FAB Molecules with a Rodent Hinge Region and a Non-Rodent CH1 Region

Abstract

The disclosure relates to novel Fab molecules comprising a modified heavy chain constant region. The modified constant region prevents the recognition of the Fab molecules by anti-Fab antibodies present in a host's serum. The disclosure further relates to methods of generating such modified Fab molecules for biological, diagnostic, pharmaceutical and other uses.

1 . A method of preparing a Fab comprising a modified heavy chain constant region, wherein the Fab comprises a CH1 domain and a hinge region in order from N- to C-terminus, comprising the steps of:
 (a) providing a Fab comprising a hinge region that is not a rodent IgG hinge; and   (b) replacing the hinge with a rodent hinge.   
     
     
         2 . The method according to  claim 1 , wherein the hinge region in step (a) is a human IgG hinge. 
     
     
         3 . The method according to  claim 2 , wherein the human IgG1 hinge comprises the amino acid sequence EPKSC (SEQ ID NO: 94). 
     
     
         4 . The method according to  claim 1 , wherein the rodent hinge is a wildtype rat IgG2a or rat IgG2b isotype. 
     
     
         5 . The method according to  claim 1 , wherein the rodent hinge comprises the amino acid sequence of any one of ERRNGGIGHKC (SEQ ID NO: 32), EERNGGIGHKC (SEQ ID NO: 93), ERRQGGIGHKC (SEQ ID NO: 34), VPREC (SEQ ID NO: 26). 
     
     
         6 . The method according to  claim 1 , wherein the rodent hinge contains not more than one cysteine. 
     
     
         7 . The method according to  claim 1 , wherein the CH1 domain is a wildtype human IgG1 CH1 domain, any allotype of a wildtype human IgG1 CH1 domain or comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of a wildtype human IgG1 CH1 domain. 
     
     
         8 . The method according to  claim 7 , wherein the CH1 domain comprises the amino acid sequence of an one of: 
       
         
           
                 
               
                   (SEQ ID NO: 1) 
                 
                   ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG 
                 
                   VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK 
                 
                   V, 
                 
                     
                 
                   (SEQ ID NO: 2) 
                 
                   ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG 
                 
                   VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRV, 
                 
                     
                 
                   (SEQ ID NO: 7) 
                 
                   ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG 
                 
                   VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK 
                 
                   I, 
                 
                     
                 
                   (SEQ ID NO: 8) 
                 
                   ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG 
                 
                   VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVEKKV, 
                 
                     
                 
                   (SEQ ID NO: 9) 
                 
                   ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG 
                 
                   VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKGV 
                 
                   or 
                 
                     
                 
                   (SEQ ID NO: 10) 
                 
                   ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG 
                 
                   VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKE 
                 
                   V. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         9 . The method according to  claim 1 , wherein the modified heavy chain constant region inhibits or prevents recognition of the Fab by anti-Fab antibodies present in a host's serum. 
     
     
         10 . The method according to  claim 1 , wherein replacing the hinge with a rodent hinge in step b) removes an epitope which is recognized by anti-Fab antibodies present in a host's serum. 
     
     
         11 . The method according to  claim 1 , where the host's serum is human serum. 
     
     
         12 . The method according to  claim 1 , wherein the modified heavy chain constant region consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 70-76. 
     
     
         13 . The method according to  claim 1 , wherein the modified heavy chain constant region comprises a CH1 domain and a hinge region in order from N- to C-terminus, wherein
 (a) the CH1 domain comprises the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKEV (SEQ ID NO: 10) and   b) the hinge region comprises the amino acid sequence ERRQGGIGHKC (SEQ ID NO: 34).   
     
     
         14 . A Fab obtainable according to the method of  claim 1 . 
     
     
         15 . A method of preventing or inhibiting recognition of a Fab by anti-Fab antibodies present in a host's serum comprising the steps of:
 (a) providing a Fab comprising a hinge region that is not a rodent IgG hinge; and   (b) replacing the hinge with a rodent hinge.   
     
     
         16 . A method for treating or preventing a disease, comprising administering to a subject a Fab comprising a modified heavy chain constant region, wherein said modified heavy chain constant region comprises a CH1 domain and a hinge region, wherein the hinge region is of a rodent IgG isotype and the CH1 domain is not of a rodent IgG isotype, so that recognition of said Fab by anti-Fab antibodies present in a host's serum is inhibited. 
     
     
         17 . The method according  claim 16 , wherein the rodent hinge region does not serve as an epitope for anti-Fab antibodies present in the host's serum. 
     
     
         18 . The method according to  claim 16 , wherein the disease is associated with the undesired presence of a target molecule of interest specifically bound by said Fab comprising the modified heavy chain constant region. 
     
     
         19 . The method according to  claim 16 , wherein the disease is an autoimmune disease, inflammatory disease, cancer, neovascular disease, infectious disease, thrombosis, myocardial infarction, and/or diabetes.