Fused G-CSF and IL-3 proteins and uses thereof

Details Fused G-CSF and IL-3 proteins and uses thereof Fused G-CSF and IL-3 proteins and uses thereof

2000-02-22

2004-05-04

Spector, Lorraine (Department: 1647)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Fusion protein or fusion polypeptide

C424S085100, C424S198100, C424S093210, C424S085200, C530S351000, C530S399000, C536S023400, C435S069700, C435S069900, C435S320100, C435S235100, C435S252300

Reexamination Certificate

active

06730303

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to multi-functional hematopoietic receptor agonists.
BACKGROUND OF THE INVENTION
Colony stimulating factors (CSFs) which stimulate the differentiation and/or proliferation of bone marrow cells have generated much interest because of their therapeutic potential for restoring depressed levels of hematopoietic stem cell-derived cells. CSFs in both human and murine systems have been identified and distinguished according to their activities. For example, granulocyte-CSF (G-CSF) and macrophage-CSF (M-CSF) stimulate the in vitro formation of neutrophilic granulocyte and macrophage colonies, respectively, while GM-CSF and interleukin-3 (IL-3) have broader activities and stimulate the formation of both macrophage, neutrophilic and eosinophilic granulocyte colonies. IL-3 also stimulates the formation of mast, megakaryocyte and pure and mixed erythroid colonies.
U.S. Pat. No. 4,877,729 and U.S. Pat. No. 4,959,455 disclose human IL-3 and gibbon IL-3 cDNAs and the protein sequences for which they code. The hIL-3 disclosed has serine rather than proline at position 8 in the protein sequence. International Patent Application (PCT) WO 88/00598 discloses gibbon- and human-like IL-3. The hIL-3 contains a Ser
8
→Pro
8
replacement. Suggestions are made to replace Cys by Ser, thereby breaking the disulfide bridge, and to replace one or more amino acids at the glycosylation sites.
U.S. Pat. No. 4,810,643 discloses the DNA sequence encoding human G-CSF.
WO 91/02754 discloses a fusion protein comprised of GM-CSF and IL-3 which has increased biological activity compared to GM-CSF or IL-3 alone. Also disclosed are nonglycosylated IL-3 and GM-CSF analog proteins as components of the multi-functional hematopoietic receptor agonist.
WO 92/04455 discloses fusion proteins composed of IL-3 fused to a lymphokine selected from the group consisting of IL-3, IL-6, IL-7, IL-9, IL-11, EPO and G-CSF.
WO 95/21197 and WO 95/21254 disclose fusion proteins capable of broad multi-functional hematopoietic properties.
GB 2,285,446 relates to the c-mpl ligand (thrombopoietin) and various forms of thrombopoietin which are shown to influence the replication, differentiation and maturation of megakaryocytes and megakaryocytes progenitors which may be used for the treatment of thrombocytopenia.
EP 675,201 A1 relates to the c-mpl ligand (Megakaryocyte growth and development factor (MGDF), allelic variations of c-mpl ligand and c-mpl ligand attached to water soluble polymers such as polyethylene glycol.
WO 95/21920 provides the murine and human c-mpl ligand and polypeptide fragments thereof. The proteins are useful for in vivo and ex vivo therapy for stimulating platelet production.
Rearrangement of Protein Sequences
In evolution, rearrangements of DNA sequences serve an important role in generating a diversity of protein structure and function. Gene duplication and exon shuffling provide an important mechanism to rapidly generate diversity and thereby provide organisms with a competitive advantage, especially since the basal mutation rate is low (Doolittle,
Protein Science
1:191-200, 1992).
The development of recombinant DNA methods has made it possible to study the effects of sequence transposition on protein folding, structure and function. The approach used in creating new sequences resembles that of naturally occurring pairs of proteins that are related by linear reorganization of their amino acid sequences (Cunningham, et al.,
Proc. Natl. Acad. Sci. U.S.A
. 76:3218-3222, 1979; Teather & Erfle,
J. Bacteriol
. 172: 3837-3841, 1990; Schimming et al.,
Eur. J. Biochem
. 204: 13-19, 1992; Yamiuchi and Minamikawa,
FEBS Lett
. 260:127-130, 1991; MacGregor et al.,
FEBS Lett
. 378:263-266). The first in vitro application of this type of rearrangement to proteins was described by Goldenberg and Creighton (
J. Mol. Biol
. 165:407-413, 1983). A new N-terminus is selected at an internal site (breakpoint) of the original sequence, the new sequence having the same order of amino acids as the original from the breakpoint until it reaches an amino acid that is at or near the original C-terminus. At this point the new sequence is joined, either directly or through an additional portion of sequence (linker), to an amino acid that is at or near the original N-terminus, and the new sequence continues with the same sequence as the original until it reaches a point that is at or near the amino acid that was N-terminal to the breakpoint site of the original sequence, this residue forming the new C-terminus of the chain.
This approach has been applied to proteins which range in size from 58 to 462 amino acids (Goldenberg & Creighton,
J. Mol. Biol
. 165:407-413, 1983; Li & Coffino,
Mol. Cell. Biol
. 13:2377-2383, 1993). The proteins examined have represented a broad range of structural classes, including proteins that contain predominantly &agr;-helix (interleukin-4; Kreitman et al.,
Cytokine
7:311-318, 1995), &bgr;-sheet (interleukin-1; Horlick et al.,
Protein Eng
. 5:427-431, 1992), or mixtures of the two (yeast phosphoribosyl anthranilate isomerase; Luger et al.,
Science
243:206-210, 1989). Broad categories of protein function are represented in these sequence reorganization studies:
Enzymes
T4 lysozyme
Zhang et al., Biochemistry
32:12311-12318, 1993; Zhang et al.,
Nature Struct. Biol. 1:434-438
(1995)
dihydrofolate
Buchwalder et al., Biochemistry
reductase
31:1621-1630, 1994; Protasova et
al., Prot. Eng. 7:1373-1377, 1995)
ribonuclease T1
Mullins et al., J. Am. Chem. Soc.
116:5529-5533, 1994; Garrett et al.,
Protein Science 5:204-211, 1996)
Bacillus &bgr;-glucanse
Hahn et al., Proc. Natl. Acad. Sci.
U.S.A. 91:10417-10421, 1994)
aspartate
Yang & Schachman, Proc. Natl. Acad.
transcarbamoylase
Sci. U.S.A. 90:11980-11984, 1993)
phosphoribosyl
Luger et al., Science 243:206-210
anthranilate
(1989; Luger et al., Prot. Eng.
isomerase
3:249-258, 1990)
pepsin/pepsinogen
Lin et al., Protein Science 4:159-
166, 1995)
glyceraldehyde-3-
Vignais et al., Protein Science
phosphate dehydro-
4:994-1000, 1995)
genase
ornithine
Li & Coffino, Mol. Cell. Biol.
decarboxylase
13:2377-2383, 1993)
yeast
Ritco-Vonsovici et al., Biochemistry
phosphoglycerate
34:16543-16551, 1995)
dehydrogenase
Enzyme Inhibitor
basic pancreatic
Goldenberg & Creighton, J. Mol.
trypsin inhibitor
Biol. 165:407-413, 1983)
Cytokines
interleukin-1&bgr;
Horlick et al., Protein Eng. 5:427-
431, 1992)
interleukin-4
Kreitman et al., Cytokine 7:311-
318, 1995)
Tyrosine Kinase
Recognition Domain
&agr;-spectrin SH3
Viguera, et al., J.
domain
Mol. Biol. 247:670-681, 1995)
Transmembrane
Protein
omp A
Koebnik & Krämer, J. Mol. Biol.
250:617-626, 1995)
Chimeric Protein
interleukin-4-
Kreitman et al., Proc. Natl. Acad.
Pseudomonas
Sci. U.S.A. 91:6889-6893, 1994).
exotoxin
The results of these studies have been highly variable. In many cases substantially lower activity, solubility or thermodynamic stability were observed (
E. coli
dihydrofolate reductase, aspartate transcarbamoylase, phosphoribosyl anthranilate isomerase, glyceraldehyde-3-phosphate dehydrogenase, ornithine decarboxylase, omp A, yeast phosphoglycerate dehydrogenase). In other cases, the sequence rearranged protein appeared to have many nearly identical properties as its natural counterpart (basic pancreatic trypsin inhibitor, T4 lysozyme, ribonuclease T1, Bacillus &bgr;-glucanase, interleukin-1&bgr;, &agr;-spectrin SH3 domain, pepsinogen, interleukin-4). In exceptional cases, an unexpected improvement over some properties of the natural sequence was observed, e.g., the solubility and refolding rate for rearranged &agr;-spectrin SH3 domain sequences, and the receptor affinity and anti-tumor activity of transposed interleukin-4-Pseudomonas exotoxin fusion molecule (Kreitman et al.,
Proc. Natl. Acad. Sci. U.S.A
. 91:6889-6893, 1994; Kreitman et al.,
Cancer Res
. 55:3357-3363, 1995).
The primary motivation for these types of studies has been to study the role of short-range and long-range interactions in protein folding and stability. Sequence rearrangements of this type

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