Conjugates with reduced adverse systemic effects

Abstract

A conjugate of an active agent and a targeting moiety having affinity for a target cell, in which the active agent has been modified by attachment of a cell membranes impermeabilizing group so that, if the active agent so modified is cleaved from the conjugate in the blood plasma instead of inside the target cell, the cell membrane-impermeabilizing group prevents or limits entry of the modified active agent into cells, thus reducing its systemic or non-specific adverse effects, including toxicity.

1 . A conjugate that has affinity for a target cell and releases inside the target cell a modified active agent that modulates one or more cellular functions of the target cell, having the structure
   T-(L-(D-Z) m ) n      
     wherein
 T is a targeting moiety that has affinity for the target cell; 
 D-Z is a modified active agent comprising a cell membrane-impermeabilizing moiety Z covalently attached to an active agent D that modulates one or more cellular functions of the target cell when inside the target cell; 
 L is a linker moiety covalently linking targeting moiety T and modified active agent D-Z, which linker moiety L is preferentially susceptible to cleavage inside the target cell to release modified active agent D-Z; 
 m is an integer ranging from 1 to 64; and 
 n is an integer ranging from 1 to 12. 
 
   
   
       2 . A conjugate according to  claim 1 , wherein targeting moiety T is a monoclonal antibody. 
   
   
       3 . A conjugate according to  claim 1 , wherein linker moiety is a lysosomal protease cleavable linker, the protease preferably being cathepsin B. 
   
   
       4 . A conjugate according to  claim 1 , wherein linker moiety L comprises a structure of the formula 
     
       
         
         
             
             
         
       
     
     wherein x is 0 or 1, AA 1  is a lysine, arginine, or citrulline amino acid side chain residue and AA 2  is a phenylalanine, valine, alanine, leucine, or isoleucine amino acid side chain residue. 
   
   
       5 . A conjugate according to  claim 1 , wherein active agent D is a cytotoxin. 
   
   
       6 . A conjugate according to  claim 5 , wherein the cytotoxin is a maytansinoid or leptomycin B. 
   
   
       7 . A conjugate according to  claim 1 , wherein cell membrane-impermeabilizing moiety Z comprises a cationic group selected from the group consisting of a primary alkyl amine group, a secondary alkyl amine group, a tertiary alkyl amine group, a quaternary alkyl or alkylaryl ammonium group, a guamidinium group, an imidazolium group, a triazolium group, and a tetrazolium group. 
   
   
       8 . A conjugate according to  claim 1 , wherein cell membrane-impermeabilizing moiety Z no comprises an anionic group selected from the group consisting of a phosphonate group, a sulfonate group, and a carboxylate group. 
   
   
       9 . A conjugate according to  claim 1 , wherein targeting moiety T has affinity for a cancer cell. 
   
   
       10 . A conjugate according to  claim 1 , wherein targeting moiety T has affinity for an osteoclast and the active agent D is bafilomycin A 1 . 
   
   
       11 . A conjugate according to  claim 10 , wherein targeting moiety T is RANKL or TNF-α. 
   
   
       12 . A conjugate according to  claim 1 , wherein the conjugate has a structure according to one of formulae I to XII: 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     wherein
 AA 1  is a lysine, arginine, or citrulline amino acid side chain residue; 
 AA 2  is a phenylalanine, valine, alamine, leucine, or isoleucine amino acid side chain residue; 
 p in formulae I to XII is an integer from 1 to 6; 
 q in formulae VII to XII is an integer from 1 to 5; 
 r in formulae VII to XII is an integer from 1 to 5; 
 —C(═O)LMB in formulae VII to XII is a leptomycin B residue, having the structure 
 
     
       
         
         
             
             
         
       
       X ⊖  in formulae IV to VI and X to XII is a pharmaceutically acceptable counteranion. 
     
   
   
       13 . A method of making a conjugate that has affinity for a target cell and releases inside the target cell a modified active agent that modulates one or more cellular functions of the target cell, comprising the steps of:
 (a) covalently attaching a cell membrane-impermeabilizing moiety Z to an active agent D that modulates one or more cellular functions of the target cell when inside the target cell, to prepare a modified active agent D-Z; and   (b) covalently linking active agent D or modified active agent D-Z to a targeting moiety T via a linker moiety L to form a conjugate of the structure
   T-(L-(D-Zt) m ) n    
   
     wherein
 targeting moiety T has affinity for the target cell; 
 linker moiety L is preferentially susceptible to cleavage inside the target cell to release modified active agent D-Z; 
 m is an integer ranging from 1 to 64; and 
 n is an integer ranging from 1 to 12. 
 
   
   
       14 . A method according to  claim 13 , wherein linking step (b) is performed before the attachment of cell membrane-impermeabilizing moiety Z to active agent D. 
   
   
       15 . A method according to  claim 13 , wherein linking step (b) is performed after the attachment of cell membrane-impermeabilizing moiety Z to active agent D. 
   
   
       16 . A method for modulating one or more target cellular functions of a target cell in a subject, comprising administering to the subject an effective amount of a conjugate that has affinity for a target cell and releases inside the target cell a modified active agent that modulates one or more cellular functions of the target cell, which conjugate has the structure
   T-(L-(D-Z) m ) n      
     wherein
 T is a targeting moiety that has affinity for the target cell; 
 D-Z is a modified active agent comprising a cell membrane-impermeabilizing moiety Z covalently attached to an active agent D that modulates one or more cellular functions of the target cell when inside the target cell; 
 L is a linker moiety covalently linking targeting moiety T and modified active agent D-Z, which linker moiety L is preferentially susceptible to cleavage inside the target cell to release modified active agent D-Z; 
 m is all integer ranging from 1 to 64; and 
 n is an integer ranging from 1 to 12. 
 
   
   
       17 . A method according to  claim 16 , wherein the target cell is a cancer cell. 
   
   
       18 . A method according to  claim 16 , wherein the target cell is an osteoclast and agent D is bafilomycin A 1 .