 , wherein the expression library contains at least 10 12  different clones.  
     
     
         34 . The method according to  claim 32 , wherein the expression library contains at least 10 12  different clones.  
     
     
         35 . The method according to  claim 31 , wherein the extracellular domains are chosen from the amino acid sequences selected from the group consisting of:  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                     
                 
                     
                   SEQ ID 1 
                   (LMP1, extracellular loop 1), 
                 
                     
                   SEQ ID 2 
                   (LMP1, extracellular loop 2), 
                 
                     
                   SEQ ID 3 
                   (LMP1, extracellular loop 3), 
                 
                     
                   SEQ ID 4 
                   (LMP2, extracellular loop 1), 
                 
                     
                   SEQ ID 5 
                   (LMP2, extracellular loop 2), 
                 
                     
                   SEQ ID 6 
                   (LMP2, extracellular loop 3), 
                 
                     
                   SEQ ID 7 
                   (LMP2, extracellular loop 4), 
                 
                     
                   SEQ ID 8 
                   (LMP2, extracellular loop 5), 
                 
                     
                   SEQ ID 9 
                   (LMP2, extracellular loop 6), 
                 
                     
                   SEQ ID 10 
                   (BARF1, SEQ 40-79), 
                 
                     
                   SEQ ID 11 
                   (BARF1, SEQ 80-154), 
                 
                     
                   SEQ ID 12 
                   (BARF1, SEQ 155-188) and 
                 
                     
                   SEQ ID 13 
                   (BARF1, SEQ 188-221). 
                 
                     
                     
                 
                     
                     
                 
             
                
                
               
               
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         36 . A method for the selection of antibody reagents capable of binding with extracellular epitopes of Epstein Barr virus (EBV) encoded membrane proteins, expressed on the outer cell surface of EBV-transformed mammalian cells, comprising the steps of: 
 A) providing the amino acid sequence of at least one open reading frame (ORF) of the EBV genome;    B) determining at least one putative extracellular domain contained within the amino acid sequence of A);    C) preparation of a peptide comprising the extracellular domain of B) or a part thereof, the peptide not comprising the full-length ORF;    D) preparation of an antibody reagent capable of specifically binding with the peptide of C);    E) reacting the antibody reagent of D) with viable EBV transformed mammalian cells;    F) detecting specific binding of the antibody reagent to the cell surface of the EBV transformed mammalian cells; and    G) selecting the detected antibody reagent of step F).    
     
     
         37 . The method according to  claim 36 , wherein the step G) further comprises the steps of: 
 G1) immunising an animal with the peptide of step C);    G2) preparing body fluid of the immunised animal, comprising antibody reagents;    G3) preparing a solid phase affinity matrix comprising the peptide bound to the said matrix;    G4) contacting the body fluid with the matrix;    G5) allowing specific binding of antibody reagents from the body fluid to the peptide bound to the matrix; and    G6) isolation of the antibody reagents, specifically bound to the peptides.    
     
     
         38 . The method according to  claim 36 , wherein the step G) further comprises the steps of: 
 G1) immunising an animal with the peptide of step C);    G2) providing B cells producing antibody reagents from the immunised animal of G1);    G3) preparing a solid phase affinity matrix comprising the peptide bound to the said matrix;    G4) contacting the B cells of G2) with the matrix;    G5) allowing specific binding of the B cells to the peptide bound to the matrix; and    G6) isolation of the B cells, specifically bound to the peptides.    
     
     
         39 . The method according to  claim 31 , wherein the peptide of C) comprises two cystein residues, where between the putative extracellular domain or part thereof is located, the said cystein residues being oxidised forming a S—S bond between the said cystein residues.  
     
     
         40 . The method according to  claim 36 , wherein the peptide of C) comprises two cystein residues, where between the putative extracellular domain or part thereof is located, the said cystein residues being oxidised forming a S—S bond between the said cystein residues.  
     
     
         41 . Use of a high-diversity library containing at least 10 9  different clones for the preparation of monoclonal antibody reagents capable of binding with extracellular epitopes of Epstein Barr virus (EBV) encoded membrane proteins, expressed on the outer cell surface of EBV-transformed mammalian cells.  
     
     
         42 . A monoclonal antibody reagent, obtainable according to the method of  claim 31 , capable of binding with extracellular epitopes of Epstein Barr virus (EBV) encoded membrane proteins, expressed on the cell surface of EBV-transformed mammalian cells.  
     
     
         43 . An antibody reagent capable of specifically binding to an amino acid sequence selected from the group consisting of SEQ ID nos. 1-13.  
     
     
         44 . An antibody reagent capable of specifically binding to an amino acid sequence having at least 80% homology with one of the amino acid sequences of SEQ ID nos. 1-13.  
     
     
         45 . The antibody reagent according to  claim 42  for use as a diagnostic or therapeutic agent in patients carrying EBV positive tumours.  
     
     
         46 . The antibody reagent according to  claim 43  for use as a diagnostic or therapeutic agent in patients carrying EBV positive tumours.  
     
     
         47 . A method for the production of targeting cells, targeted to cells expressing LMP1, LMP2 or BARF1 comprising the steps of: 
 transfecting the cells with genetic information for the antibody reagent according to  claim 43;     expressing said antibody reagent on the surface of the said targeting cells.    
     
     
         48 . A method for the production of targeting viral particles, targeted to cells expressing LMP1, LMP2 or BARF1, comprising the steps of: 
 cloning the genetic information for the antibody reagent according to any of the claims  11 - 13  into the viral genome,    expressing the said antibody on the viral coat of the said viral particles.    
     
     
         49 . Use of an antibody reagent according to  claim 40  for the preparation of a medicament, in particular for treatment of EBV-infection.  
     
     
         50 . A method for diagnosing EBV infected cells, especially EBV+malignant cells and EBV mediated diseases, comprising the steps of: 
 taking a sample of cells of an individual, suspected of carrying said infected cells of disease;    contacting an antibody reagent according to  claim 40  with the said cells;    reacting the antibody reagent with the said cells, forming a complex of antibody reagent, bound to an EBV infected cell; and    assaying for the presence of the said complex.    
     
     
         51 . A method for specifically eliminating cells expressing LMP1, LMP2 or BARF1 or parts thereof on their cell surface ex vivo, comprising the steps of: 
 isolating a number of cells from an individual;    contacting the isolated cells with an antibody reagent according to  claim 40;  and    negatively selecting the cells expressing LMP1, LMP2 or BARF1 or parts thereof on their cell surface, wherein cells not expressing LMP1, LMP2 or BARF1 on their cell surface survive.    
     
     
         52 . The method according to  claim 51 , wherein the surviving cells are reimplanted into the individual.  
     
     
         53 . The method according to  claim 51 , wherein the step of isolating further comprises isolating cells from the bone marrow of the individual.