Protein-active agent conjugates and method for preparing the same

Abstract

The invention provides protein-active agent conjugates having an amino acid motif that can be recognized by an isoprenoid transferase. The invention also provides compositions containing the conjugates, as well as methods for making the conjugates and compositions. The invention further provides methods for using the conjugates to deliver the active agent to a target cell, as well as methods for using the conjugates to treat a subject in need thereof (e.g., a subject in need of the active agent).

1 . A protein-active agent conjugate comprising a protein having an amino acid motif that can be recognized by an isoprenoid transferase, wherein the active agent is covalently linked to the protein at the amino acid motif. 
     
     
         2 . The protein-active agent conjugate of  claim 1 , wherein the protein comprises a modification selected from the group consisting essentially of:
 (i) a deletion in the carboxy terminus of the protein;   (ii) an oligopeptide or polypeptide addition in the carboxy terminus of the protein; and   (iii) a deletion in the carboxy terminus of the protein and an oligopeptide or polypeptide addition in the carboxy terminus of the protein;   wherein the modification is attached to the amino acid motif.   
     
     
         3 . The protein-active agent conjugate of  claim 1 , wherein the protein is an antibody or a fragment of an antigenic polypeptide. 
     
     
         4 . The protein-active agent conjugate of  claim 3 , wherein the protein is a monoclonal antibody. 
     
     
         5 . The protein-active agent conjugate of  claim 4 , wherein at least one light chain and/or at least one heavy chain of the monoclonal antibody comprises an amino acid region having the amino acid motif. 
     
     
         6 . The protein-active agent conjugate of  claim 1 , wherein the isoprenoid transferase is FTase or GGTase. 
     
     
         7 . The protein-active agent conjugate of  claim 1 , wherein the active agent is a drug, a toxin, an affinity ligand, a detection probe, or a combination thereof. 
     
     
         8 . The protein-active agent conjugate of  claim 1 , wherein the amino acid motif is CAAX, XXCC, XCXC, or CXX, wherein C represents cysteine, A represents an aliphatic amino acid, and X represents an amino acid that determines a substrate specificity of the isoprenoid transferase. 
     
     
         9 . The protein-active agent conjugate of  claim 1 , wherein the amino acid motif is covalently linked to the active agent via at least one linker. 
     
     
         10 . The protein-active agent conjugate of  claim 9 , wherein the linker is an isoprenyl derivative that can be recognized by the isoprenoid transferase. 
     
     
         11 . The protein-active agent conjugate of  claim 9 , wherein the linker is represented by the following formula (I): 
       
         
           
           
               
               
           
         
         wherein, 
         P 1  and Y is independently a group containing a first functional group (FG1), the FG1 being selected from the group consisting of: acetylene, azide, aldehyde, hydroxylamine, hydrazine, ketone, nitrobenzofurazan (NBD), dansyl, fluorescein, biotin, and Rhodamin, 
         L 1  is (CH 2 ) r X q (CH 2 ) p , 
         X is oxygen, sulfur, —NR 1 —, —C(O)NR 1 —, —NR 1 C(O)—, —NR 1 SO 2 —, —SO 2 NR 1 —, —(CH═CH)—, or acetylene, 
         R 1  is hydrogen, C 1-6  alkyl, C 1-6  alkyl aryl, or C 1-6  alkyl heteroaryl, 
         r and p is independently an integer of 0 to 6, 
         q is an integer of 0 to 1, and 
         n is an integer of 1 to 4. 
       
     
     
         12 . The protein-active agent conjugate of  claim 9 , wherein the active agent is attached to a group containing a second functional group (FG2) that can react with the FG1, wherein the FG2 is selected from the group consisting of: acetylene, hydroxylamine, azide, aldehyde, hydrazine, ketone, and amine. 
     
     
         13 . The protein-active agent conjugate of  claim 12 , wherein the active agent is attached to the group containing an FG2 via —(CH 2 ) r X q (CH 2 ) p — or —[ZCH 2 CH 2 O(CH 2 CH 2 O) w CH 2 CH 2 Z]—, in which
 X is oxygen, sulfur, —NR 1 —, —C(O)NR 1 —, —NR 1 C(O)—, —NR 1 SO 2 —, or —SO 2 NR 1 —, 
 Z is oxygen, sulfur or NR 1 , 
 R 1  is hydrogen, C 1-6  alkyl, C 1-6  alkyl aryl, or C 1-6  alkyl heteroaryl, 
 r and p is independently an integer of 0 to 6, 
 q is an integer of 0 to 1, and 
 w is an integer of 0 to 6. 
 
     
     
         14 . The protein-active agent conjugate of  claim 13 , wherein the —(CH 2 ) r X q —CH 2 ) p — or —[ZCH 2 CH 2 O(CH 2 CH 2 O) w CH 2 CH 2 Z]— is attached to (i) a peptide(s) that can be cleaved by cathepsin B or (ii) a glucuronide that can be cleaved by β-glucuronidase. 
     
     
         15 . The protein-active agent conjugate of  claim 14 , wherein the peptide that can be cleaved by cathepsin B is 
       
         
           
           
               
               
           
         
       
     
     
         16 . The protein-active agent conjugate of  claim 14 , wherein the glucuronide that can be cleaved by β-glucuronidase is 
       
         
           
           
               
               
           
         
       
     
     
         17 . A method for preparing the protein-active agent conjugate of  claim 1 , the method comprising:
 (a) expressing a protein having an amino acid motif that can be recognized by an isoprenoid transferase;   (b) enzymatically reacting, using the isoprenoid transferase, the expressed protein with at least one isosubstrate having a first functional group (FG1), thereby producing a functionalized protein;   (c) attaching a second functional group (FG2) to an active agent, thereby producing a functionalized active agent; and   (d) reacting the functionalized protein with the functionalized active agent, thereby producing the protein-active agent conjugate of  claim 1 .   
     
     
         18 . The method of  claim 17 , wherein the amino acid motif is in the carboxy terminus of the protein. 
     
     
         19 . The method of  claim 17 , wherein the amino acid motif is CAAX, XXCC, XCXC, or CXX, wherein C represents cysteine, A represents an aliphatic amino acid, and X represents an amino acid that determines the substrate specificity of the isoprenoid transferase. 
     
     
         20 . The method of  claim 19 , wherein the amino acid motif is CAAX, and wherein the method further comprises removing AAX from the amino acid motif after step (b). 
     
     
         21 . The method of  claim 17 , wherein the FG2 is attached to the active agent by at least one linker. 
     
     
         22 . The method of  claim 17 , wherein the reaction between the functionalized protein and the functionalized active agent is click chemistry reaction or a hydrazone and/or oxime formation. 
     
     
         23 . The method of  claim 22 , wherein the FG1 is an azide group and the FG2 is an acetylene group, or wherein the FG1 is an acetylene group and the FG2 is an azide group. 
     
     
         24 . The method of  claim 22 , wherein the FG1 is an aldehyde or ketone group and the FG2 is a hydrazine or hydroxylamine, or wherein the FG1 is hydrazine or hydroxylamine and the FG2 is an aldehyde or ketone. 
     
     
         25 . A method for preparing the protein-active agent conjugate of  claim 1 , the method comprising:
 (a) expressing a protein having an amino acid motif that can be recognized by an isoprenoid transferase;   (b) attaching an isosubstrate of an isoprenoid transferase to an active agent; and   (c) enzymatically reacting, using the isoprenoid transferase, the expressed protein with the active agent attached to the isosubstrate.   
     
     
         26 . The method of  claim 25 , wherein the amino acid motif is in the carboxy terminus of the protein. 
     
     
         27 . The method of  claim 25 , wherein the amino acid motif is CAAX, XXCC, XCXC, or CXX, wherein C represents cysteine, A represents an aliphatic amino acid, and X represents an amino acid that determines the substrate specificity of the isoprenoid transferase. 
     
     
         28 . The method of  claim 25 , wherein the isosubstrate is attached to the active agent by at least one linker. 
     
     
         29 . A composition comprising the protein-active agent conjugate of  claim 1 . 
     
     
         30 . A method for delivering an active agent to a target cell in a subject, the method comprising administering the protein-active agent conjugate of  claim 1  or a composition comprising the protein-active agent conjugate to the subject. 
     
     
         31 . A method of treating a subject in need thereof, the method comprising administering a therapeutically effective amount of the protein-active agent conjugate of  claim 1  or a composition comprising the protein-active agent conjugate to the subject.