Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-05-11
2003-02-04
Spector, Lorraine (Department: 1846)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S442000, C435S007100, C530S351000, C424S085600
Reexamination Certificate
active
06514729
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to novel interferon-beta activity (IbA) proteins and nucleic acids. The invention further relates to the use of the IbA proteins in the treatment of interferon-beta (INF-&bgr;) related disorders.
BACKGROUND OF THE INVENTION
Human Interferons (IFNs) are members of a biologically potent family of cytokines. Originally, IFNs were identified as agents produced and secreted by virus-infected cells which can protect cells against further viral infections. However, in addition to this antiviral effect, IFNs can elicit many other changes in cellular behavior, including effects on cellular growth and differentiation and modulation of the immune system [e.g., see Lengyel, Annu. Rev. Biochem. 51:251-82 (1982); Gresser and Tovey, Biochim. Biophys. Acta 516(2):231-47 (1978); Gresser et al., Nature New Biol. 231(18):20-1 (1971); Dolei et al., J. Gen. Virol. 46(1):227-36 (1980); Gresser, Cell Immunol 34(2):406-15 (1977)]. By virtue of their antigenic, biological and physico-chemical properties, IFNs are classified into three groups, INF-&agr; (leukocyte), INF-&bgr; (fibroblast) and INF-&ggr; (immune) [Stewart, J. Infect. Dis. 142(4):643 (1980)].
In humans, the IFN-&agr; subtype encompass a multigene family of about 20 genes, encoding proteins of 166-172 amino acids that are all closely related. In contrast to this diversity, there is only one human interferon-beta (IFN-&bgr;) gene, also encoding a protein of 166 amino acids. IFN-&bgr; has low homology to the IFN-&agr; family and is an N-linked glycoprotein [Knight, Proc. Natl. Acad. Sci. U.S.A. 73(2):520-523 (1976)]. There is also only one human IFN-&ggr; gene that encodes a polypeptde of 143 amino acids that is glycosylated and forms a dimer in its native state. IFN-&ggr; shows only slight structural similarities to IFN-&agr; or to IFN-&bgr;.
All IFN-&agr; and IFN-&bgr; (also commonly referred to as type I interferon family) appear to bind to a common high affinity cell surface receptor, a 130 kD glycoprotein that is widely distributed on different cell types and that is distinct from the one bound by IFN-&ggr;. Type-I interferons are recognized by a complex containing the receptor subunits ifnar1 and ifnar2 and their associated Janus tyrosine kinases, Tyk2 and Jak1, that activate the transcription factors STAT1 and STAT2, leading to the formation of the transcription factor complex ISGF3 [interferon-stimulated gene factor 3; Li et al., Biochemie 80(8-9):703-20 (1998); Nadeau et al., J. Biol. Chem. 274(7):4045-52 (1999)]. Three distinct modes of IFN/receptor complex interaction are known: (i) INF-&agr; with ifnar1 and ifnar2; (ii) IFN-&bgr; with ifnar1 and ifnar2; and (iii) IFN-&bgr; with ifnar2 alone [Lewerenz et al., J. Mol. Biol. 282(3):585-99(1998)]. While Lewerenz et al. suggest that INF-&agr; and IFN-&bgr; interact with their receptors in different ways and as such may also signal differently, the events responsible for biological activity beyond receptor binding are poorly understood.
As might be predicted for such a large family of cytokines with almost ubiquitously distributed receptors, IFNs display varied physiological roles. Production of IFN-&agr; or IFN-&bgr; is induced by infection, including viral infection or the presence of foreign cell types and antigens. It is not clear what specific molecules are responsible for induction, but double-stranded RNA and cytokines can be good inducers. There is much overlap between different cell types in both the inducers and the species of IFN that is induced. The major cell types that produce IFNs are: lymphocytes, monocytes and macrophages (for IFN-&agr;); fibroblasts and some epithelial cells and lymphoblastoid cells (for IFN-&bgr;); and activated T lymphocytes (for IFN-&ggr;).
In addition to the ‘classical’ anti-viral activities that all IFNs elicit in their target cells, the biological consequences of IFN binding to its receptor can include inhibition of cell proliferation, induction of cell differentiation, changes in cell morphology, enhancement of histocompatibility antigen expression on many cells and stimulation of immunoglobulin-Fc receptor expression on macrophages. B lymphocytes can be induced to increase antibody production by low concentration of IFN-&agr; or IFN-&bgr;. An additional effect of IFN-&agr; and IFN-&bgr; is activation of natural killer cells that may be responsible for the destruction of virus-infected cells or tumor cells in vivo. Overall, IFNs seem to be of great importance as part of the body's defense against foreign organisms, foreign antigens and abnormal cell types (Clemens, in Cytokines, BIOS Scientific Publishers Limited, 1991; De Maeyer et al., in Interferons and Other Regulatory Cytokines, Wiley, New York, 1988).
INF-&agr; and IFN-&bgr; were among the first of the cytokines to be produced by recombinant DNA technology. For example, the amino acid and nucleotide sequence of human IFN-&bgr; [Tanaguchi et al., Gene 10(1):11-15 (1980); Houghton et al., Nucleic Acids Res. 8(13):2885-94 (1980)] made it possible to produce recombinant human IFN-&bgr; in e.g., mammalian, insect, and yeast cells and in
E. coli
, that is free from viruses and other contaminants from human sources [e.g., Ohno and Taniguchi, Nucleic Acids Res. 10(3):967-77 (1982); Smith et al., Mol. Cell. Biol. 3(12):2156-65 (1983); Demolder et al., J. Biotechnol. 32(2):179-89 (1994); Dorin et al., U.S. Pat. No. 5,814,485 (1998); Konrad et al., U.S. Pat. No. 4,450,103 (1984)].
IFNs have been shown to have therapeutic value in conditions such as inflammatory, viral, and malignant diseases [e.g., see Desmyter et al., Lancet 2(7987):645-7 (1976); Makower and Wadler, Semin. Oncol. 26(6):663-71 (1999); Sturzebecher et al., J. Interferon Cytokine Res. 19(11):1257-64 (1999); Zein, Cytokines Cell. Mol. Ther. 4(4):229-41 (1998; Musch et al., Hepatogastroeneterology 45(24):2282-94 (1998); Wadler et al., Cancer J. Sci. Am. 4(5):331-7 (1998)]. IFN-&bgr; is a marketed drug (Betaseron, manufactured by Berlex and Avonex, manufactured by Biogen) that has been approved for use in treatment of multiple sclerosis (MS) [Arnason, Biomed Pharmacother 53(8):344-50, (1999); Comi et al., Mult. Scler. 1(6):317-20 (1996); Aappos, Lancet 353(9171):2242-3 (1999)]. IFN-&bgr; seems to reduce the number of attacks suffered by patients with relapsing and remitting MS. Betaseron, a recombinant IFN-&bgr; expressed in
E. coli
, consists of 165 amino acids (missing the initial methionine) and is genetically engineered so that it contains a serine at position 17, to replace a cysteine. It is a nonglycosylated form of IFN-&bgr;. Avonex is a human IFN-&bgr;, consisting of 166 amino acids that is produced by recombinant DNA techniques in CHO cells. This is a glycosylated form of IFN-&bgr;. Also, recent studies show promising IFN efficacy in treating certain viral diseases, such as Hepatitis B or C, and cancer.
Most cytokines, including IFN-&bgr;, have relatively short circulation half-lives since they are produced in vivo to act locally and transiently. To use IFN-&bgr; as an effective systemic therapeutic, one needs relatively large doses and frequent administrations. Frequent parenteral administrations are inconvenient and painful. Further, toxic side effects are associated with IFN-&bgr; administration which are so severe that some multiple sclerosis patents cannot tolerate the treatment. These side effects are probably associated with administration of a high dosage. In clinical studies it has been found that some patients produce antibodies to IFN-&bgr;, which neutralize its biological activity.
Furthermore, it has been observed that dimers and oligomers of microbially produced IFN-&bgr; are formed in
E. coli
, rendering purification and separation of IFN-&bgr; laborious and time consuming. It also necessitates several additional steps in purification and isolation procedures such as reducing the protein during purification and reoxidizing it to restore it to its original conformation, the
Jiang Dong
Kosslrk Renee M.
Silva Robin M.
Spector Lorraine
Trecartin Richard F.
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