Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-11-05
2002-06-18
Eyler, Yvonne (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C514S002600, C424S085400
Reexamination Certificate
active
06407209
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to purified proteins induced in human cells by interferon &agr; or &bgr;, RNAs, DNAs and hybrid vectors coding for said proteins, hosts transformed with such a hybrid vector, processes for the preparation and purification of these proteins, DNAs, vectors and hosts, monoclonal antibodies specific to these proteins, monoclonal antibody derivatives, hybridoma cell lines secreting these monoclonal antibodies, the use of the monoclonal antibodies and their derivatives in the qualitative and quantitative determination of these proteins, test kits containing the monoclonal antibodies, and pharmaceutical preparations containing said proteins.
BACKGROUND OF THE INVENTION
Interferons are a class of naturally occurring proteins which show promise in the defense against viral infections and in tumor growth control. They seem to interfere with cellular functions necessary for viral replication and to inhibit cellular growth by pleiotropic effects which are not yet understood at the molecular level. Furthermore, interferons stimulate the activity of natural killer (NK) cells, and within a complicated network of lymphokine interactions, modulate the activity of macrophages, B- and T-cells.
A set of induced proteins may be involved in the antiviral, antiproliferative and immunomodulatory activities of interferons. In mammalian cells interferons induce the synthesis of several proteins that are not detected or do exist at much lower concentrations in untreated cells. Some of these induced proteins have been widely studied [review: P. Lengyel, Annu. Rev. Biochem. 51, 251 (1982)], but are still poorly characterized. Interferon-treated cells of both human and mouse origin contain elevated levels of 2′,5′-oligoiso-adenylate synthetase and protein kinase activities. The synthesis and properties of the mouse protein Mx induced by interferon &agr; and &bgr; have been studied in detail [P. Staeheli et al., Cell 44, 147 (1986)]. This protein is associated with a highly efficient and specific antiviral resistance to influenza viruses [M. A. Horisberger et al., Proc. Natl. Acad. Sci. USA 80, 1910 (1983); M. A. Horisberger & H. K. Hochkeppel, J. Biol. Chem. 260, 1730 (1985)]. A related human protein was detected in interferon-induced human peripheral blood lymphocytes and fibroblasts [P. Staeheli & O. Haller, Mol. Cell. Biol. 5, 2150 (1985)]. A molecular weight of 80 kDa (kilo-Dalton) was found, and the protein was predominantly localized in the cell cytoplasm, but otherwise the protein was not further characterized or isolated.
The fast progress in recombinant DNA technology in recent years provides the general methods for the preparation of proteins in large amounts independent of the primary natural sources of such compounds. Identification of an mRNA or a DNA coding for the desired polypeptide is crucial for the success of this approach. If (partial) amino acid sequence information is available, a chemically synthetized nucleic acid probe may lead to the isolation of coding mRNA or DNA from a mixture of mRNA derived from cells producing the desired polypeptides or from a DNA library, respectively. Although many examples for the isolation of an mRNA or DNA coding for a desired polypeptide have so far become known and the general procedure has been described in principle, each new specific problem requires adaption of the technique to the particular case.
Once a complementary or genomic DNA coding for the desired polypeptide is at hand, preparation of suitable expression vectors, transformation of hosts with these vectors, fermentation of transformed hosts and isolation of the expressed polypeptide follows standard procedures. Here again, these procedures must be adapted to the particular problem in order to get stable incorporation of the DNA and sufficiently high expression of the desired polypeptide in a chosen host organism, and acceptable yields of pure, biologically active isolated protein.
Furthermore recombinant DNA technology allows one to produce polypeptide variants by mutating or otherwise altering the coding DNA incorporated in a host organism, thereby enlarging the potential applications of an active principle found in a single polypeptide structure in nature.
Proteins induced by interferon are important in diagnosis, disease management and therapy in two respects: On one hand they may exert some of the beneficial properties such as antiviral or antiproliferative activities ascribed to interferons, but without the unwanted side effects of interferons, on the other hand they may be valuable indicators of the cell response to an interferon therapy. Antibodies to such interferon-induced proteins allow the qualitative and quantitative determination of these proteins and therefore are indispensable means in the surveillance of a therapy with these proteins or with interferons.
Polyclonal and monoclonal antibodies to the mouse Mx protein induced by interferons are known [P. Staeheli & O. Haller, Mol. Cell. Biol. 5, 2150 (1985)]. One of these shows also a weak cross-reactivity to a human interferon-induced protein, however, non-specific cross-reaction cannot be excluded.
OBJECT OF THE INVENTION
It is an object of the present invention to provide pure proteins related to or identical with those found in human cells induced by interferon &agr; or &bgr;. The problem of industrial synthesis of such proteins can be solved by the methods of recombinant DNA technology. A further object of the present invention is therefore to provide mRNAs, DNAs and hybrid vectors coding for said proteins, and hosts transformed with such a vector.
Further objects are processes for the preparation and purification of said proteins, mRNAs, DNAs and hybrid vectors, and processes for tie preparation of hosts containing said hybrid vectors.
It is another object of the invention to provide monoclonal antibodies as diagnostic means for the qualitative and quantitative determination of said proteins, hybridomas secreting such antibodies, and processes for the preparation of these antibodies and hybridomas, furthermore pharmaceutical preparations containing said proteins.
DESCRIPTION OF THE INVENTION
The invention relates to essentially pure proteins, characterized by
(1) their presence in human cells induced by interferon &agr; or &bgr;, but not in untreated cells to a reasonable extent,
(2) a molecular weight of approximately 78 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophotesis (SDS-PAGE),
(3) an isoelectric point of approximately 6.3,
(4) a partial N-terminal amino acid sequence
(SEQ ID NO.1)
5 10
Val-Val-Ser-Glu-Val-Asp-Ile-Ala-Lys-Ala-.
In particular, these proteins are found in Namalwa or human embryonic foreskin cells treated with a recombinant interferon &agr;/B, &agr;/D, &agr;/F or interferon &agr;/B-D hybrids.
Particularly preferred is a protein with a partial N-terminal amino acid sequence
5 10 15
(SEQ ID NO.2)
Val-Val-Ser-Glu-Val-Asp-Ile-Ala-Lys-Ala-Asp-Pro-Ala-Ala-Ala-Ser-
20 25 30
His-Pro-Leu-Leu-Leu-Asn-Gly-Asp-Ala-Thr-Val-Ala-Gln-Lys-Asn-Pro-
35 &emsp
Content Jean
Hochkeppel Heinz-Kurt
Horisberger Michel Andre
Andres Janet L.
Eyler Yvonne
Ferraro Gregory D.
Novartis AG
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