Long-acting polypeptides and methods of producing same

Abstract

A polypeptide and polynucleotides encoding same comprising one carboxy-terminal peptide (CTP) of chorionic gonadotrophin attached to an amino terminus of a cytokine and two carboxy-terminal peptides (CTP) of chorionic gonadotrophin attached to a carboxy terminus of a cytokine are disclosed. Pharmaceutical compositions comprising the polypeptide and polynucleotides of the invention and methods of using same are also disclosed.

What is claimed is: 
     
         1 . A method of treating or ameliorating a disease in a subject in need of cytokine therapy, comprising administering to said subject a therapeutically effective amount of a pharmaceutical composition comprising a polypeptide comprising a cytokine attached to a single chorionic gonadotropin carboxy terminal peptide (CTP) at the amino terminus of said cytokine, and two chorionic gonadotropin carboxy terminal peptides (CTP) attached to the carboxy terminus of said cytokine, thereby treating said subject in need of a cytokine therapy. 
     
     
         2 . The method of  claim 1 , wherein the sequence of at least one CTP consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 17 and SEQ ID NO: 18. 
     
     
         3 . The method of  claim 1 , wherein said at least one CTP is glycosylated. 
     
     
         4 . The method of  claim 1 , wherein said at least one CTP is truncated. 
     
     
         5 . The method of  claim 1 , wherein at least one CTP is optionally attached to said cytokine via a linker. 
     
     
         6 . The method of  claim 5 , wherein the linker is a peptide bond. 
     
     
         7 . The method of  claim 1 , wherein said attaching produces a CTP-modified cytokine polypeptide, wherein when said polypeptide is a precursor polypeptide consisting of a cytokine attached to a single chorionic gonadotropin carboxy terminal peptide (CTP) at the amino terminus of said cytokine, and two chorionic gonadotropin carboxy terminal peptides (CTP) attached to the carboxy terminus of said cytokine, and a signal peptide attached to the amino terminus of said one CTP, and when said polypeptide is a mature polypeptide, said polypeptide consists of a cytokine attached to a single chorionic gonadotropin carboxy terminal peptide (CTP) at the amino terminus of said cytokine, and two chorionic gonadotropin carboxy terminal peptides (CTP) attached to the carboxy terminus of said cytokine and said polypeptide lacks a signal peptide. 
     
     
         8 . The method of  claim 7 , wherein said signal peptide consists of an amino acid sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 64. 
     
     
         9 . The method of  claim 1 , wherein said treating or ameliorating a disease in said subject induces growth, induces weight gain, induces erythropoiesis, treats or ameliorates anemia, provides an anti-viral activity, or provides an anti-proliferative activity, or any combination thereof, in said subject. 
     
     
         10 . The method of  claim 1 , wherein said subject is a human subject. 
     
     
         11 . The method of  claim 1 , wherein said disease is a cancer. 
     
     
         12 . The method of  claim 11 , wherein said cancer is hairy cell leukemia, malignant melanoma, Kaposi's sarcoma, bladder cancer, chronic myelocytic leukemia, kidney cancer, carcinoid tumors, non-Hodgkin's lymphoma, ovarian cancer, or skin cancers. 
     
     
         13 . The method of  claim 1 , wherein said pharmaceutical composition comprises a pharmaceutical formulation comprising a buffer, a tonicity agent, and the polypeptide comprising the cytokine attached to a single chorionic gonadotropin carboxy terminal peptide (CTP) at the amino terminus of said cytokine, and two chorionic gonadotropin carboxy terminal peptides (CTP) attached to the carboxy terminus of said cytokine. 
     
     
         14 . The method of  claim 13 , wherein said formulation comprises a liquid formulation or a topical formulation. 
     
     
         15 . The method of  claim 13 , wherein said buffer is citrate 
     
     
         16 . The method of  claim 13 , wherein said tonicity agent is NaCl. 
     
     
         17 . The method of  claim 13 , wherein said composition is at a pH of about 4-7.2. 
     
     
         18 . The method of  claim 13 , wherein said composition is a liquid. 
     
     
         19 . The method of  claim 13 , wherein said composition is administered once weekly or once bi-weekly. 
     
     
         20 . The method of  claim 13 , wherein said composition is formulated in a form suitable for an intramuscular (IM) injection, a subcutaneous (SC) injection, or an intravenous (IV) injection.