Drug – bio-affecting and body treating compositions – Lymphokine
Reexamination Certificate
1999-06-25
2002-03-19
Mertz, Prema (Department: 1646)
Drug, bio-affecting and body treating compositions
Lymphokine
C435S007200, C424S185100, C424S192100, C514S002600
Reexamination Certificate
active
06358505
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to human G-CSF receptor agonists with activity on hematopoietic cell differentiation and expansion.
BACKGROUND OF THE INVENTION
The human blood-forming (hematopoietic) system replaces a variety of white blood cells (including neutrophils, macrophages, and basophils/mast cells), red blood cells (erythrocytes) and clot-forming cells (megakaryocytes/platelets). The hematopoietic systems of the average male has been estimated to produce on the order of 4.5×10
11
granulocytes and erythrocytes every year, which is equivalent to an annual replacement of total body weight (Dexter et al.,
BioEssays,
2;154-158, 1985).
It is believed that small amounts of certain hematopoietic growth factors account for the differentiation of a small number of progenitor “stem cells” into the variety of blood cell lines, for the tremendous proliferation of those lines, and for the ultimate differentiation of mature blood cells from those lines. Because the hematopoietic growth factors are present in extremely small amounts, the detection and identification of these factors has relied upon an array of assays which as yet only distinguish among the different factors on the basis of stimulative effects on cultured cells under artificial conditions.
U.S. Pat. No. 4,999,291 discloses DNA and methods for making G-CSF the disclosure of which is incorporated herein by reference in it entirety.
U.S. Pat. No. 4,810,643 relates to DNA and methods of making G-CSF and Cys to Ser substitution variants of G-CSF.
Kuga et al. (
Biochem.+Biophys. Res. Comm.
159:103-111, 1989) made a series of G-CSF variants to partially define the structure-function relationship. Kuga et al. found that internal and C-terminal deletions abolished activity, while N-terminal deletions of up to 11 amino acids and amino acid substitutions at positions 1, 2 and 3 were active.
Watanabe et al. (
Anal. Biochem.
195:38-44, 1991) made a variant to study G-CSF receptor binding in which amino acids 1 and 3 were changed to Tyr for radioiodination of the protein. Watanabe et al. found this Tyr
1
, Tyr
3
G-CSF variant to be active.
WO 95/27732 describes, but does not show that the molecule has biological activity, a circularly permuted G-CSF ligand with a breakpoint at positions 68/69 creating a circularly permuted G-CSF ligand with a new N-terminus at the original position 69 of G-CSF and a new C-terminus at the original position 68 of G-CSF. WO 95/27732 also discloses circularly permuted GM-CSF, IL-2 and IL-4.
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, 1996). 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 fusion
molecule
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 trying 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 convert a subset of interactions that are long-range in the original sequence into short-range interactions in the new sequence, and vice versa. The fact that many of the
Braford-Goldberg Sarah
Feng Yiping
Klein Barbara
McKearn John
McWherter Charles
Bauer S. Christopher
Mertz Prema
Murphy Joseph F.
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