Method for the production of fusion proteins in transgenic mammal milk

Abstract

Desirable fusion proteins can be produced in and purified from the milk of transgenic animals. The peptides are made as fusion proteins with a suitable fusion partner such as human alpha-fetoprotein. The fusion partner protein acts to promote and increase the half-life of the overall molecule as well as having therapeutic effects on its own. The fusion protein is typically produced through the use of transgenic animals and can be purified away from the now the milk or other bodily fluid of such an animal by an affinity purification method. A particular advantage of producing peptides via this route, in addition to the obvious advantages of high yield and biocompatibility, is that specific post-translational modifications, such as carboxy terminal amidation, can be performed in the mammary gland. Biologically active polypeptides comprising a therapeutically active polypeptide fused to human alpha-fetoprotein fragment or a variant thereof, methods for the preparation thereof, nucleotide sequences encoding such fusion polypeptides, expression cassettes comprising such nucleotide sequences, self-replicating plasmids containing such expression cassettes, and pharmaceutical compositions containing said fusion polypeptides.

1 . A bifunctional fusion protein, encoded by a transgene DNA construct,
 wherein the bifunctional fusion protein comprises a first polypeptide domain which has a desired bioactivity, and a second polypeptide domain which has a desired bioactivity,   wherein each of said first and said second polypeptide domains retain their desired bioactivity,   wherein the first polypeptide domain is human alpha-fetoprotein or a fragment thereof having the biological activity of human alpha-fetoprotein.   
     
     
         2 . (canceled) 
     
     
         3 . The fusion protein of  claim 1 , wherein said second polypeptide domain is human IFN-β or a fragment thereof having the biological activity of IFN-β. 
     
     
         4 . The fusion protein of  claim 1 , wherein said second polypeptide domain is selected from a group consisting of: antithrombin III, lactoferrin, ferritin, calcitonin, urokinase, Platelet Factor 4, alpha-fetoprotein, alpha-1-antitrypsin, C-1 esterase inhibitor, decorin, prolactin, tau interferon, alpha interferon, ferritin, prolactin, CFTR, Muellerian Inhibitory Substance, blood Factor X, blood Factor VIII, truncated ATIII, and erthyropoietin. 
     
     
         5 . (canceled) 
     
     
         6 . An isolated polynucleotide encoding the amino acid sequence of SEQ. ID NO: 4 linked to an amino acid sequence selected from the group consisting of SEQ. ID NOs: 10-25. 
     
     
         7 . (canceled) 
     
     
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         21 . (canceled) 
     
     
         22 . A recombinant DNA vector comprising the nucleic acid sequence encoding the fusion protein of  claim 1 . 
     
     
         23 . A host cell transformed with said recombinant DNA vector of  claim 22 . 
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . A fusion protein produced by a method comprising:
 (a) expressing the fusion protein of  claim 1  by a cell; and   (b) recovering the protein.   
     
     
         27 . (canceled) 
     
     
         28 . A method of treating a disease condition comprising administering an effective amount of the fusion protein of  claim 1 . 
     
     
         29 . The method of  claim 28 , wherein said disease condition is selected from the group consisting of a viral infection, cancer, human cancer, tumor cell, rheumatoid arthritis, multiple sclerosis, osteoporosis, psoriasis, myasthenia gravis, muscular dystrophy, insulin-dependent diabetes mellitus, systemic lupus erythematosus, hairy cell leukemia, chronic myelogenous leukemia, cutaneous T cell lymphoma carcinoid tumor, renal cell carcinoma, squamous epithelial tumors of the head and neck, multiple myeloma, malignant melanoma, hepatitis, B, hepatitis C, low grade non-Hodgkin lymphoma, human skin disease or injury, photoaging damage, rhinitis, sunburn, dermatitis and burns. 
     
     
         30 . (canceled) 
     
     
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         43 . (canceled) 
     
     
         44 . (canceled) 
     
     
         45 . (canceled) 
     
     
         46 . (canceled) 
     
     
         47 . A fusion protein produced by a method comprising:
 (a) expressing the fusion protein of  claim 1  by a transgenic animal; and   (b) recovering the protein.   
     
     
         48 . A method for the production of transgenic animals capable of producing the fusion protein of  claim 1  comprising:
 transfecting a non-human mammalian cell-line with a transgene DNA construct encoding the fusion protein of  claim 1 ;   selecting a cell or cell line(s) in which said transgene DNA construct has been inserted into the genome of that cell or cell-line; and   performing a first nuclear transfer procedure to generate a first transgenic animal heterozygous for the transgene DNA construct.   
     
     
         49 . (canceled) 
     
     
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         57 . An offspring of the first transgenic animal produced by the method of  claim 48 . 
     
     
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         64 . A transgenic animal produced by the method of  claim 48 . 
     
     
         65 . (canceled) 
     
     
         66 . (canceled) 
     
     
         67 . Milk derived from the transgenic animal of  claim 64  or its offspring. 
     
     
         68 . (canceled) 
     
     
         69 . The fusion protein of  claim 1 , wherein said human alpha-fetoprotein or fragment thereof has a high plasma half-life. 
     
     
         70 . (canceled) 
     
     
         71 . (canceled) 
     
     
         72 . (canceled) 
     
     
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         74 . The fusion protein of  claim 1 , wherein said fusion protein further comprises a peptide linker. 
     
     
         75 . The fusion protein of  claim 1 , wherein said fusion protein comprises a secretion signal sequence. 
     
     
         76 . The fusion protein of  claim 3 , wherein said human IFN-β is fused to the N-terminal end of said human alpha-fetoprotein or fragment thereof. 
     
     
         77 . The fusion protein of  claim 3 , wherein said human IFN-β is fused to the C-terminal end of said human alpha-fetoprotein or fragment thereof. 
     
     
         78 . An isolated nucleic acid sequence as shown in SEQ. ID NO: 1. 
     
     
         79 . The isolated polynucleotide of  claim 6 , further comprising a nucleic acid sequence encoding an amino acid linking sequence. 
     
     
         80 . (canceled) 
     
     
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