Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1998-09-17
2001-11-20
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S004000, C435S212000, C435S219000, C435S226000
Reexamination Certificate
active
06319681
ABSTRACT:
TECHNICAL FIELD
The present invention relates to mammalian tumor necrosis factor-&agr; (TNF-&agr;) convertase enzymes. More particularly, it relates to bovine, human and other TNF-&agr; convertases, isolated nucleic acids and recombinant vectors encoding the enzymes, methods for making the enzymes, fragments or fusion proteins thereof using recombinant DNA methodology or chemical synthesis, and to methods for using the enzymes in screening systems to identify TNF-&agr; convertase inhibitors for the treatment of various diseases, and nucleic acids encoding a TNF-&agr; convertase. This invention further relates to antibodies, both polyclonal and monoclonal, which specifically bind to the TNF-&agr; convertases, and to fragments and fusion proteins of the TNF-&agr; convertases of the invention.
BACKGROUND OF THE INVENTION
TNF-&agr;, also known as cachectin, is a 17 kDa (kilodalton) protein produced by cells of the monocyte/macrophage lineage, and by other cells. A variety of biological effects, both beneficial and deleterious, have been attributed to TNF-&agr;. TNF-&agr; is beneficial, e.g., in that it is believed to be a part of host anti-tumor defenses. It also produces detrimental effects, however, including, e.g., cardiovascular (shock, ARDS, capillary leakage syndrome), renal (nephritis, acute tubal necrosis), and gastrointestinal (ischemia, colitis, hepatic necrosis) effects, and effects on the central nervous system (fever, anorexia, altered pituitary hormone secretion). In view of the foregoing, a consensus view has developed that TNF-&agr; is a key mediator of inflammation (including inflammatory diseases such as arthritis) and mammalian responses to injury, invasion by pathogens, and neoplasia.
The biosynthesis of human TNF-&agr; proceeds by way of a membrane-bound precursor containing 233 amino acid residues [Wang et al.,
Science
228:149-154 (1985); Muller et al.,
Nature
335:265-267 (1987)], which is processed during cellular activation by cleavage of a 76-residue peptide to produce the mature, secreted form of TNF-&agr;. The enzyme(s) responsible for this cleavage, called TNF-&agr; convertase, has until the present invention been elusive for most mammalian species.
A putative TNF-&agr; convertase, called PR-3, has been isolated and cloned from human neutrophils, and it has been suggested that this enzyme can be used in screens to identify TNF-&agr; convertase inhibitors. See International Patent Applications Publication Numbers WO 94/00555 and WO 95/24501. This enzyme, however, is not believed to be the physiologically relevant human TNF-&agr; convertase because it is a serine protease, whereas the relevant enzyme is believed to be a metalloproteinase. Moreover, the source of the serine protease, neutrophils, is not believed to be important in the production of TNF-&agr;, and the serine protease does not cleave the precursor form of TNF-&agr; (proTNF-&agr;) at the point expected for the physiologically relevant human enzyme.
Mohler et al. [Nature 70:218 (1994)] have partially purified another TNF-&agr; convertase from the human monocytic cell line THP-1. This preparation, however, was very impure, and little could be said about the nature of the TNF-&agr; convertase in the crude protein mixture of Mohler et al.
In view of the important role of TNF-&agr; in many disease processes, there is a need for agents that can selectively block the biosynthesis of mature, secreted TNF-&agr;. The search for such agents would be greatly facilitated by the availability of substantially pure mammalian TNF-&agr; convertases.
SUMMARY OF THE INVENTION
The present invention fills the foregoing need by providing materials and methods for identifying specific inhibitors of TNF-&agr; convertase. More particularly, this invention provides substantially pure mammalian TNF-&agr; convertases capable of converting proTNF-&agr; to the mature, secreted form. This invention further provides isolated or recombinant nucleic acids encoding mammalian TNF-&agr; convertases, and recombinant vectors and host cells comprising such nucleic acids.
This invention further provides a method for making a mammalian TNF-&agr; convertase, comprising culturing a host cell comprising a nucleic acid encoding a mammalian TNF-&agr; convertase under conditions in which the nucleic acid is expressed. In some embodiments, the method further comprises isolation of the TNF-&agr; convertase from the culture.
This invention also provides polypeptides comprising a fragment of a TNF-&agr; convertase having an amino acid sequence corresponding to the sequence of at least about 8 contiguous residues of the complete enzyme sequence. Preferably, the polypeptides comprise at least about 12, more preferably at least about 20, and most preferably at least about 30 such residues.
Still further, this invention provides fusion proteins comprising a TNF-&agr; convertase or a polypeptide thereof covalently linked to a fusion partner.
The present invention also provides antibodies, both polyclonal and monoclonal, that specifically bind to one or more of the TNF-&agr; convertases or to a polypeptide thereof. Also provided are anti-idiotypic antibodies, both monoclonal and polyclonal, which specifically bind to the foregoing antibodies.
This invention still further provides a method of treatment comprising administering to a mammal afflicted with a medical condition caused or mediated by TNF-&agr;, an effective amount of an antibody, or an antigen-binding fragment thereof, that specifically binds to a mammalian TNF-&agr; convertase, and pharmaceutical compositions comprising such antibodies or fragments and pharmaceutically acceptable carriers.
The present invention also provides a method for identifying an inhibitor of a mammalian TNF-&agr; convertase, comprising:
(a) contacting a mammalian TNF-&agr; convertase in the presence of substrate with a sample to be tested for the presence of an inhibitor of the convertase; and
(b) measuring the rate of cleavage of the substrate;
whereby an inhibitor of the TNF-&agr; convertase in the sample is identified by measuring substantially reduced cleavage of the substrate, compared to what would be measured in the absence of such inhibitor.
In a preferred embodiment, the contacting of the convertase with the sample in the presence of substrate occurs on the surface of a mammalian host cell comprising one or more nucleic acids encoding a mammalian TNF-&agr; convertase and a substrate of the convertase.
REFERENCES:
patent: WO 96/41624 (1996-12-01), None
patent: WO 95/24501 (1997-10-01), None
patent: 91/02540 (1991-03-01), None
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Dalie Barbara
Fan Xuedong
Lundell Daniel
Lunn Charles A.
Tan Jimmy C.
Achutamurthy Ponnathapu
Schering Corporation
Thampoe Immac J.
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