Modified Relaxin Polypeptides and Their Uses

Abstract

Modified relaxin polypeptides and their uses thereof are provided

What is claimed is: 
     
         1 - 99 . (canceled) 
     
     
         100 . A modified relaxin polypeptide comprising a non-naturally encoded amino acid, wherein: (a) the relaxin polypeptide comprises a relaxin A chain polypeptide and a relaxin B chain polypeptide, wherein said relaxin A chain polypeptide has a sequence at least 95% identical to SEQ ID NO: 4, and said relaxin B chain polypeptide has a sequence at least 95% identical to SEQ ID NO: 5 or SEQ ID NO: 6, and said non-naturally encoded amino acid is substituted in said A chain polypeptide at residue 1; and (b) said non-naturally encoded amino acid is linked to a linker or polymer, wherein said non-naturally encoded amino acid comprises a first functional group and the linker or polymer comprises a second functional group, wherein the first functional group and second functional group are not identical and each comprise a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group, and the resultant covalent linkage created by the reaction of the first and second functional groups comprises a triazole or an oxime linkage. 
     
     
         101 . An isolated nucleic acid encoding for a modified relaxin polypeptide according to  claim 100 , wherein: (a) said isolated nucleic acid comprises a selector codon that encodes said non-naturally encoded amino acid; (b) the encoded modified relaxin polypeptide comprises a relaxin A chain polypeptide having a sequence at least 95% identical to SEQ ID NO: 4; and (c) said non-naturally encoded amino acid occurs at residue 1 of the A chain of SEQ ID NO:4 or the corresponding position of said modified relaxin polypeptide, and said non-naturally encoded amino acid comprises a functional group selected from a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group. 
     
     
         102 . A method of treating a patient comprising a disorder modulated by relaxin comprising administering to the patient a therapeutically effective amount of a modified relaxin polypeptide comprising a non-naturally encoded amino acid, wherein:
 (a) the relaxin polypeptide comprises a relaxin A chain polypeptide and a relaxin B chain polypeptide, wherein said relaxin A chain polypeptide has a sequence at least 95% identical to SEQ ID NO: 4, and said relaxin B chain polypeptide has a sequence at least 95% identical to SEQ ID NO: 5 or SEQ ID NO: 6, and said non-naturally encoded amino acid is substituted in said A chain polypeptide at residue 1; and   (b) said non-naturally encoded amino acid is linked to a linker or polymer, wherein said non-naturally encoded amino acid comprises a first functional group and the linker or polymer comprises a second functional group, wherein the first functional group and second functional group are not identical and each comprise a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group, and the resultant covalent linkage created by the reaction of the first and second functional groups comprises a triazole or an oxime linkage.   
     
     
         103 . The method of  claim 102 , wherein the disorder is selected from atherosclerosis, Type 1 diabetes, Type 2 diabetes, coronary artery disease, scleroderma, stroke, diastolic dysfunction, familial hypercholesterolemia, isolated systolic hypertension, primary hypertension, secondary hypertension, left ventricular hypertrophy, arterial stiffness associated with long-term tobacco smoking, arterial stiffness associated with obesity, arterial stiffness associated with age, systemic lupus erythematosus, preeclampsia, and hypercholesterolemia. 
     
     
         104 . The method of  claim 102 , wherein the administration of the modified relaxin modulates at least one of angiotensin-II (Ang-II)-mediated vasoconstriction, endothelin-1 (ET-1) mediated vasoconstriction, nitric oxide (NO) production, angiogenesis, and platelet aggregation. 
     
     
         105 . The method of  claim 102 , wherein the non-naturally encoded amino acid in the administered modified relaxin is linked to a linker, polymer, or biologically active molecule by an oxime linkage. 
     
     
         106 . The method of  claim 102 , wherein said non-naturally encoded amino acid in the administered modified relaxin has the structure: 
       
         
           
           
               
               
           
         
         wherein the R group is a substituent other than the side chain found in alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine. 
       
     
     
         107 . The method of  claim 102 , wherein said non-naturally encoded amino acid in the administered modified relaxin is selected from a para-substituted, ortho-substituted, or meta-substituted phenylalanine comprising a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group. 
     
     
         108 . The method of  claim 102 , wherein said non-naturally encoded amino acid in the administered modified relaxin comprises para-acetyl-L-phenylalanine or para-azido-L-phenylalanine. 
     
     
         109 . The method of  claim 102 , wherein said administered modified relaxin polypeptide is recombinant, is produced in a eukaryotic host cell, is produced in a non-eukaryotic host cell, or is produced by in vitro translation. 
     
     
         110 . The method of  claim 102 , wherein said non-naturally encoded amino acid in the administered modified relaxin polypeptide is linked to a polymer. 
     
     
         111 . The method of  claim 110 , wherein said polymer is a water soluble polymer. 
     
     
         112 . The method of  claim 111 , wherein said water soluble polymer comprises poly(ethylene glycol) having an average molecular weight of between 0.1 kDa and 100 kDa. 
     
     
         113 . A method of treatment according to  claim 102 , wherein the administered modified relaxin polypeptide comprises:
 (a) a relaxin A chain polypeptide and a relaxin B chain polypeptide, wherein the relaxin A chain polypeptide comprises SEQ ID NO: 4 containing a substitution of an amino acid with a non-naturally encoded amino acid at residue 1 and the relaxin B chain polypeptide comprises SEQ ID NO: 6 or SEQ ID NO: 5, and   (b) said non-naturally encoded amino acid comprises para-acetyl-L-phenylalanine and said non-naturally encoded amino acid is linked to a linker, polymer, or biologically active molecule.   
     
     
         114 . The method of  claim 113 , wherein the non-naturally encoded amino acid in the in the administered modified relaxin polypeptide is linked to a polymer. 
     
     
         115 . The method of  claim 114 , wherein said polymer is a water soluble polymer. 
     
     
         116 . The method of  claim 115 , wherein said polymer comprises poly(ethylene glycol) having an average molecular weight of between 0.1 kDa and 100 kDa. 
     
     
         117 . The method of  claim 113 , wherein said non-naturally encoded amino acid in the in the administered modified relaxin polypeptide comprises para-acetyl-L-phenylalanine. 
     
     
         118 . The method of  claim 113 , wherein said relaxin B chain polypeptide in the in the administered modified relaxin polypeptide comprises SEQ ID NO: 6.