Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1993-10-20
2001-04-17
Marschel, Ardin H. (Department: 1631)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120, C536S024310
Reexamination Certificate
active
06218520
ABSTRACT:
TECHNICAL FIELD
The invention is related to human therapeutics and regulation of the complement cascade. More specifically, it concerns the recombinant production of human membrane cofactor protein (MCP) (Sequence ID No. 2) which is an important factor in the regulation of complement cascade.
BACKGROUND ART
The complement system is capable of tissue and cell destruction and is therefore a major element of the defense system against invasion by foreign tissue. However, control of this system is necessary in order to prevent destruction of autologous cells. A large number of proteins. which are involved in control of the complement cascade have been described.
Most relevant to the present invention is the group which controls the C3 convertase stage of the cascade and binds to fragments of either C3 or C4 or both. This group includes serum proteins such as C4-binding protein and factor H and membrane proteins such as C3b receptor, C3d/Epstein-Barr virus receptor, decay-accelerating factor (DAF), and the protein of the invention, membrane cofactor protein (MCP)(Sequence ID No. 2). Reviews of these various factors and their role in complement cascade regulation can be found in Holers, B. M., et al.,
Immunol Today
(1985) 6:188; Ross, G. D., et al.,
Adv Immunol
(1985) 37:217; Atkinson, J. P., et al.,
Immunol Today
(1987) 8:212.
Much is known concerning these regulatory proteins, except for MCP. They are each composed of multiple repeats of an approximately 60-amino acid consensus sequence composed of conserved cys, pro, gly, trp, leu/ile/val, and tyr/phe residues (Reid, K., et al.,
Immunol Today
(1986) 7:230). The genes encoding these proteins have been localized to the long arm of human chromosome 1, band lq32 and form a multigene family designated the regulator of complement activation (RCA) gene cluster. As will be shown below, MCP is also a member of this family.
A member of this family particularly related to the MCP (Sequence ID No. 2) of the invention is the decay-accelerating factor (DAF) which was identified on human platelets by Yu, G.H., et al.,
J Clin Invest
(1986) 78:494-501. DAF is present on virtually all peripheral blood cells, including erythrocytes, granulocytes, T and B lymphocytes, monocytes, and platelets; in addition, soluble forms of DAF have been found in extracellular fluids and tissue culture supernatants. The gene encoding DAF has been cloned and sequenced (by Medof, M. E., et al.,
Proc Natl Acad Sci USA
(1987) 84:2007-2011; and by Caras, I. W., et al.,
Science
(1987) 238:1280-1283). It has been shown that the membrane and soluble secreted forms of DAF result from differential splicing of the mRNA encoding these proteins with the soluble form having a longer C-terminus, but a C-terminus which lacks the membrane binding region associated with the membrane DAF, as described in PCT application W089/01041.
MCP was initially identified by iC3/C3b affinity chromatography on surface-labeled peripheral blood cells and designated gp45-70 to describe the range of M
r
obtained on SDS-PAGE (Cole, J. L., et al.,
Proc Natl Acad Sci USA
(1985) 82:859). MCP was partially purified from the human mononuclear cell lines and shown to have a cofactor activity but no decay accelerating function (Seya, T. J., et al.,
J Exp Med
(1986) 163:837). MCP is absent from erythrocytes, but present as a membrane-bound protein on human T and B lymphocytes, granulocytes, monocytes, platelets, endothelial cells, epithelial cells, and fibroblasts; on most of these cells it occurs in polymorphic forms of molecular weight 63 kd and 58 kd, as determined by SDS-PAGE. These appear to result from a two allelic system encoding MCP (Ballard, L., et al.,
J Immunol
(1987) 138:3850-3855). The MCP found by immunoprecipitation on the membranes of granulocytes appears, however, not to exhibit this polymorphism (Seya, T., et al.,
Eur J Immunol
(1988) 18:1289-1294). The occurrence of MCP on a wide range of host cells is consistent with a role in protecting host cells from damage by complement (Seya, T. L., et al.,
Complement
(1987) 4:225).
The previously purified MCP has been utilized to prepare a polyclonal rabbit antiserum monospecific for this protein. The antisera were raised in rats by repetitive injections of MCP purified as described by Seya, T., et al.,
J Exp Med
(1986) (supra), in complete Freund's adjuvant. These antisera have been used to identify MCP in extracts from various membranes.
The present invention provides a more highly purified form of this protein and the capacity to produce it recombinantly, thus providing practical quantities for therapeutic use.
DISCLOSURE OF THE INVENTION
Human membrane cofactor protein (MCP)(Sequence ID No. 2) is a significant protector of host tissue from autologous destruction by the complement system. Practical quantities of this protein and antibodies specifically immunoreactive with it are made available by recombinant production of human MCP.
Accordingly, in one aspect, the invention is directed to purified and isolated human MCP and to human MCP produced recombinantly. In other aspects, the invention is directed to recombinant materials and methods which result in the manufacture of useful quantities of this protein. Also an aspect of the invention are antibodies immunoreactive with the protein which are useful in diagnosis of disorders associated with reduced or elevated amounts of MCP. Further, the invention includes genetic probes useful in detecting polymorphisms of the MCP-encoding gene (Sequence ID No. 1), and in obtaining DNA encoding corresponding MCP in other species.
In still further aspects, the invention is directed to pharmaceutical compositions containing the MCP of the invention and to methods of treating or ameliorating inflammatory and autoimmune conditions mediated by an excess or misdirection of complement activity.
In still another aspect, the invention is directed to methods to diagnose abnormalities in the immune system, specifically the presence or absence of autoimmune disease by assessing the levels of MCP present on peripheral blood cells and to a method to predict the probability of recurrent miscarriage by testing elevated levels of MCP in the placenta.
REFERENCES:
New England Biolabs Catalog(publ. 1986/87 by New England Biolabs, Beverly, MA, USA) p. 60.*
Maniatis et al., Molecular Cloning, A Laboratory Manual (publ. 1982 by Cold Spring Harbor Labs., Cold Spring Harbor, NY, USA) pp. 387-389.
Arnall Golden & Gregory LLP
Marschel Ardin H.
Washington University
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