DERIVATIVES OF GROWTH HORMONE AND RELATED PROTEINS, AND METHODS OF USE THEREOF

• US 20110172151A1
• Filed: 09/21/2010
• Published: 07/14/2011
• Est. Priority Date: 07/14/1997
• Status: Active Grant

• Current Assignee: Bolder Biotechnology, Inc.
• Original Assignee: Bolder Biotechnology, Inc.
• Inventors: Cox, George N. III, Doherty, Daniel H.
• Granted Patent: US 8,148,500 B2
• Time in Patent Office: Days
• US Class Current: 514/9.7
• CPC Class Codes: A61K 38/193 Colony stimulating factors [CSF]
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: C07K 14/475 Growth factors; Growth regulators
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• CPC Class Codes: C07K 14/535 Granulocyte CSF; Granulocyte-macrophage CSF
• CPC Class Codes: C07K 14/5431 IL-11
• CPC Class Codes: C07K 14/61 Growth hormone [GH], i.e. somatotropin

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 proteins or substituting cysteine residues for non-essential 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. Also disclosed are therapeutic methods for using the cysteine variants of the invention.

Claims

1-40. -40. (canceled)

41. A method for preparing a recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) protein comprising the steps of:

(a) obtaining an Escherichia coli (E. coli) host cell transformed with a plasmid encoding a GM-CSF protein fused to the E. coli STII signal sequence;

(b) growing the E. coli host cell under conditions that cause the host cell to express the GM-CSF protein and secrete the GM-CSF protein into the E. coli periplasm;

(c) lysing the cells;

(d) isolating the insoluble GM-CSF protein from the lysed cells;

(e) denaturing and reducing the insoluble GM-CSF protein; and

(f) refolding the GM-CSF protein into a biologically active form.

42. The method of claim 41 further comprising isolating the refolded GM-CSF protein.

43. The method of claim 42 wherein said GM-CSF protein is isolated by column chromatography.

44. The method of claim 41, wherein the GM-CSF protein is human GM-CSF.

45. The method of claim 41, wherein the GM-CSF protein is a human GM-CSF cysteine mutein.

46. The method of claim 45, wherein the GM-CSF cysteine mutein comprises a cysteine residue substituted for an amino acid selected from the group consisting of A1, A3, S5, S7, N27, T32, A33, E51, R67, S69, E93, T94, T98, Q99, T102, E123, V125, Q126, and E127.

47. The method of claim 45, wherein the GM-CSF cysteine mutein comprises a cysteine residue added preceding the first amino acid of the mature protein.

48. The method of claim 45, wherein the GM-CSF cysteine mutein comprises a cysteine residue added following the last amino acid of the protein.

49. The method of claim 45, wherein the GM-CSF cysteine mutein comprises a cysteine residue substituted for A3.

50. The method of claim 45, wherein the GM-CSF cysteine mutein comprises a cysteine residue substituted for an amino acid selected from the group consisting of A1, S5 and S7.

51. The method of claim 45, wherein the GM-CSF cysteine mutein comprises a cysteine residue substituted for an amino acid selected from the group consisting of T32, A33 and E51.

52. The method of claim 45, wherein the GM-CSF cysteine mutein comprises a cysteine residue substituted for an amino acid selected from the group consisting of R67 and S69.

53. The method of claim 45, wherein the GM-CSF cysteine mutein comprises a cysteine residue substituted for an amino acid selected from the group consisting of E93, T94, T98, Q99 and T102.

54. The method of claim 45, wherein the GM-CSF cysteine mutein comprises a cysteine residue substituted for an amino acid selected from the group consisting of N27 and E123.

55. The method of claim 45, wherein the GM-CSF protein comprises a cysteine residue substituted for an amino acid selected from the group consisting of V125, Q126 and E127.

56. The method of claim 42, wherein the isolated GM-CSF protein is modified with polyethylene glycol.

57. The method of claim 56, wherein the polyethylene glycol is a cysteine-reactive polyethylene glycol.

58. The method of claim 42, wherein the isolated GM-CSF protein is modified with a cysteine-reactive moiety.

59. The method of claim 45, wherein the GM-CSF cysteine mutein is modified with polyethylene glycol