Antibodies specific for dkk-1

Abstract

Antibodies specific for Dkk-1, an inhibitor of the osteoanabolic Wnt/LRP5 signaling pathway, are described. The antibodies, which inhibit binding of Dkk-1 to LRP5, are useful in compositions for stimulating bone growth, in particular, compositions for treating bone disorders which result in a loss in bone, for example, osteoporosis.

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) an LC CDR1 with at least 80% sequence identity to SEQ ID NO:12, 
 (ii) an LC CDR2 with at least 80% sequence identity to SEQ ID NO:13, and 
 (iii) an LC CDR3 with at least 80% sequence identity to SEQ ID NO:14; 
   (b) one or more heavy chain (HC) CDRs selected from the group consisting of
 (i) an HC CDR1 with at least 80% sequence identity to SEQ ID NO:9, 
 (ii) an HC CDR2 with at least 80% sequence identity to SEQ ID NO:10, and 
 (iii) an TIC CDR3 with at least 80% sequence identity to SEQ ID NO:11; 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 light chain (LC) complementary determining regions (CDRS) selected from the group consisting of
 (i) an LC CDR1 with at least 90% sequence identity to SEQ ID NO:12, 
 (ii) an LC CDR2 with at least 90% sequence identity to SEQ ID NO:13, and 
 (iii) an LC CDR3 with at least 90% sequence identity to SEQ ID NO:14; 
   (b) one or more heavy chain (HC) CDRs selected from the group consisting of
 (i) an HC CDR1 with at least 90% sequence identity to SEQ ID NO:9, 
 (ii) an TIC CDR2 with at least 90% sequence identity to SEQ ID NO:10, and 
 (iii) an HC CDR3 with at least 90% sequence identity to SEQ ID NO:11; 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.   
     
     
         3 . The isolated antibody or immunologically functional fragment of  claim 2 , that comprises:
 (a) one or more light chain (LC) complementary determining regions (CDRS) selected from the group consisting of
 (i) an LC CDR1 with the sequence as set forth in SEQ ID NO:12, 
 (ii) an LC CDR2 with the sequence as set forth in SEQ ID NO:13, and 
 (iii) an LC CDR3 with the sequence as set forth in SEQ ID NO:14; 
   (b) one or more heavy chain (HC) CDRs selected from the group consisting of
 (i) an HC CDR1 with the sequence as set forth in SEQ ID NO:9, 
 (ii) an HC CDR2 with the sequence as set forth in SEQ ID NO:10, and 
 (iii) an HC CDR3 with the sequence as set forth in SEQ ID NO:11; 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.   
     
     
         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:14 or the HC CDR3 with the amino acid sequence of SEQ ID NO: 11. 
     
     
         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:14 and the HC CDR3 with the amino acid sequence of SEQ ID NO:11. 
     
     
         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 Kd of about 269 pM 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 (V L ) having at least 80% sequence identity with SEQ ID NO:4;   (b) heavy chain variable region (V H ) having at least 80% sequence identity with SEQ ID NO:8; or   (c) a V L  of (a) and a V H  of (b).   
     
     
         17 . The isolated antibody or immunologically functional fragment of  claim 16 , that consists of two identical V H  and two identical V L . 
     
     
         18 . The isolated antibody or immunologically functional fragment of  claim 16 , wherein the V L  has at least 90% sequence identity with SEQ ID NO:4; and the V H  has at least 90% sequence identity with SEQ ID NO:8. 
     
     
         19 . The isolated antibody or immunologically functional fragment of  claim 18 , that consists of two identical V H  and two identical V L . 
     
     
         20 . The isolated antibody or immunologically functional fragment of  claim 16 , wherein the V L  has at least 95% sequence identity with SEQ ID NO:4; and the V H  has at least 95% sequence identity with SEQ ID NO:8. 
     
     
         21 . The isolated antibody or immunologically functional fragment of  claim 20  that consists of two identical V H  and two identical V L . 
     
     
         22 . The isolated antibody or immunologically functional fragment of  claim 21  wherein the V L  has the amino acid sequence of SEQ ID NO:4; and the V H  has the amino acid sequence of SEQ ID NO:8. 
     
     
         23 . The isolated antibody or immunologically functional fragment of  claim 22  that consists of two identical V H  and two identical V L . 
     
     
         24 . The isolated antibody or immunologically functional fragment of  claim 22  that comprises:
 (a) a light chain comprising the amino acid sequence of SEQ ID NO:3; 
 (b) a heavy chain comprising the amino acid sequence of SEQ ID NO:7; or 
 (c) a light chain comprising the amino acid sequence of SEQ ID NO:3 and a heavy chain comprising the amino acid sequence of SEQ ID NO:7. 
 
     
     
         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′).sub.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:35 and having a tertiary structure established by a disulfide bond between cysteine residues 220 and 245, wherein the antibody binds to an epitope comprising a loop consisting of the amino acids between cysteine residues 201 and 210 of SEQ ID NO:35. 
     
     
         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′).sub.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 the heavy chains consist of the amino acid sequence set forth in SEQ ID NO:3 and the light chains consist of the amino acid sequence set forth in SEQ BD NO:7. 
     
     
         35 . The isolated antibody or immunologically functional fragment of  claim 34  that dissociates from the Dkk-1 polypeptide with a Kd of about 269 pM or less. 
     
     
         36 . A nucleic acid encoding (a) a light chain CDR with the amino acid sequence as set forth in SEQ ID NO:14; and/or (b) a heavy chain CDR with the amino acid sequence as set forth in SEQ ID NO:11, wherein the nucleic acid encodes an antibody or an immunologically functional fragment thereof. 
     
     
         37 . A nucleic acid comprising a sequence that encodes the V H , the V L  or both the V H  and the V L  of the antibody or immunologically active fragment of  claim 16 . 
     
     
         38 . A nucleic acid comprising a nucleic acid segment encoding the V H , V L  or both the V H  and the V L  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 in an individual comprising administering to the individual 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, bone disorders, and skin diseases. 
     
     
         45 . The method of  claim 44 , wherein the disease is selected from the group consisting of rheumatoid arthritis, psoriatic arthritis, and osteoarthritis. 
     
     
         46 . The method of  claim 44 , wherein the disease is osteoporosis. 
     
     
         47 . A method of stimulating growth of bone in an individual comprising administering to the individual an effective amount of the antibody or immunologically active fragment thereof of  claim 1 .