Lefty, lefty derivatives and uses thereof

Abstract

The disclosure relates to Lefty derivatives and the uses of Lefty polypeptides as antagonists of the function of certain ligands such as Nodal, GDF-8 (Myostatin), and GDF-11. These derivatives may be fused to other functional heterologous proteins such as IgG, especially the Fc portion of IgG. According to the disclosure, Lefty polypeptides are useful in the treatment of a variety of disorders, including, for example, neuronal diseases, muscle and bone conditions, and metabolic disorders.

1 . A recombinant Lefty derivative polypeptide comprising an amino acid sequence as set forth in the formula: -A-X-B-,
 wherein A consists essentially of an amino acid sequence at least 85% identical to the sequence of Region 2 of SEQ ID NO:1;   wherein B consists essentially of an amino acid sequence at least 85% identical to the sequence of Region 4 of SEQ ID NO:1;   wherein X consists of zero, one or more than one amino acid; and   wherein the recombinant, Lefty derivative polypeptide binds to one or more of Nodal, myostatin and GDF-11.   
     
     
         2 . The recombinant Lefty derivative polypeptide of  claim 1 , wherein the recombinant Lefty derivative polypeptide comprises an amino acid sequence that is at least 85% identical to the cystine knot portion of a human Lefty polypeptide. 
     
     
         3 . The recombinant Lefty derivative polypeptide of  claim 1 , wherein the recombinant Lefty derivative polypeptide comprises an amino acid sequence that is at least 85% identical to a human Lefty polypeptide sequence selected from the group consisting of: amino acids 22-353 of SEQ ID NO:1 and amino acids 22-353 of SEQ ID NO:2, wherein one or both RXXR cleavage sequences are altered so as to prevent cleavage at the altered sequence. 
     
     
         4 . The recombinant Lefty derivative polypeptide of  claim 1 , wherein A consists of an amino acid sequence at least 95% identical to the sequence of Region 2 of SEQ ID NO:1 and wherein B consists of an amino acid sequence at least 95% identical to the sequence of Region 4 of SEQ ID NO:1. 
     
     
         5 . The recombinant Lefty derivative polypeptide of  claim 1 , wherein A is selected from the group consisting of: CRQEMYIDLQGMKWAKNWVLEPPG FLAYECVGT (SEQ ID NO: 5) and CRQEMYIDLQGMKWAENWVLEPPGFLAYECVGT (SEQ ID NO: 7), and wherein B is selected from the group consisting of: 
       
         
           
                 
               
                   (SEQ ID NO: 6) 
                 
                 
                 
               
                     
                   CIASETASLPMIVSIKEGGRTRPQVVSLPNMRVQKC 
                 
                     
                   and 
                 
                     
                     
                 
                 
               
                   (SEQ ID NO: 8) 
                 
                 
                 
               
                     
                   CIASETDSLPMIVSIKEGGRTRPQVVSLPNMRVQKC. 
                 
             
                
               
            
             
                
                
                
               
            
             
                
               
            
             
                
               
            
           
         
       
     
     
         6 . The recombinant Lefty derivative polypeptide of  claim 1 , wherein X comprises an amino acid sequence that has low immunogenicity. 
     
     
         7 . The recombinant Lefty derivative polypeptide of  claim 1 , wherein X comprises a glycosylation site. 
     
     
         8 . The recombinant Lefty derivative polypeptide of  claim 1 , wherein the length of X is between 0-50 amino acids. 
     
     
         9 . The recombinant Lefty derivative polypeptide of  claim 1 , wherein X comprises a dimerization domain. 
     
     
         10 . The recombinant Lefty derivative polypeptide of  claim 1 , wherein the polypeptide is fused to an additional domain. 
     
     
         11 . The recombinant Lefty derivative polypeptide of  claim 10 , wherein the additional domain is a dimerization domain. 
     
     
         12 . The recombinant Lefty derivative polypeptide of  claim 10 , wherein the additional domain is fused to the carboxyl or amino terminus of the Lefty polypeptide. 
     
     
         13 . The recombinant Lefty derivative polypeptide of  claim 11 , wherein the dimerization domain comprises a Lefty propeptide sequence. 
     
     
         14 . The recombinant Lefty derivative polypeptide of  claim 11 , wherein the dimerization domain comprises an immunoglobulin Fab constant domain. 
     
     
         15 . The recombinant Lefty derivative polypeptide of  claim 14 , wherein said immunoglobulin Fab constant domain is selected from an immunoglobulin heavy chain constant region and an immunoglobulin light chain constant region. 
     
     
         16 . The recombinant Lefty derivative polypeptide of  claim 11 , wherein the dimerization domain is a leucine zipper domain. 
     
     
         17 . The recombinant Lefty derivative polypeptide of  claim 16 , wherein said leucine zipper domain comprises at least four leucine heptads. 
     
     
         18 . The recombinant Lefty derivative polypeptide of  claim 17 , wherein said leucine zipper domain is selected from the group consisting of a Fos and a Jun leucine zipper domain. 
     
     
         19 . The recombinant Lefty derivative polypeptide of  claim 11 , further comprising a linker sequence interposed between and covalently joining the Lefty polypeptide and the dimerization domain. 
     
     
         20 . The recombinant Lefty derivative polypeptide of  claim 1 , wherein X comprises a domain that binds Nodal, myostatin and/or GDF-11. 
     
     
         21 . The recombinant Lefty derivative polypeptide of  claim 1 , wherein the additional domain is a domain that binds Nodal, myostatin and/or GDF-11. 
     
     
         22 . The recombinant Lefty derivative polypeptide of  claim 21 , wherein the domain that binds Nodal, myostatin and/or GDF-11 inhibits the binding of Nodal, myostatin and/or GDF-11 to a Type I receptor. 
     
     
         23 . The recombinant Lefty derivative polypeptide of  claim 22  wherein the domain that binds Nodal, myostatin and/or GDF-11 competitively inhibits the binding of Nodal, myostatin and/or GDF-11 to a Type I receptor selected from the group consisting of ALK4 and ALK7. 
     
     
         24 . The recombinant Lefty derivative polypeptide of  claim 22 , wherein the domain that binds Nodal, myostatin and/or GDF-11 is selected from the group consisting of:
 (a) an extracellular portion of ALK4;   (b) an extracellular portion of ALK7;   (c) an antigen-binding portion of an antibody that binds Nodal, myostatin and/or GDF-11; and   (d) a randomized polypeptide that has been selected for binding to Nodal, myostatin and/or GDF-11.   
     
     
         25 . The recombinant Lefty derivative polypeptide of  claim 1 , wherein said modified Lefty polypeptide inhibits signaling mediated by a protein selected from an ActRII receptor, myostatin, Nodal, and GDF-11 in a cell. 
     
     
         26 . The recombinant Lefty derivative polypeptide of  claim 1 , wherein said modified Lefty polypeptide comprises a heterogenous sequence that mediates secretion of the recombinant Lefty derivative polypeptide. 
     
     
         27 . The recombinant Lefty derivative polypeptide of  claim 26 , wherein said heterogenous sequence that mediates secretion of the recombinant Lefty derivative polypeptide is a honey bee melatin leader sequence. 
     
     
         28 . A recombinant polynucleotide comprising a nucleotide sequence encoding a Lefty derivative polypeptide of  claim 1 . 
     
     
         29 . The recombinant polynucleotide of  claim 28 , further comprising a promoter sequence operably linked to the nucleotide sequence encoding the Lefty derivative polypeptide. 
     
     
         30 . A cell transformed with a recombinant polynucleotide of  claim 29 . 
     
     
         31 . The cell of  claim 30 , wherein the cell is a mammalian cell. 
     
     
         32 . The cell of  claim 31 , wherein the cell is a human cell. 
     
     
         33 . A method of making a recombinant Lefty derivative polypeptide, comprising:
 a) culturing a cell of  claim 30  under conditions suitable for expression of the recombinant Lefty derivative polypeptide; and   b) recovering the recombinant Lefty derivative polypeptide so expressed.   
     
     
         34 . An isolated Lefty polypeptide complex comprising:
 a) a first Lefty polypeptide; and   b) a second Lefty polypeptide,   wherein the first and second Lefty polypeptides are associated to form a complex, and wherein the complex binds to a TGF-β family member selected from the group consisting of: myostatin, Nodal and GDF-11.   
     
     
         35 . The Lefty polypeptide complex of  claim 34 , wherein the polypeptide complex is a homodimer. 
     
     
         36 . A pharmaceutical preparation comprising a modified Lefty polypeptide of  claim 1 . 
     
     
         37 . A method for inhibiting the activity of GDF-11 and/or myostatin in vivo, the method comprising administering to the subject an effective amount of a Lefty polypeptide. 
     
     
         38 . A method for treating a subject having a disorder associated with muscle loss or insufficient muscle growth, comprising administering to the subject an effective amount of a composition comprising a Lefty polypeptide. 
     
     
         39 . The method of  claim 38 , wherein the subject has a condition selected from muscle atrophy, ALS, and a muscle wasting disorder. 
     
     
         40 . The method of  claim 39 , wherein the muscle wasting disorder is selected from the group consisting of cachexia, anorexia, DMD syndrome, BMD syndrome, AIDS wasting syndrome, muscular dystrophies, neuromuscular diseases, motor neuron diseases, diseases of the neuromuscular junction, and inflammatory myopathies. 
     
     
         41 . A method for treating a subject having a disorder associated with neurodegeneration, comprising administering to the subject an effective amount of a composition comprising a Lefty polypeptide. 
     
     
         42 . The method of  claim 41 , wherein the disorder is selected from the group consisting of Alzheimer's Disease (AD), Parkinson's Disease (PD), Amyotrophic Lateral Sclerosis (ALS), Huntington's disease (HD). 
     
     
         43 . A method for inhibiting the activity of GDF-11 and/or myostatin in vitro, the method comprising administering to the cells an effective amount of a Lefty polypeptide. 
     
     
         44 . A method for decreasing the body fat content or reducing the rate of increase in body fat content in a subject, comprising administering to a subject in need thereof an effective amount of a Lefty polypeptide. 
     
     
         45 . A method for treating a disorder associated with undesirable body weight gain in a subject, comprising administering to a subject in need thereof an effective amount of a Lefty polypeptide. 
     
     
         46 . The method of  claim 45 , wherein said disorder is selected from the group consisting of obesity, non-insulin dependent diabetes mellitus (NIDDM), cardiovascular disease, cancer, hypertension, osteoarthritis, stroke, respiratory problems, and gall bladder disease. 
     
     
         47 . The method of  claim 37  wherein the Lefty polypeptide is selected from a wildtype Lefty polypeptide or fragments thereof, a recombinant Lefty derivative polypeptide, and a dimerized Lefty polypeptide.