Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-09-23
2001-12-18
Zitomer, Stephanie (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S287200, C536S023100, C536S023520, C536S024300, C536S024310
Reexamination Certificate
active
06331396
ABSTRACT:
BACKGROUND OF THE INVENTION
Interferons (IFNS) are a family of related cytokines that mediate a range of diverse functions including antiviral, antiproliferative, antitumor and immunomodulatory activities. The pleiotropic activities of IFNs are mediated primarily through the transcriptional regulation of many downstream effector genes. IFNs bind to their cognate receptors and initiate a signaling cascade, involving the JAK-family of tyrosine kinases and STAT-family of transcription factors, that leads to the transcriptional induction of a number of IFN-stimulated genes (ISGs). IFN actions are largely mediated by the proteins encoded by ISGs, the best studied of which include the dsRNA-activated protein kinase (PKR), the 2′-5′ oligoadenylate (2-5A) synthetases and 2-5A-dependent ribonuclease (RNaseL), and the Mx proteins. The other well-characterized category of ISGs include the STAT and IRF families of transcription factors which are involved in the regulation of both ISG and IFN gene expression. However, the biological functions for many ISGs, including 6-16, 9-27 and the ISG-54 gene family, remain unclear despite, in some cases, extensive investigation of the 5′ regulatory regions of those genes.
ISGs exhibit unique patterns of transcriptional inducibility by different types of IFNs. Type I IFNs, representing the IFN-alpha (&agr;), -beta (&bgr;), -omega (&ohgr;), and -tau (&tgr;) genes, are grouped together since they bind to common Type I IFN receptor, while IFN-gamma (&ggr;) is designated as Type II IFN since it is the unique ligand for the distinct Type II IFN receptor. In general, engagement of the Type I IFN receptor results in activation of the multimeric transcription factor, ISGF3, and leads to the transcriptional initiation of gene promoters containing IFN-Stimulated Response Elements (ISRE). Type II IFN receptor stimulation typically activates STAT1 homodimers which can regulate transcription from gene promoters containing IFN-&ggr; Activated Sites (GAS). In accordance with this model, the differential inducibility of certain ISGs by Type I, Type II IFN, or by both, can be attributed to the presence of canonical ISRE or GAS elements.
Despite their common utilization of the same receptor complex, distinct Type I IFN proteins can mediate different biological responses. Numerous studies, using recombinant or purified natural, IFN-&agr; or IFN-&bgr; proteins, have shown that individual Type I IFNs can be distinguished by their unique profiles of specific biological activities including antiviral and antiproliferative finctions, natural killer cell stimulation, and receptor binding affinities. While the molecular mechanisms underlying these differences are not yet known, a likely possibility involves the differential expression of ISGs by the various Type I IFNs. The best documented example of this concept involves betaRI (&bgr;RI), a chemokine gene whose expression is preferentially inducible by IFN-&bgr; but not by IFN-&ggr; nor several IFN-&agr; subtypes.
Considerable efforts have been devoted to exploit the therapeutic potential of IFNs. Currently both IFN-&agr; and IFN-&bgr; are used to treat patients with serious diseases. Presently, IFN-&agr; is used in the treatment of chronic myelogenous leukemia (CML), hairy cell leukemia, renal cell carcinoma, melanoma, hepatitis C virus infections, Kaposi sarcoma and hepatitis B infections. IFN-&bgr; is used for the treatment of multiple sclerosis. Unfortunately, patients who have been treated with the currently available forms of IFN-&agr; and IFN-&bgr;, which include purified naturally occurring IFNs and recombinant IFNs, have experienced serious side-effect including including fever, malaise, myalgia, and depression.
Accordingly, it is desirable to have new methods and tools for identifying new therapeutic agents (hereinafter referred to as “IFN mimics”) which upregulate the ISGs that cause desirable effects in patients but which do not upregulate the ISGs that cause the detrimental side-effects associated with treating patients with IFNs. It is also desirable to have methods and tools which can be used to identify agents that selectively inhibit upregulation of the ISGs that cause the detrimental side-effects which result from treatment with IFNs, particularly IFN-&agr; and IFN-&bgr;. It is also desirable to have a method for establishing patient sensitivity to interferon before beginning treatment protocols. It is also desirable to have methods and tools for monitoring the efficacy of treatment of patients with the currently used interferons and with any new interferon-like agents which are being developed. It is also desirable to have new treatment protocols which can make interferon therapy for cancer more effective. It is also desirable to have new methods and tools for identifying the biochemical pathways by which IFNs mediate their therapeutic effects.
SUMMARY OF THE INVENTION
The present invention provides novel methods and model systems for identifying and characterizing new therapeutic agents, particularly proteins, which mimic the activity of all interferons, Type I interferons, IFN-&agr;, IFN &bgr;, IFN-&tgr;, IFN-&ohgr;, and IFN-&ggr;,. The method comprises administering an interferon selected from the group consisting of IFN-&agr;, IFN &bgr;, IFN-&tgr;, IFN-&ohgr;, IFN-&ggr;, and combinations thereof to cultured cells, administering the candidate agent to a duplicate culture of cells; and measuring the effect of the candidate agent and the interferon on the transcription or translation of one or, preferably, a plurality of the interferon stimulated genes or the interferon repressed genes (hereinafter referred to as “ISG's” and “IRGs”, respectively). The model system is an array with gene probes that hybridize with from about 100 to about 5000 ISG and IRG transcripts.
In another aspect the present invention provides new methods for identifying and characterizing new therapeutic agents which block the activity of all interferons, Type I interferons, select Type I interferons, or IFN-&ggr;. The method comprises administering an interferon selected from the group consisting of IFN-&agr;, IFN &bgr;, IFN-&tgr;, IFN-&ohgr;, IFN-&ggr;, and combinations thereof to cultured cells, administering the interferon and the candidate agent to a duplicate cell culture; and assaying for changes in the levels of transcription of one or, preferably, a plurality of ISGs and IRGs, particularly those new ISGs and IRGs which are listed in Table 2 and Table 3, respectively.
In another aspect the present invention provides new methods for identifying and characterizing new agents which inhibit the transcription or translation of a new ISG that has been uncovered by the present study. The method comprises administering the interferon which has been shown to stimulate transcription of the new ISG to a set of cultured cells, administering the interferon and the candidate inhibitor to a duplicate set of cultured cells; and measuring the levels of mRNA or the levels of protein or factor encoded by the ISG gene in the two sets of cultured cells. This method is especially useful for identifying inhibitors for those ISGs whose transcription is increased by 2-fold or greater by treatment with the interferon.
In another aspect, the present invention provides a method of determining the sensitivity of a patient to prospective interferon therapy. The method comprises exposing cell samples obtained from a patient who is a candidate for interferon therapy to the interferon and measuring the levels of one or, preferably, a plurality of the ISG and IRG transcripts that are produced in response to the exposure. An increase in the levels of ISG transcripts and a decrease in the level of IRG transcripts in the interferon treated cells relative to the levels found in untreated control cells indicates that the patient is responsive to treatment with the interferon. A finding of no or little difference in the levels of ISGs and IRGs transcripts in the interferon-treated cells and control cells indicates that the patient is not respo
Der Sandy
Silverman Robert H.
Williams Bryan R. G.
Calfee Halter & Griswold LLP
Forman B J
The Cleveland Clinic Foundation
Zitomer Stephanie
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