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
2001-10-24
2004-03-09
Spector, Lorraine (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C530S351000, C424S085700, C424S085400, C536S023520
Reexamination Certificate
active
06703225
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a novel interferon-&agr; (hereinafter referred to as IFN-&agr;). More preferably, the present invention relates to a novel human IFN-&agr; and its derivative having an unprecedentedly high specific activity, and a gene thereof, as well as medical uses of said IFN-&agr; and its derivative.
BACKGROUND ART
IFN is a generic term for proteins having anti-viral activity, among which those produced from leukocytes or lymphoblastic cells by stimulation with virus or double stranded nucleic acids are termed as IFN-&agr;. IFN-&agr; has a variety of activities including anti-viral activity and a cellular growth-suppressing activity, which activities have been found to be useful in a variety of diseases such as hepatitis type B, hepatitis type C, and cancer.
Analysis of base sequences of IFN-&agr; genes cloned from a variety of DNA libraries have revealed that IFN-&agr; has several subtypes (Science 209: 1343-7 (1980), Gene 11: 181-6 (1980), Nature 290: 20-26 (1981), Nature 313: 698-700 (1985), J. Invest. Dermatol. 83: 128s-136s (1984)). For example, for the main subtype gene of IFN-&agr;2, three types(&agr;2a, &agr;2b, and &agr;2c) have been identified (J. Interferon Res. 2: 575-85 (1982), J. Interferon Res. 13: 227-31 (1993), J. Biol. Chem. 268: 12565-9 (1993), Acta Virol. 38: 101-4 (1994), Biochim. Biophys. Acta. 1264: 363-8 (1995)). In addition, there are currently known nearly 20 types of subtype genes including IFN-&agr;1a, -&agr;1b, -&agr;4a, -&agr;4b, -&agr;5, -&agr;6, etc.
On the other hand, vigorous efforts have been made in structural analysis of proteins in stead of genes, that is to purify each subtype of natural IFN-&agr; and then to analyze its primary structure. A group in Wellcome, for example, made an attempt on structural analysis using a mixture of two fractions separated by gel filtration of purified IFN derived from Namalwa cells, human lymphoblastic cells, and, as a result, have demonstrated the structure, though not complete, of IFN-&agr;1 and IFN-&agr;2 (Nature 287: 408-11 (1980)). As a result of intensive efforts to purify Namalwa cell-derived IFN subtypes , Zoon et al. of FDA have successfully isolated several subtypes and revealed their partial structure, anti-viral activity, cellular growth-suppressing activity, and NK cell-inducing activity (Infect. Immun. 34: 1068-70 (1981), J. Biol. Chem. 267: 15210-6 (1992), J. Biol. Chem. 268: 12591-5 (1993)). Furthermore, in the analysis of the primary structure for one major subtype, they have demonstrated that it was IFN-&agr;2b (J. Biol. Chem. 267: 15210-6 (1992)).
As stated above, IFN-&agr; has various subtypes, of which base sequences and amino acid sequences are being elucidated, though the structure and physical properties of all subtypes have not been revealed.
DISCLOSURE OF THE INVENTION
The present invention intends to provide a novel IFN-&agr; and its gene. Thus, the present invention intends to provide a novel human IFN-&agr;, its derivative having an unprecedentedly high specific activity, a gene encoding them, and a pharmaceutical agent comprising said IFN-&agr; and its derivative as active ingredient.
The inventors of the present invention have attempted to isolate major subtypes contained in IFN-&agr; derived from human natural-type lymphoblastic cells (hereinafter referred to as HLBI). Thus, the inventors have found that the subtypes can be easily separated by means of a reverse-phase HPLC that utilizes &mgr;Bondasphere column and Vydac™-C4 column and thereby have successfully isolated and purified 12 major subtypes contained in HLBI.
From the analysis of the N-terminal amino acid sequence and the primary structure of the isolated subtypes, it was found that a novel IFN-&agr; subtype was contained in addition to the existing IFN-&agr;1, &agr;2b, &agr;5, &agr;7, &agr;8, &agr;14, &agr;17 and &agr;21. The inventors of the present invention have termed this novel IFN-&agr; subtype as IIIe.
On these subtypes, anti-viral activity against Sindbis virus was determined using human-derived cultured cells, FL cells, and it was found that the anti-viral activity of a major subtype IFN-&agr;2b was 1.67×10
8
u/mg whereas the novel IFN-&agr; subtype IIIe had the highest and unprecedentedly high specific activity of 4.3-5.2×10
8
u/mg.
Furthermore, the identification of the entire amino acid sequence of the subtype IIIe revealed that the primary structure of the subtype IIIe was similar to an amino acid sequence deduced from the sequence of IFN-&agr;10a (=−&agr;C) gene as reported in Nature Mar. 5, 1981; 290, 20-26, but had a novel amino acid sequence in which the amino acid at position 19 was Ala in stead of Gly. The cloning of said IIIe gene also revealed that it is different from IFN-&agr;10a by three bases on the base sequence level.
As described above, the IFN-&agr; subtype IIIe of the present invention has an unprecedentedly high specific activity, and thereby its dosage can possibly be reduced compared to commercially available recombinant human IFN-&agr;2a, recombinant human IFN-&agr;2b, etc. Furthermore, it is expected to exhibit effectiveness on cases with HCV-Genotype II, high virus level etc. on which conventional IFN is believed to be not very effective.
The present invention was completed based on the above findings.
Thus, the present invention relates to the following (1) to (13):
(1) DNA comprising the base sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2, or DNA encoding a protein comprising the amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4;
(2) DNA encoding a derivative of human interferon-&agr;, said DNA being selected from
(A) DNA hybridizing to the DNA according to the above (1) under a stringent condition, and
(B) DNA encoding a protein in which one or a plurality of amino acid residues of a protein encoded by the DNA according to the above (1) have been replaced, deleted, and/or added,
wherein the protein encoded by said DNA has the following characteristics (a) and (b):
(a) having a specific activity higher than 4.0×10
8
units/mg as measured by an anti-viral activity assay on Sindbis virus using the FL cell, a human-derived cultured cell; and
(b) migrating as a band with an apparent molecular weight of 20 kDa-23 kDa on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction treatment;
(3) The DNA according to the above (2) which encodes a protein comprising an amino acid sequence in which 1-5 amino acid residues in the amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4 have been replaced, deleted, and/or added;
(4) An expression vector having the DNA according to any one of the above (1)-(3);
(5) A transformant transformed with the expression vector according to the above (4);
(6) A method of producing a recombinant human interferon-&agr; or its derivative, which method comprises culturing the transformant according to the above (5) and recovering the expressed recombinant human interferon-&agr; or its derivative;
(7) A human interferon-&agr; or its derivative which is encoded by the DNA according to any one of the above (1)-(3) or produced by the production method according to the above (6);
(8) A human interferon-&agr; comprising the amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4;
(9) A human interferon-&agr; or its derivative according to the above (7) or (8) or a pharmaceutically acceptable salt thereof for use as active ingredient of a pharmaceutical composition;
(10) A pharmaceutical composition comprising the human interferon-&agr; or its derivative according to the above (7) or (8) or a pharmaceutically acceptable salt thereof as active ingredient together with a pharmaceutically acceptable carrier or excipient;
(11) The pharmaceutical composition according to the above (10) which is for treatment of viral diseases;
(12) The pharmaceutical composition according to the above (10) which is for treatment of cancer;
(13) A method of treating viral diseases or cancer which method comprises administering to a mammal including a human an e
Asakura Akira
Fukuda Yuki
Futatsugi Tetsuaki
Kojima Shin-ichi
Ota Yuko
Andres Janet L.
Spector Lorraine
Sughrue & Mion, PLLC
Sumitomo Pharmaceuticals Company Limited
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