Bispecific antibodies comprising a modified c-terminal crossfab fragment

Abstract

The present invention relates to bispecific antibodies comprising a modified C-terminal crossfab fragment that have reduced or no reactivity against preexisting antidrug antibodies.

1 . A bispecific antigen binding molecule, comprising a first Fab fragment and a second Fab fragment capable of specific binding to a first antigen, and a third Fab fragment capable of specific binding to a second antigen, and an Fc domain composed of a first and a second subunit capable of stable association, wherein
 (a) the first Fab fragment capable of specific binding to a first antigen is fused at its C-terminus to the N-terminus of the first Fc domain subunit;   (b) the second Fab fragment capable of specific binding to a first antigen is fused at its C-terminus to the N-terminus of the second Fc domain subunit,   (c) the third Fab fragment capable of specific binding to a second antigen is a cross-fab fragment, wherein the constant domains CH1 and CL are replaced by each other and the N-terminus of the VH domain is fused to the C-terminus of one of Fc domain subunits, and wherein the CH1 domain of the cross-fab fragment terminates with an amino acid sequence selected from the group consisting of EPKSCS (SEQ ID NO:1), EPKSCG (SEQ ID NO:2), EPKSCD (SEQ ID NO:3), EPKSCDK (SEQ ID NO:4) and EPKSCDKTHL (SEQ ID NO:5).   
     
     
         2 . The bispecific antigen binding molecule of  claim 1 , wherein the bispecific antigen binding molecule has reduced or no reactivity towards pre-existing anti-drug antibodies. 
     
     
         3 . The bispecific antigen binding molecule of  claim 1 , wherein the CH1 domain of the crossfab fragment terminates with the amino acid sequence of EPKSCS (SEQ ID NO:1). 
     
     
         4 . The bispecific antigen binding molecule of  claim 1 , wherein the CH1 domain of the crossfab fragment terminates with the amino acid sequence of EPKSCG (SEQ ID NO:2). 
     
     
         5 . The bispecific antigen binding molecule of  claim 1 , wherein the CH1 domain of the crossfab fragment terminates with the amino acid sequence selected from the group consisting of EPKSCD (SEQ ID NO:3), EPKSCDK (SEQ ID NO:4) and EPKSCDKTHL (SEQ ID NO:5). 
     
     
         6 . The bispecific antigen binding molecule of  claim 1 , wherein the CH1 domain of the crossfab fragment terminates with the amino acid sequence of EPKSCD (SEQ ID NO:3). 
     
     
         7 . The bispecific antigen binding molecule of  claim 1  wherein the bispecific antigen binding molecule binds bivalently to the first antigen and monovalently to the second antigen. 
     
     
         8 . The bispecific antigen binding molecule of  claim 1 , comprising
 (a) two heavy chains, each heavy chain comprising a VH and CH1 domain of a Fab fragment capable of specific binding to the first antigen and a Fc domain subunit,   (b) two light chains, each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to the first antigen, and   (c) a cross-fab fragment capable of specific binding to the second antigen comprising a VL-CH1 chain and a VH-CL chain, wherein the VH-CL chain is connected to the C-terminus of one of the two heavy chains of (a).   
     
     
         9 . The bispecific antigen binding molecule of  claim 1 , wherein the antigen binding molecule comprises a fourth Fab fragment capable of specific binding to the first antigen, wherein the fourth Fab fragment is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the VH-CH1 chain of the first Fab fragment capable of specific binding to the first antigen. 
     
     
         10 . The bispecific antigen binding molecule of  claim 1  wherein the bispecific antigen binding molecule binds trivalently to the first antigen and monovalently to the second antigen. 
     
     
         11 . The bispecific antigen binding molecule of  claim 1 , comprising
 (a) a heavy chain comprising a VH-CH1 chain of the first Fab fragment capable of specific binding to the first antigen fused at its N-terminus to the VH-CH1 chain of the fourth Fab fragment capable of specific binding to the first antigen, optionally via a peptide linker, a Fc domain subunit, and a VH-CL chain of the third Fab fragment capable of specific binding to the second antigen fused to the C-terminus of the Fc region subunit, optionally via a peptide linker,   (b) a heavy chain comprising a VH-CH1 domain of the second Fab fragment capable of specific binding to first antigen and a Fc domain subunit,   (c) three light chains, each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to the first antigen, and   (d) a light chain comprising a VL and CH1 domain of the third Fab fragment capable of specific binding to the second antigen.   
     
     
         12 . The bispecific antigen binding molecule of  claim 1 , comprising
 (a) a heavy chain comprising a VH-CH1 chain of the first Fab fragment capable of specific binding to the first antigen fused at its N-terminus to the VH-CH1 chain of the fourth Fab fragment capable of specific binding to the first antigen, optionally via a peptide linker, a Fc domain subunit,   (b) a heavy chain comprising a VH-CH1 domain of the second Fab fragment capable of specific binding to first antigen and a Fc region subunit, and a VH-CL chain of the third Fab fragment capable of specific binding to the second antigen fused to the C-terminus of the Fc domain subunit, optionally via a peptide linker,   (c) three light chains, each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to the first antigen, and   (d) a light chain comprising a VL and CH1 domain of a Fab fragment capable of specific binding to the second antigen.   
     
     
         13 . The bispecific antigen binding molecule of  claim 1 , wherein the antigen binding molecule comprises a fifth Fab fragment capable of specific binding to the first antigen, wherein the fifth Fab fragment is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the VH-CH1 chain of the second Fab fragment capable of specific binding to the first antigen. 
     
     
         14 . The bispecific antigen binding molecule of  claim 1 , wherein the bispecific antigen binding molecule binds tetravalently to the first antigen and monovalently to the second antigen. 
     
     
         15 . The bispecific antigen binding molecule of  claim 1 , comprising
 (a) a heavy chain comprising a VH-CH1 chain of a first Fab fragment capable of specific binding to the first antigen fused at its N-terminus to the VH-CH1 chain of the fourth Fab fragment capable of specific binding to the first antigen, optionally via a peptide linker, a Fc domain subunit, and a VH-CL chain of the third Fab fragment capable of specific binding to the second antigen fused to the C-terminus of the Fc region subunit, optionally via a peptide linker,   (b) a heavy chain comprising a VH-CH1 domain of the second Fab fragment capable of specific binding to first antigen fused at its N-terminus to the VH-CH1 chain of the fifth Fab fragment capable of specific binding to the first antigen, optionally via a peptide linker, a Fc domain subunit,   (c) three light chains, each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to the first antigen, and   (d) a light chain comprising a VL and CH1 domain of the third Fab fragment capable of specific binding to the second antigen.   
     
     
         16 . The bispecific antigen binding molecule of  claim 1 , wherein the Fc domain is an IgG, particularly an IgG1 Fc domain or an IgG4 Fc domain and/or wherein the Fc domain comprises one or more amino acid substitution that reduces the binding affinity of the antibody to an Fc receptor and/or effector function. 
     
     
         17 . The bispecific antigen binding molecule of  claim 1 , wherein the Fc domain is of human IgG1 subclass with the amino acid mutations L234A, L235A and P329G (EU numbering according to Kabat). 
     
     
         18 . The bispecific antigen binding molecule of  claim 1 , wherein the first subunit of the Fc domain comprises knobs and the second subunit of the Fc domain comprises holes according to the knobs into holes method. 
     
     
         19 . The bispecific antigen binding molecule of  claim 1 , wherein the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering according to Kabat) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S and Y407V (EU numbering according to Kabat). 
     
     
         20 . An isolated nucleic acid encoding the bispecific antigen binding molecule of  claim 1 . 
     
     
         21 . An expression vector comprising the isolated nucleic acid of  claim 20 . 
     
     
         22 . A host cell comprising the isolated nucleic acid of  claim 20 . 
     
     
         23 . A method of producing a bispecific antigen binding molecule, comprising culturing the host cell of  claim 22  under conditions suitable for the expression of the bispecific antigen binding molecule, and isolating the bispecific antigen binding molecule. 
     
     
         24 . A pharmaceutical composition comprising the bispecific antigen binding molecule of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         25 . (canceled)