Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues
Reexamination Certificate
1999-08-20
2002-04-02
Huff, Sheela (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
C530S324000, C530S350000, C530S380000, C530S385000, C530S386000, C530S387100, C530S387200, C530S387900, C530S388100, C530S388150, C530S388200, C530S388250, C530S388700, C424S009100, C424S130100, C424S131100, C424S133100, C424S134100, C514S001000, C514S002600, C435S004000, C435S007100, C435S041000, C435S069100
Reexamination Certificate
active
06365712
ABSTRACT:
BACKGROUND OF THE INVENTION
Ligation of the T-cell receptor initiates a cascade of intracellular signaling events resulting in the proliferation and differentiation of the activated cell. Many of the phenotypic changes which define T-cell activation result from new gene transcription (Ullman, et al.,
Annu. Rev. Immunol.
8:421-452 (1990)). Activation-induced changes in cell surface proteins resulting from a primary stimulus play a particularly important role in regulating downstream proliferative and differentiative responses. Events mediated at the cell surface include binding of soluble factors and interactions with other cells and extracellular matrix. In vivo, activated T cells play an instrumental role in the propagation of immunologically mediated inflammation (Brezinschek, et al.,
J. Immunol.,
154:3062-77 (1995)).
The development and progression of inflammation is dependent upon the infiltration of leukocytes into the affected tissues. The accumulation of leukocytes into tissues involves receptor-mediated interactions with the endothelial cell lining of postcapillary venules, extravasation, and migration toward and localization within the inflammatory site (Shimizu, et al.,
FASEB J.
5:2292-2299 (1992)). A large body of work has shown that the combinatorial use of multiple adhesion and chemoattractant receptors appears to regulate selection of subclasses of leukocytes emigrating at inflammatory sites as well as the distinctive recirculation behavior of lymphocyte subsets (Springer,
Cell
76:301-314 (1994)). Little is known about the range of receptor-ligand interactions in leukocytes that regulate their localization within the tissue microenvironment following extravasation.
Once at the site of inflammation, immune cells undergo additional phenotypic changes that contribute to eliminating the foreign antigen and to amplifying the inflammatory response. Various soluble mediators of inflammation such as prostaglandins, leukotrienes, complement fragments, platelet-activating factors, chemokines, and formyl peptides, among others (Murphy,
Annu. Rev. Immunol.
12:593-633 (1994)) bind to specific receptors that are part of a very large and diverse class of receptors that span the membrane seven times (7TM receptors). 7TM receptors, also called G protein coupled receptors, transduce signals following ligand binding via their association with heterotrimeric G proteins (Martens, PROGRESS IN BRAIN RESEARCH, Joose, et al., (eds.), pp. 201-214 (1992)). Receptor coupled G protein activation in turn regulates a variety of enzymes (such as adenyl cyclase, phospholipase Cb, phosphoinositide 3-kinase), ion channels and transporters (Neer,
Cell
80:249-257 (1995)).
The family of 7TM receptors is probably the largest receptor family known, a with hundreds of receptors cloned to date. The receptors bind a wide structural array of ligands including various types of hormones, neurotransmitters, lipids, peptides, and odorants (Spiegel, G PROTEINS, Spiegel., et al., (eds.), R. G. Landes Co., Austin. pp. 6-17 (1994)). The defining feature and the areas of greatest homology among the 7TM receptors are in the seven transmembrane regions (Probst, et al.,
DNA and Cell Biol.
11:1-20 (1992)). Some residues are found in virtually all 7TM receptors and may mediate evolutionarily-conserved tertiary structural requirements for functional activity. Other residues are conserved among subfamilies that bind similar ligands and have been shown to contribute to ligand binding and/or specificity (Savarese & Fraser,
Biochem. J.
283:1-19 (1992)). In the case of the glucagon/calcitonin receptor subfamily, relatedness based on sequence identity is apparent despite the diversity of the peptides that bind to these receptors (Attwood & Findlay,
Protein Eng.
7:195-203 (1994)).
The structural features required for ligand binding and receptor activation have been investigated and found to vary according to ligand and receptor subfamily (Coughlin,
Curr. Opin. in Cell Biol.
6:191-197 (1994)). Many small ligands such as 11-cis-retinal, serotonin, and acetylcholine, bind within the cavity formed by the receptors' transmembrane domains (Baldwin,
Curr. Opin. in Cell Biol.
6:180-190 (1994); Dohlman, et al.,
Annu. Rev. Biochem.
283:1-19 (1992); and Savarese & Fraser,
Biochem. J.
283:1-19 (1992)). Other ligands such as peptides and glycoprotein hormones require amino-terminal exodomains and most likely some portion of the extracellular loops for binding, but signaling requires the seven membrane spans (Holtmann, et al.,
J. Biol. Chem.
270:14394-14398 (1995) and Nagayama, et al.,
Proc. Nat'l Acad. Sci. (USA)
88:902-905 (1991)). A remarkable signaling mechanism has been described for the thrombin receptor in which thrombin cleaves its receptor's amino-terminal extension to create a new receptor amino terminus that functions as a tethered ligand and activates the receptor through interactions with the interhelical pocket (Vu, et al.,
Cell
64:1057-1068 (1991)).
Hamann, et al. (
J. Immunol.
155:1942-1950 (1995)) report the isolation of a glycoprotein designated CD97. Seven hydrophobic segments within CD97 suggest that this glycoprotein is a 7TM molecule. CD97 is induced on the surface of most leukocytes upon activation. In its mature form, Hamann, et al. indicate that CD97 is a single chain glycoprotein of 722 amino acids in length with a molecular weight of 75 to 85 kDa.
SUMMARY OF THE INVENTION
The present invention relates to the previously unrecognized a subunit of CD97. The &agr; subunit binds to the &bgr; subunit of CD97 to form an &agr;&bgr; heterodimer. The &agr; subunit is localized extracellularly on T-cells. Upon activation, expression of the &agr; subunit is dramatically increased and shed into the external medium. The &agr; subunit plays a role in angiogenesis, inflammation, and atherosclerosis. Detection and inhibition of &agr; subunit expression provides diagnostic and therapeutic methods for these disease states.
In one aspect, the present invention relates to an isolated protein comprising a soluble CD97 &agr; subunit. The soluble CD97 &agr; subunit is selected from the group consisting of &agr;1, &agr;2, and &agr;3. The &agr;1, &agr;2 and &agr;3 subunits are related in that they all originate as a proprotein with the &bgr; subunit (
FIG. 1
) and are processed in the endoplasmic reticulum or early golgi to a specific &agr; subunit with an identifying number of EGF repeats and a non-covalently linked &bgr; subunit.
The &agr;3 subunit has a molecular weight of about 45 kDa in non-glycosylated form, has an EGF-like repeat selected from the group consisting of EGF-1 (SEQ ID NO:1), EGF-2 (SEQ ID NO:2), and EGF-5 (SEQ ID NO:5), and is immunologically cross-reactive to an antibody that is specifically reactive to the protein of SEQ ID NO:6. The &agr;2 subunit has a molecular weight of about 50 kDa in non-glycosylated form, has an EGF-like repeat selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:5, and is immunologically cross-reactive to an antibody that is specifically reactive to the protein of SEQ ID NO:6. The &agr;1 subunit has a molecular weight of about 55 kDa in non-glycosylated form, has an EGF-like repeat selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:5, and is immunologically cross-reactive to an antibody that is specifically reactive to the protein of SEQ ID NO:6. In some embodiments the &agr;1 subunit further comprises an EGF-like repeat selected from the group consisting of EGF-3 (SEQ ID NO:3), and EGF-4 (SEQ ID NO:4). In other embodiments the &agr;2 subunit further comprises EGF-like repeat SEQ ID NO:3. Conveniently, the isolated protein is recombinantly produced.
In another aspect, the present invention relates to an isolated nucleic acid encoding a soluble CD97 &agr; subunit protein. The CD97 &agr; subunit protein is selected from the group consisting of &agr;1, &agr;2, and &agr;3. The &agr;3 subunit has a molecular weight of about 45 kDa in non-glycosylated form, has an EGF-like repeat selected from the gro
Harris Alana M.
Huff Sheela
The United States of America as represented by the Secretary of
Townsend and Townsend / and Crew LLP
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