Bispecific anti-vegf/anti-ang-2 antibodies and their use in the treatment of ocular vascular diseases

Abstract

The present invention relates to bispecific antibody against human vascular endothelial growth factor (VEGF/VEGF-A) and against human angiopoietin-2 (ANG-2) of human IgG1 or IgG4 subclass with mutations I253A, H310A, and H435A, methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.

1 . A method for the reduction of the viscosity of an antibody wherein the antibody comprises a constant heavy chain region of human IgG1 or human IgG4 subclass(derived from human origin and) wherein the method comprises
 the modification of the antibody constant heavy chain region of human IgG1 or human IgG4 subclass with the mutations I253A, H310A, and H435A (numbering according to EU Index of Kabat).   
     
     
         2 . The method of  claim 1 , wherein the antibody is a bispecific antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, wherein
 i) said first antigen-binding site specifically binding to VEGF comprises in the heavy chain variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and in the light chain variable domain a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:6; and   ii) said second antigen-binding site specifically binding to ANG-2 comprises in the heavy chain variable domain a CDR3H region of SEQ ID NO: 9, a CDR2H region of, SEQ ID NO: 10, and a CDR1H region of SEQ ID NO: 11, and in the light chain variable domain a CDR3L region of SEQ ID NO: 12, a CDR2L region of SEQ ID NO: 13, and a CDR1L region of SEQ ID NO: 14, and wherein   iii) the bispecific antibody comprises a constant heavy chain region of human IgG1 or human IgG4 subclass (derived from human origin and) comprising the mutations I253A, H310A, and H435A (numbering according to EU Index of Kabat).   
     
     
         3 . The method of  claim 2 , wherein the bispecific antibody comprises a constant heavy chain region of human IgG1 subclass (derived from human origin and) comprising the mutations I253A, H310A, and H435A (numbering according to EU Index of Kabat) and further comprising the mutations L234A , L235A and P329G (numbering according to EU Index of Kabat). 
     
     
         4 . An antibody obtained by the method of any one of  claims 1  to  3 . 
     
     
         5 . A bispecific antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, wherein
 i) said first antigen-binding site specifically binding to VEGF comprises in the heavy chain variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and in the light chain variable domain a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:6; and   ii) said second antigen-binding site specifically binding to ANG-2 comprises in the heavy chain variable domain a CDR3H region of SEQ ID NO: 9, a CDR2H region of, SEQ ID NO: 10, and a CDR1H region of SEQ ID NO: 11, and in the light chain variable domain a CDR3L region of SEQ ID NO: 12, a CDR2L region of SEQ ID NO: 13, and a CDR1L region of SEQ ID NO: 14, and wherein   iii) the bispecific antibody comprises a constant heavy chain region of human IgG1 or human IgG4 subclass (derived from human origin and) comprising the mutations I253A, H310A, and H435A (numbering according to EU Index of Kabat)   
     
     
         6 . The bispecific antibody according to  claim 5 , wherein
 i) said first antigen-binding site specifically binding to VEGF comprises as heavy chain variable domain VH an amino acid sequence of SEQ ID NO: 7, and as light chain variable domain VL an amino acid sequence of SEQ ID NO: 8, and   ii) said second antigen-binding site specifically binding to ANG-2 comprises as heavy chain variable domain VH an amino acid sequence of SEQ ID NO: 15, and as light chain variable domain VL an amino acid sequence of SEQ ID NO: 16.   
     
     
         7 . The bispecific antibody according to any one of  claims 5  to  6 , wherein the constant heavy chain region under iii) is of IgG1 subclass 
     
     
         8 . The bispecific antibody according to  claim 6 , wherein the constant heavy chain region of IgG1 subclass further comprises the mutations L234A , L235A and P329G (numbering according to EU Index of Kabat) 
     
     
         9 . The bispecific antibody according to any one of  claims 5  to  6 , wherein the constant heavy chain region under iii) is of IgG4 subclass 
     
     
         10 . The bispecific antibody according to  claim 9 , wherein the constant heavy chain region of IgG4 subclass further comprises the mutations S228P and L235E (numbering according to EU Index of Kabat) 
     
     
         11 . The bispecific antibody according to  claim 9 , wherein the constant heavy chain region of IgG4 subclass further comprises the mutations S228P , L235E and P329G (numbering according to EU Index of Kabat) 
     
     
         12 . A pharmaceutical composition comprising an antibody according to any one of  claims 4  and  5  to  11 . 
     
     
         13 . A nucleic acid encoding a bispecific antibody according to any one of  claims 5  to  11 . 
     
     
         14 . Expression vector containing said nucleic acid according  claim 13  capable of expressing said nucleic acid in a prokaryotic or eukaryotic host cell. 
     
     
         15 . A prokaryotic or eukaryotic host cell comprising a vector according to  claim 14 . 
     
     
         16 . A method for the preparation of a bispecific antibody according to any one of  claims 5  to  11 
 comprising the steps of 
 a) transforming a host cell with vectors comprising nucleic acid molecules encoding said antibody; 
 b) culturing the host cell under conditions that allow synthesis of said antibody molecule; and 
 c) recovering said antibody molecule from said culture. 
 
     
     
         17 . A bispecific antibody obtained by the method of  claim 16 . 
     
     
         18 . A bispecific, bivalent antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, characterized in comprising the amino acid sequences of SEQ ID NO: 25, of SEQ ID NO: 26, of SEQ ID NO: 27, and of SEQ ID NO: 28. 
     
     
         19 . A bispecific, bivalent antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, characterized in comprising the amino acid sequences of SEQ ID NO: 21, of SEQ ID NO: 22., of SEQ ID NO: 23., and of SEQ ID NO: 24. 
     
     
         20 . A bispecific, bivalent antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, characterized in comprising the amino acid sequences of SEQ ID NO: 29, of SEQ ID NO: 30, of SEQ ID NO: 31, and of SEQ ID NO: 32. 
     
     
         21 . A method of treatment of patient suffering from ocular vascular diseases by administering an antibody according to any one of  claims 4  and  5  to  11  to a patient in the need of such treatment. 
     
     
         22 . The method of  claim 21 , wherein the antibody is administered via intravitreal application.