Chimeric anti-VEGF-D antibodies and humanized anti-VEGF-D antibodies and methods of using same

Abstract

The present invention relates to materials and methods for modulating angiogenesis and lymphangiogenesis. The compositions of the invention provide chimeric and/or humanized VEGF-D antibody substances, antibodies, polypeptides and fragments thereof useful for modulating angiogenesis and lymphangiogenesis in a subject.

1 . A chimeric or humanized antibody substance according to  claim 2  comprising: 
 (a) an antibody that specifically binds Vascular Endothelial Growth Factor-D (VEGF-D), comprising 
 i) a light chain variable region comprising complementarity determining regions (L-CDR), wherein at least one of said L-CDR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-49;  
 ii) a heavy chain variable region comprising complementarity determining regions (H-CDR), wherein at least one of said H-CDR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-52;  
 iii) a human antibody light chain constant region; and  
 iv) a human antibody heavy chain constant region; or  
   (b) a fragment of (a) that binds VEGF-D.    
     
     
         2 . A chimeric or humanized antibody substance that binds VEGF-D.  
     
     
         3 . A chimeric or humanized antibody substance according to  claim 2  comprising: 
 (a) an antibody that specifically binds Vascular Endothelial Growth Factor-D (VEGF-D), comprising 
 i) a light chain variable region comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 37;  
 ii) a heavy chain variable region comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 39;  
 iii) a human antibody light chain constant region; and  
 iv) a human antibody heavy chain constant region; or  
   (b) a fragment of (a) that binds VEGF-D.    
     
     
         4 . A chimeric or humanized antibody substance according to  claim 2  comprising 
 (a) an antibody that specifically binds Vascular Endothelial Growth Factor-D (VEGF-D), comprising 
 i) heavy chain complementarity determining regions from a mouse antibody and framework regions from non-murine source, wherein the heavy chain variable region (VH) comprises complementarity determining regions (CDR) with the amino acid sequences:  
 H-CDR1 set out in SEQ ID NO: 50  
 H-CDR2 set out in SEQ ID NO: 51  
 H-CDR3 set out in SEQ ID NO: 52  
 ii) light chain complementarity determining regions from a mouse antibody and framework regions from non-murine source, wherein the light chain variable region (VL) comprises complementarity determining regions (CDR) with the amino acid sequences:  
 L-CDR1: set out in SEQ ID NO: 47  
 L-CDR2: set out in SEQ ID NO: 48  
 L-CDR3: set out in SEQ ID NO: 49;  
 iii) a human antibody light chain constant region; and  
 iv) a human antibody heavy chain constant region; or  
   (b) a fragment of (a) that binds VEGF-D.    
     
     
         5 . The antibody substance of  claim 4  wherein the framework regions of the heavy chain variable region and the framework regions of the light chain variable region comprise framework regions from a human antibody.  
     
     
         6 . The antibody substance of  claim 4  wherein the framework regions of the heavy chain variable region and the framework regions of the light chain variable region are chemically altered by amino acid substitution to be more homologous to a human antibody sequence.  
     
     
         7 . The antibody substance of  claim 5  wherein from 1 to 20 native framework region residues of the murine heavy chain variable region have been altered by amino acid substitution and wherein from 1 to 20 light chain variable region have been altered by amino acid substitution.  
     
     
         8 . The antibody substance of  claim 1  wherein the light chain constant region is a kappa or lambda light chain.  
     
     
         9 . The antibody substance of  claim 1  wherein the heavy chain constant region is selected from the group consisting of a constant region from an IgM chain, an IgG chain, an IgA chain, an IgE chain, an IgD chain, fragments thereof, and combinations thereof.  
     
     
         10 . The antibody substance of  claim 1  wherein the heavy chain constant region comprises an IgG chain selected from the group consisting of IgG1, IgG2, IgG3, IgG4, fragments thereof, and combinations thereof.  
     
     
         11 . The antibody substance of  claim 10  wherein the constant region comprises at least one of CH1, CH2, and CH3 regions of a human IgG1 heavy chain constant region.  
     
     
         12 . The antibody substance of  claim 1  that comprises a Fab fragment of the humanized antibody.  
     
     
         13 . The antibody substance of  claim 1  that is a monoclonal antibody.  
     
     
         14 . A chimeric or humanized monoclonal antibody according to  claim 2  which specifically binds to Vascular Endothelial Growth Factor-D (VEGF-D), the monoclonal antibody comprising: 
 complementarity determining regions (CDR) selected from the group consisting of: the CDR sequences set forth in SEQ ID NOs: 47-52 and variants of said sequences with 1 or 2 amino acid substitutions; and    constant regions of light and heavy chains, said constant region being of human origin, wherein the biological function of specific binding to said VEGF-D is preserved.    
     
     
         15 . A chimeric or humanized monoclonal antibody according to  claim 14  wherein the amino acid substitutions are conservative substitutions.  
     
     
         16 . A chimeric or humanized monoclonal antibody according to  claim 2  that specifically binds to Vascular Endothelial Growth Factor-D (VEGF-D), the monoclonal antibody comprising complementarity determining regions (CDR) of non-human origin from SEQ ID NOS: 37 and 39 and constant regions of light and heavy chains, said constant region being of human origin, wherein the biological function of specific binding to said VEGF-D is preserved.  
     
     
         17 . A chimeric or humanized antibody according to  claim 2  having binding specificity for Vascular Endothelial Growth Factor-D (VEGF-D), said humanized antibody comprising a light chain complementarity determining region amino acid sequences set forth in SEQ ID NOs: 47-49.  
     
     
         18 . A chimeric or humanized antibody according to  claim 17 , wherein said antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 37.  
     
     
         19 . A chimeric or humanized antibody according to  claim 17  that further comprises a heavy chain variable region from an antibody having binding specificity for VEGF-D.  
     
     
         20 . A chimeric or humanized antibody according to  claim 2  having binding specificity for Vascular Endothelial Growth Factor-D (VEGF-D), said humanized antibody comprising a heavy chain variable region comprising complementarity determining region amino acid sequences of SEQ ID NOs: 50-52.  
     
     
         21 . A chimeric or humanized antibody according to  claim 20 , wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 39.  
     
     
         22 . A chimeric or humanized antibody according to  claim 20  that further comprises a light chain variable region from an antibody having binding specificity for VEGF-D.  
     
     
         23 . A purified polypeptide according to  claim 24  that binds VEGF-D, comprising an antigen binding region of a VEGF-D antibody, wherein the antigen binding region comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 39, and wherein the antibody is humanized.  
     
     
         24 . A purified polypeptide that binds VEGF-D.  
     
     
         25 . A purified polypeptide according to  claim 24  comprising: 
 (a) the amino acid sequence of SEQ ID NO: 37 fused to the amino acid sequence of SEQ ID NO: 39, or    (b) fragments of (a) that include at least a portion of SEQ ID NO: 37 and SEQ ID NO: 39,    wherein the polypeptide binds VEGF-D.    
     
     
         26 . (canceled)  
     
     
         27 . A purified polypeptide according to  claim 24  comprising 
 (a) a polypeptide comprising complementarity determining regions from a mouse antibody and framework regions from non-murine source, wherein the heavy chain variable region (VH) comprises complementarity determining regions (CDR) with the amino acid sequences:    H-CDR1 set out in SEQ ID NO: 50    H-CDR2 set out in SEQ ID NO: 51    H-CDR3 set out in SEQ ID NO: 52; fused to    a polypeptide comprising complementarity determining regions from a mouse antibody and framework regions from non-murine source, wherein the light chain variable region (VL) comprises complementarity determining regions (CDR) with the amino acid sequences:    L-CDR1 set out in SEQ ID NO: 47    L-CDR2 set out in SEQ ID NO: 48    L-CDR3 set out in SEQ ID NO: 49; or    (b) fragments of (a) that include at least one of said CDR,    wherein the polypeptide binds VEGF-D.    
     
     
         28 - 31 . (canceled)  
     
     
         32 . A chimeric or humanized antibody according to  claim 2  that binds VEGF-D, 
 wherein the light chain variable region CDR comprise amino acid sequences at least 90% identical to the L-CDR1, L-CDR2, or L-CDR sequences set out in SEQ ID NO: 47-49; and    wherein the heavy chain variable region CDR comprise an amino acid sequence at least 90% identical to the H-CDR1, H-CDR2, or H-CDR3 sequences set out in SEQ ID NO: 50-52.    
     
     
         33 . A purified chimeric or humanized antibody substance, antibody, polypeptide, or fragment according to  claim 2  that inhibits VEGF-D binding to VEGFR-2 or to VEGFR-3.  
     
     
         34 . A purified chimeric or humanized antibody substance, antibody, polypeptide, or fragment according to  claim 2  that inhibits VEGF-D binding to VEGFR-2.  
     
     
         35 . A purified chimeric or humanized antibody substance, antibody, polypeptide, or fragment according to  claim 2  that inhibits VEGF-D stimulation of endothelial cell growth.  
     
     
         36 . A composition comprising a purified chimeric or humanized antibody substance, antibody, polypeptide, or fragment according to  claim 2  in a pharmaceutically acceptable carrier.  
     
     
         37 . An isolated polynucleotide comprising a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 37, 39, 47, 48, 49, 50, 51 or 52.  
     
     
         38 - 44 . (canceled)  
     
     
         45 . An isolated polynucleotide according to  claim 37  comprising a nucleotide sequence that encodes a light chain polypeptide of a chimeric or humanized VEGF-D antibody or antigen-binding fragment thereof, wherein the light chain polypeptide comprises L-CDR sequences set forth in SEQ ID NOs: 47-49.  
     
     
         46 . (canceled)  
     
     
         47 . An isolated polynucleotide according to  claim 37  comprising a nucleotide sequence that encodes a heavy chain polypeptide of a chimeric or humanized VEGF-D antibody or antigen-binding fragment thereof, wherein the heavy chain polypeptide comprises a H-CDR sequences set forth in SEQ ID NOs: 50-52.  
     
     
         48 . An isolated polynucleotide according to  claim 47 , wherein the polynucleotide further comprises a nucleotide sequence that encodes a light chain polypeptide of a chimeric or humanized VEGF-D antibody or antigen-binding fragment thereof, wherein the light chain polypeptide comprises L-CDR sequences set forth in SEQ ID NOs: 47-49.  
     
     
         49 - 50 . (canceled)  
     
     
         51 . An isolated polynucleotide comprising a nucleotide sequence that encodes the antibody substance, antibody, polypeptide, or fragment according to  claim 2 .  
     
     
         52 . A polynucleotide comprising a nucleotide sequence encoding a chimeric or humanized VEGF-D antibody or fragment thereof, wherein said antibody or fragment is immunospecific for VEGF-D, and wherein the antibody comprises at least one complementary determining region (CDR1, CDR2, CDR3) of the light chain variable region from the VEGF-D specific antibody VD1/4A5 and at least one complementary determining region (CDR1, CDR2, CDR3) of the heavy chain variable of the VEGF-D specific monoclonal antibody VD1/4A5.  
     
     
         53 . An expression vector comprising a polynucleotide according to  claim 37 .  
     
     
         54 . A host cell transformed or transfected with a polynucleotide according to  claim 37 .  
     
     
         55 . A host cell transformed or transfected with the vector of  claim 53 , wherein the cell expresses the antibody substance, antibody, or polypeptide encoded by the polynucleotide.  
     
     
         56 . A method for producing an antibody substance, antibody, or polypeptide that specifically binds VEGF-D, comprising culturing a host cell according to  claim 55  in a culture medium and recovering the antibody substance, antibody, or polypeptide from the cell or the medium.  
     
     
         57 - 59 . (canceled)  
     
     
         60 . A method for inhibiting VEGF-D mediated cell growth, migration, or differentiation, comprising administering to a human subject an antibody substance, antibody, polypeptide, or fragment according to  claim 1 , in an amount effective to inhibit VEGF-D interaction with VEGFR-2 or VEGFR-3.  
     
     
         61 - 65 . (canceled)