Method for preparing multiple antigen glycopeptide carbohydrate conjugates

Abstract

The present invention relates to a method for preparing carbohydrate T cell epitope conjugates of formula (I): M(T-B) n (I) wherein M, T, B and n are as defined in claim 1.

1 . A method for preparing a carbohydrate T cell epitope conjugate of formula (I):
   M(T-B) n   (I)
   wherein:   M is a dendrimerie poly-Lysine core;   T is a T cell epitope;   B is a carbohydrate B cell epitope comprising at least one carbohydrate residue (b);   n is an integer and represents the number of -T-B groups covalently bonded to M;   Said method comprising the steps of:   i) coupling a protected carbohydrate B cell epitope (B Pr ) which hydroxyl groups of the carbohydrate residue (b) are protected with a protecting group (Pr), with a compound M(T) n  thereby forming a carbohydrate T cell epitope conjugate M(T-B Pr ) n , said protecting group (Pr) being selected from the group consisting of allyl, p-methoxybenzyl (PMB), t-butyldimethylsilyl (TBDMS), benzyloxymethyl (BOM), levulinyl (Lev), benzoyl (Bz), 2,5-difluorobenzoyl, chloroacetyl, benzyl (Bn) or an acetyl (Ac), or forming with two hydroxyl groups to which it is attached a C 5 -C 6  isopropylidene ketal or a C 5 -C 6  cyclic alkylcarbonate;   and   ii) removing the protecting groups Pr from the obtained conjugate M(T-B Pr ) n  thereby obtaining the carbohydrate T cell epitope conjugate M(T-B) n .   
     
     
         2 . The method of  claim 1 , wherein M(T-B) n  is selected from the following formulae (Ia) or (Ib): 
       
         
           
           
               
               
           
         
         Wherein K is a lysine residue, and 
         T and B are as defined in  claim 1 . 
       
     
     
         3 . The method of  claim 1 , wherein M is (K) 2 K-βAla-OH of formula: 
       
         
           
           
               
               
           
         
       
     
     
         4 . The method of  claim 1 , wherein T is a T cell epitope comprising a peptide. 
     
     
         5 . The method of  claim 4 , wherein T is or comprises a CD8 +  or a CD4 +  T cell epitope. 
     
     
         6 . The method of  claim 5 , wherein T is or comprises a CD4 +  T cell epitope. 
     
     
         7 . The method of  claim 6 , wherein T is or comprises a poliovirus (PV) fragment protein, a tetanus toxin fragment or a PADRE peptide. 
     
     
         8 . The method of  claim 7 , wherein T is a peptide consisting of QYIKANSKFIGITEL (SEQ ID NO: 1). 
     
     
         9 . The method of  claim 1 , wherein the carbohydrate residue (b) is attached to an amino acid, peptide or lipid residue. 
     
     
         10 . The method of  claim 1 , wherein B is or comprises a carbohydrate residue selected from the group consisting of:
 α-GalNAc-Ser,   α-GalNAc-Thr,   β-GalNAc-Ser,   β-GalNAc-Thr,   β-Gal-(1-3)-α-GalNAc-Ser,   β-Gal-(1-3)-α-GalNAc-Thr,   (α-GalNAc-Ser/Thr) 2 ,   (α-GalNAc-Ser/Thr) 3 , and   (α-GalNAc-Ser/Thr) 6 ,   
     
     
         11 . The method of  claim 10 , wherein B is or comprises the residue (α-GalNAc-Ser/Thr)3. 
     
     
         12 . The method of  claim 1 , wherein the protecting group Pr is benzyl (Bn) or acetyl (Ac). 
     
     
         13 . The method of  claim 1 , wherein step i) comprises the steps of:
 a) coupling a first protected carbohydrate residue (b Pr ) which hydroxyl groups are protected with a protecting group (Pr) as defined in any of the preceding claims with a compound M(T) n  thereby forming a carbohydrate T cell epitope conjugate M(T-b Pr ) n ; and   b) repeating step a) with further protected carbohydrate residues (b Pr ) up to obtaining a protected carbohydrate conjugate of formula (II) M(T-B Pr ) n      
     
     
         14 . The method of  claim 1 , wherein Pr is benzyl. 
     
     
         15 . The method of  claim 14 , wherein benzyl groups are removed in the presence of TfOH or H 2 . 
     
     
         16 . The method of  claim 1 , wherein Pr is acetyl. 
     
     
         17 . The method of  claim 16 , wherein acetyl groups are removed in the presence of hydrazine or MeONa. 
     
     
         18 . A carbohydrate T cell epitope conjugate of formula (II):
   M(T-B Pr ) n ,  (II)
   wherein   M is a dendrimeric poly-Lysine core;   T is a T cell epitope;   B Pr , is a protected carbohydrate B cell epitope comprising at least one carbohydrate residue (b) which hydroxyl groups are protected by a Pr group as defined in  claim 1 ; and   n is an integer and represents the number of -T-B groups covalently bonded to M.   
     
     
         19 . The carbohydrate T cell epitope conjugate of  claim 18 , wherein T is or comprises a peptide. 
     
     
         20 . The carbohydrate T cell epitope conjugate of  claim 18 , wherein Pr is benzyl (Bn) or acetyl (Ac). 
     
     
         21 . The carbohydrate T cell epitope conjugate of  claim 18 , wherein B Pr , is a protected (α-GalNAc-Ser/Thr) 3 . 
     
     
         22 . The carbohydrate T cell epitope conjugate of  claim 18 , wherein M is HO-βAla-K(K) 2 . 
     
     
         23 . The carbohydrate T cell epitope conjugate of  claim 18 , wherein T is QYIKANSKFIGITEL (SEQ ID NO:1). 
     
     
         24 . A use of a carbohydrate T cell epitope conjugate M(T-B Pr ), of  claim 18  for preparing a carbohydrate peptide conjugate M(T-B) n  as defined in  claim 1 .