Targeted protein silencing using chimeras between antibodies and ubiquitination enzymes

Abstract

The present invention relates to an isolated chimeric molecule comprising a degradation domain including a eukaryotic U-box motif and a targeting domain capable of immuno specifically directing the degradation domain to a substrate where the targeting domain is heterologous to the degradation domain. A linker couples the degradation domain to the targeting domain. Also disclosed are compositions as well as methods of treating a disease, substrate silencing, screening agents for therapeutic efficacy against a disease, and methods of screening for disease biomarkers.

What is claimed: 
     
         1 . An isolated chimeric molecule comprising:
 a degradation domain comprising a eukaryotic U-box motif;   a targeting domain capable of immunospecifically directing said degradation domain to a substrate, wherein said targeting domain is heterologous to said degradation domain; and   a linker coupling said degradation domain to said targeting domain.   
     
     
         2 . The chimeric molecule of  claim 1 , wherein said degradation domain lacks an endogenous substrate recognition region. 
     
     
         3 . The chimeric molecule of  claim 1 , wherein said U-box motif comprises a modified binding region which inhibits or decreases binding to said substrate compared to said U-box motif without the modified binding region. 
     
     
         4 . The chimeric molecule of  claim 3 , wherein the modification is a mutation or deletion of said binding region. 
     
     
         5 . The chimeric molecule of  claim 1 , wherein said U-box motif permits proteolysis of said substrate. 
     
     
         6 . The chimeric molecule of  claim 1 , wherein said U-box motif possesses a cell-type specific or tissue specific ligase function. 
     
     
         7 . The chimeric molecule of  claim 6 , wherein said ligase function is cell-type specific and the cell-type is selected from the group consisting of skin cells, muscle cells, epithelial cells, endothelial cells, stem cells, umbilical vessel cells, corneal cells, cardiomyocytes, aortic cells, corneal epithelial cells, somatic cells, fibroblasts, keratinocytes, melanocytes, adipose cells, bone cells, osteoblasts, airway cells, microvascular cells, mammary cells, vascular cells, chondrocytes, placental cells, hepatocytes, glial cells, epidermal cells, limbal stem cells, periodontal stem cells, bone marrow stromal cells, hybridoma cells, kidney cells, pancreatic islets, articular chondrocytes, neuroblasts, lymphocytes, and erythrocytes. 
     
     
         8 . The chimeric molecule of  claim 1 , wherein said U-box motif is selected from the group consisting of E3A, UBR5 (EDD1), CHIP (STUB1), STUB, UBE3A, UBE3B, UBE3C, UBE4A (UFD2b), UBE4B (UFD2a), UBOX5 (UIP5), UBR5, ACT1 (TRAF3IP2), PUB19, PRP19 (PRPF19, SNEV), CYC4 (PPIL2, Cyp-60), and WDSUB1. 
     
     
         9 . The chimeric molecule of  claim 1 , wherein said targeting domain binds said substrate. 
     
     
         10 . The chimeric molecule of  claim 1 , wherein said substrate is an intracellular substrate. 
     
     
         11 . The chimeric molecule of  claim 1 , wherein said targeting domain is an antibody, polyclonal antibody, monoclonal antibody, recombinant antibody, antibody fragment, Fab′, F(ab′)2, Fv, scFv, tascFvs, bis-scFvs, sdAb, V H , V L , V., scFvD10, scFv13R4, scFvD10, humanized antibody, chimeric antibody, complementary determining region (CDR), IgA antibody, IgD antibody, IgE antibody, IgG antibody, IgM antibody, nanobody, intrabody, unibodiy, or a minibody. 
     
     
         12 . The chimeric molecule of  claim 1 , wherein said substrate is selected from the group consisting of β-galactosidase, gpD, Hsp70, post-translationally modified proteins, fibrillin, huntingtin, tumorigenic proteins, p53, Rb, adhesion proteins, receptors, cell-cycle proteins, checkpoint proteins, HFE, ATP7B, prion proteins, viral proteins, bacterial proteins, parasitic proteins, fungal proteins, DNA binding proteins, metabolic proteins, regulatory proteins, structural proteins, enzymes, immunogenic proteins, auto-immunogenic proteins, immunogens, antigens, and pathogenic proteins. 
     
     
         13 . The chimeric molecule of  claim 1 , wherein said linker is a polypeptide linker of sufficient length to prevent the steric disruption of binding between said targeting domain and said substrate. 
     
     
         14 . The chimeric molecule of  claim 1 , wherein said linker is not cleavable. 
     
     
         15 . The chimeric molecule of  claim 1 , wherein said linker is enzymatically or hydrolytically cleavable. 
     
     
         16 . A composition comprising:
 the chimeric molecule of  claim 1 ; and   a pharmaceutically-acceptable carrier.   
     
     
         17 . The composition of  claim 16  further comprising:
 a second agent selected from the group consisting of an anti-inflammatory agent, an antidiabetic agent, a hypolipidemic agent, a chemotherapeutic agent, an antiviral agent, an antibiotic, a metabolic agent, a small molecule inhibitor, a protein kinase inhibitor, adjuvants, apoptotic agents, a proliferative agent, and organotropic targeting agents, and any combination thereof. 
 
     
     
         18 . A method of treating a disease comprising:
 administering said composition of  claim 16  to a subject having said disease, wherein the subject to whom said composition is administered has an increased expression level of said substrate compared to a subject not afflicted with said disease.   
     
     
         19 . The method of  claim 18 , wherein said disease is selected from the group consisting of cancer, metastatic cancer, stroke, ischemia, peripheral vascular disease, alcoholic liver disease, hepatitis, cirrhosis, Parkinson's disease, Alzheimer's disease, cystic fibrosis diabetes, ALS, pathogenic diseases, idiopathic diseases, viral diseases, bacterial, diseases, prionic diseases, fungal diseases, parasitic diseases, arthritis, wound healing, immunodeficiency, inflammatory disease, aplastic anemia, anemia, genetic disorders, congenital disorders, type 1 diabetes, type 2 diabetes, gestational diabetes, high blood glucose, metabolic syndrome, lipodystrophy syndrome, dyslipidemia, insulin resistance, leptin resistance, atherosclerosis, vascular disease, hypercholesterolemia, hypertriglyceridemia, non-alcoholic fatty liver disease, overweight, and obesity. 
     
     
         20 . The method of  claim 18 , wherein the administering is carried out orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, transdermally, or by application to mucous membranes. 
     
     
         21 . A method for substrate silencing, the method comprising:
 selecting a substrate to be silenced;   providing said chimeric molecule of  claim 1 ; and   contacting said substrate and said chimeric molecule under conditions effective to permit the formation of a substrate-molecule complex, wherein said complex mediates the degradation of said substrate to be silenced.   
     
     
         22 . The method of  claim 21 , wherein said substrate is selected from the group consisting of β-galactosidase, gpD, Hsp70, post-translationally modified proteins, fibrillin, huntingtin, tumorigenic proteins, p53, Rb, adhesion proteins, receptors, cell-cycle proteins, checkpoint proteins, HFE, ATP7B, prion proteins, viral proteins, bacterial proteins, parasitic proteins, fungal proteins, DNA binding proteins, metabolic proteins, regulatory proteins, structural proteins, enzymes, immunogenic proteins, auto-immunogenic proteins, immunogens, antigens, and pathogenic proteins. 
     
     
         23 . A method of screening agents for therapeutic efficacy against a disease, said method comprising:
 providing a biomolecule whose presence is mediated by a disease state;   providing a test agent comprising (i) a degradation domain comprising a eukaryotic U-box motif, (ii) an targeting domain capable of immunospecifically directing said degradation domain to said biomolecule, wherein said targeting domain is heterologous to said degradation domain, and (iii) a linker coupling said degradation domain to said targeting domain;   contacting said biomolecule and said test agent under conditions effective for the test agent to facilitate degradation of the biomolecule;   determining the level of said biomolecule as a result of said contacting; and   identifying said test agent which, based on said determining, decreases the level of said biomolecule as being a candidate for therapeutic efficacy against said disease.   
     
     
         24 . The method of  claim 23 , wherein said identifying is carried out with respect to a standard biomolecule level in a subject not afflicted with said disease. 
     
     
         25 . The method of  claim 23 , wherein said identifying is carried out with respect to the biomolecule level absent said contacting. 
     
     
         26 . The method of  claim 23 , wherein said U-box motif is selected from the group consisting of E3A, UBR5 (EDD1), CHIP (STUB1), STUB, UBE3A, UBE3B, UBE3C, UBE4A (UFD2b), UBE4B (UFD2a), UBOX5 (UIP5), UBR5, ACT1 (TRAF3IP2), PUB19, PRP19 (PRPF19, SNEV), CYC4 (PPIL2, Cyp-60), and WDSUB1. 
     
     
         27 . The method of  claim 23 , wherein said targeting domain is an antibody, polyclonal antibody, monoclonal antibody, recombinant antibody, antibody fragment, Fab′, F(ab′)2, Fv, scFv, tascFvs, bis-scFvs, sdAb, V H , V L , V., scFvD10, scFv13R4, scFvD10, humanized antibody, chimeric antibody, complementary determining region (CDR), IgA antibody, IgD antibody, IgE antibody, IgG antibody, IgM antibody, nanobody, intrabody, unibodiy, or a minibody. 
     
     
         28 . The method of  claim 23 , wherein said biomolecule is selected from the group consisting of β-galactosidase, gpD, Hsp70, post-translationally modified proteins, fibrillin, huntingtin, tumorigenic proteins, p53, Rb, adhesion proteins, receptors, cell-cycle proteins, checkpoint proteins, HFE, ATP7B, prion proteins, viral proteins, bacterial proteins, parasitic proteins, fungal proteins, DNA binding proteins, metabolic proteins, regulatory proteins, structural proteins, enzymes, immunogenic proteins, auto-immunogenic proteins, immunogens, antigens, and pathogenic proteins. 
     
     
         29 . The method of  claim 23 , wherein said linker is a polypeptide linker of sufficient length to prevent the steric disruption of binding between said targeting domain and said biomolecule. 
     
     
         30 . The method of  claim 23 , wherein said biomolecule is associated with cancer, metastatic cancer, stroke, ischemia, peripheral vascular disease, alcoholic liver disease, hepatitis, cirrhosis, Parkinson's disease, Alzheimer's disease, cystic fibrosis diabetes, ALS, pathogenic diseases, idiopathic diseases, viral diseases, bacterial, diseases, prionic diseases, fungal diseases, parasitic diseases, arthritis, wound healing, immunodeficiency, inflammatory disease, aplastic anemia, anemia, genetic disorders, congenital disorders, type 1 diabetes, type 2 diabetes, gestational diabetes, high blood glucose, metabolic syndrome, lipodystrophy syndrome, dyslipidemia, insulin resistance, leptin resistance, atherosclerosis, vascular disease, hypercholesterolemia, hypertriglyceridemia, non-alcoholic fatty liver disease, overweight, or obesity, and any combination thereof. 
     
     
         31 . A method of screening for disease biomarkers, said method comprising:
 providing a sample of diseased cells expressing one or more ligands;   providing a plurality of chimeric molecules comprising (i) a degradation domain comprising a eukaryotic U-box motif, (ii) a targeting domain capable of immunospecifically directing said degradation domain to said one or more ligands, wherein said targeting domain is heterologous to said degradation domain, and (iii) a linker coupling said degradation domain to said targeting domain;   contacting said sample and said plurality of chimeric molecules under conditions effective for the diseased cells to fail to proliferate in the absence of the chimeric molecule;   determining which of said chimeric molecules permit the diseased cells to proliferate; and   identifying, as biomarkers for the disease, the ligands which bind to the chimeric molecules determined to permit diseased cells to proliferate.   
     
     
         32 . The method of  claim 31 , wherein said disease is selected from the group consisting of cancer, metastatic cancer, stroke, ischemia, peripheral vascular disease, alcoholic liver disease, hepatitis, cirrhosis, Parkinson's disease, Alzheimer's disease, cystic fibrosis diabetes, ALS, pathogenic diseases, idiopathic diseases, viral diseases, bacterial, diseases, prionic diseases, fungal diseases, parasitic diseases, arthritis, wound healing, immunodeficiency, inflammatory disease, aplastic anemia, anemia, genetic disorders, congenital disorders, type 1 diabetes, type 2 diabetes, gestational diabetes, high blood glucose, metabolic syndrome, lipodystrophy syndrome, dyslipidemia, insulin resistance, leptin resistance, atherosclerosis, vascular disease, hypercholesterolemia, hypertriglyceridemia, non-alcoholic fatty liver disease, overweight, and obesity. 
     
     
         33 . The method of  claim 31 , wherein said U-box motif is selected from the group consisting of E3A, UBR5 (EDD1), CHIP (STUB1), STUB, UBE3A, UBE3B, UBE3C, UBE4A (UFD2b), UBE4B (UFD2a), UBOX5 (UIP5), UBR5, ACT1 (TRAF3IP2), PUB19, PRP19 (PRPF19, SNEV), CYC4 (PPIL2, Cyp-60), and WDSUB1. 
     
     
         34 . The method of  claim 31 , wherein said linker is a polypeptide linker of sufficient length to prevent the steric disruption of binding between said targeting domain and said ligand. 
     
     
         35 . The method of  claim 31 , wherein said targeting domain is an antibody, polyclonal antibody, monoclonal antibody, recombinant antibody, antibody fragment, Fab′, F(ab′)2, Fv, scFv, tascFvs, bis-scFvs, sdAb, V H , V L , V nar , scFvD10, scFv13R4, scFvD10, humanized antibody, chimeric antibody, complementary determining region (CDR), IgA antibody, IgD antibody, IgE antibody, IgG antibody, IgM antibody, nanobody, intrabody, unibodiy, or a minibody.