Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
2000-04-07
2003-04-01
Spector, Lorraine (Department: 1646)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C536S023500, C530S350000, C435S069100
Reexamination Certificate
active
06541232
ABSTRACT:
TECHNICAL FIELD
The invention is generally directed toward signal transduction pathways associated with inflammation, and more particularly toward IL-1/TNF-&agr;-activated kinases (ITAK).
BACKGROUND OF THE INVENTION
Interleukin-1 (IL-1) and tumor necrosis factor-&agr; (TNF-&agr;) are two cytokines produced systemically and locally in response to infection, injury or immunological challenge. Based upon studies in which the action of one (or the other) cytokine has been specifically blockaded, or in which purified cytokines have been administered, IL-1 and TNF-&agr; have been implicated in a number of disease processes. For example, IL-1 has been implicated in inflammatory diseases including rheumatoid arthritis and other degenerative joint diseases, inflammatory bowel disease, type I diabetes, psoriasis, Alzheimer's disease, and allergy. Overproduction of TNF-&agr; has likewise been implicated in diseases such as reperfusion injury, rheumatoid arthritis, cardiovascular disease, infectious disease such as HIV infection and HIV-induced neuropathy, allergic/atopic diseases, inflammatory disease/autoimmunity, malignancy, transplant difficulties including organ transplant rejection or graft-versus-host disease, cachexia, and congenital, dermatologic, neurologic, renal, toxicity and metabolic/idiopathic diseases. A particular case where the two cytokines are thought to act synergistically is in the induction of the Systemic Inflammatory Response Syndrome.
Because the consequences of uncontrolled production of IL-1 and TNF-&agr; can be severe, considerable effort has been expended on therapies that would limit the production or activity of one, or preferably both, of the cytokines. The prevailing therapy has been to administer proteins that bind specifically to the circulating cytokines, thus preventing them from interacting with their cellular receptors. Typically these protein-based therapeutics are antibodies or ‘soluble’ receptors (i.e., recombinant versions of the natural cellular receptors which lack transmembrane and signaling domains). An additional protein-based therapeutic is the IL-1 receptor antagonist protein (IL-1ra), which competes for binding to the same cellular receptors as the agonist forms of IL-1, but does not elicit a cellular signal.
The effectiveness of all three types of protein-based therapy is limited because occupation of even a very small number of IL-1 or TNF-&agr; receptors by IL-1 or TNF-&agr; generates a cellular response (and therefore the harmfull effects described above). It is therefore necessary to maintain relatively high levels of anti-cytokine antibody, soluble receptor or antagonist protein in order to drive the equilibrium in favor of complex formation (i.e., to effectively prevent binding of IL-1 or TNF-&agr; to their respective receptors). Another drawback to such protein-based therapeutics is that each therapeutic is selective for only one of the two cytokines. Thus, large doses of a multitude of therapeutics must be administered to a patient in order to attempt to control IL-1 and TNF-&agr; production.
Although the biological effects of TNF-&agr; and IL-1 are quite similar, the structures of the cytokines, and the structure of their receptors, are very different. IL-1 and TNF-&agr; appear to have overlapping biological activities because the binding of each cytokine to its receptor appears to affect similar post-receptor signal transduction pathways. Many details of these pathways are unclear.
For example, although both cytokines activate the transcription factors NF-&kgr;B and AP-1, which leads to the regulated transcription of a wide variety of genes, the particular receptor-proximal effector molecules that regulate this process is unclear. Additionally, both cytokines have been reported to cause the activation of sphingomyelinases and phospholipases that generate, respectively, ceramide and arachidonic acid. Both cytokines also activate members of the mitogen-activated protein kinase (MAPK) family including ERK1, ERK2, and the stress-activated kinases JNK-1 and p38. This family of kinases is activated, to varying extent, by a wide range of hormones, growth factors, heavy metals, protein synthetic inhibitors and ultraviolet light and therefore activation of such kinases cannot be considered unique to the IL-1/TNF-&agr; signal transduction pathway.
In addition to -items activated by both IL-1 and TNF-&agr;, IL-1 has been reported to specifically activate the IL-1 receptor associated kinase, IRAK, (Cao, Henzel and Gao,
Science
271:1128 (1996)). The cytoplasmic domains of TNF receptors have also been reported to interact with other signal transduction molecules such as TRAF1 and TRAF2, FADD, MORT and TRADD. Such TNF-&agr; receptor-interacting proteins also appear capable of interacting with an extended receptor family, including those that mediate quite distinct cellular responses such as the T- and B-cell activator CD40 and a mediator of apoptosis, fas. (Tewari and Dixit,
Curr. Opin. Genet. Dev
. 6:39, 1996; Lee et al.,
J. Exp. Med
. 183:669, 1996).
While certain cellular responses may be elicited by IL-1, TNF-&agr;, or other mediators, the only known signaling event that appears to be uniquely induced by IL-1 or TNF-&agr;, but no other defined stimulus, is a protein serine/threonine kinase activity that could be detected in vitro by its ability to phosphorylate &bgr;-casein. Guesdon et al.,
J. Biol. Chem
. 268:4236 (1993);
Biochem. J
. 304:761 (1994). This &bgr;-casein kinase activity was induced in fibroblasts and other connective-tissue derived cells by IL-1 and TNF-&agr; but not by 21 other agents tested. The structure of the &bgr;-casein kinase was not elucidated in this report.
However, there has gone unmet a need for substances and/or methods that provide either repression or stimulation of intracellular effects of both IL-1 and TNF-&agr;. There has also gone unmet a need for substances and methods that provide interaction(s) with the post-receptor pathway(s) of IL-1 and TNF-&agr;, as well as substances and methods that provide opportunities to detect agonists and/or antagonists to IL-1 or TNF-&agr;, including single compounds that act as an agonist or antagonist to both IL-1 and TNF-&agr;. The present invention provides these and other related advantages.
SUMMARY OF THE INVENTION
The present invention provides nucleic acid and amino acid sequences of protein kinases, preferably human, that interact with at least one post-receptor intracellular pathway of both IL-1 and TNF-&agr;. Such kinases are referred to herein as IL-1/TNF-&agr;-activated kinase (ITAK). Such kinases are induced as enzymatically active kinases capable of phosphorylating specific substrate proteins by treatment of suitable cells with IL-1 or TNF-&agr;. The present invention further provides compositions and methods for the isolation and purification of nucleic acid molecules encoding ITAK. Also disclosed herein are methods for expressing and purifying ITAK, as well as specific assays for the detection of inhibitors or stimulators of ITAK activity, which would have utility as antagonists or agonists of IL-1 and TNF-&agr;.
In addition, the present invention is directed to isolated nucleic acids encoding ITAK and to vectors, including expression vectors, capable of expressing ITAK, preferably from a cDNA encoding ITAK. The present invention includes host cells containing such expression vectors, and processes for producing ITAK by culturing such host cells under conditions conducive to expression of ITAK, and preferably the purification of ITAK, including in industrial quantities. In part due to such purification of ITAK, the invention is also directed to antibodies, preferably monoclonal antibodies, specific for ITAK.
The present invention is also directed to assays utilizing ITAK to screen for potential inhibitors or stimulators of ITAK activity, for example as a means of blocking a signal transduced in response to IL-1 or TNF-&agr;. Further, methods of using ITAK in the design of inhibitors of ITAK activity are also disclosed.
In particular, in one as
Anderson Dirk M.
Bird Timothy A.
Sims John E.
Virca G. Duke
Immunex Corporation
Lazar-Wesley Elaine
Smith Julie K.
Spector Lorraine
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