Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Lymphokines – e.g. – interferons – interlukins – etc.
Reexamination Certificate
1995-06-07
2002-10-01
Eyler, Yvonne (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Lymphokines, e.g., interferons, interlukins, etc.
C530S387900, C530S387100, C530S389100, C530S388100, C530S300000, C435S069100, C514S002600
Reexamination Certificate
active
06458932
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel interferon-&agr;/&bgr; binding proteins, capable of modulating the activity of various IFN-&agr; subtypes, as well as IFN-&bgr;. More particularly, this invention relates to the cloning of DNA molecules coding for these proteins, their expression in host cells and to antibodies against these proteins.
Grandparent U.S. application Ser. No. 08/115,741 now abandoned describes a soluble IFN-&agr; receptor protein of molecular weight of about 45,000, identified by Western blotting with monoclonal anti-IFN-&agr; receptor antibodies. The above application also describes a different soluble IFN-&agr; binding protein, having a molecular weight of about 40,000, that was identified by crosslinking with
125
I-IFN-&agr; and immuno-precipitation with anti-IFN-&agr; monoclonal antibodies. When obtained from serum, this species had a molecular weight of 50K. The aforesaid 40,000 IFN-&agr; binding protein, (hereinafter “urinary IFNAB-BP” or “IFNAB-BPII”) is obtained from urine in a homogenous state and has a sequence that differs from any other known protein. The IFNAB-BP binds to and blocks the activity of a variety of different IFN-&agr; subtypes, as well as IFN-&bgr;. In this respect, the binding characteristics of IFNAB-BP is significantly different from a previously described cell surface interferon receptor that binds only human interferon alpha B.
In accordance with the present invention, two cDNA molecules coding for precursors of IFNAB-BP are cloned and their sequence is determined. Both are probably derived from the same gene, e.g., by alternative splicing. Production of two recombinant proteins, designated IFNAB-BPI and IFNAB-BPII, in mammalian and other host cells is also described. Polyclonal and monoclonal antibodies directed against IFNAB-BP and useful for blocking the IFN receptor, for immunoassays and immunopurification of IFNAB-BPI and IFNAB-BPII are also disclosed.
IFNAB-BPI and IFNAB-BPII are capable of modulating the activity of type I interferons, i.e., the various subtypes of interferon-&agr;, as well as interferon-&bgr;. Thus they may inhibit undesired effects of type I interferons.
BACKGROUND OF THE INVENTION
Type I interferons (IFNs) (IFN-&agr;, IFN-&bgr; and IFN-&ohgr;) constitute a family of structurally related cytokines, usually defined by their ability to confer resistance to viral infections. Many other biological activities of type I IFNs have been reported, including inhibition of cell proliferation, induction of class I MHC antigens and several other immunoregulatory activities (1). IFN-&agr; and IFN-&bgr; are useful for the treatment of several viral diseases, including hepatitis-C (2,3) and viral warts (4,5), as well as certain malignancies such as hairy cell leukemia (6), chronic myelogenous leukemia (7) and Kaposi's sarcoma (8).
IFN-&agr; was detected in sera of various patients having autoimmune diseases such as systemic lupus erythematosus (9), as well as AIDS patients (10). IFN-&agr; was implicated in the progression of juvenile diabetes (11). There has also been a report that increased expression of IFN-A in the white matter microglia may contribute to Alzheimer's disease pathology (51). Further, IFN-&agr; therapy has been shown in some cases to lead to undesired side effects, including fever and neurological disorders (12). Hence there are pathological situations in which neutralization of IFN-&agr; activity may be beneficial to the patient.
As in the case of other cytokines, IFN-&agr; exerts its biological activities by binding to a cell surface receptor, which is specific for all IFN-&agr; subtypes, as well as for IFN-&bgr; (13). A human IFN-&agr; receptor was identified and cloned from Daudi cells (14). The cloned receptor has a single transmembrane domain, an extracellular and an intracellular domain. When expressed in murine cells, this receptor confers responsiveness to human IFN-&agr;&bgr;, but not significantly to other IFN-&agr; and IFN-&bgr; species, indicating that additional components may be involved in the response to IFN&bgr; and to various IFN-&agr; subtypes.
Several other studies indicate that there are additional components or receptor subunits involved in the binding of IFN-&agr; and IFN-&bgr; (15-17). Furthermore, it was reported that the already described receptor (14) is involved in binding of all IFN-&agr; and IFN-&bgr; species (18).
Cytokine binding proteins (soluble cytokine receptors) correspond to the extracellular ligand binding domains of their respective cell surface cytokine receptors. They are derived either by alternative splicing of a pre-mRNA common to the cell surface receptor, or by proteolytic cleavage of the cell surface receptor. Such soluble receptors have been described in the past, including among others, the soluble receptors of IL-6 and IFN-&ggr; (19-21), TNF (22-24), IL-1 (25-27), IL-4 (25,28), IL-2 (29,30), IL-7 (31) and IFN-alpha (32).
SUMMARY OF THE INVENTION
The present invention provides DNA molecules encoding the IFN-&agr;/&bgr; binding protein. Such DNA molecules actually encode two distinct proteins, IFNAB-BPI and IFNAB-BPII, probably derived from the same pre-mRNA by alternative splicing, to yield two mRNA molecules, one having a size of about 1.5 kb and the other a size of about 4.5 kb, each of which encodes one of the binding proteins, the 1.5 kb mRNA encoding the IFNAB-BPI and the 4.5 kb mRNA encoding the IFNAB-BP. The term IFNAB-PB corresponds to both IFNAB-BPI and IFNAB-BPII. Urinary IFNAB-BP is identified as IFNAB-BPII.
Accordingly, the present invention provides a DNA molecule encoding an IFN-&agr;/&bgr; binding protein selected from IFNAB-BPI, IFNAB-BPII, fused proteins and muteins of IFNAB-BPI and IFNAB-BPII, their functional derivatives and their active fractions.
The invention further provides replicable expression vehicles containing said DNA molecules, hosts transformed therewith and proteins produced by such transformed hosts. The term “DNA molecules” includes genomic DNA, cDNA, synthetic DNA and combinations thereof.
The invention also relates to DNA molecules which hybridize under stringent conditions to the above DNA molecules and encode proteins having the same biological activity as the IFNAB-BPs.
The present invention also provides methods for preparation in host cells capable of production of a functional IFNAB-BPI and IFNAB-BPII, fused proteins, muteins or active fractions thereof.
The present invention also provides the recombinant IFNAB-BPI and IFNAB-BPII, fused proteins, muteins or active fractions thereof, and salts of all of same, and pharmaceutical compositions containing IFNAB-BPI or IFNAB-BPII, fused proteins, muteins, active fractions thereof, or salts of all of same.
IFNAB-BPI and IFNAB-BPII inhibit the biological activities of natural human leukocyte and fibroblast interferons, as well as recombinant human IFN-&agr;2, IFN-&agr;&bgr;, IFN-&agr;C and IFN-&bgr;. IFNAB-BPI corresponds to a novel transmembrane protein which is the ligand-binding IFN-&agr;/&bgr; receptor. IFNAB-BPII is a soluble receptor, essentially corresponding to the extracellular, ligand-binding domain of IFNAB-BPI.
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Novick, Daniela et al, “The Human Interferon alpha/beta Receptor: Characterization and Molecular Cloning”,Cell, vol. 77, pp. 391-400 (May 6, 1994).
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Novick et al., Purification of soluble cytokine receptors form normal human urine by ligand
Cohen Batya
Novick Daniela
Rubinstein Menachem
Basi Nirmal S.
Browdy and Neimark
Eyler Yvonne
Yeda Research and Development Co. Ltd.
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