Synthetic resins or natural rubbers -- part of the class 520 ser – Synthetic resins – Mixing of two or more solid polymers; mixing of solid...
Patent
1990-08-30
1992-10-06
Nutter, Nathan M.
Synthetic resins or natural rubbers -- part of the class 520 ser
Synthetic resins
Mixing of two or more solid polymers; mixing of solid...
530351, 530410, 530413, 514 12, C08L 8900, C07G 700, A61K 3702
Patent
active
051532654
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a chemical modification of biologically active colony stimulating factor-1 (CSF-1) that alters the chemical and/or physiological properties of this protein. More specifically, this invention relates to selective conjugation of CSF-1 to polymers to increase the circulating half-life of the protein in mammals.
Colony stimulating factor-1 (CSF-1) (also known as M-CSF) is one of several proteins that are capable of stimulating colony formation by bone marrow cells plated in semisolid culture medium. CSF-1 is distinguished from other colony stimulating factors by its ability to stimulate the formation of predominantly macrophage colonies. Other CSFs stimulate the production of colonies that consist of neutrophilic granulocytes and macrophages, exclusively neutrophilic granulocytes, or neutrophilic and eosinophilic granulocytes and macrophages. A review of these CSFs has been published by Dexter, T. M., Nature (1984) 309:746, and by Vadas, M. A., J. Immunol (1983) 130:793. There is currently no routine in vivo assay that is known to be specific for CSF-1 activity.
CSF-1 has been purified from native sources (see, e.g., Csejtey et al., Biochem. Biophys. Res. Comm. (1986) 138:238 and PCT publication No. WO 86/04587 published Aug. 14, 1986 regarding immunoaffinity chromatography of native CSF-1 to enable partial amino acid determinations). CSF-1 has also been produced from recombinant DNA using two apparently related cDNA clones: (1) a "short" form that encodes a monomeric protein of 224 amino acids preceded by a 32-amino acid signal sequence (Kawasaki et al., Science (1985) 230:292-296); and (2) a "long" form, encoding a monomeric protein of 522 amino acids, also preceded by the 32-amino acid signal sequence. The long form has been cloned and expressed by two groups, as disclosed in European Patent Publication No. 0272779 published Jun. 29, 1988 and Ladner et al. EMBO J. (1987) 6:2693, each incorporated herein by reference; and in Wong et al. Science (1987) 235:1504- 1509, and PCT WO87/06954 published Nov. 19, 1987. (The DNA and amino acid sequences for these two clones are shown in FIGS. 1 and 2, respectively.) Both the long and short forms of CSF-1 are described by Clark and Kamen, Science (1987) 236:1229-1237. An "intermediate" form that encodes a monomeric protein of 406 amino acids preceded by a 32-amino acid signal sequence has also been recently reported (Cerretti et al. (1988) Mol. Immunol. 25:761-770).
The long and short forms of the CSF-1-encoding DNA appear to arise from a variable splice junction at the upstream portion of exon 6 of the genomic CSF-1-encoding DNA. When CSF-1 is expressed in certain eucaryotic cells from either the long or short cDNA forms, it is secreted as a dimeric glycoprotein and appears to be variably processed at the C-terminus and/or variably glycosylated. Consequently, CSF-1 proteins of varying molecular weights are found when the reduced monomeric form is subjected to Western analysis.
The amino acid sequences of the long and short forms, as predicted from the DNA sequence of the isolated clones and by their relationship to the genomic sequence, are identical in the first 149 amino acids at the N-terminus after signal peptide cleavage, and diverge thereafter as a result of the insertion in the longer clone of an additional 894 bp fragment (encoding 298 additional amino acids) before the codon encoding amino acid 150. Therefore, both the shorter and longer forms of the gene encode regions of identical sequence at the C-terminus, as well as at the N-terminus. Biologically active protein has been recovered when truncated cDNAs encoding only the first 145 or 147 amino acids of the mature short form (European Patent Publication No. 0261592 published Mar. 30, 1988; Cerretti et al. supra or the first 190 or 221 amino acids of the mature longer form, are expressed in eucaryotic cells.
Recombinant CSF-1 was expressed in E. coli by modifying a short clone cDNA originally described by Kawasaki et al., Science (1985) 230:291 to code for proteins that contained 1)
REFERENCES:
patent: 4094744 (1978-06-01), Hartdegen et al.
patent: 4847325 (1989-07-01), Shadle et al.
Aldwin Lois
Katre Nandini
Koths Kirston E.
Laird Walter J.
Moreland Margaret
Cetus Corporation
Halluin Albert P.
McGarrigle Philip L.
McLaughlin Jane R.
Nutter Nathan M.
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