Treatment methods using dkk-1 antibodies

Abstract

The present invention provides antibodies and immunologically functional fragments thereof that specifically bind Dkk-1 polypeptides. The subject antibodies and fragments bind with high affinity to a conformational epitope located in the carboxy region of the Dkk-1 protein. Methods for preparing such antibodies or fragments thereof as well as physiologically acceptable compositions containing the antibodies or fragments are also provided. Use of the antibodies and fragments to treat various diseases including bone disorders, inflammatory diseases, neurological diseases, ocular diseases, renal diseases, pulmonary diseases and skin diseases are also disclosed.

1 . An isolated antibody or immunologically functional fragment thereof, comprising:
 (a) one or more light chain (LC) complementary determining regions (CDRs) selected from the group consisting of:
 (i) a LC CDR1 with at least 80% sequence identity to SEQ ID NO:70; 
 (ii) a LC CDR2 with at least 80% sequence identity to SEQ ID NO:72; and 
 (iii) a LC CDR3 with at least 80% sequence identity to SEQ ID NO:74; 
   (b) one or more heavy chain (HC) CDRs selected from the group consisting of
 (i) a HC CDR1 with at least 80% sequence identity to SEQ ID NO:76; 
 (ii) a HC CDR2 with at least 80% sequence identity to SEQ ID NO:78; and 
 (iii) a HC CDR3 with at least 80% sequence identity to SEQ ID NO:80; 
   
       or
 (c) one or more LC CDRs of (a) and one or more HC CDRs of (b),
 wherein the antibody or immunologically functional fragment thereof can specifically bind a Dkk-1 polypeptide. 
 
 
     
     
         2 . The isolated antibody or immunologically functional fragment of  claim 1 , that comprises:
 (a) one or more LC CDRs selected from the group consisting of:
 (i) a LC CDR1 with at least 90% sequence identity to SEQ ID NO:70 
 (ii) a LC CDR2 with at least 90% sequence identity to SEQ ID NO:72; and 
 (iii) a LC CDR3 with at least 90% sequence identity to SEQ ID NO:74; 
   (b) one or more HC CDRs selected from the group consisting of
 (i) a HC CDR1 with at least 90% sequence identity to SEQ ID NO:76 
 (ii) a HC CDR2 with at least 90% sequence identity to SEQ ID NO:78; and 
 (iii) a HC CDR3 with at least 90% sequence identity to SEQ ID NO:80; or 
   (c) one or more LC CDRs of (a) and one or more HC CDRs of (b).   
     
     
         3 . The isolated antibody or immunologically functional fragment of  claim 2 , that comprises:
 (a) one or more LC CDRs selected from the group consisting of:
 (i) a LC CDR1 with the amino acid sequence as set forth in SEQ ID NO:70 
 (ii) a LC CDR2 with the amino acid sequence as set forth in SEQ ID NO:72; and 
 (iii) a LC CDR3 with the amino acid sequence as set forth in SEQ ID NO:74; 
   (b) one or more HC CDRs selected from the group consisting of
 (i) a HC CDR1 with the amino acid sequence as set forth in SEQ ID NO:76 
 (ii) a HC CDR2 with the amino acid sequence as set forth in SEQ ID NO:78; and 
 (iii) a HC CDR3 with the amino acid sequence as set forth in SEQ ID NO:80; or 
   (c) one or more LC CDRs of (a) and one or more HC CDRs of (b).   
     
     
         4 . The isolated antibody or immunologically functional fragment of  claim 3  that comprises the LC CDR3 with the amino acid sequence of SEQ ID NO:74 or the HC CDR3 with the amino acid sequence of SEQ ID NO:80. 
     
     
         5 . The isolated antibody or immunologically functional fragment of  claim 4  that comprises the LC CDR3 with the amino acid sequence of SEQ ID NO:74 and the HC CDR3 with the amino acid sequence of SEQ ID NO:80. 
     
     
         6 . The isolated antibody or immunologically functional fragment of  claim 1  that comprises at least two CDRs from the CDRs listed in (a) and (b). 
     
     
         7 . The isolated antibody or immunologically functional fragment of  claim 6  that comprises at least three CDRs from the CDRs listed in (a) and (b). 
     
     
         8 . The isolated antibody or immunologically functional fragment of  claim 7  that comprises at least four CDRs from the CDRs listed in (a) and (b). 
     
     
         9 . The isolated antibody or immunologically functional fragment of  claim 8  that comprises at least five CDRs from the CDRs listed in (a) and (b). 
     
     
         10 . The isolated antibody or immunologically functional fragment of  claim 9  that comprises all six of the CDRs listed in (a) and (b). 
     
     
         11 . The isolated antibody or immunologically functional fragment of  claim 1  that is a domain antibody. 
     
     
         12 . The isolated antibody or immunologically functional fragment of  claim 1  that dissociates from the Dkk-1 polypeptide with a k d  of 5×10 −4  s −1  or less. 
     
     
         13 . The isolated antibody or immunologically functional fragment of  claim 1  that is a monoclonal antibody. 
     
     
         14 . The isolated antibody or immunologically functional fragment of  claim 1  that is a scFv, a Fab, a Fab′ or a (Fab′) 2 . 
     
     
         15 . The isolated antibody or immunologically functional fragment of  claim 1  that is a human or humanized antibody. 
     
     
         16 . An isolated antibody or immunologically functional fragment thereof, comprising:
 (a) a light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO:84, 28 or 32;   (b) heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO:91, 36, 40, 44, 48, 52, 56, 60, 64 or 68; or   (c) a VL of (a) and a VH of (b).   
     
     
         17 . The isolated antibody or immunologically functional fragment of  claim 16 , that consists of two identical VH and two identical VL. 
     
     
         18 . The isolated antibody or immunologically functional fragment of  claim 16 , wherein
 the VL has at least 90% sequence identity with SEQ ID NO:84, 28 or 32; and   the VH has at least 90% sequence identity with SEQ ID NO:91, 36, 40, 44, 48, 52, 56, 60, 64 or 68.   
     
     
         19 . The isolated antibody or immunologically functional fragment of  claim 18 , that consists of two identical VH and two identical VL. 
     
     
         20 . The isolated antibody or immunologically functional fragment of  claim 16 , wherein
 the VL has at least 95% sequence identity with SEQ ID NO:84, 28 or 32; and   the VH has at least 95% sequence identity with SEQ ID NO:91, 36, 40, 44, 48, 52, 56, 60, 64, or 68.   
     
     
         21 . The isolated antibody or immunologically functional fragment of  claim 20  that consists of two identical VH and two identical VL. 
     
     
         22 . The isolated antibody or immunologically functional fragment of  claim 21  wherein
 the VL has the amino acid sequence of SEQ ID NO:84, 28 or 32; and 
 the VH has the amino acid sequence of SEQ ID NO:91, 36, 40, 44, 48, 52, 56, 60, 64 or 68. 
 
     
     
         23 . The isolated antibody or immunologically functional fragment of  claim 22  that consists of two identical VH and two identical VL. 
     
     
         24 . The isolated antibody or immunologically functional fragment of  claim 22  that comprises
 a light chain comprising the amino acid sequence of SEQ ID NO:82, 26, or 30; 
 a heavy chain comprising the amino acid sequence of SEQ ID NO:89, 34, 38, 42, 46, 50, 54, 58, 62, or 66; or 
 a light chain comprising the amino acid sequence of SEQ ID NO:82, 26, or 30 and a heavy chain comprising the amino acid sequence of SEQ ID NO:89, 34, 38, 42, 46, 50, 54, 58, 62, or 66. 
 
     
     
         25 . The isolated antibody or immunologically functional fragment of  claim 24  that consists of two identical light chains and two identical heavy chains. 
     
     
         26 . The isolated antibody or immunologically functional fragment of  claim 16  that is a monoclonal antibody. 
     
     
         27 . The isolated antibody or immunologically functional fragment of  claim 16  that is a scFv, a Fab, a Fab′ or a (Fab′) 2 . 
     
     
         28 . The isolated antibody or immunologically functional fragment of  claim 16  that is a human or humanized antibody. 
     
     
         29 . An isolated antibody or an immunologically functional fragment thereof that specifically binds a mature human Dkk-1 protein consisting of amino acids 32-266 of SEQ ID NO:2, wherein said antibody binds to an epitope comprising two loops, said loops being formed by disulfide bonds between 220 and 237 of SEQ ID NO:2 and between cysteine residues 245 and 263 of SEQ ID NO:2. 
     
     
         30 . The isolated antibody or immunologically functional fragment of  claim 29  that is a monoclonal antibody. 
     
     
         31 . The isolated antibody or immunologically functional fragment of  claim 29  that is a scFv, a Fab, a Fab′ or a (Fab′) 2 . 
     
     
         32 . The isolated antibody or immunologically functional fragment of  claim 29  that is a human or humanized antibody. 
     
     
         33 . An antibody or an immunologically functional fragment thereof that competes with an antibody of  claim 24  for specific binding to a Dkk-1 polypeptide. 
     
     
         34 . The isolated antibody or immunologically functional fragment of  claim 33  that competes with an antibody that consists of two identical heavy chains and two identical light chains, wherein said heavy chains consist of amino acids 20-465 of SEQ ID NO:12 and said light chains consist of amino acids 21-234 of SEQ ID NO:10. 
     
     
         35 . The isolated antibody or immunologically functional fragment of  claim 34  that dissociates from the Dkk-1 polypeptide with a k d  of 5×10 −4  s −1  or less. 
     
     
         36 . A nucleic acid encoding
 (a) a light chain CDR with the amino acid sequence as set forth in SEQ ID NO:70, 72 and/or 74; and/or   (b) a heavy chain CDR with the amino acid sequence as set forth in SEQ ID NO:76, 78 and/or 80,   wherein the nucleic acid encodes an antibody or an immunologically functional fragment thereof.   
     
     
         37 . A nucleic acid comprising a sequence that encodes the VH, the VL or both the VH and the VL of the antibody or immunologically active fragment of  claim 16 . 
     
     
         38 . A nucleic acid comprising a nucleic acid segment encoding the VH, VL or both the VH and the VL of the antibody or immunologically active fragment of  claim 22 . 
     
     
         39 . An expression vector comprising the nucleic acid of  claim 37 . 
     
     
         40 . An isolated cell comprising the expression vector of  claim 39 . 
     
     
         41 . A method of producing an antibody or an immunologically active fragment thereof comprising the step of culturing a cell according to  claim 40 . 
     
     
         42 . A composition comprising an antibody or immunologically functional fragment thereof according to  claim 1  and a component selected from the group consisting of a buffer, a pharmaceutically acceptable diluent, a carrier, a solubilizer, an emulsifier and a preservative. 
     
     
         43 . A composition comprising an antibody or immunologically functional fragment thereof according to  claim 16  and a component selected from the group consisting of a buffer, a pharmaceutically acceptable diluent, a carrier, a solubilizer, an emulsifier and a preservative. 
     
     
         44 . A method of treating a disease comprising administering to a patient in need thereof an effective amount of the antibody or immunologically active fragment thereof of  claim 1 , wherein the disease is selected from the group consisting of arthritis, diseases responsive to stem cell renewal, inflammatory diseases, neurological diseases, ocular diseases, renal diseases, pulmonary diseases, and skin diseases. 
     
     
         45 . The method of  claim 44 , wherein the disease is rheumatoid arthritis, psoriatic arthritis, and osteoarthritis. 
     
     
         46 . The method of  claim 44 , wherein the disease
 (a) is responsive to stem cell renewal and is selected from the group consisting of diabetes, chronic heart failure and diseases of the muscle;   (b) is an inflammatory disease selected from the group consisting of Crohn's disease, colitis, and inflammatory bowel disease;   (c) is a neurological disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, and Huntington's disease;   (d) is an ocular disease selected from the group consisting of macular degeneration and retinopathies;   (e) is a renal disease selected from the group consisting of end stage renal disease, chronic renal disease, glomerulonephritis, tubulointerstitial nephritis, and IgA nephropathy;   (f) is a pulmonary disease selected from the group consisting of chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and cystic fibrosis; or   (g) is a skin disease resulting from chemotherapy-induced damage to the intestinal epithelium.