Compositions and methods for the preparation of protease resistant human growth hormone glycosylation mutants
The present invention relates to protease resistant mutants of human growth hormone, which contain newly introduced proteolysis resistant mutations and N-linked or O-linked glycosylation site(s), such that these recombinantly produced polypeptides have glycosylation patterns distinctly different from that of the naturally occurring human growth hormone. The polynucleotide coding sequences for the mutants, expression cassettes comprising the coding sequences, cells expressing the mutants, and methods for producing the mutants are also disclosed. Further disclosed are pharmaceutical compositions comprising the mutants and method for using the mutants.
1 . An isolated nucleic acid comprising a polynucleotide sequence encoding a mutant human growth hormone, wherein said mutant human growth hormone comprises a member selected from a protease recognition site comprising a proteolysis resistant mutation not present in wild type human growth hormone, an N-linked glycosylation site mutation not present in wild type human growth hormone, an O-linked glycosylation site mutation not present in wild-type human growth hormone, and combinations thereof.
2 . The nucleic acid of claim 1 , wherein said wild-type human growth hormone has the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
3 . The nucleic acid of claim 1 , wherein said O-linked glycosylation site mutation is proximate a proline residue.
4 . The nucleic acid of claim 3 , wherein said proline residue is located at a position which is a member selected from position 2, 5, 37, 48, 59, 89, 113, 140, 190, and combinations thereof, of a member selected from SEQ ID NO:1 and SEQ ID NO:2.
5 . The nucleic acid of claim 1 , wherein the mutant human growth hormone comprises an amino acid sequence which is a member selected from SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 80, 81, 82, 83, 84, 85, 86, 87, 88 and 89.
6 . The nucleic acid of claim 1 , wherein the mutant human growth hormone comprises more than one glycosylation site mutation.
7 . An expression cassette comprising the nucleic acid of claim 1 .
8 . A cell comprising the nucleic acid of claim 1 .
9 . A mutant human growth hormone, comprising a mutation which is a member selected from a protease recognition site comprising a proteolysis resistant mutation not present in wild type human growth hormone, an N-linked glycosylation site mutation not present in wild type human growth hormone, an O-linked glycosylation site mutation not present in wild-type human growth hormone, and combinations thereof.
10 . The mutant human growth hormone of claim 9 , wherein said wild-type human growth hormone has an amino acid sequence which is a member selected from SEQ ID NO:1 and SEQ ID NO:2.
11 . The mutant human growth hormone of claim 9 , wherein said glycosylation site mutation is proximate a proline residue.
12 . The mutant human growth hormone of claim 11 , wherein said proline residue is located at a position which is a member selected from position 2, 5, 37, 48, 59, 89, 113, 140, 190, and combinations thereof, of a member selected from SEQ ID NO:1 and SEQ ID NO:2.
13 . The mutant human growth hormone of claim 9 , comprising an amino acid sequence which is a member selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 80, 81, 82, 83, 84, 85, 86, 87, 88, and 89.
14 . The mutant human growth hormone of claim 9 , wherein the mutant human growth hormone comprises more than one glycosylation site mutation.
15 . The mutant human growth hormone of claim 9 , comprising a water-soluble polymer attached to said glycosylation site mutation through a glycosyl linker.
16 . The mutant human growth hormone of claim 15 , wherein said glycosyl linker is an intact glycosyl linker.
17 . The mutant human growth hormone of claim 16 , wherein said intact glycosyl linker is a member selected from a galactosyl, an N-acetylgalactosyl and a sialic acid residue.
18 . The mutant human growth hormone of claim 15 , wherein said water-soluble polymer is poly(ethylene glycol).
19 . The mutant human growth hormone of claim 15 , wherein said O-glycosylation site mutation comprises an amino acid which is member selected from threonine and serine and said glycosyl linker is covalently attached to a member selected from said threonine and said serine.
20 . The mutant human growth hormone of claim 9 , further comprising a water-soluble polymer covalently bound to a member selected from an amino acid which is said protease resistant mutation, an amino acid residue proximate said amino acid which is said protease resistant mutation and combinations thereof.
21 . The mutant human growth hormone of claim 20 , wherein said water-soluble polymer is a poly(ethylene glycol).
22 . A pharmaceutical formulation comprising a mutant human growth hormone according to claim 9 and a pharmaceutically acceptable carrier.
23 . A method of treating a subject in need of supplementation of endogenously produced human growth hormone, said method comprising administering to said subject a therapeutically effective amount of a mutant human growth hormone according to claim 9 .
24 . A method for making a mutant human growth hormone comprising a mutation which is a member selected from a protease recognition site comprising a proteolysis resistant mutation not present in wild type human growth hormone, an N-linked glycosylation site mutation not present in wild type human growth hormone, an O-linked glycosylation site mutation not present in wild-type human growth hormone, and combinations thereof, said method comprising:
(a) transfecting a cell capable of expressing said mutant human growth hormone with a nucleic acid encoding said mutant human growth hormone; and
(b) expressing said mutant human growth hormone.
25 . The method according to claim 24 , further comprising:
(c) contacting said mutant human growth hormone with a sugar donor and an enzyme for which said sugar donor is a substrate under conditions appropriate to transfer a sugar moiety from said donor to said glycosylation site.
26 . The method according to claim 25 , wherein said sugar moiety is a modified sugar moiety.
27 . The method according to claim 26 , wherein said sugar moiety is modified by a water-soluble polymer covalently attached thereto.
28 . The method according to claim 27 , wherein said water-soluble polymer is a poly(ethylene glycol).
29 . The method according to claim 25 , wherein said sugar moiety is a member selected from a galactosyl, N-acetylgalactosyl and a sialic acid moiety.
30 . The method according to claim 24 , further comprising:
(d) contacting said mutant human growth hormone with an activated water-soluble polymer under conditions appropriate to form a covalent bond between a water-soluble polymer moiety of said activated water-soluble polymer and an amino acid residue of said mutant human growth hormone.
31 . The method according to claim 30 , wherein said amino acid residue is a member selected from said protease resistant mutation, an amino acid residue proximate to said mutation and combinations thereof.
32 . The method of claim 24 , wherein said wild-type human growth hormone has an amino acid sequence which is a member selected from SEQ ID NO:1 and SEQ ID NO:2.
33 . The method of claim 24 , wherein said O-glycosylation site mutation is proximate a proline residue.
34 . The method of claim 33 , wherein the proline residue is located at position which is a member selected from 2, 5, 37, 48, 59, 89, 113, 140, 190, and combinations thereof, of a member selected from SEQ ID NO:1 and SEQ ID NO:2.
35 . The method of claim 24 , wherein the mutant human growth hormone comprises an amino acid sequence which is a member selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 80, 81, 82, 83, 84, 85, 86, 87, 88, and 89.
36 . The method of claim 24 , wherein the mutant human growth hormone comprises more than one glycosylation site mutation.
37 . The method of claim 33 wherein said O-glycosylation mutant is a member selected from threonine and serine.
38 . The method according to claim 25 , said method further comprising:
(d) prior to step (c), said mutant human growth hormone is isolated.