Stable and soluble antibodies inhibiting vegf

Abstract

The present invention relates to soluble and stable anti-VEGF imunobinders comprising CDRs from rabbit monoclonal antibodies. Said antibodies are designed for the diagnosis and/or treatment of VEGF-mediated disorders. The hybridomas, nucleic acids, vectors and host cells for expression of the recombinant antibodies of the invention, methods for isolating them and the use of said antibodies in medicine are also disclosed.

1 . A humanized antibody or antigen-binding fragment thereof comprising a variable heavy chain (VH), and a variable light chain (VL), wherein:
 the VH comprises CDRH1, CDRH2 and CDRH3 sequences of SEQ ID NO: 8, SEQ ID NO: 20 and SEQ ID NO: 32, respectively, and the VL comprises CDRL1, CDRL2, and CDRL3 sequences of SEQ ID NO: 43, SEQ ID NO: 55 and SEQ ID NO: 66,   wherein the antibody or antigen-binding fragment thereof binds human VEGF 165  with an affinity (K d ) of ≤1×10 −9 M.   
     
     
         2 . The humanized antibody or antigen-binding fragment of  claim 1 , wherein the fragment is an scFv, a Fab fragment, a Fab′ fragment, or a F(ab′) 2  fragment. 
     
     
         3 . A composition comprising the humanized antibody or antigen-binding fragment of  claim 1 , and a pharmaceutically acceptable carrier. 
     
     
         4 . The composition of  claim 3 , formulated for topical, intraocular, oral, nasal, rectal or parental administration. 
     
     
         5 . The humanized antibody or antigen-binding fragment of  claim 1  comprising a heavy chain variable region variable region framework sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 164. 
     
     
         6 . The humanized antibody or antigen-binding fragment of  claim 5 , wherein the heavy chain variable region framework comprises a sequence having 100% identity to SEQ ID NO: 164. 
     
     
         7 . The humanized antibody or antigen-binding fragment of  claim 1 , comprising a light chain variable region framework sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 87. 
     
     
         8 . The humanized antibody or antigen-binding fragment of  claim 7 , wherein the light chain variable region framework comprises a sequence having 100% identity to SEQ ID NO: 87. 
     
     
         9 . A method of treating a VEGF-mediated disease in a subject, comprising administering to the subject in need thereof the humanized antibody or antigen-binding fragment of  claim 1 , wherein the VEGF-mediated disease is age-related macular degeneration. 
     
     
         10 . A method of inhibiting human VEGF-mediated endothelial cell proliferation and vascular permeability in a subject having a human VEGF-mediated disease, comprising administering to the subject a humanized antibody or antigen-binding fragment of  claim 1 , such that the subject is treated for a VEGF-mediated disease. 
     
     
         11 . An isolated nucleic acid molecule encoding the humanized antibody or antigen-binding fragment thereof of  claim 1 . 
     
     
         12 . The isolated nucleic acid molecule of  claim 11 , wherein the antibody or antigen-binding fragment comprises a variable heavy chain having an amino acid sequence at least 95% identical to the sequence of SEQ ID NO: 164 and wherein the antibody or antigen-binding fragment comprises a variable light chain having an amino acid sequence at least 95% identical to the sequence of SEQ ID NO: 87. 
     
     
         13 . The isolated nucleic acid molecule of  claim 12 , wherein the variable heavy chain comprises an amino acid sequence of SEQ ID NO: 164 and the variable light chain comprises an amino acid sequence of SEQ ID NO: 87. 
     
     
         14 . The isolated nucleic acid molecule of  claim 13 , wherein the heavy chain variable region and the light chain variable region are linked by the sequence of SEQ ID NO: 181. 
     
     
         15 . An expression vector comprising the nucleic acid molecule of  claim 11 . 
     
     
         16 . An expression vector comprising the nucleic acid molecule of  claim 12 . 
     
     
         17 . An expression vector comprising the nucleic acid molecule of  claim 13 . 
     
     
         18 . An expression vector comprising the nucleic acid molecule of  claim 14 . 
     
     
         19 . A host cell comprising the expression vector of  claim 15 . 
     
     
         20 . A host cell comprising the expression vector of  claim 16 . 
     
     
         21 . A host cell comprising the expression vector of  claim 17 . 
     
     
         22 . A host cell comprising the expression vector of  claim 18 . 
     
     
         23 . The humanized antibody or antigen-binding fragment of  claim 1 , wherein the antibody or antigen-binding fragment comprises a variable heavy chain comprises the sequence of SEQ ID NO: 164 and wherein the antibody or antigen-binding fragment comprises a variable light chain comprising the sequence of SEQ ID NO: 87. 
     
     
         24 . The humanized antibody or antigen-binding fragment of  claim 23 , further comprising a linker sequence having the sequence of SEQ ID NO: 181 linking the heavy chain variable region and the light chain variable region. 
     
     
         25 . The antigen-binding fragment of  claim 24 , which is a single-chain antibody (scFv). 
     
     
         26 . The antigen-binding fragment of  claim 25 , comprising an N-terminal methionine derived from a start codon present in an expression vector that expresses the antigen-binding fragment. 
     
     
         27 . The humanized antibody or antigen-binding fragment of  claim 23 , wherein the antibody or antigen-binding fragment thereof binds human VEGF 165  with an affinity (K d ) of ≤1×10 −10  M. 
     
     
         28 . A composition comprising the humanized antibody or antigen-binding fragment of  claim 27 , and a pharmaceutically acceptable carrier. 
     
     
         29 . A method of treating a VEGF-mediated disease in a mammal, comprising administering to a subject the composition of  claim 28 , wherein the VEGF-mediated disease is age-related macular degeneration. 
     
     
         30 . The method of  claim 29 , wherein the mammal is a human.