Analogs of macrophage stimulating protein

Details Analogs of macrophage stimulating protein Analogs of macrophage stimulating protein

1999-04-22

2001-06-19

Chan, Christina Y. (Department: 1644)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S325000, C435S320100, C536S023500

Reexamination Certificate

active

06248560

ABSTRACT:

FIELD OF THE INVENTION
The invention generally relates to analogs of macrophage stimulating protein, or MSP. More particularly, the invention relates to analogs of MSP which promote multimer formation and have enhanced biological activity.
BACKGROUND OF THE INVENTION
Macrophage Stimulating Protein, or MSP, has been previously identified as an activity present in mammalian blood plasma which makes mouse peritoneal macrophages responsive to chemoattractants such as complement C5a (Leonard et al. Exp. Cell. Res. 102, 434 (1976); Leonard et al. Exp. Cell Res. 114, 117 (1978). MSP was purified from human serum as described in U.S. Pat. No. 5,219,991 and the DNA sequence encoding human MSP was reported in U.S. Pat. No. 5,315,000. MSP is synthesized in a prepro form which is secreted as a single chain polypeptide. The pro form is proteolytically cleaved to form a disulfide-linked heterodimer having an &agr; and &bgr; chain of molecular weights 53 kDa and 25 kDa, respectively. The heterodimer is the biologically active form of MSP. It has not been established which protease is responsible for the in vivo activation of MSP, but several proteases, such as human plasma kallikrein are reported to efficiently activate MSP in vitro (Wang et al. J. Biol. Chem. 269, 3436-3440 (1994)).
MSP is a member of a family of proteins having triple disulfide loop structures, or kringle domains (Donate et al. Protein Science 3, 2378-2394 (1994)). Family members include plasminogen and hepatocyte growth factor (HGF). MSP also exhibits sequence homology to both plasminogen and HGF and its proteolytic activation occurs at Arg-Val residues which are also conserved in other family members.
A variety of in vitro biological activities have been reported for MSP. MSP was initially purified based upon stimulation of a chemotactic response of mouse resident peritoneal macrophages (Leonard et al., supra) and was believed to play a role in cell motility. MSP stimulated megakaryocyte maturation and thrombocyte production from isolated bone marrow preparations (PCT Application No. WO96/14082). The in vivo activity of MSP remains to be elucidated.
Recently, it has been reported that MSP is a ligand for RON, a cell membrane protein tyrosine kinase which is a member of the c-met family of protein tyrosine kinases (Wang et al. Science 266, 117-119 (1994); Gaudino et al. EMBO J. 13, 3524-3532 (1994); Ronsin et al. Oncogene 8, 1195-1202 (1993)). The expression of RON in human tissues and cell lines was examined (Gaudino et al., supra) and RON was found to be expressed in colon, skin, lung and bone marrow, and in granulocytes and adherent monocytes. Epithelial cell lines derived from gastric, pancreatic and mammary carcinoma, and hematopoietic cell lines also showed RON expression. MSP induced tyrosine phosphorylation of RON and stimulated DNA synthesis in a mammary carcinoma cell line. These observations suggest that MSP may act on a variety of cell types. MSP promotes colony formation by mouse colon crypts as shown in co-owned U.S. Pat. No. 5,814,308, suggesting that MSP may be useful in protecting and regenerating the intestinal epithelium.
In view of the useful biological activities exhibited by MSP. it is desirable to find forms of MSP which have enhanced biological activity. Such forms could provide a more favorable therapeutic regimen in that they can be administered at lower dosages and/or less frequently than MSP.
SUMMARY OF THE INVENTION
The invention provides for analogs of MSP which have increased heterodimer formation and enchanced biological activity compared to recombinant human MSP. The analogs of the present invention are constructed by substituting a cysteine residue with another amino acid such that interchain disulfide bonds will form efficiently and promote MSP heterodimer formation. The resulting MSP analog forms heterodimers of one &agr; (kringle-containing) chain and one &bgr; (serine protease) chain to a greater extent than recombinant human MSP. DNA sequences encoding MSP analogs, expression vectors comprising the DNA sequences and modified host cells which express MSP analogs are also provided by the invention. Pharmaceutical compositions of MSP analogs may be used to treat conditions treatable by MSP including gastrointestinal and hematopoietic disorders.


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