Glycoprotein Hormone Analogs

Abstract

This invention relates to the field of glycoprotein hormone analogs and their uses as agonists, antagonists, targeting vectors, and immunogens. In particular, this invention describes a method for stabilizing a heterodimer that permits the preparation of functional glycoprotein hormone analogs. The analogs of present invention comprise at least one alpha subunit polypeptide and at least one beta subunit polypeptide, wherein the seatbelt region of the beta subunit is linked to the alpha subunit. The invention also provides for a beta subunit polypeptide wherein the C-terminal amino acid is from residue 10 to residue 20 of the seatbelt region.

1 . A glycoprotein hormone analog capable of binding to a receptor selected from the group consisting of luteinizing hormone receptor, follicle stimulating hormone receptor, and thyroid stimulating hormone receptor, said analog comprising at least one α subunit polypeptide and at least one β subunit polypeptide, said β subunit comprising a seatbelt region comprising 1 to 20 consecutive amino acid residues,
 wherein said α subunit comprises a first amino acid residue, and said seatbelt region comprises a second amino acid residue, and said first said second amino acid residues are covalently linked by a first covalent bond;   wherein said first amino acid residue corresponds to an amino acid residue selected from the group consisting of Glu9, Thr11, Leu 12, Phe33, Arg35, Tyr37, Thr39, Pro40, Leu41, Arg42, Ser43, Val53, Thr54, Ser55, Glu56, Ser57, Thr58, His83, Ser85, Thr86, Tyr89, and Ser92 of SEQ ID NO: 7; and   wherein said second amino acid residue is selected from the group consisting of seatbelt residues 11 to 18.   
     
     
         2 . A glycoprotein hormone analog capable of binding to a receptor selected from the group consisting of luteinizing hormone receptor, follicle stimulating hormone receptor, and thyroid stimulating hormone receptor, said analog comprising at least one α subunit polypeptide and at least one β subunit polypeptide, wherein said α subunit comprises a first amino acid residue, said seatbelt region comprises a second amino acid residue, wherein said first and said second amino acid residues are covalently linked by a first covalent bond, and wherein the C-terminal amino acid of said β subunit polypeptide is from seatbelt residue 10 to seatbelt residue 20. 
     
     
         3 . The analog of  claim 2  wherein said first amino acid residue corresponds to an amino acid residue selected from the group consisting of Glu9, Thr11, Leu12, Phe33, Arg35, Tyr37, Thr39, Pro40, Leu41, Arg42, Ser43, Val53, Thr54, Ser55, Glu56, Ser57, Thr58, His83, Ser85, Thr86, Tyr89, and Ser92 of SEQ ID NO: 7; and
 wherein said second amino acid residue is selected from the group consisting of seatbelt residues 11 to 18.   
     
     
         4 . The analog of  claim 1  wherein said first amino acid residue and said second amino acid residue are both mutated. 
     
     
         5 . The analog of  claim 4  wherein said first amino acid residue and said second amino acid residue are both mutated to cysteine residues and first covalent bond is a disulfide bond. 
     
     
         6 . The analog of  claim 1  wherein residue 20 of said seatbelt region is not covalently linked to a distal portion of said β subunit. 
     
     
         7 . The analog of  claim 6  wherein said residue 20 has been mutated from a cysteine residue to another amino acid residue. 
     
     
         8 . The analog of  claim 1  wherein said second amino acid residue of said β subunit polypeptide is the C-terminal residue of said β subunit polypeptide. 
     
     
         9 . The analog of  claim 1  wherein said first amino acid residue corresponds to Thr86 of SEQ ID NO: 7 and said second amino acid residue is seatbelt residue 12. 
     
     
         10 . The analog of  claim 1 , wherein said α subunit polypeptide has reduced glycosylation relative to a native α subunit polypeptide. 
     
     
         11 . The analog of  claim 10 , wherein said α subunit polypeptide comprises an α2 loop which has reduced glycosylation relative to an α2 loop of a native β subunit polypeptide. 
     
     
         12 . The analog of  claim 11  wherein said α subunit polypeptide comprises a mutation of at least one asparagine residue relative to a native α subunit polypeptide. 
     
     
         13 . The analog of  claim 1 , wherein said α subunit polypeptide and β subunit polypeptide are linked by a peptide bond, wherein said peptide bond is between the C-terminus of said α subunit polypeptide and the N-terminus of said β subunit polypeptide. 
     
     
         14 . The analog of  claim 13 , wherein said analog comprises a cleavage site in between said α subunit and said β subunit. 
     
     
         15 . The analog of  claim 14 , wherein said cleavage site is selected from the group consisting of a furin cleavage site, thrombin cleavage site, Factor Xa cleavage site, and enterokinase cleavage site. 
     
     
         16 . The analog of  claim 1 , wherein said α subunit polypeptide has an amino acid sequence comprising a sequence selected from the group consisting of SEQ ID NO: 1, 2, 9, 10, 11, 12, 17, 23, 24, 26, 27, 28, 29, 39, 40, 41, 54, 56, 61, 64, and 66. 
     
     
         17 . The analog of  claim 1 , wherein said β subunit polypeptide has an amino acid sequence comprising a sequence selected from the group consisting of SEQ ID NO: 3, 4, 6, 13, 14, 15, 16, 18, 19, 20, 21, 22, 25, 30, 31, 32, 33, 35, 36, 37 46, 47, 48, 51, 53, 55, 57, and 60. 
     
     
         18 . The analog of  claim 1 , wherein said β subunit polypeptide and said α subunit polypeptide are covalently linked via a second covalent bond. 
     
     
         19 . The analog of  claim 18  wherein said α subunit comprises a third amino acid residue, said β subunit polypeptide comprises a fourth amino acid residue, and said third and said fourth amino acid residues are covalently linked via a said second covalent bond. 
     
     
         20 . The analog of  claims 19  wherein said third amino acid residue and said fourth amino acid residue are both mutated. 
     
     
         21 . The analog of  claim 20  wherein said third amino acid residue and said fourth amino acid residue are both mutated to cysteine residues and said second covalent bond is a disulfide bond. 
     
     
         22 . The analog of  claims 20  or  21  wherein said third residue of said α subunit polypeptide is selected from the group consisting of Gln5, Tyr37, and Arg35. 
     
     
         23 . The analog of  claim 20  wherein said fourth residue of said β subunit polypeptide is selected from the group consisting of Leu5, Arg6, Arg8, Ile33, and Ala35. 
     
     
         24 . A targeting compound comprising an analog of  claim 1 , wherein said targeting compound is complexed with an active agent. 
     
     
         25 . The targeting compound of  claim 24 , wherein said active agent is selected from the group consisting of imaging agents, antigens, antibodies, oligonucleotides, antisense oligonucleotides, genes, gene correcting hybrid oligonucleotides, aptameric oligonucleotides, triple-helix forming oligonucleotides, ribozymes, signal transduction pathway inhibitors, tyrosine kinase inhibitors, DNA-modifying agents, therapeutic genes, systems for therapeutic gene delivery, drugs, hormones, analgesics, anti-migraine agents, anti-coagulant agents, cardiovascular agents, anti-hypertensive agents, vasodilator agents, sedatives, narcotic antagonists, chelating agents, anti-diuretic agents, chemotherapeutic agents, and apoptosis-inducing agents. 
     
     
         26 . The targeting compound of  claim 24 , wherein said active agent is covalently linked to said analog. 
     
     
         27 . A method of treating a disease or condition in a subject comprising administering an effective dose of a formulation comprising an analog of  claim 1  to said subject. 
     
     
         28 . The method of  claim 27  wherein said disease or condition is selected from the group consisting of polycystic ovary syndrome and infertility. 
     
     
         29 . A nucleic acid comprising a polynucleotide encoding an α subunit polypeptide of  claim 1 . 
     
     
         30 . A nucleic acid comprising a polynucleotide encoding β subunit polypeptide of  claim 1 . 
     
     
         31 . A vector comprising a nucleic acid of  claim 29 . 
     
     
         32 . A vector comprising a nucleic acid of  claim 30 . 
     
     
         33 . A cell comprising a nucleic acid of  claim 29 . 
     
     
         34 . A cell comprising a nucleic acid of  claim 30 . 
     
     
         35 . A pharmaceutical composition comprising an analog of  claim 1  in admixture with a pharmaceutically acceptable carrier. 
     
     
         36 . A method of inducing follicle development in fish comprising administering an effective dose of a formulation comprising an analog of  claim 1  to said fish.