Derivatives of growth hormone and related proteins
AssigneeBolder Biotechnology, Inc.
InventorGeorge N. Cox, III
Life of this patent
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Abstract
The growth hormone supergene family comprises greater than 20 structurally related cytokines and growth factors. A general method is provided for creating site-specific, biologically active conjugates of these proteins. The method involves adding cysteine residues to non-essential regions of the prr teins or substituting cysteine residues for non-ssential amino acids in the proteins using site-directed mutagenesis and then covalently coupling a cysteine-reactive polymer or other type of cysteine-reactive moiety to the proteins via the added cysteine residue. Disclosed herein are preferred sites for adding cysteine residues or introducing cysteine substitutions into the proteins, and the proteins and protein derivatives produced thereby.
Claims87
A cysteine variant of growth hormone of SEQ ID NO:1, wherein a cysteine residue is substituted for at least one amino acid located in at least one region of growth hormone selected from the group consisting of:
- the C-D loop, the first three or last three amio acids in helix A, the first three or last three amino acids in helix B, the first three amino acids in helix C, the first three o r last three a mno acids in helix D, the amino acids preceding helix A corresponding to amino acids 1-5 of SEQ ID NO:1, and the amino acids prclowing helix D corresponding to amino acids 184-191 of SEQ ID NO:1;
- wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to growth hormone.
A cysteine variant of growth hormone of SEQ ID NO:1, wherein said variant contains the four native cysteines, C53, C165, C182 and C189, and wherein a cysteine residue is substituted for at least one amnmo acid located in at least one region of growth hormone selected from the group consisting of:
- the B loop, the C-D loop, the first three or last three amino acids in helix A, the first three or last three amino acids in helix B, the first three amino acids in helix C, the first three or last three amino acids in helix D, the amino acids preceding helix A corresponding to amino acids 1-5 of SEQ ID NO:1, and the amino acids following helix D corresponding to amino acids 184-191 of SEQ ID NO:1;
- wherein said variant has biological activity in vitro as meastured by proliferation of a cell line that proliferates in response to growth hormone.
A cysteine variant of growth hormone of SEQ ID NO:1, wherein a cysteine residue is substituted for at least one amino acid located in the region of growth hormone preceding helix A corresponding to amino acids 1-5 of SEQ ID NO:1;
- wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to growth hormone.
A cysteine variant of growth hormone of SEQ ID NO:1, wherein a cysteine residue is substituted for at least one amino acid located in the region of growth hormone following helix D corresponding to amino acids 184-191 of SEQ ID NO:1;
- wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to growth hormone.
A cysteine variant of growth hormone of SEQ ID NO:1, wherein a cysteine residue is substituted for at least one amino acid of growth hormone selected from the group consisting of A34, K38, E39, Q40, S43, N47, P48, Q49, T50, S51, S55 and T60;
- wherein said variant has biolgical activity in vitro as measured by proliferation of a cell line that proliferates in response to growth hormone.
A cysteine variant of growth hormone of SEQ ID NO:1, wherein a cysteine residue is substituted for at least one amino acid located in the C-D loop of growth hormone;
- wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to growth hormone.
A cysteine variant of growth hormone of SEQ ID NO:1, wherein said variant contains the four native cysteines, C53, C165, C182 and C189, and wherein a cysteine residue is substituted for at least one amino acid located in the B-C loop of growth hormone;
- wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to growth hormone.
A cysteine variant of growth hormone of SEQ ID NO:1, wherein a cysteine residue is substituted for at least one arnino acid located in at least one region of growth hormone selected frorn the group consisting of the first three amino acids in helix A or the last three amino acids in helix A;
- wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to growth hormone.
A cysteine variant of growth hormone of SEQ ID NO:1, wherein a cysteine residue is substituted for at least one amino acid located in at least one region of growth hormone selected from the group consisting of the first three amino acids in helix B or the last three amino acids in helix B;
- wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to growth hormone.
A cysteine variant of growth hormone of SEQ ID NO:1, wherein a cysteine residue is substituted for at least one of the first three amino acids in helix C of growth hormone;
- wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to growth hormone.
A cysteine variant of growth hormone of SEQ ID NO:1, wherein a cysteine residue is substituted for at least one amino acid located in at least one region of growth hormone selected from the group consisting of the first three amino acids in helix D or the last three amino acids in helix D;
- wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to growth hormone.
A cysteine variant of growth hormone of SEQ ID NO:1, wherein a cysteine residue is substituted for E129 of growth hormone;
- wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to growth hormone.
A cysteine variant of growth hormone of SEQ ID NO:1, wherein a cysteine residue is substituted for at least one amino acid located in at least one region of growth hormone selected from the group consisting of:
- the C-D loop, the first three or last three amino acids in helix A, the first three or last three amino acids in helix B, the first three amino acids in helix C, the first three or last thiee amino acids in helix D, the amino acids preceding helix A corresponding to amino acids 1-5 of SEQ ID NO:1, and the amino acids following helix D corresponding to amino acids 184-191 of SEQ ID NO:1;
- and
- wherein said growth hormone cysteine variant comprises one or more additional mutations tlat cause the protein to act as a growth hormone antagonist as measured in vitro by the ability ofthe variant to antagonize growth hormone biological activity using a cell line that proliferates in response to growth hormone.
A cysteine variant of growth hormone of SEQ ID NO:1, wherein said variant contains the four native cysteines, C53, C165, C182 and C189, and wherein a cysteine residue is substituted for at least one amino acid located in at least one region of growth hormone selected from the group consisting of:
- the B-C loop, the C-D loop, the first three or last three amino acids in helix A, the first tree or last three amino acids in helix B. the first three amino acids in helix C, the first three or last three a mino acids in helix D, the amino acids preceding helix A corresponding to amino acids 1-5 of SEQ ID NO:1, and the amino acids following helix D corresponding to amino acids 184-191 of SEQ ID NO:1;
- and
- wherein said growth hormone cysteine variant comprises onc or more additional mutations that cause the protein to act as a growth hormone antagonist as measured in vitro by the ability of the variant to antagonize growth hormone biological activity using a cell line that proliferates in response to growth hormone.
A cysteine variant of growth hormone of SEQ ID NO:1, wherein a cysteine residue is substituted for at least one amino acid located in the region of growth honnone preceding helix A corresponding to amino acids 1-5 of SEQ ID NO:1;
- and
- wherein said growth hormone cysteine variant comprises one or more additional mutations that cause the protein to act as a growth hormone antagonist as measured in vitro by the ability of the variant to antagonize growth hormone biological activity using a cell line that proliferates in response to growth hormone.
A cysteine variant of growth hormone of SEQ ID NO:1, wherein a cysteine residue is substituted for at least one amino acid located in the region of growth hormone following helix D corresponding to amino acids 184-191 of SEQ ID NO:1;
- and
- wherein said growth hormone cysteine variant comprises one or more additional mutations that cause the protein to act as a growth hormone antagonist as measured in vitro by the ability of the variant to antagonize growth hormone biological activity using a cell line that proliferates in response to growth hormone.
A cysteine variant of growth hormone of SEQ ID NO:1, wherein a cysteine residue is substituted for at least one amino acid of growth hormone selected from the group consisting of A34, K38, E39, Q40, S43, N47, P48, Q49, T50, S51, S55 and T60;
- and
- wherein said growth hormone cysteine variant comprises one or more additional mutations that cause the protein to act as a growth hormone antagonist as measured in vitro by the ability of the varian to antagonize growth hormone biological activity using a cell line that proliferates in response to growth hormone.
A cysteine variant of growth hormone of SEQ ID NO:1, wherein a cysteine residue is substituted for at least one amino acid located in the C-D loop of growth hormone;
- and
- wherein said growth hormone cysteine variant comprises one or more additional mutations that cause the protein to act as a growth hormone antagonist as measured in vitro by the ability of the variant to antagonize growth hormone biological activity using a cell line that proliferates in response to growth hormone.
A cysteine variant of growth hormone of SEQ ID NO:1, wherein said variant contains the four native cysteines, C53, C165, C182 and C 189, and wherein a cysteine residue is substituted for at least one amino acid located in the B-C loop of growth hormone;
- and
- wherein said growth hormone cysteine variant comprises one or more additional mutations that cause the protein to act as a growth hormone antagonist as measured in vitro by the ability of the variant to antagonize growth hormone biological activity using a cell line that proliferates in response to growth hormone.
A cysteine variant of growth hormone of SEQ ID NO:1, wherein a cysteinc residue is substituted for at least one amino acid located in at least one region of growth hormone selected from the group consisting of the first three amino acids in helix A or the last three amino acids in helix A;
- and
- wherein said growth hormone cysteine variant comprises one or more additional mutations that cause the protein to act as a growth hormone antagonist as measured in vitro by the ability of the variant to antagonize growth hormone biological activity using a cell line that proliferates in response to growth hormone.
A cysteine variant of growth hormone of SEQ ID NO:1, wherein a cysteine residue is substituted for at least one amino acid located in at least one region of growth hormone selected from the group consisting of the first three amino acids in helix B or the last three amino acids in helix B;
- and
- wherein said growth hormone cysteine variant comprises one or more additional mutations that cause the protein to act as a growth hormone antagonist as measured in vitro by the ability of the variant to antagonize growth hormone biological activity using a cell line that proliferates in response to growth hormone.
A cysteine variant of growth hormone of SEQ ID NO:1, wherein a cysteine residue is substituted for at least one of the first three amino acids in helix C of growth hormone;
- and
- wherein said growth hormone cysteine variant comprises one or more additional mutations that cause the protein to act as a growth hormone antagonist as measured in vitro by the ability of the variant to antagonize growth hormone biological activity using a cell line that proliferates in response to growth hormone.
A cysteine variant of growth hormone of SEQ ID NO:1, wherein a cysteine residue is substituted for at least one amino acid located in at least one region of growth hormone selected from the group consisting of the first three amino acids in helix D or the last three amino acids in, helix D;
- and
- wherein said growth hormone cysteine variant comprises one or more additional mutations that cause the protein to act as a growth hormone antagonist as measured in vitro by the ability of the variant to antagonize growth hormone biological activity using a cell line that proliferates in response to growth hormone.
A cysteine variant of growth hormone of SEQ ID NO:1, wherein a cysteine residue is substituted for E129 of growth hormone;
- wherein said growth hormone cysteine variant comprises one or more additional mutations that cause the protein to act as a growth hormone antagonist as measured in vitro by the ability of the variant to antagonize growth hormone biological activity using a cell line that proliferates in response to growth hormone.
Dependencies are inferred from the claim text.