Modified transferrin fusion proteins comprising duplicate transferrin amino or carboxy terminal domains
Fusion proteins of transferrin and other therapeutic moieties 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 transferrin fusion protein comprising a transferrin (Tf) moiety and at least one therapeutic protein or peptide, wherein the transferrin moiety comprises two or more transferrin N domains.
2 . A transferrin fusion protein of claim 1 , wherein the Tf moiety consists of two Tf N domains.
3 . A transferrin fusion protein of claim 1 , wherein the Tf moiety comprises one Tf N domain and one Tf C domain that has been modified to substantially comprise the sequence of a Tf N domain.
4 . A transferrin fusion protein of claim 1 , wherein the Tf moiety is unglycosylated.
5 . A fusion protein of claim 4 , wherein the serum half-life of the therapeutic protein or peptide is increased over the serum half-life of the therapeutic protein or peptide in an unfused state.
6 . A fusion protein of claim 1 , wherein the therapeutic protein or peptide is fused to the C-terminal end of the Tf moiety.
7 . A fusion protein of claim 1 , wherein the therapeutic protein or peptide is fused to the N-terminal end of the Tf moiety.
8 . A fusion protein of claim 1 , wherein the therapeutic protein or peptide is inserted into at least one loop of the Tf moiety.
9 . A fusion protein of claim 1 , wherein the Tf moiety has reduced affinity for a TfR.
10 . The fusion protein of claim 1 , wherein the Tf moiety is lacto transferrin (lactoferrin).
11 . A fusion protein of claim 9 , wherein the TF moiety does not bind a TfR.
12 . A fusion protein of claim 1 , wherein the Tf moiety has reduced affinity for iron.
13 . A fusion protein of claim 12 , where the Tf moiety does not bind iron.
14 . A fusion protein of claim 1 , wherein said Tf moiety comprises at least one mutation that prevents glycosylation.
15 . A fusion protein of claim 14 , wherein the Tf protein is lacto transferrin (lactoferrin).
16 . A fusion protein of claim 1 , which is expressed in the presence of tunicamycin
17 . A fusion protein of claim 1 , wherein a linker peptide links the therapeutic protein or peptide to Tf
18 . A fusion protein of claim 1 , wherein the Tf protein have 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 N domain residue 94 to about residue 96 of SEQ ID NO: 3, about residue 245 to about residue 247 of SEQ ID NO: 3, and about residue 316 to about residue 318 of SEQ ED NO: 3.
20 . A fusion protein of claim 1 , wherein said Tf protein has at least one amino acid substitution, deletion or addition at a position in SEQ ID NO: 3 selected from the group consisting of N domain residues Asp 63, Gly 65, Tyr 95, Tyr 188, Lys 206, His 207, His 249, Thr 120, Arg 124, Ala 126, and Gly 127.
21 . A fusion protein of claim 8 , wherein the therapeutic protein or peptide replaces at least one loop.
22 . A fusion protein of claim 3 , wherein the C domain residues corresponding to amino acids 413-427 of SEQ ID NO: 3 are replaced with N domain residues corresponding to amino acids 86-96 of SEQ ID NO: 3.
23 . A fusion protein of claim 3 , wherein the C domain residues corresponding to amino acids 610-620 of SEQ ID NO: 3 are replaced with N domain residues corresponding to amino acids 276-283 of SEQ ID NO: 3.
24 . A transferrin fusion protein comprising a transferrin (Tf) moiety and at least one therapeutic protein or peptide, wherein the transferrin moiety comprises two or more transferrin C domains that are modified to inhibit or prevent glycosylation and Tf receptor binding.
25 . A transferrin fusion protein of claim 24 , wherein the T moiety has been modified to inhibit or prevent iron binding.
26 . A nucleic acid molecule encoding a fusion protein of claim 1 or 24 .
27 . A vector comprising a nucleic acid molecule of claim 25 .
28 . A host cell comprising a vector of claim 27 .
29 . A host cell comprising a nucleic acid molecule of claim 26 .
30 . A method of expressing a Tf fusion protein comprising culturing a host cell of claim 28 under conditions which express the encoded fusion protein.
31 . A method of expressing a Tf fusion protein comprising culturing a host cell of claim 29 under conditions which express the encoded fusion protein.
32 . A host cell of claim 28 , wherein the cell is prokaryotic or eukaryotic.
33 . A host cell of claim 29 , wherein the cell is prokaryotic or eukaryotic.
34 . A host cell of claim 32 , wherein the cell is a yeast cell.
35 . A host cell of claim 33 , wherein the cell is a yeast cell.
36 . A transgenic animal comprising a nucleic acid molecule of 26 .
37 . A method of producing a Tf fusion protein comprising isolating a fusion protein from a transgenic animal of claim 36 .
38 . A method of claim 37 , wherein the Tf moiety is a lactoferrin moiety.
39 . A method of claim 38 , wherein the fusion protein is isolated from a biological fluid from the transgenic animal.
40 . A method of claim 39 , wherein the fluid is serum or milk.
41 . A method of treating a disease or disease symptom in a patient, comprising the step of administering a fusion protein of claim 1 or 24 .