Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-08-20
2002-07-02
Kemmerer, Elizabeth (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S069100, C435S252300, C435S320100, C536S023510, C536S023400
Reexamination Certificate
active
06414118
ABSTRACT:
BACKGROUND OF THE INVENTION
Interferon (IFNs) are a family of cytokines with diverse biological functions, including antitumor, immunomodulatory, antiviral and antiparasitic actions. At present, the IFN family includes more than 20 different proteins. Initially, IFN's were classified by source as leucocyte, fibroblast, or immune IFNs. Initially, IFNs were also grouped into two types with Type 1 comprising fibroblast and leucocyte IFNs and type II comprising immune IFNs. Based on their genetic similarities and differences, the type I IFNs are currently divided into the following three classes: IFN-&agr;, IFN-&bgr;, and IFN-&ohgr;. The type II IFNs are classified as IFN-&ggr;.
IFNs act by binding to specific cell receptors, which are found on the surface of most cells, and causing the translocation to the nucleus of cytoplasmic transcription factors that enhance or suppress the expression of specific genes. The products of these interferon-stimulated genes (hereinafter referred to as ISGs) are primarily polypeptides that act as mediators of the biological activities associated with the respective IFN. IFN-&agr; and IFN-&bgr; bind to the same receptor and activate transcription of ISGs through the assembly and translocation from the cytoplasm to the nucleus of ISGF3, a multisubunit transcription factor that interacts with a cis-acting DNA regulatory element termed the interferon-stimulated response element or ISRE.
Transcription of the ISGs stimulated by IFN-&agr; and IFN-&bgr; occurs in the absence of ongoing protein synthesis, and is therefore mediated directly by the interaction of either IFN-&agr; or IFN-&bgr; with their common receptor.
IFN-&bgr; is a particularly useful cytokine which has been shown to be effective in decreasing the relapse rate, relapse severity, progression of neurological disability and development of new brain lesions, as observed with brain magnetic resonance imaging, in relapsing-remitting multiple sclerosis patients. Unfortunately, IFN-&bgr; can cause side effects in such patients. Moreover, a substantial number of the patients treated with IFN-&bgr; develop neutralizing antibodies. In addition, IFN-&bgr; must be administered by injection. There are also concerns that IFN-&bgr; may not penetrate certain tissue compartments. Accordingly, efforts are currently underway to identify other compounds, particularly small molecular weight molecules, that induce an IFN-&bgr; type response, hereinafter referred to as “IFN-&bgr; mimics”. These efforts are directed at identifying IFN-&bgr; mimics that can be administered orally and that freely permeate tissues. The efforts are also directed at identifying IFN-&bgr; mimics that are less toxic than naturally-occurring or recombinant forms of IFN-&bgr;.
In certain instances, such as autoimmune diseases, IFN-&agr; or IFN-&bgr; can induce a deleterious inflammatory reaction in an individual. Accordingly efforts are also underway to identify compounds, hereinafter referred to as “IFN-&bgr; inhibitors”, that block the IFN-&bgr; type components of this reaction.
The efforts to identify IFN-&bgr; mimics and IFN-&bgr; inhibitors have been hampered by the fact that there are no known ISG products whose synthesis is selectively induced by IFN-&bgr; as compared to IFN-&agr; and thus can be used to give the full measure of an IFN-&bgr; type response. Accordingly, it is desirable to have an ISG product whose synthesis is selectively induced by IFN-&bgr; as compared to IFN-&agr;. It is also desirable to have a system that can be used to identify compounds that selectively induce an IFN-&bgr; type response as compared to an IFN-&agr; type response. An in vitro system that can be used to screen IFN-&bgr; mimics and that can be used to assess the potency of the IFN-&bgr; mimics is especially desirable. It is also desirable to have a system that can be used to screen IFN-&bgr; inhibitors.
SUMMARY OF THE INVENTION
The present invention provides a system for screening IFN-&bgr; mimics or IFN-&bgr; inhibitors. The system comprises a polynucleotide that comprises a DNA sequence encoding a unique chemokine promoter, hereinafter referred to as the “Beta R1 promoter”, operatively linked to a reporter gene that encodes an assayable product.
The present invention also provides a method of employing the system to assay the concentration of IFN-&bgr; in a sample and to screen putative IFN-&bgr; mimics. In one embodiment, the method comprises the steps of providing cultured cells transfected with a polynucleotide construct that comprises a DNA sequence that encodes the Beta R1 promoter operatively linked to a reporter gene that encodes an assayable product; treating the transfected cells with the putative IFN-&bgr; mimic or the sample; detecting the assayable product produced by the treated cells. Synthesis of the assayable product by the cells treated with the putative IFN-&bgr; mimic or the sample is indicative of the IFN-&bgr; response. In another embodiment, the method comprises the steps of treating a group of cultured cells with the putative IFN-&bgr; mimic or sample and detecting the presence of the Beta R1 transcript in the treated cells; wherein the presence of the Beta R1 transcript in the treated cells is indicative of the IFN-&bgr; response.
The present invention also provides an isolated Beta R1 polypeptide, whose production is selectively induced by IFN-&bgr; as compared to IFN-&agr;. The Beta R1 polypeptide is an inducible CXC, non-ELR chemokine whose expression is stimulated in T cells, astrocytes, and fibroblasts that have been exposed to IFN-&bgr;. The present invention also provides an isolated polynucleotide. that encodes the Beta R1 polypeptide.
REFERENCES:
patent: 5707829 (1998-01-01), Jacobs et al.
“Characterization of &bgr;-R1, a Gene that is Selectively Induced by Interferon &bgr; (IFN-&bgr;) compared with IFN-&agr;*”, Rani, et al.,The Journal of Biological Chemistry;vol. 271, No. 37, Sep. 13, 1996; pp 22878-22884.
GenBank Accession, U59286; Oct. 24, 1996.
Rani Sandhya
Ransohoff Richard
Calfee Halter & Griswold LLP
DeBerry Regina M.
Kemmerer Elizabeth
The Cleveland Clinic Foundation
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