Drug – bio-affecting and body treating compositions – Lymphokine
Patent
1992-06-25
1994-10-25
Wityshyn, Michael G.
Drug, bio-affecting and body treating compositions
Lymphokine
530351, A61K 4505
Patent
active
053587073
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The control of blood cell formation is mediated by a group of glycoproteins called colony stimulating factors. One such protein is granulocyte-macrophage colony stimulating factor (GM-CSF), which stimulates progenitor cells to form granulocyte, macrophage and eosinophil colonies on semisolid medium [Lopez et al., J. Clin. Invest. 78: 1220 (1986); Metcalf, Blood 67: 257 (1986)]. GM-CSF can stimulate the initial cell divisions of erythroid progenitor cells without altering their ultimate ability to form mature red cells [Sieff et al., Science , 230: 1171 (1985)]. It also can stimulate colon), formation in the HL60 and KG-1 human myeloid leukemic cell lines [Tomonaga et al., Blood 67: 31 (1986)]and has a direct effect on neutrophil function [Lopez et al., J. Clin. Invest. 78: 1220 (1986)].
The cloning and sequencing of complementary DNAs encoding human [Lee et al., Proc. Natl. Acad. Sci. USA 82: 4360 (1985); Wong et al., Science 228: 810 (1985); Cantrell et al., Proc. Natl. Acad. Sci. USA 82: 6250 (1985)], gibbon [Wong et al., supra and murine [Gough et al., Nature 309: 763 (1984)]GM-CSF have been reported. Greenberg et al. [Current Microbiol. 17: 321 (1988)]have described the expression of biologically active, mature human GM-CSF in an E. Coli secretory expression system. The isolation of natural GM-CSF from cultured human [U.S. Pat. No. 4,438,032; Gasson et al., Science 230: 1171 (1985); Burgess et al., Blood 69: 43 (1987)], mouse [Burgess et al., Exp. Hematol. 9: 893 (1981); Sparrow et al., Proc. Natl. Acad. Sci. USA 82: 292 (1985)] and rat [Wu et al., Exp. Hematol. 12: 267 (1984)] cells has also been reported.
Mature human GM-CSF consists of about 127 amine acid residues, four of which are methionine residues. The mature forms of mouse and gibbon GM-CSF appear to contain only three methionine residues. GM-CSF from various species is also known to contain a number of cysteine residues, and the human protein contains four.
Methionine and cysteine are often important to the overall conformation and biological activity of a protein. The side chains of methionine residues are hydrophobic and tend to associate with other hydrophobic residues in the stable conformation of a protein. The sulfhydryl groups of cysteines are often joined in disulfide linkages (as cystine), which also serve to maintain conformation.
Under oxidizing conditions, the character of methionine and cysteine residues changes markedly. The sulfur in methionine is converted from a thio ether to a highly polar sulfoxide or sulfone. If a given methionine was associated with other hydrophobic residues, there is a likelihood that the hydrophilic oxidized form will no longer do so. Oxidation of cysteine residues can give rise to unnatural intra- and inter-chain disulfide bridges.
The possible overall effect of the oxidation of a protein can thus be a dramatic change in conformation (3-dimensional shape) which can result in a strong reduction or complete loss of biological activity. Whether a given protein will be thus altered by oxidation cannot presently be predicted with any reasonable degree of certainty.
SUMMARY OF THE INVENTION
It has surprisingly been found that the methionine residues of GM-CSF can be oxidized to a high degree while retaining a significant amount of biological activity, as determined in an in vitro bioassay. This invention provides such oxidized variants of GM-CSF.
More particularly, this invention provides biologically active oxidized variants of GM-CSF having at least one oxidized methionine residue, which variants are essentially free of unoxidized GM-CSF and other oxidized variants. Variants are provided in which one, two, three or four oxygen atoms are introduced into the methionine residues of GM-CSF. At least about 30% of the biological activity of the unoxidized protein is retained in these variants. Some of the variants are essentially as active as unoxidized GM-CSF.
This invention further provides methods for making and characterizing the oxidized GM-CSF variants.
The oxidized GM-CSF
REFERENCES:
patent: 5116943 (1992-05-01), Koths et al.
patent: 5200327 (1993-04-01), Garvin et al.
Fukita, et al., Chem. Abstracts 113:35453d (1990).
Gough, et al., Nature 309:763 (1984).
Greenberg, et al., Current Microbiol. 17:321 (1988).
Ohgami, et al., J. Biotechnol. 12:219 (1989).
Reichert Paul
Seelig Gail F.
Trotta Paul P.
Dulak Norman C.
Kanstad Steinar V.
Lunn Paul G.
Sayala C.
Schering Corporation
LandOfFree
Oxidized variants of GM-CSF does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Oxidized variants of GM-CSF, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Oxidized variants of GM-CSF will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-133585