Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2000-11-29
2002-06-18
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S219000, C435S226000, C435S004000, C435S252300, C435S320100, C536S023200
Reexamination Certificate
active
06406877
ABSTRACT:
FIELD OF THE INVENTION
The invention is directed to purified and isolated TNF-&agr; converting enzyme, the nucleic acids encoding such enzyme, processes for production of recombinant TNF-&agr; convertases, pharmaceutical compositions containing such enzymes, and their use in various assays and therapies.
BACKGROUND OF THE INVENTION
Tumor necrosis factor-&agr; (TNF-&agr;, also known as cachectin) is a mammalian protein capable of inducing a variety of effects on numerous cell types. TNF-&agr; was initially characterized by its ability to cause lysis of tumor cells and is produced by activated cells such as mononuclear phagocytes, T-cells, B-cells, mast cells and NK cells. There are two forms of TNF-&agr;, a type II membrane protein of relative molecular mass 26,000 (26 kD) and a soluble 17 kD form generated from the cell-bound protein by proteolytic cleavage. TNF-&agr; is a principal mediator of the host response to gram-negative bacteria. Lipopolysaccharide (LPS, also called endotoxin), derived from the cell wall of gramnegative bacteria, is a potent stimulator of TNF-&agr; synthesis. Because the deleterious effects which can result from an over-production or an unregulated-production of TNF-&agr; are extremely serious, considerable efforts have been made to control or regulate the serum level of TNF-&agr;. An important part in the effort to effectively control serum TNF-&agr; levels is the understanding of the mechanism of TNF-&agr; biosynthesis.
The mechanism by which TNF-&agr; is secreted has not previously been elucidated. Kriegler et al.
Cell
, 53:45 (1988) conjectuued that TNF-&agr; “secretion” is due to the converting of the 26 kD membrane-bound molecule by a then unknown proteolytic enzyme or protease. Scuderi et. al.,
J. Immunology
, 143:168 (1989), suggested that the release of TNF-&agr; from human leukocyte cells is dependent on one or more serine proteases, e.g., a leukocyte elastase or trypsin. A serine protease inhibitor, p-toluenesulfonyl-L-arginine methyl ester, was found to suppress human leukocyte TNF-&agr; release in a concentration-dependent manner. Scuderi et al. suggested that an arginine methyl ester competes for the arginine-binding site in the enzyme's reactive center and thereby blocks hydrolysis. The lysine and phenylalanine analogs of the inhibitor reportedly failed to mimic the arginine methyl ester. However, it was never shown that this compound acted by inhibiting a protease that cleaves the 26 kD TNF. More recently, it has been reported that metalloprotease inhibitors block the release of TNF from THP-1 cells See Mohler et al.,
Nature
370:218 (1994); Gearing et al.,
Nature
, 370:555 (1994); and McGeehan et al.,
Nature
, 370:568 (1994).
Most, but not all, proteases recognize a specific amino acid sequence. Some proteases primarily recognize residues located N-terminal of the cleaved bond, some recognize residues located C-terminal of the cleaved bond, and some proteases recognize residues on both sides of the cleaved bond. Metalloprotease enzymes utilize a bound metal ion, generally Zn
2+
, to catalyze the hydrolysis of the peptide bond. Metalloproteases are implicated in joint destruction (the matrix metalloproteases), blood pressure regulation (angiotensin converting enzyme), and regulation of peptide-hormone levels (neutral endopeptidase-24.11).
SUMMARY OF THE INVENTION
The invention pertains to biologically active TNF-&agr; converting enzyme (“TACE”) as an isolated and purified polypeptide. In addition, the invention is directed to isolated nucleic acids encoding TACE and to expression vectors comprising a cDNA encoding TACE. Within the scope of this invention are host cells that have been transfected or transformed with expression vectors that comprise a cDNA encoding TACE, and processes for producing TACE by culturing such host cells under conditions conducive to expression of TACE. By virtue of the purification of TACE, antibodies, and in particular, monoclonal antibodies against TACE are an aspect of the invention. In addition, assays utilizing TACE to screen for potential inhibitors thereof, and methods of using TACE as a therapeutic agent for the treatment of diseases mediated by cell-bound TNF-&agr; or other molecules are encompassed by the invention. Further, methods of using TACE in the design of inhibitors thereof are also an aspect of the invention.
The isolated and purified metalloprotease of the invention is capable of converting TNF-&agr; from the 26 kd membrane-bound form to the 17 kD form, and which has a molecular weight of between approximately 66 kD and approximately 97 kD. The cDNA sequence of TACE is shown in SEQ ID NO:1. The isolated and purified TNF-&agr; converting enzyme (“TACE”) comprises amino acids 18-824 of SEQ ID NO:2.
Inhibition of the TACE inhibits release of TNF-&agr; into the serum and other extracellular spaces. TACE inhibitors would therefore have clinical utility in treating conditions characterized by over-production or upregulated production of TNF-&agr;. A particularly useful TACE inhibitor for certain pathological conditions would selectively inhibit TACE while not affecting TNF-&bgr; (also known as lymphotoxin) serum levels. The over-production or unregulated production of TNF-&agr; has been implicated in certain conditions and diseases, for example, Systemic Inflammatory Response Syndrome, reperfusion injury, cardiovascular disease, infectious disease such as HIV infection and HIV neuropathy, obstetrical or gynecological disorders, inflammatory disease/auto-immunity, allergic/atopic diseases, malignancy, transplants including organ transplant rejection or graft-versus-host disease, cachexia, congenital, dermatologic, neurologic, renal, toxicity, and metabolic/idiopathic diseases.
Inhibitors of TACE would prevent the cleavage of cell-bound TNF-&agr; thereby reducing the level of TNF-&agr; in serum and tissues. The present invention encompasses such an embodiment and comprises a method of inhibiting the cleavage of TNF-&agr; from cell membranes in a mammal comprising administering to such mammal an effective amount of a compound that inhibits the TNF-&agr; proteolytic activity of an enzyme comprising the sequence of amino acids from 18 to 671 through 824 of SEQ ID NO:2. In addition, the invention comprises a method for treating a mammal having a disease characterized by an overproduction or an upregulated production of TNF-&agr;, comprising administering to the mammal a composition comprising an effective amount of a compound that inhibits the TNF-&agr; proteolytic activity of an enzyme comprising the sequence of amino acids 18-824 of SEQ ID NO:2. Such inhibitors would be of significant clinical utility and could be potential therapeutics for treating the above-listed TNF-&agr;-related disorders. Isolation and purification of TACE would provide a significant advancement in the effort to develop inhibitors of such enzyme, and the treatment of TNF-associated diseases, and indeed, could lead to use of TACE itself as a therapeutic agent for certain physiological disorders. For example, in addition to TNF-&agr;, other cytokines as well as cytokine receptors and several adhesion proteins may be released from the cell surface by TACE or related proteases. TACE may be administered to modulate or remove cell surface cytokines, cytokine receptors and adhesion proteins involved in tumor cell growth, inflammation, or fertilization.
DETAILED DESCRIPTION OF THE INVENTION
A cDNA encoding human TNF-&agr; converting enzyme (“TACE”) has been isolated and is disclosed in SEQ ID NO:1. This discovery of the cDNA encoding human TACE enables construction of expression vectors comprising nucleic acid sequences encoding TACE; host cells transfected or transformed with the expression vectors; biologically active human TACE as isolated and purified proteins; and antibodies immunoreactive with TACE.
Isolated and purified TACE polypeptides according to the invention are useful for detecting the TACE-inhibiting activity of a molecule. In such a method involving routine and conventional techniques, a molecule of unk
Black Roy A.
Cerretti Douglas P.
March Carl J.
Rauch Charles
Achutamurthy Ponnathapu
Immunex Corporation
Pak Yong
Sprunger Suzanne A.
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