Drug – bio-affecting and body treating compositions – Lymphokine
Reexamination Certificate
1997-10-21
2003-12-09
Kunz, Gary (Department: 1647)
Drug, bio-affecting and body treating compositions
Lymphokine
C424S198100, C530S351000, C530S399000, C514S002600, C514S012200
Reexamination Certificate
active
06660257
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to human flt3 receptor agonists. These flt3 receptor agonists retain one or more activities of native flt3 ligand and may also show improved hematopoietic cell-stimulating activity and/or an improved activity profile which may include reduction of undesirable biological activities associated with native flt3 ligand and/or have improved physical properties which may include increased solubility, stability and refold efficiency.
BACKGROUND OF THE INVENTION
Colony stimulating factors 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. Colony stimulating factors 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. Certain factors such as flt3 ligand are able to predominately affect stem cells.
Tyrosine kinase receptors are growth factor receptors that regulate the proliferation and differentiation of a number of cell. Certain tyrosine kinase receptors function within the hematopoietic system. Flt3 ligand (Rosnet et al.,
Oncogene
, 6:1641-1650, 1991) and flk-2 (Matthews et al.,
Cell
, 65:1143-1152, 1991) are forms of a tyrosine kinase receptor that is related to c-fms and c-kit receptors. The flk-2 and flt3 receptors are similar in amino acid sequence and vary at two amino acid residues in the extracellular domain and diverge in a 31 amino acid segment located near the C-terminus.
flt3 ligand is a hematopoietic growth factor which has the property of being able to regulate the growth and differentiation of hematopoietic progenitor and stem cells. Because of its ability to support the growth and proliferation of progenitor cells, flt3 receptor agonists have potential for therapeutic use in treating hematopoietic disorders such as aplastic anemia and myelodysplastic syndromes. Additionally, flt3 receptor agonists will be useful in restoring hematopoietic cells to normal amounts in those cases where the number of cells has been reduced due to diseases or to therapeutic treatments such as radiation and chemotherapy.
WO 94/28391 discloses the native flt3 ligand protein sequence and a cDNA sequence encoding the flt3 ligand, methods of expressing flt3 ligand in a host cell transfected with the cDNA and methods of treating patients with a hematopoietic disorder using flt3 ligand.
U.S. Pat. No. 5,554,512 is directed to human flt3 ligand as an isolated protein, DNA encoding the flt3 ligand, host cells transfected with cDNAs encoding flt3 ligand and methods for treating patients with flt3 ligand.
WO 94/26891 provides mammalian flt3 ligands, including an isolate that has an insertion of 29 amino acids, and fragments there of.
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 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;-
Feng Yiqing
Mc Kearn John P.
McWherter Charles A.
Minnerly John C.
Minster Nancy I.
Bauer S. Christopher
Kunz Gary
Pharmacia Corporation
Pharmacia Corporation
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