Modified Human Four Helical Bundle Polypeptides and Their Uses
Modified human four helical bundle (4HB) polypeptides and uses thereof are provided.
1 . A four helical bundle (4HB) polypeptide comprising one or more non-naturally encoded amino acids wherein said non-naturally encoded amino acids are substituted at one or more positions in the A-B loop, B-C loop, or C-D loop.
2 . The 4HB polypeptide of claim 1 , wherein the 4HB polypeptide further comprises one or more amino acid substitutions made within the N-terminus, A helix, B helix, C helix, D helix, C-terminus, or, when present, the E helix or D-E loop.
3 . The 4HB polypeptide of claim 1 , wherein the 4HB polypeptide comprises one or more amino acid substitutions, additions or deletions that modulate affinity of the 4HB polypeptide for a 4HB receptor.
4 . The 4HB polypeptide of claim 1 , wherein the 4HB polypeptide comprises one or more amino acid substitutions, additions or deletions that increase the stability or solubility of the 4HB polypeptide.
5 . The 4HB polypeptide of claim 1 , wherein the 4HB polypeptide comprises one or more amino acid substitution, addition or deletion that increases the expression of the 4HB polypeptide in a recombinant host cell or synthesized in vitro.
6 . The 4HB polypeptide of claim 1 , wherein the 4HB polypeptide comprises one or more amino acid substitution, addition or deletion that increases protease resistance of the 4HB polypeptide.
7 . The 4HB polypeptide of claim 1 , wherein the non-naturally encoded amino acid is selectively reactive toward a linker, polymer, or biologically active molecule.
8 . The 4HB polypeptide of claim 14 , wherein the non-naturally encoded amino acid comprises a carbonyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group.
9 . The 4HB polypeptide of claim 14 , wherein the 4HB polypeptide comprises one or more post-translational modifications.
10 . The 4HB polypeptide of claim 1 , selected from the group consisting of granulocyte cell stimulating factor (G-CSF), erythropoietin, interferon, and growth hormone.
11 . The 4HB polypeptide of claim 1 , wherein the 4HB polypeptide is linked to a linker, polymer, or biologically active molecule.
12 . The 4HB polypeptide of claim 11 , wherein the 4HB polypeptide is linked to a water soluble polymer.
13 . The 4HB polypeptide of claim 1 , wherein the 4HB polypeptide is linked to a multifunctional polymer, bifunctional linker, or at least one additional 4HB polypeptide.
14 . The 4HB polypeptide of claim 13 , wherein the bifunctional linker or bifunctional polymer is linked to a second polypeptide.
15 . The 4HB polypeptide of claim 14 , wherein the second polypeptide is a 4HB polypeptide.
16 . The 4HB polypeptide of claim 11 , wherein the water soluble polymer comprises a poly (ethylene glycol) moiety.
17 . The 4HB polypeptide of claim 11 , wherein said water soluble polymer is linked to a non-naturally encoded amino acid present in said 4HB polypeptide.
18 . The 4HB polypeptide of claim 1 , wherein the 4HB polypeptide further comprises one or more free amide or free sulfhydryl groups.
19 . The 4HB polypeptide of claim 18 , wherein the free amide or free sulfhydryl groups are present in naturally occurring amino acids.
20 - 83 . (canceled)
84 . A polypeptide from the growth hormone supergene family comprising one or more non-naturally encoded amino acids wherein said non-naturally encoded amino acids are substituted at one or more positions in the D-E loop.
85 . The polypeptide of claim 84 , wherein the polypeptide further comprises one or more amino acid substitutions made within the A helix, B helix, C helix, D helix, E helix, N-terminus, or C-terminus.