Site-specific labeling methods and molecules produced thereby

Abstract

The present invention provides methods of site-specific labeling of antibodies, using proteins having 4′-phosphopantetheinyl transferase activity that catalyze post-translational modification of peptide sequences (“peptide tags”) incorporated into one or more specific sites of an antibody of interest. Enzymatic labeling enables quantitative and irreversible covalent modification of a specific serine residue within the peptide tags incorporated into the antibody, and thus creates desirable antibody conjugates.

1 . A modified antibody or an antigen binding fragment thereof, which comprises at least one peptide tag that is a substrate of 4′-phosphopantetheinyl transferase, and is located within the structural loop of said antibody or antigen binding fragment thereof. 
     
     
         2 . The modified antibody or antigen binding fragment thereof of  claim 1 , wherein said 4′-phosphopantetheinyl transferase is Sfp, AcpS,  T. maritima  PPTase, human PPTase or a mutant or homolog form thereof that retains the 4′-phosphopantetheinyl transferase activity. 
     
     
         3 . The modified antibody or antigen binding fragment thereof of  claim 1 , wherein the peptide tag is selected from the group consisting of: 
       
         
           
                 
                 
                 
               
                     
                   GDSLSWLLRLLN, 
                   (SEQ ID NO: 1) 
                 
                     
                     
                 
                     
                   GDSLSWL, 
                   (SEQ ID NO: 2) 
                 
                     
                     
                 
                     
                   GDSLSWLVRCLN, 
                   (SEQ ID NO: 3) 
                 
                     
                     
                 
                     
                   GDSLSWLLRCLN, 
                   (SEQ ID NO: 4) 
                 
                     
                     
                 
                     
                   GDSLSWLVRLLN, 
                   (SEQ ID NO: 5) 
                 
                     
                     
                 
                     
                   GDSLSWLLRSLN, 
                   (SEQ ID NO: 6) 
                 
                     
                     
                 
                     
                   GSQDVLDSLEFIASKLA, 
                   (SEQ ID NO: 7) 
                 
                     
                     
                 
                     
                   VLDSLEFIASKLA, 
                   (SEQ ID NO: 8) 
                 
                     
                     
                 
                     
                   DSLEFIASKLA, 
                   (SEQ ID NO: 9) 
                 
                     
                     
                 
                     
                   GDSLDMLEWSLM, 
                   (SEQ ID NO: 10) 
                 
                     
                     
                 
                     
                   GDSLDMLEWSL, 
                   (SEQ ID NO: 11) 
                 
                     
                     
                 
                     
                   GDSLDMLEWS, 
                   (SEQ ID NO: 12) 
                 
                     
                     
                 
                     
                   GDSLDMLEW, 
                   (SEQ ID NO: 13) 
                 
                     
                     
                 
                     
                   DSLDMLEW, 
                   (SEQ ID NO: 14) 
                 
                     
                     
                 
                     
                   GDSLDM, 
                   (SEQ ID NO: 15) 
                 
                     
                     
                 
                     
                   LDSVRMMALAAR, 
                   (SEQ ID NO: 16) 
                 
                     
                     
                 
                     
                   LDSLDMLEWSLR, 
                   (SEQ ID NO: 17) 
                 
                     
                     
                 
                     
                   DSLEFIASKL, 
                   (SEQ ID NO: 18) 
                 
                     
                     
                 
                     
                   DSLEFIASK, 
                   (SEQ ID NO: 19) 
                 
                     
                     
                 
                     
                   DVLDSLEFI, 
                   (SEQ ID NO: 20) 
                 
                     
                   and 
                     
                 
                     
                     
                 
                     
                   VLDSLEFIAS. 
                   (SEQ ID NO: 21) 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         4 . The modified antibody or antigen binding fragment thereof of  claim 1 , wherein the peptide tag is located within the structural loop of VH, VL, CH1, CH2, CH3, or C L  region of the antibody or antigen binding fragment thereof. 
     
     
         5 . The modified antibody or antigen binding fragment thereof of  claim 1 , wherein said the peptide tag is located within the structural loop of the CH1 region of the antibody or antigen binding fragment thereof. 
     
     
         6 . The modified antibody or antigen binding fragment thereof of  claim 1 , wherein said peptide tag is inserted between any two amino acids that are listed in Table 1. 
     
     
         7 . The modified antibody or antigen binding fragment thereof of  claim 1 , wherein the peptide tag is inserted between amino acid residues 2 and 3 of the VH or VL domain, or between amino acid residue 110 and 111 of the light chain, or between 119 and 120, or between 120 and 121, or between 135 and 136, or between 136 and 137, or between 138 and 139, or between 164 and 165, or between 165 and 166, or between 194 and 195 of the CH1 domain, or between 388 and 389, or between 445 and 446, or between 446 and 447 of the CH3 domain of a parental antibody or antigen binding fragment thereof. 
     
     
         8 . The modified antibody or antigen binding fragment thereof of  claim 1 , wherein the peptide tag is inserted between amino acid residue 110 and 111 of the light chain, or between 119 and 120, or between 120 and 121, or between 135 and 136, or between 136 and 137, or between 138 and 139, or between 165 and 166 of the CH1 domain, or between 388 and 389 of the CH3 domain of a parental antibody or antigen binding fragment thereof. 
     
     
         9 . The modified antibody or antigen binding fragment thereof of  claim 1 , wherein the peptide tag is grafted between amino acid residues 62 to 64 or 62 to 65 of the V H  domain, or between amino acid residues 133 and 138 of the CH1 domain, or between 189 and 195 of the CH1 domain, or between 190 and 197 of the CH1 domain. 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . The modified antibody or antigen binding fragment thereof of  claim 1 , wherein the enzyme having 4′-phosphopantetheinyl transferase activity is Sfp and the peptide tag is GDSLSWLLRLLN (SEQ ID NO:1), GDSLSWLVRCLN (SEQ ID NO:3), GDSLSWLLRCLN (SEQ ID NO:4), GDSLSWLVRLLN (SEQ ID NO:5), GDSLSWLLRSLN (SEQ ID NO:6), GSQDVLDSLEFIASKLA (SEQ ID NO:7), VLDSLEFIASKLA (SEQ ID NO:8), DSLEFIASKLA (SEQ ID NO:9), GDSLDMLEWSLM (SEQ ID NO:10), GDSLDMLEWSL (SEQ ID NO:11), GDSLDMLEWS (SEQ ID NO:12), GDSLDMLEW (SEQ ID NO:13), DSLDMLEW (SEQ ID NO:14), LDSLDMLEWSLR (SEQ ID NO:17), DSLEFIASKL (SEQ ID NO:18), DSLEFIASK (SEQ ID NO:19), or DSLEFIAS (SEQ ID NO:22). 
     
     
         13 . The modified antibody or antigen binding fragment thereof of  claim 1 , wherein the enzyme having 4′-phosphopantetheinyl transferase activity is Sfp and the peptide tag is GDSLSWLLRLLN (SEQ ID NO:1), GDSLSWL (SEQ ID NO:2), DSLEFIASKLA (SEQ ID NO:9), GDSLDMLEWSLM (SEQ ID NO:10), DSLEFIASKL (SEQ ID NO:18), or DSLEFIASK (SEQ ID NO:19). 
     
     
         14 . The modified antibody or antigen binding fragment thereof of  claim 1 , wherein the antibody or antigen binding fragment thereof is an isotype selected from IgG, IgM, IgE and IgA. 
     
     
         15 . The modified antibody or antigen binding fragment thereof of  claim 1 , wherein the antibody or antigen binding fragment thereof is a subtype of IgG selected from IgG1, IgG2, IgG3 and IgG4. 
     
     
         16 . The modified antibody or antigen binding fragment thereof of  claim 1 , wherein the antibody or antigen binding fragment thereof is a human or humanized antibody or antigen binding fragment thereof. 
     
     
         17 . The modified antibody or antigen binding fragment thereof of  claim 16 , wherein the antibody or antigen binding fragment thereof is an anti-HER2 antibody or anti-HER2 antibody fragment. 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . (canceled) 
     
     
         27 . (canceled) 
     
     
         28 . (canceled) 
     
     
         29 . (canceled) 
     
     
         30 . (canceled) 
     
     
         31 . (canceled) 
     
     
         32 . (canceled) 
     
     
         33 . (canceled) 
     
     
         34 . (canceled) 
     
     
         35 . (canceled) 
     
     
         36 . (canceled) 
     
     
         37 . (canceled) 
     
     
         38 . (canceled) 
     
     
         39 . (canceled) 
     
     
         40 . The modified antibody or antigen binding fragment thereof according to  claim 1  for use as a medicament. 
     
     
         41 . The modified antibody or antigen binding fragment thereof according to  claim 1  for use in the treatment of cancer, an inflammatory disease, or an infectious disease. 
     
     
         42 . The modified antibody or antigen binding fragment thereof according to  claim 1  for use in the treatment of cancer. 
     
     
         43 . (canceled) 
     
     
         44 . (canceled) 
     
     
         45 . A nucleic acid encoding the modified antibody or antigen binding fragment thereof of  claim 1 . 
     
     
         46 . A host cell comprising the nucleic acid of  claim 45 . 
     
     
         47 . (canceled) 
     
     
         48 . (canceled) 
     
     
         49 . (canceled) 
     
     
         50 . (canceled) 
     
     
         51 . (canceled) 
     
     
         52 . (canceled) 
     
     
         53 . (canceled) 
     
     
         54 . (canceled) 
     
     
         55 . (canceled) 
     
     
         56 . (canceled)