Antibodies and methods for wnt pathway-related diseases

Abstract

The transmembrane E3 ubiquitin ligases ZNRF3 and RNF43 are negative regulators of β-catenin and the Wnt signaling pathway in eukaryotic cells. The activity of ZNRF3 can be modulated by antibody binding to its extracellular domain, thus causing an increase in Wnt signaling. The ZNRF3 antagonizing antibodies can be used to treat diseases with low Wnt signaling, such as short bowel syndrome, osteoporosis, diabetes, neurodegenerative diseases, and mucositis. In addition, the antagonizing antibodies of the invention can be used to enhance Wnt signaling for tissue repair and wound healing.

We claim: 
     
         1 . An isolated antibody or antigen binding fragment thereof that specifically binds to the extracellular domain of a transmembrane E3 ubiquitin ligase, wherein:
 (a) the transmembrane E3 ubiquitin ligase is selected from the group consisting of ZNRF3 and RNF43; and   (b) binding of the antibody to the extracellular domain of the transmembrane E3 ubiquitin ligase, wherein the transmembrane E3 ubiquitin ligase is on the surface of a eukaryotic cell, increases Wnt signaling in the eukaryotic cell.   
     
     
         2 . The isolated antibody or antigen binding fragment of  claim 1 , wherein the increased Wnt signaling can be detected in an in vitro assay. 
     
     
         3 . The isolated antibody or antigen binding fragment of  claim 1 , wherein the antibody or antigen binding fragment comprises at least one genetically engineered polypeptide. 
     
     
         4 . The isolated antibody or antigen binding fragment of  claim 1 , wherein the antibody or antigen binding fragment comprises three regions from the amino terminus of the antibody or antigen binding fragment to the carboxyl terminus:
 (a) a first region of having a peptide sequence selected from the group consisting of SEQ ID NO: 31; SEQ ID NO: 37 SEQ ID NO: 43; SEQ ID NO: 49; SEQ ID NO: 55; SEQ ID NO: 61; SEQ ID NO: 91; SEQ ID NO: 97; SEQ ID NO: 103; SEQ ID NO: 109; SEQ ID NO: 115; SEQ ID NO: 121; SEQ ID NO: 127; SEQ ID NO: 133; SEQ ID NO: 139; SEQ ID NO: 145; SEQ ID NO: 151 and SEQ ID NO: 157;   (b) a second region having a peptide sequence selected from the group consisting of SEQ ID NO: 32; SEQ ID NO: 38; SEQ ID NO: 44; SEQ ID NO: 50; SEQ ID NO: 56; SEQ ID NO: 62; SEQ ID NO: 92; SEQ ID NO: 98; SEQ ID NO: 104; SEQ ID NO: 110; SEQ ID NO: 116; SEQ ID NO: 122; SEQ ID NO: 128; SEQ ID NO: 134; SEQ ID NO: 140; SEQ ID NO: 146; SEQ ID NO: 152; and SEQ ID NO: 158; and   (c) a third region having a peptide sequence selected from the group consisting of SEQ ID NO: 33; SEQ ID NO: 39; SEQ ID NO: 45; SEQ ID NO: 51; SEQ ID NO: 57; SEQ ID NO: 63; SEQ ID NO: 93; SEQ ID NO: 99; SEQ ID NO: 105; SEQ ID NO: 111; SEQ ID NO: 117; SEQ ID NO: 123; SEQ ID NO: 129; SEQ ID NO: 135; SEQ ID NO: 141; SEQ ID NO: 147; SEQ ID NO: 153; and SEQ ID NO: 159.   
     
     
         5 . The isolated antibody or antigen binding fragment of  claim 1 , wherein the antibody or antigen binding fragment comprises three regions from the amino terminus of the antibody or antigen binding fragment to the carboxyl terminus:
 (a) a first region of having a peptide sequence selected from the group consisting of SEQ ID NO: 34; SEQ ID NO: 40; SEQ ID NO: 46; SEQ ID NO: 52; SEQ ID NO: 58; SEQ ID NO: 64; SEQ ID NO: 94; SEQ ID NO: 100; SEQ ID NO: 106; SEQ ID NO: 112; SEQ ID NO: 118; SEQ ID NO: 124; SEQ ID NO: 130; SEQ ID NO: 136; SEQ ID NO: 142; SEQ ID NO: 148; SEQ ID NO: 154; and SEQ ID NO: 160;   (b) a second region having a peptide sequence selected from the group consisting of SEQ ID NO: 35; SEQ ID NO: 41; SEQ ID NO: 47; SEQ ID NO: 53; SEQ ID NO: 59; SEQ ID NO: 65; SEQ ID NO: 95; SEQ ID NO: 101; SEQ ID NO: 107; SEQ ID NO: 113; SEQ ID NO: 119; SEQ ID NO: 125; SEQ ID NO: 131; SEQ ID NO: 137; SEQ ID NO: 143; SEQ ID NO: 149; SEQ ID NO: 155; and SEQ ID NO: 161; and   (c) a third region having a peptide sequence selected from the group consisting of SEQ ID NO: 36; SEQ ID NO: 42; SEQ ID NO: 48; SEQ ID NO: 54; SEQ ID NO: 60; SEQ ID NO: 66; SEQ ID NO: 96; SEQ ID NO: 102; SEQ ID NO: 108; SEQ ID NO: 114; SEQ ID NO: 120; SEQ ID NO: 126; SEQ ID NO: 132; SEQ ID NO: 138; SEQ ID NO: 144; SEQ ID NO: 150; SEQ ID NO: 156; and SEQ ID NO: 162.   
     
     
         6 . The isolated antibody or antigen binding fragment of  claim 1 , wherein:
 (a) the antibody or antigen binding fragment comprises one or more polypeptides;   (b) at least one of the polypeptides comprises three regions from the amino terminus of the antibody or antigen binding fragment to the carboxyl terminus:
 (1) a first region of having a peptide sequence selected from the group consisting of SEQ ID NO: 31; SEQ ID NO: 37 SEQ ID NO: 43; SEQ ID NO: 49; SEQ ID NO: 55; SEQ ID NO: 61; SEQ ID NO: 91; SEQ ID NO: 97; SEQ ID NO: 103; SEQ ID NO: 109; SEQ ID NO: 115; SEQ ID NO: 121; SEQ ID NO: 127; SEQ ID NO: 133; SEQ ID NO: 139; SEQ ID NO: 145; SEQ ID NO: 151 and SEQ ID NO: 157; 
 (2) a second region having a peptide sequence selected from the group consisting of SEQ ID NO: 32; SEQ ID NO: 38; SEQ ID NO: 44; SEQ ID NO: 50; SEQ ID NO: 56; SEQ ID NO: 62; SEQ ID NO: 92; SEQ ID NO: 98; SEQ ID NO: 104; SEQ ID NO: 110; SEQ ID NO: 116; SEQ ID NO: 122; SEQ ID NO: 128; SEQ ID NO: 134; SEQ ID NO: 140; SEQ ID NO: 146; SEQ ID NO: 152; and SEQ ID NO: 158; and 
 (3) a third region having a peptide sequence selected from the group consisting of SEQ ID NO: 33; SEQ ID NO: 39; SEQ ID NO: 45; SEQ ID NO: 51; SEQ ID NO: 57; SEQ ID NO: 63; SEQ ID NO: 93; SEQ ID NO: 99; SEQ ID NO: 105; SEQ ID NO: 111; SEQ ID NO: 117; SEQ ID NO: 123; SEQ ID NO: 129; SEQ ID NO: 135; SEQ ID NO: 141; SEQ ID NO: 147; SEQ ID NO: 153; and SEQ ID NO: 159; and 
   (b) at least one of the polypeptides comprises three regions three regions from the amino terminus of the antibody or antigen binding fragment to the carboxyl terminus:
 (1) a first region of having a peptide sequence selected from the group consisting of SEQ ID NO: 34; SEQ ID NO: 40; SEQ ID NO: 46; SEQ ID NO: 52; SEQ ID NO: 58; SEQ ID NO: 64; SEQ ID NO: 94; SEQ ID NO: 100; SEQ ID NO: 106; SEQ ID NO: 112; SEQ ID NO: 118; SEQ ID NO: 124; SEQ ID NO: 130; SEQ ID NO: 136; SEQ ID NO: 142; SEQ ID NO: 148; SEQ ID NO: 154; and SEQ ID NO: 160; 
 (2) a second region having a peptide sequence selected from the group consisting of SEQ ID NO: 35; SEQ ID NO: 41; SEQ ID NO: 47; SEQ ID NO: 53; SEQ ID NO: 59; SEQ ID NO: 65; SEQ ID NO: 95; SEQ ID NO: 101; SEQ ID NO: 107; SEQ ID NO: 113; SEQ ID NO: 119; SEQ ID NO: 125; SEQ ID NO: 131; SEQ ID NO: 137; SEQ ID NO: 143; SEQ ID NO: 149; SEQ ID NO: 155; and SEQ ID NO: 161; and 
 (3) a third region having a peptide sequence selected from the group consisting of SEQ ID NO: 36; SEQ ID NO: 42; SEQ ID NO: 48; SEQ ID NO: 54; SEQ ID NO: 60; SEQ ID NO: 66; SEQ ID NO: 96; SEQ ID NO: 102; SEQ ID NO: 108; SEQ ID NO: 114; SEQ ID NO: 120; SEQ ID NO: 126; SEQ ID NO: 132; SEQ ID NO: 138; SEQ ID NO: 144; SEQ ID NO: 150; SEQ ID NO: 156; and SEQ ID NO: 162. 
   
     
     
         7 . The isolated antibody or antigen binding fragment of  claim 1 , wherein the peptide sequence of the antibody or antigen binding fragment has at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 67; SEQ ID NO: 69; SEQ ID NO: 71; SEQ ID NO: 73; SEQ ID NO: 75; SEQ ID NO: 77; SEQ ID NO: 79; SEQ ID NO: 81; SEQ ID NO: 83; SEQ ID NO: 85; SEQ ID NO: 87; and SEQ ID NO: 89. 
     
     
         8 . The isolated antibody or antigen binding fragment of  claim 1 , wherein the peptide sequence of the antibody or antigen binding fragment has at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 68; SEQ ID NO: 70; SEQ ID NO: 72; SEQ ID NO: 74; SEQ ID NO: 76; SEQ ID NO: 78; SEQ ID NO: 80; SEQ ID NO: 82; SEQ ID NO: 84; SEQ ID NO: 86; SEQ ID NO: 88; and SEQ ID NO: 90. 
     
     
         9 . The isolated antibody or antigen binding fragment of  claim 1 , wherein:
 (a) the antibody or antigen binding fragment comprises one or more polypeptides;   (b) at least one of the polypeptides has at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 67; SEQ ID NO: 69; SEQ ID NO: 71; SEQ ID NO: 73; SEQ ID NO: 75; SEQ ID NO: 77; SEQ ID NO: 79; SEQ ID NO: 81; SEQ ID NO: 83; SEQ ID NO: 85; SEQ ID NO: 87; and SEQ ID NO: 89; and   (b) at least one of the polypeptides has at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 68; SEQ ID NO: 70; SEQ ID NO: 72; SEQ ID NO: 74; SEQ ID NO: 76; SEQ ID NO: 78; SEQ ID NO: 80; SEQ ID NO: 82; SEQ ID NO: 84; SEQ ID NO: 86; SEQ ID NO: 88; and SEQ ID NO: 90.   
     
     
         10 . The isolated antibody or antigen binding fragment of  claim 1 , for use as a medicament in the treatment of a disease or other indication that will benefit from an increase in Wnt signaling. 
     
     
         11 . The isolated antibody or antigen binding fragment of  claim 10 , wherein the disease or other indications that will benefit from an increase in Wnt signaling are selected from the group consisting of mucositis short bowel syndrome, bacterial translocation in the gastrointestinal mucosa, enterotoxigenic or enteropathic infectious diarrhea, celiac disease, non-tropical sprue, lactose intolerance, other conditions where dietary exposures cause blunting of the mucosal villi and malabsorption, atrophic gastritis, osteoporosis, bone fracture, metabolic disease, diabetes, neurodegenerative disease, melanoma and conditions requiring tissue regeneration, tissue repair or wound healing. 
     
     
         12 . The isolated antibody or antigen binding fragment of  claim 1 , wherein the antibody or antigen binding fragment further binds specifically to another cell surface protein to increase Wnt signaling in the eukaryotic cell, wherein the other cell surface protein is selected from the group consisting of LGR4, LGR5 and LGR6. 
     
     
         13 . The antibody or antigen binding fragment of  claim 1 , in a pharmaceutically acceptable carrier. 
     
     
         14 . An isolated antibody or antigen binding fragment thereof that specifically binds to the extracellular domain of a transmembrane E3 ubiquitin ligase, for use as a medicament in the treatment of a disease or other indication that will benefit from a decrease in Wnt signaling in a subject, wherein:
 (a) the transmembrane E3 ubiquitin ligase is selected from the group consisting of ZNRF3 and RNF43; and   (b) binding of the antibody or antigen binding fragment to the extracellular domain of the transmembrane E3 ubiquitin ligase blocks the R-spondin-stimulated Wnt signaling in the subject.   
     
     
         15 . The isolated antibody or antigen binding fragment of  claim 14 , wherein the disease or other indication is selected from the group consisting of cancer, sclerosteosis, idiopathic pulmonary fibrosis, and cardiac hypertrophy. 
     
     
         16 . A soluble extracellular domain of a transmembrane E3 ubiquitin ligase in a pharmaceutically acceptable carrier, for use as a medicament in the treatment of a disease or other indication that will benefit from a decrease in Wnt signaling in a subject, wherein:
 (a) the transmembrane E3 ubiquitin ligase is selected from the group consisting of ZNRF3 and RNF43; and   (b) the soluble extracellular domain of a transmembrane E3 ubiquitin ligase specifically binds to R-spondin to block R-spondin-stimulated Wnt signaling in the subject.   
     
     
         17 . The soluble extracellular domain of  claim 16 , wherein the disease or other indication is selected from the group consisting of cancer, sclerosteosis, idiopathic pulmonary fibrosis, and cardiac hypertrophy. 
     
     
         18 . The isolated antibody or antigen binding fragment of  claim 1 , for use as a medicament in the treatment of type II diabetes mellitus, where the antibody or antigen binding fragment is administered to a subject with type II diabetes mellitus:
 (a) before the administration of a dipeptidyl peptidase-4 (DPP-4) inhibitor to the subject; or   (b) concurrent with the administration of a DPP-4 inhibitor to the subject; or   (c) before the administration of a DPP-4 inhibitor to the subject and then concurrent with the administration of a DPP-4 inhibitor to the subject   
     
     
         17 . The isolated antibody or antigen binding fragment of  claim 18 , wherein the DPP-4 inhibitor is vildagliptin (Galvus®).