Modified transferrin fusion proteins

Abstract

Modified fusion proteins of transferrin and therapeutic proteins or peptides including soluble toxin receptors, with increased serum half-life or serum stability are disclosed. Preferred fusion proteins include those modified so that the transferrin moiety exhibits no or reduced glycosylation, binding to iron and/or binding to the transferrin receptor.

1 . A fusion protein comprising a modified transferrin (Tf) protein fused to a therapeutic protein or peptide, wherein the Tf protein exhibits reduced glycosylation, reduced metal binding, or reduced receptor binding.  
     
     
         2 . A fusion protein of  claim 1 , wherein the therapeutic protein or peptide is selected from the group comprising β-interferon (IFN), glucagon-like peptide (GLP-1), erythropoietin mimetic peptide (EMP1), T-20, and soluble toxin receptor.  
     
     
         3 . A fusion protein of  claim 2 , wherein the soluble toxin receptor is synaptotagmin I.  
     
     
         4 . A fusion protein of  claim 1 , wherein the therapeutic protein or peptide or the soluble toxin receptor is fused to the C-Terminal end of Tf.  
     
     
         5 . A fusion protein of  claim 1 , wherein the therapeutic protein or peptide or the soluble toxin receptor is fused to the N-terminal end of Tf.  
     
     
         6 . A fusion protein of  claim 1 , wherein the therapeutic protein or peptide or the soluble toxin receptor is inserted into at least one loop of the Tf.  
     
     
         7 . A fusion protein of  claim 1 , wherein the Tf protein has reduced affinity for a transferrin receptor (TfR).  
     
     
         8 . A fusion protein of  claim 1 , wherein the Tf protein is lactoferrin.  
     
     
         9 . A fusion protein of  claim 7 , wherein the Tf protein does not bind a TfR.  
     
     
         10 . A fusion protein of  claim 1 , wherein the Tf protein has reduced affinity for iron.  
     
     
         11 . A fusion protein of  claim 10 , wherein the Tf protein does not bind iron.  
     
     
         12 . A fusion protein of  claim 1 , wherein said Tf protein comprises at least one mutation that prevents glycosylation.  
     
     
         13 . A fusion protein of  claim 12 , wherein the Tf protein is lactoferrin.  
     
     
         14 . A fusion protein of  claim 1 , which is expressed in the presence of tunicamycin.  
     
     
         15 . A fusion protein of  claim 1 , wherein said Tf protein comprises a portion of the N domain of a Tf protein, a bridging peptide and a portion of the C domain of a Tf protein.  
     
     
         16 . A fusion protein of  claim 15 , wherein the bridging peptide links the therapeutic protein or peptide to Tf  
     
     
         17 . A fusion protein of  claim 15 , wherein the therapeutic protein or peptide is inserted between an N and a C domain of Tf protein.  
     
     
         18 . A fusion protein of  claim 1 , wherein the Tf protein comprises at least one amino acid substitution, deletion or addition in the hinge region.  
     
     
         19 . A fusion protein of  claim 18 , wherein said hinge region is selected from the group consisting of about residue 94 to about residue 96, about residue 245 to about residue 247, about residue 316 to about residue 318, about residue 425 to about residue 427, about residue 581 to about residue 582 and about residue 652 to about residue 658.  
     
     
         20 . A fusion protein of  claim 1 , wherein said Tf protein has at least one amino acid substitution, deletion or addition at a position selected from the group consisting of Asp 63, Gly 65, Tyr 95, Tyr 188, Lys 206, His 207, His 249, Asp 392, Tyr 426, Tyr 514, Tyr 517, His 585, Thr 120, Arg 124, Ala 126, Gly 127, Thr 452, Arg 456, Ala 458 and Gly 459.  
     
     
         21 . A fusion protein of  claim 6 , wherein the therapeutic protein or peptide replaces at least one loop of Tf.  
     
     
         22 . A fusion protein of  claim 12 , wherein the glycosylation site is selected from the group consisting of an amino acid residue corresponding to amino acids N413, N61 1.  
     
     
         23 . A fusion protein of  claim 7  or  9 , wherein the Tf comprises at least one amino acid substitution, deletion or addition at an amino acid residue corresponding to an amino acid selected from the group consisting of Asp 63, Gly 65, Tyr 95, Tyr 188, Lys 206, His 207, His 249, Asp 392, Tyr 426, Tyr 514, Tyr 517, His 585, Thr 120, Arg 124, Ala 126, Gly 127, Thr 452, Arg 456, Ala 458 and Gly 459.  
     
     
         24 . A fusion protein of  claim 4 , wherein the Tf C-terminal proline residue is deleted.  
     
     
         25 . A fusion protein of  claim 4 , wherein the Tf C-terminal cysteine loop is deleted.  
     
     
         26 . A fusion protein of  claim 1 , wherein the serum half-life of the therapeutic protein or peptide is increased over the serum half-life of the therapeutic protein or peptide or soluble toxin receptor in an unfused state.  
     
     
         27 . A fusion protein of  claim 1 , wherein the Tf protein does not bind a TfR.  
     
     
         28 . A fusion protein of  claim 1 , wherein said Tf protein exhibits reduced or no glycosylation.  
     
     
         29 . A fusion protein of  claim 28 , comprising at least one mutation that prevents glycosylation.  
     
     
         30 . A nucleic acid molecule encoding a fusion protein of either  claim 1 .  
     
     
         31 . A vector comprising a nucleic acid molecule of  claim 30 .  
     
     
         32 . A host cell comprising a vector of  claim 31 .  
     
     
         33 . A host cell comprising a nucleic acid molecule of  claim 30 .  
     
     
         34 . A method of expressing a Tf fusion protein comprising culturing a host cell of  claim 32  under conditions which express the encoded fusion protein.  
     
     
         35 . A method of expressing a Tf fusion protein comprising culturing a host cell of  claim 33  under conditions which express the encoded fusion protein.  
     
     
         36 . A host cell of  claim 32 , wherein the cell is prokaryotic or eukaryotic.  
     
     
         37 . A host cell of  claim 33 , wherein the cell is prokaryotic or eukaryotic.  
     
     
         38 . A host cell of  claim 36 , wherein the cell is a yeast cell.  
     
     
         39 . A host cell of  claim 37 , wherein the cell is a yeast cell.  
     
     
         40 . A transgenic animal comprising a nucleic acid molecule of 30.  
     
     
         41 . A method of producing a Tf fusion protein comprising isolating a fusion protein from a transgenic animal of  claim 40 .  
     
     
         42 . A method of  claim 41 , wherein the Tf fusion protein comprises lactoferrin.  
     
     
         43 . A method of  claim 42 , wherein the fusion protein is isolated from a biological fluid from the transgenic animal.  
     
     
         44 . A method of  claim 42 , wherein the fluid is serum or milk.  
     
     
         45 . A method of treating a disease or disease symptom in a patient, comprising the step of administering a fusion protein of  claim 1 .  
     
     
         46 . The fusion protein of  claim 1 , wherein the Tf protein has a N-terminal domain at each end of the protein.  
     
     
         47 . The fusion protein of  claim 46 , wherein the therapeutic protein or peptide or the soluble toxin receptor is fused to each N-terminal domain of the Tf protein.  
     
     
         48 . The fusion protein of  claim 2 , wherein the soluble toxin receptor binds specifically to a toxin.  
     
     
         49 . A fusion protein of  claim 2 , wherein the therapeutic protein or peptide is an analog of β-IFN, GLP-1, erythropoietin mimetic peptide (EMP1), T-20, or soluble toxin receptor wherein the analog is effective in treating, preventing, or ameliorating a disease, condition or disorder.  
     
     
         50 . A pharmaceutical composition comprising the fusion protein of claims  1 ,  2  or  3 , and a carrier.  
     
     
         51 . A method of treating a subject comprising administering to the subject a therapeutically effective amount of a fusion protein of  claim 1 .  
     
     
         52 . A method of  claim 51 , wherein the subject is suffering from multiple sclerosis, brain tumor, skin cancer, hepatitis B, or hepatitis C, and wherein the fusion protein comprises β-IFN or an analog thereof.  
     
     
         53 . A method of  claim 52 , wherein the subject is suffering from multiple sclerosis.  
     
     
         54 . A method of  claim 51 , wherein the subject is suffering from elevated level of glucose as compared to a healthy subject and wherein the fusion protein comprises GLP-1 or an analog thereof.  
     
     
         55 . A method of  claim 54 , wherein the elevated level of glucose is associated with diabetes.  
     
     
         56 . A method of  claim 55 , wherein the diabetes is Type II diabetes.  
     
     
         57 . A method of  claim 51 , wherein the subject is suffering from low or defective red blood cell production as compared to a healthy subject and wherein the fusion protein comprises EMP1 or an analog thereof.  
     
     
         58 . A method of  claim 57 , wherein the low or defective red blood cell production is associated with anemia, P-thalassemia, pregnancy or menstrual disorders, rheumatoid arthritis, AIDS, and cancer.  
     
     
         59 . A method of  claim 51 , wherein the subject is suffering from a disease caused by the transmission of a retrovirus and wherein the therapeutic peptide is an inhibitor of virus entry.  
     
     
         60 . A method of  claim 59 , wherein the retrovirus is a human retrovirus.  
     
     
         61 . A method of  claim 60 , wherein the human retrovirus is selected from the group consisting of HIV-1, HIV-2, and the human T-lymphocyte virus I (HTLV-1), HTLV-II, and HTLV-III.  
     
     
         62 . A method of  claim 59 , wherein the retrovirus is a non-human retrovirus.  
     
     
         63 . A method of  claim 62 , wherein the non-human retrovirus is selected from the group consisting of bovine leukosis virus, feline sarcoma and leukemia viruses, simian sarcoma and leukemia viruses, and sheep progress pneumonia viruses.  
     
     
         64 . A method of  claim 59 , wherein the inhibitor is T-20, T-1249 or an analog thereof.  
     
     
         65 . A method of  claim 59 , wherein the disease is AIDS.  
     
     
         66 . A method of preventing or treating a disease or condition associated with a toxin comprising administering to the subject a therapeutically effective amount of a fusion protein comprising a modified Tf protein fused to a soluble toxin receptor, wherein the modified Tf protein exhibits reduced glycosylation, reduced metal binding, or reduced receptor binding.  
     
     
         67 . A method of  claim 66 , wherein the soluble toxin receptor is selected from the group consisting of anthrax toxin receptor, botulinum toxin receptor, and diptheria toxin receptor.  
     
     
         68 . A method of  claim 67 , wherein the soluble botulinum toxin receptor is amino acids 1-53 (SEQ ID NO: 4) of synaptotagmin.  
     
     
         69 . A fusion protein of  claim 1 , wherein a therapeutic protein or peptide is inserted in one or more of the transferrin loops.  
     
     
         70 . A fusion protein of  claim 69 , wherein a therapeutic protein is inserted in each of the 5 transferrin loops.  
     
     
         71 . A fusion protein of  claim 2 , wherein the GLP-1 further comprises an additional amino acid at the N-terminus.  
     
     
         72 . The fusion protein of  claim 71 , wherein the amino acid is Gly.  
     
     
         73 . The fusion protein of  claim 71 , wherein the GLP-1 analog is fused to modified transferrin protein at the N-terminal end.  
     
     
         74 . A method of regulating glucose level in a subject comprising administering to the subject a therapeutically effective amount of a fusion protein comprising a modified Tf protein fused to GLP-1 or analog thereof, wherein the modified Tf protein exhibits reduced glycosylation, reduced metal binding, or reduced receptor binding.  
     
     
         75 . A fusion protein of  claim 1 , wherein the therapeutic protein or peptide is inserted into the N-domain of mTf at one or more of the sites selected from the group consisting of Asp33, Asn55, Asn 75, Asp9O, Gly257, Lys280, His289, Ser298, Ser105, Glu141, Asp166, Gln184, asp197, Lys217, Thr231, and Cys241.  
     
     
         76 . A fusion protein of  claim 1 , wherein the therapeutic protein or peptide is inserted into the C-domain of mTf at one or more sites corresponding to Asp33, Asn55, Asn 75, Asp9O, Gly257, Lys280, His289, Ser298, Ser105, Glu141, Asp166, Gln184, asp197, Lys217, Thr231, or Cys241.  
     
     
         77 . A fusion protein of  claim 75 , wherein the therapeutic protein or peptide is further inserted into the mTf at one or more sites corresponding to Asp33, Asn55, Asn 75, Asp90, Gly257, Lys280, His289, Ser298, Ser105, Glu141, Asp166, Gln184, asp197, Lys217, Thr231, or Cys241,  
     
     
         78 . A fusion protein of  claim 2 , wherein the therapeutic peptide is EMP1 and wherein EMP1 is inserted into the N-domain of mTf at one or more sites selected from the group consisting of His289 and Asp166.  
     
     
         79 . A fusion protein of  claim 2 , wherein the therapeutic peptide is EMP1 and wherein EMP1 is inserted into the C-domain of mTf at one or more sites corresponding to His289 or Asp166.  
     
     
         80 . A fusion protein of  claim 78 , wherein the therapeutic peptide is further inserted into the C-domain of mTf at one or more sites corresponding to His289 or Asp166.  
     
     
         81 . A fusion protein of  claim 1 , wherein mTf is further modified by deletion of the C-terminus Pro.  
     
     
         82 . A fusion protein of  claim 81 , wherein mTf is further modified by deletion of Arg-Arg adjacent to the C-terminus Pro.  
     
     
         83 . A fusion protein of  claim 1 , wherein the mTf is further modified by removing the disulfide bond between Cys402 and Cys674.  
     
     
         84 . A fusion protein of  claim 83 , wherein the disulfide bond is removed by mutating Cys402 and Cys674 into Gly residues.  
     
     
         85 . A fusion protein of  claim 82 , wherein th0.e mTf is further modified by mutating Cys402 and Cys674 into Gly residues.  
     
     
         86 . A fusion protein of  claim 1 , wherein the therapeutic peptide or protein is inserted into one or more of the loops selected from the group consisting of N 1 (286-292), N 2 (162-170), C 1 (489-495), and C 2 (623-628).