U.S. patent application number 10/757511 was filed with the patent office on 2004-07-15 for novel interferon-alpha.
This patent application is currently assigned to SUMITOMO PHARMACEUTICALS COMPANY LIMITED. Invention is credited to Asakura, Akira, Fukuda, Yuki, Futatsugi, Tetsuaki, Kojima, Shin-ichi, Ota, Yuko, Sagara, Shinsuke.
Application Number | 20040137009 10/757511 |
Document ID | / |
Family ID | 11602962 |
Filed Date | 2004-07-15 |
United States Patent
Application |
20040137009 |
Kind Code |
A1 |
Kojima, Shin-ichi ; et
al. |
July 15, 2004 |
Novel interferon-alpha
Abstract
A novel human IFN-.alpha. subtype and its derivative having an
unprecedentedly high specific activity, DNA encoding these
proteins, an expression vector having said DNA, a transformant
transformed with said expression vector, a method of producing the
above human IFN-.alpha. and its derivative, and medical uses of the
above human IFN-.alpha. and its derivative.
Inventors: |
Kojima, Shin-ichi;
(Kobe-shi, JP) ; Asakura, Akira; (Kawanishi-shi,
JP) ; Futatsugi, Tetsuaki; (Otsu-shi, JP) ;
Ota, Yuko; (Nishinomiya-shi, JP) ; Fukuda, Yuki;
(Nishinomiya-shi, JP) ; Sagara, Shinsuke; (Osaka,
JP) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
SUMITOMO PHARMACEUTICALS COMPANY
LIMITED
|
Family ID: |
11602962 |
Appl. No.: |
10/757511 |
Filed: |
January 15, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10757511 |
Jan 15, 2004 |
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09889035 |
Oct 24, 2001 |
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6703225 |
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09889035 |
Oct 24, 2001 |
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PCT/JP00/00015 |
Jan 5, 2000 |
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Current U.S.
Class: |
424/186.1 ;
530/350 |
Current CPC
Class: |
C07K 14/56 20130101;
A61P 35/00 20180101; A61K 38/00 20130101; A61P 31/12 20180101 |
Class at
Publication: |
424/186.1 ;
530/350 |
International
Class: |
C07K 014/005; A61K
039/12 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 12, 1999 |
JP |
11/005138 |
Claims
1. A purified polypeptide comprising an amino acid sequence as set
forth in SEQ ID NO: 3 or SEQ ID NO: 4.
2. A purified polypeptide comprising an amino acid sequence as set
forth in SEQ ID NO: 3 or SEQ ID NO: 4, wherein from one to five
amino acid residues have been replaced, deleted, and/or added, and
wherein the polypeptide has the following characteristics (a) and
(b): (a) a specific activity higher than 4.0.times.108 units/mg in
an anti-Sindbis virus assay on cultured FL cells; and (b) an
apparent molecular weight of 20 kDa-23 kDa as determined by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after
treatment with a reducing agent.
3. A pharmaceutical composition comprising the polypeptide of claim
1 or claim 2, and a pharmaceutically acceptable carrier or
excipient.
4. The pharmaceutical composition of claim 3, which is for
treatment of a viral disease.
5. The pharmaceutical composition of claim 3, which is for
treatment of cancer.
6. A method for treating a viral disease in a mammal comprising
administering to said mammal in need of treatment an effective
amount of the polypeptide of claim 1 or 2.
7. The method of claim 6, wherein said mammal is a human.
8. A method for treating cancer in a mammal comprising
administering to said mammal in need of treatment an effective
amount of the polypeptide of claim 1 or claim 2.
9. The method of claim 8, wherein said mammal is a human.
10. A recombinant polypeptide produced by a process comprising: (a)
transforming a cell with an expression construct, said expression
construct comprising a nucleotide sequence operably linked to a
promoter, said nucleotide sequence encoding the polypeptide with an
amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4,
thereby creating a transformant; (b) culturing said transformant;
and (c) recovering expressed polypeptide.
11. A recombinant polypeptide produced by a process comprising: (a)
transforming a cell with an expression construct, said expression
construct comprising a nucleotide sequence operably linked to a
promoter; said nucleotide sequence encoding the polypeptide with an
amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4,
wherein from one to five amino acid residues have been replaced,
deleted, and/or added, and wherein the polypeptide has the
following characteristics (1) and (2): (1) a specific activity
higher than 4.0.times.108 units/mg in an anti-Sindbis virus assay
on cultured FL cells; and (2) an apparent molecular weight of 20
kDa-23 kDa as determined by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) after treatment with a reducing
agent; thereby creating a transformant; (b) culturing said
transformant; and (c) recovering expressed polypeptide.
12. A pharmaceutical composition comprising the recombinant
polypeptide of claim 10 or claim 11, and a pharmaceutically
acceptable carrier or excipient.
13. The pharmaceutical composition of claim 12, which is for
treatment of a viral disease.
14. The pharmaceutical composition of claim 12, which is for
treatment of cancer.
15. A method for treating a viral disease in a mammal comprising
administering to said mammal in need of treatment an effective
amount of the recombinant polypeptide of claim 10 or claim 11.
16. The method of claim 15, wherein said mammal is a human.
17. A method for treating cancer in a mammal comprising;
administering to said mammal in need of treatment an effective
amount of the recombinant polypeptide of claim 10 or claim 11.
18. The method of claim 17, wherein said mammal is a human.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This is a divisional of application Ser. No. 09/889,035,
filed Jul. 11, 2001, which is a National Stage Application filed
under .sctn. 371 of PCT Application No. PCT/JP00/00015, filed Jan.
5, 2000; the above noted prior applications are all hereby
incorporated by reference.
TECHNICAL FIELD
[0002] The present invention relates to a novel interferon-.alpha.
(hereinafter referred to as IFN-.alpha.). More preferably, the
present invention relates to a novel human IFN-.alpha. and its
derivative having an unprecedentedly high specific activity, and a
gene thereof, as well as medical uses of said IFN-.alpha. and its
derivative.
BACKGROUND ART
[0003] 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-.alpha.. IFN-.alpha. 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.
[0004] Analysis of base sequences of IFN-.alpha. genes cloned from
a variety of DNA libraries have revealed that IFN-.alpha. 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-.alpha.2, three types (.alpha.2a, .alpha.2b,
and .alpha.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-.alpha.1a,
-.alpha.1b, -.alpha.4a, .alpha.4b, -.alpha.5, -.alpha.6, etc.
[0005] 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-.alpha. 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-.alpha.1
and IFN-.alpha.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-.alpha.2b (J. Biol. Chem. 267: 15210-6 (1992)).
[0006] As stated above, IFN-.alpha. 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
[0007] The present invention intends to provide a novel IFN-.alpha.
and its gene. Thus, the present invention intends to provide a
novel human IFN-.alpha., its derivative having an unprecedentedly
high specific activity, a gene encoding them, and a pharmaceutical
agent comprising said IFN-.alpha. and its derivative as active
ingredient.
[0008] The inventors of the present invention have attempted to
isolate major subtypes contained in IFN-.alpha. 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
.mu.Bondasphere column and Vydac-C4 column and thereby have
successfully isolated and purified 12 major subtypes contained in
HLBI.
[0009] 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-.alpha. subtype was contained in addition to the existing
IFN-.alpha.1, .alpha.2b, .alpha.5, .alpha.7, .alpha.8, .alpha.14,
.alpha.17 and .alpha.21. The inventors of the present invention
have termed this novel IFN-.alpha. subtype as IIIe.
[0010] 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-.alpha.2b was 1.67.times.10.sup.8 u/mg whereas the novel
IFN-.alpha. subtype IIIe had the highest and unprecedentedly high
specific activity of 4.3-5.2.times.10.sup.8 u/mg.
[0011] 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-.alpha.10a (=-.alpha.C) gene as reported in
Nature 1981 Mar. 5; 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-.alpha.10a by three bases on the base sequence level.
[0012] As described above, the IFN-.alpha. 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-.alpha.2a, recombinant
human IFN-.alpha.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.
[0013] The present invention was completed based on the above
findings.
[0014] Thus, the present invention relates to the following (1) to
(13):
[0015] (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;
[0016] (2) DNA encoding a derivative of human interferon-.alpha.,
said DNA being selected from
[0017] (A) DNA hybridizing to the DNA according to the above (1)
under a stringent condition, and
[0018] (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,
[0019] wherein the protein encoded by said DNA has the following
characteristics (a) and (b):
[0020] (a) having a specific activity higher than
4.0.times.10.sup.8 units/mg as measured by an anti-viral activity
assay on Sindbis virus using the FL cell, a human-derived cultured
cell; and
[0021] (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;
[0022] (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;
[0023] (4) An expression vector having the DNA according to any one
of the above (1)-(3);
[0024] (5) A transformant transformed with the expression vector
according to the above (4);
[0025] (6) A method of producing a recombinant human
interferon-.alpha. or its derivative, which method comprises
culturing the transformant according to the above (5) and
recovering the expressed recombinant human interferon-.alpha. or
its derivative;
[0026] (7) A human interferon-.alpha. 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);
[0027] (8) A human interferon-a comprising the amino acid sequence
as set forth in SEQ ID NO: 3 or SEQ ID NO: 4;
[0028] (9) A human interferon-.alpha. 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;
[0029] (10) A pharmaceutical composition comprising the human
interferon-.alpha. 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;
[0030] (11) The pharmaceutical composition according to the above
(10) which is for treatment of viral diseases;
[0031] (12) The pharmaceutical composition according to the above
(10) which is for treatment of cancer;
[0032] (13) A method of treating viral diseases or cancer which
method comprises administering to a mammal including a human an
effective amount of the human interferon-.alpha. or its derivative
according to the above (7) or (8) or a pharmaceutically acceptable
salt thereof.
BEST MODE FOR CARRYING OUT THE INVENTION
[0033] The DNA of the present invention encodes a novel human
IFN-.alpha. and its derivative, and specifically there can be
mentioned 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.
[0034] Also encompassed in the scope of the present invention is
DNA that hybridizes to the DNA described above under a stringent
condition, or DNA encoding a derivative of a human
interferon-.alpha. selected from DNA encoding a protein in which
one or a plurality of amino acid residues of the protein encoded by
the above DNA have been replaced, deleted, and/or added, wherein
the protein encoded by said DNA has the characteristics of (a)
having a specific activity higher than 4.0.times.10.sup.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. The DNA of the present invention will now be
sequentially explained below.
[0035] 1) DNA Encoding the IFN-.alpha. Subtype IIIe
[0036] Among the above DNA, "DNA comprising the base sequence as
set forth in SEQ ID NO: 1 or SEQ ID NO: 2" and "DNA encoding a
protein comprising the amino acid sequence as set forth in SEQ ID
NO: 3 or SEQ ID NO: 4" are DNA encoding the human-derived
IFN-.alpha. subtype IIIe of the present invention. Among them, the
base sequence as set forth in SEQ ID NO: 1 and the amino acid
sequence as set forth in SEQ ID NO: 3 are a base sequence and an
amino acid sequence corresponding to the full-length subtype IIIe
including the signal peptide, and the base sequence as set forth in
SEQ ID NO: 2 and the amino acid sequence as set forth in SEQ ID NO:
4 are a base sequence and an amino acid sequence corresponding to
the full-length mature type subtype IIIe including no signal
peptide.
[0037] Said DNA can be cloned by the PCR method described in
Example 3 below. As templates for performing PCR, genomic DNA or
cDNA derived, for example, from the Namalwa cell (ATCC No. CRL-1432
etc.) may be used, and as primers, a primer comprising the base
sequence, for example, as set forth in SEQ ID NO: 6 and SEQ ID NO:
7 may be mentioned.
[0038] Furthermore, cloning may also be performed by modifying
amino acids based on the known IFN-.alpha. subtypes reported in
Nature 290: 20-26 (1981), etc. Said cloning may be readily
performed by a person skilled in the art according to Molecular
Cloning 2nd Ed., Cold Spring Harbor Laboratory Press (1989)
etc.
[0039] 2) DNA Encoding a Derivative of IFN-.alpha. Subtype IIIe
[0040] Among the above DNA, "DNA that hybridizes to the DNA of the
subtype IIIe under a stringent condition" and "DNA encoding a
protein in which one or a plurality of amino acid residues of the
amino acid sequence of the subtype IIIe have been replaced,
deleted, and/or added" mean DNA encoding a protein having a
structure similar to the subtype IIIe such as an artificially
constructed, so-called modified protein, an allele mutant present
in the living body, and an IFN-.alpha. subtype similar to IIIe.
Hereinbelow, protein having such a structure similar to the subtype
IIIe will be termed as a "derivative."
[0041] As used herein, as a method of producing "DNA encoding
protein in which one or a plurality of amino acid residues of the
amino acid sequence of the subtype IIIe have been replaced,
deleted, and/or added," there can be known methods such as
site-directed mutagenesis and the PCR method, which may be easily
performed by a person skilled in the art according to Nucleic Acid
Res. 10: 6487 (1982), Methods in Enzymology 100: 448 (1983),
Molecular Cloning 2nd Ed., Cold Spring Harbor Laboratory Press
(1989), PCR A Practical Approach, IRL Press, pp. 200 (1991),
etc.
[0042] As the number of amino acid residues to be modified, there
can be mentioned those numbers that may be replaced, deleted,
and/or added by known methods such as the site-directed mutagenesis
mentioned above. Since IFN-.alpha. is a relatively small protein of
which mature type comprises 166 amino acids, the number of amino
acid residues to be modified is preferably 10 or less, and more
preferably 5 or less. For sites that are important for activity
expression, modification is preferably substitution to conservative
amino acids.
[0043] As used herein, as a method of producing "DNA that
hybridizes to the DNA of the subtype IIIe under a stringent
condition," there can be mentioned known methods such as a PCR
method, and a hybridization method. Specifically, it may be
performed according to the method described in the above Molecular
Cloning.
[0044] As used herein, "under a stringent condition" means a
condition in which hybridization is performed at 42.degree. C. in a
solution containing 6.times.SSC (20.times.SSC represents 333 mM
sodium citrate and 333 mM NaCl), 0.5% SDS, and 50% formamide,
followed by washing at 68.degree. C. in a solution of 0.1.times.SSC
and 0.5% SDS, a condition as described in the above-mentioned
Molecular Cloning, or the like. More preferably, there can be
mentioned a condition in which hybridization occurs only for those
that are different from the DNA of the subtype IIIe by about 1-5
amino acids.
[0045] Among the above DNA, the DNA encoding a protein which has
the following characteristics can be the DNA of the present
invention: (a) having a specific activity higher than
4.0.times.10.sup.8 units/mg as measured by an anti-viral activity
assay on Sindbis virus using human-derived cultured cells, FL
cells; 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.
[0046] Whether the protein encoded by the candidate DNA that can be
the DNA of the present invention satisfies the above requirement
(a) can be evaluated by performing an anti-viral activity assay as
described below.
[0047] Thus, 45,000-60,000 FL cells (The National Institute of
Health, ATCC etc.) prepared in a 10 v/v % bovine calf serum-Eagle's
minimum essential medium are inoculated into each well of a
microtiter plate, which is incubated in a 5% carbon dioxide
incubator at 37.degree. C. for 20 hours. Then 100 .mu.l of the
candidate IFN sample is added to each well and incubated at
37.degree. C. for 6 hours. The culture liquid is discarded and
10.sup.5-10.sup.6 PFU of Sindbis virus (The National Institute of
Health, ATTC etc.) per well is added, and incubated at 37.degree.
C. for 2 days. The cells are stained in a 0.02 w/v % Neutral red-5
v/v % bovine calf serum-Eagle's minimum essential medium, and the
degree of cytopathic effect is determined by the amount of the dye
incorporated.
[0048] As methods of calculating titer, the following method may be
mentioned. Thus, the dye incorporated into the cell is eluted with
an acidified 30 v/v % ethanol and absorbance is determined at a
wavelength of 545 m.mu.. The experimental titer of the sample and
the standard (The National Institute of Health) are calculated from
the dilution factor of the sample exhibiting 50% of the absorbance
of the dye incorporated into the normal cell and that of the
standard. The titer of the standards and the experimental titer are
used to determine a correction factor, which is used to correct the
experimental titers of the samples to obtain the titers of the
samples. In the above activity assay, those having a specific
activity higher than 4.0.times.10.sup.8 units/mg are included in
the scope of the present invention.
[0049] Whether the protein encoded by the candidate DNA satisfies
the above requirement (b) can be detected by subjecting the
candidate protein to reduction treatment with 2-mercaptoethanol
followed by a normal sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and then staining with Coomassie
brilliant blue. The density of the SDS-PAGE gel at this time is
preferably about 3.5% for the concentration gel and about 15% for
the separation gel. Electrophoresis is preferably performed at
about 50 mA. In the SDS-PAGE as described above, those having an
apparent molecular weight of 20 kDA-23 kDa are included in the
scope of the present invention.
[0050] By recombinant DNA technology using the DNA of the present
invention, the protein of the present invention, that is, the novel
human IFN-.alpha. subtype IIIe and its derivative can be produced
in large quantities. In order to produce the recombinant human
IFN-.alpha. and its derivative of the present invention by
expressing the DNA of the present invention, methods are used, for
example, based on many textbooks and references including the above
Molecular Cloning. For human IFN-.alpha.2a and -.alpha.2b, their
recombinant types have already been produced and are commercially
available. Based on the production method for known IFN-.alpha.,
the novel human IFN-.alpha. and its derivative of the present
invention can be produced in large quantities (see Japanese
Examined Patent Publication (Kokoku) No. 63-63198, Japanese
Examined Patent Publication (Kokoku) No. 3-21151, Nucleic Acids
Res. 8: 4057 (1981), Nature 287: 411 (1980), Proc. Natl. Acad. Sci.
USA 77: 5230 (1980), and the like).
[0051] Specifically, by optionally adding a regulatory gene such as
a promoter sequence (for example, trp, lac, T7, and SV40 early
promoter) that controls transcription to the upstream of the DNA to
be expressed, which is then integrated into a suitable vector (for
example, PBK-CMV, pCAGGS, and pZeoSV), it is possible to construct
an expression vector that is replicated and expressed in the host
cell. Then said expression vector is introduced into a suitable
host cell to obtain a transformant. As the host cell, there can be
mentioned a prokaryote such as Escherichia coli, a unicellular
eukaryote such as yeast, a multicellular eukaryote such as an
insect or an animal, and the like. As the method of introducing an
expression vector into a host cell, a known method can be used such
as the calcium phosphate method, the DEAE-dextran method, and the
electric pulse method. By culturing the thus obtained transformant
in a culture medium suitable for said transformant by a standard
method, the desired recombinant human IFN-.alpha. and its
derivative of the present invention can be produced. The
recombinant human IFN-.alpha. and its derivative of the present
invention obtained in this manner can be isolated and purified by a
common biochemical method using, for example, anti-IFN-.alpha.
antibody.
[0052] Furthermore, they can also be obtained by purifying a
subtype obtained by using as raw material the human natural type
lymphoblast-derived IFN-.alpha. (HLBI) (manufactured by Sumitomo
Pharmaceutical Co., Ltd.) as described in Example 1 below.
[0053] The novel human IFN-.alpha. subtype IIIe and its derivative
of the present invention thus obtained are encoded by the above DNA
of the present invention, and they are proteins produced by the
expression of the latter. As a specific example, there may be
illustrated the novel human IFN-.alpha. subtype IIIe of the present
invention comprising the amino acid sequence as set forth in SEQ ID
NO: 3 or SEQ ID NO: 4.
[0054] The novel IFN-.alpha. and its derivative of the present
invention can be used as active ingredient of pharmaceutical
agents. Thus the present invention also intends to provide a
pharmaceutical composition comprising a novel IFN-.alpha. and its
derivative or a pharmaceutically acceptable salt thereof together
with a pharmaceutically acceptable carrier or excipient, and
optionally with another therapeutic and/or preventive agent.
[0055] It is conventionally known that IFN-.alpha. has a variety of
effects including an anti-viral effect, a cellular
growth-suppressing effect, a natural killer cell-activating effect,
and the like. Accordingly, the novel IFN-.alpha. of the present
invention is also expected to be able to treat various diseases
based on these effects.
[0056] As the indicated diseases, cancer (malignant tumor), viral
diseases, and immunological diseases may be mentioned, and
specifically there can be mentioned kidney cancer, renal-cell
carcinoma, breast cancer, bladder cancer, basal cell carcinoma,
head and neck cancer, cervical dysplasia, skin carcinoma, Kaposi's
sarcoma, malignant melanoma, non-Hodgkin lymphoma, infant
hemangioma, chronic granulomatosis, type B chronic hepatitis, type
C chronic hepatitis (active, non-active), herpes infections
(genital herpes, corneal herpes inflammation, oral herpes
inflammation, etc.), chronic myelocytic leukemia (CML), adult T
cell leukemia, hairy cell leukemia, hairy cell leukemia, T cell
leukemia virus (HTLV-1) myelopathy, multiple myeloma, lymphoma,
subacute sclerosing panencephalitis (SSPE), Sjogren's syndrome,
condyloma acuminata, AIDS, multiple sclerosis (MS), stomatitis,
genital wart, intravaginal wart, erythrocytosis, thrombocythemia,
psoriasis, mycosis fungoides, sudden deafness, senile disciform
macular degeneration, Paget's disease, and the like.
[0057] Since the IFN-.alpha. and its derivative of the present
invention has a specific activity higher than the conventional
ones, they are expected to be effective on cases with HCV-Genotype
II, high virus level etc. on which conventional IFN is said to be
not very effective.
[0058] The novel interferon-.alpha. and its derivative of the
present invention or a pharmaceutically acceptable salt thereof may
be administered as a pharmaceutical composition via an oral or
parenteral (for example, intravenous, subcutaneous or intramuscular
injection, local, transrectal, transdermal, or nasal) route. As
compositions for oral administration, there can be mentioned, for
example, tablets, capsules, pills, granules, powders, liquids, and
suspensions, and as compositions for parenteral administration,
there can be mentioned, for example, aqueous or oily agents for
injection, ointments, creams, lotions, aerosols, suppositories, and
adhesives. It is also possible to prepare sustained release
minipellet formulations and implant them near the affected area, or
to gradually administer them to the affected area on a continuous
basis using an osmotic pump. These formulations may be prepared
using conventionally known technology, and may contain non-toxic
and inert carriers or excipients that are commonly used in the
field of pharmaceutics.
[0059] The above pharmaceutical compositions may be manufactured by
blending the active ingredient of the present invention with
pharmaceutically acceptable conventional carriers, excipients,
binders, stabilizers, buffers, solution adjuvants, or tonicity
agents. When used as injections, there may be added buffers,
solution adjuvants, tonicity agents etc..
[0060] The dosage and the frequency of administration may vary
depending on the condition and history of the disease, age and
weight of the patient, dosage form, etc., but when they are
administered to adults (body weight 60 kg) via a parenteral (for
example, intravenous) route, they are generally prepared, as
appropriate, in the range of 0.001-1 mg per day, preferably
0.005-0.5 mg, and most preferably 0.010-0.2 mg, and administered in
single or several divided doses.
[0061] The present invention will now be explained in further
details hereinbelow with reference to examples, but it should noted
that the present invention will not be limited by these examples in
any way.
EXAMPLE 1
Isolation of Each IFN-.alpha. Subtype, and the Measurement of its
Physical Properties and Biological Activity
[0062] In order to elucidate the physical properties and partial
structures in detail of major natural-type IFN-.alpha. subtypes
contained in HLBI, IFN-.alpha. subtypes were isolated. It was found
that each subtype can be isolated by a reverse phase HPLC method
using the .mu.Bondasphere (5.mu., C4, 300A) column manufactured by
Water's and the RP-304 column (Vydac-C4 column) manufactured by
Bio-Rad that have slightly different subtype-separation
characteristics from each other, and therefore the present method
was routinely used.
[0063] After extensive study, 12 IFN-.alpha. subtypes each being
different from one another were isolated and then each IFN-.alpha.
subtype was subjected to purity analysis using the reductive
SDS-PAGE method. Furthermore, each IFN-.alpha. subtype was
subjected to amino acid analysis, molecular weight determination by
the reductive SDS-PAGE method, and the amino acid sequencing of the
amino terminal to clarify its physical properties. In addition, in
order to elucidate the biological activity, the FL (amniotic) cell,
a human-derived cultured cell, was used as a test cell and its
anti-viral activity on Sindbis virus was determined. The
experimental method and result are described below.
Experimental Method
1) IFN-.alpha. Samples
[0064] A total of two lots, a subpool lot No. PS/199 (50 ml) and a
subpool lot No. PS/200 (50 ml) that are intermediate products of
HLBI were used as the samples for isolation of IFN-.alpha.
subtypes.
2) Fractionation of IFN-.alpha. Sample (Lot No. PS/199)
[0065] After thawing, the sample was centrifuged at a high speed of
12,000 rpm at 4.degree. C. for 10 minutes, and 45 ml of the
supernatant obtained was used. After passing 45 ml of the
supernatant at a flow rate of 3 ml/min through the Water's
.mu.Bondasphere (5.mu., C4, 300A) column (19.phi..times.150 mm)
preequilibrated with a 0.1% TFA solution, 15 ml of the 0.1% TFA
solution was run at a flow rate of 3 ml/min, and then the column
was extensively washed by passing 24 ml of the 0.1% TFA solution at
a flow rate of 8 ml/min. Then by a gradient method in which
acetonitrile concentration was increased, IFN-.alpha. subtypes were
eluted.
[0066] The eluate was monitored at A280 nm to fractionate at 4
ml/Fr. RP-HPLC was performed under the following condition.
RP-HPLC Condition
[0067] High performance liquid chromatography instrument: LC6A
manufactured by Shimadzu
[0068] Column: Water's .mu.Bondasphere (5.mu., C4, 300A) column
(19.phi..times.150 mm)
[0069] Flow rate: 8 ml/min
[0070] Eluent A: 0.1% TFA, eluent B: 0.1% TFA-95% acetonitrile
[0071] Gradient elution condition: As described in Table 1
1 TABLE 1 Time: min 0 3 35 75 95 105 115 115.1 Solution 0 30 46 52
55 60 100 0 B %
[0072] Detection: A280 nm, 0.04 aufs
[0073] Fractionation: 4 ml/Fr
[0074] 3) Fractionation of the IFN-.alpha. Sample (Lot No.
PS/200)
[0075] After thawing, the sample was centrifuged at a high speed of
12,000 rpm at 4.degree. C. for 10 minutes, and 45 ml of the
supernatant obtained was used. After passing 45 ml of the
supernatant at a flow rate of 3 ml/min through the Water's
.mu.Bondasphere (5.mu., C4, 300A) column (19.phi..times.150 mm)
preequilibrated with a 0.1% TFA solution, 15 ml of the 0.1% TFA
solution was run at a flow rate of 3 ml/min, and then the column
was extensively washed by passing 24 ml of the 0.1% TFA solution at
a flow rate of 8 ml/min. Then by a gradient method in which
acetonitrile concentration was increased, IFN-.alpha. subtypes were
eluted.
[0076] The eluate was monitored at A280 nm to fractionate at 4
ml/Fr. RP-HPLC was performed under the above condition 2).
4) Isolation of IFN-.alpha. Subtypes
[0077] Fractions obtained in the above 2) and 3) were subjected to
purification by the reverse phase HPLC method using the RP-304
column manufactured by Bio-Rad. An equal amount of a 0.1% TFA
solution was added to each fraction, and then equilibrated with the
0.1% TFA solution. After running at a flow rate of 1 ml/min through
the Bio-Rad's RP-304 (5.mu., C4, 300A) column (4.6.phi..times.250
mm), the column was extensively washed by passing 15 ml of the 0.1%
TFA solution at a flow rate of 1 ml/min. Then by a gradient method
in which acetonitrile concentration was increased, IFN-.alpha.
subtypes were eluted. The eluate was monitored at A220 nm to
fractionate at 0.3 or 0.4 ml/Fr. RP-HPLC was performed for each
fraction under the following condition. Furthermore, each
IFN-.alpha. subtype was chromatographed until a single peak was
obtained on RP-HPLC.
RP-HPLC Condition
[0078] High performance liquid chromatography instrument: 5000 LC
manufactured by Varian
[0079] Column: RP-304 (5.mu., C4, 300A) column (4.6.phi..times.250
mm) manufactured by Bio-Rad
[0080] Flow rate: 1 ml/min
[0081] Eluent A: 0.1% TFA, eluent B: 0.1% TFA-95% acetonitrile
[0082] Gradient elution condition: As described in Table 2
2 TABLE 2 Time: min 0 3 5 45 65 Solution 0 40 46 50 52 B %
[0083] Detection: A220 nm, 0.64-2.56 aufs
[0084] Fractionation: 0.3 or 0.4 ml/Fr
5) Purity Analysis by the Reverse Phase HPLC of IFN-.alpha.
Subtypes
[0085] The above isolated IFN-.alpha. subtypes were subjected to
purity analysis by a reverse phase HPLC method using the Bio-Rad's
RP-304 (5.mu., C4, 300A) column.
RP-HPLC Condition
[0086] High performance liquid chromatography instrument: LC4A
manufactured by Shimadzu
[0087] Column: RP-304 manufactured by Bio-Rad (5.mu., C4, 300A)
column (4.6.phi..times.250 mm)
[0088] Flow rate: 1 ml/min
[0089] Eluent A: 0.1% TFA-40% acetonitrile, eluent B: 0.1% TFA-50%
acetonitrile
[0090] Gradient elution condition: As described in Table 3
3 TABLE 3 Time: min 0 2 42 42.1 Solution 0 20 100 0 B %
[0091] Detection: A220 nm
6) Purity Analysis by the SDS-PAGE Method of the IFN-.alpha.
Subtype
[0092] The above isolated IFN-.alpha. subtypes were subjected to
purity analysis by the SDS-PAGE method. After about 0.1-0.3 .mu.g
of each IFN-.alpha. subtype was concentrated without heating by a
speedvac concentrator under reduced pressure, 2-mercaptoethanol was
added and then reduced at 100.degree. C. for 1.5 minutes. An
electrophoresis gel having a concentrating gel concentration of
3.5% and a separating gel concentration of 15% was used. After
preelectrophoresis at 40 mA, SDS-PAGE analysis was performed by
electrophoresing at 50 mA. After the electrophoresis, it was
stained with a CBB solution.
7) Analysis of the Amino Acid Composition of IFN-.alpha.
Subtypes
[0093] To 20-100 .mu.l of the IFN-.alpha. subtype solutions
isolated above, 1 nmol of Nle was added as an internal standard,
and concentrated by a speedvac concentrator under reduced pressure.
0.2 ml of constant boiling hydrochloric acid containing 0.1%
thioglycolic acid was added, and then sealed under reduced pressure
and hydrolyzed at 110.degree. C. for 24 hours. Using the automatic
amino acid analyzer model 835 manufactured by Hitachi Seisakusho,
the constituent amino acids that formed were analyzed by the OPA
hypo method, and amino sugars and Trp were analyzed by the OPA
method. Based on the analytical result of amino acid composition,
the protein concentration of each IFN-.alpha. subtype was
calculated.
8) Amino Terminal-Amino Acid Sequencing of IFN-.alpha. Subtypes
[0094] The above isolated IFN-.alpha. subtypes were subjected to
automatic Edman degradation using the gas-phase type protein
sequencer model 477A manufactured by Applied Biosystems, and the
PTH-amino acids that formed were identified using the PTH-amino
acid analyzer model 120A manufactured by Applied Biosystems.
9) Anti-Viral Activity of IFN-.alpha. Subtypes
[0095] As the test cell for anti-viral activity, the FL (amniotic)
cell, a human-derived cultured cell, was used. The cell was
provided by the National Institute of Health. The medium used was a
MEM medium containing 10% fetal calf serum. Cell culture was
performed at 37.degree. C. under 5% CO2. The virus used was Sindbis
virus (SBV) provided from the National Institute of Health. SBV was
used to prepared a virus stock using developing chicken eggs. As
the standard IFN for anti-viral activity determination, the
national standard Lot. J-501 (obtained from the National Institute
of Health) was used. Specific activity was expressed per mg of
protein with the concentration that inhibits 50% of the virus being
defined as one unit. The detailed method of measurement is shown
below.
[0096] First, 45,000-60,000 FL cells prepared in a 10 v/v % bovine
calf serum-Eagle's minimum essential medium were inoculated into
each well of a microtiter plate, which was incubated in a 5% carbon
dioxide incubator at 37.degree. C. for 20 hours. Then 100 .mu.l of
each IFN sample was added to each well and incubated at 37.degree.
C. for 6 hours. The culture liquid was discarded and
10.sup.5-10.sup.6 PFU of Sindbis virus per well was added, and
incubated at 37.degree. C. for 2 days. The cells were stained in a
0.02 w/v % Neutral red -5 v/v % bovine calf serum-Eagle's minimum
essential medium, and the degree of cytopathic effect was
determined by the amount of the dye incorporated.
[0097] Titer was calculated as follows. Thus, the dye incorporated
into the cell was eluted with an acidified 30 v/v % ethanol and
absorbance was determined at a wavelength of 545 m.mu.. The
experimental titer of the sample and the standard were calculated
from the dilution factor of the sample exhibiting 50% of the
absorbance of the dye incorporated into the normal cell and that of
the standard. The titer of the standard and its experimental titer
were used to determine a correction factor, which was used to
correct the experimental titers of the sample to obtain the titers
of the samples.
Experimental Results and Discussion
1) Isolation of IFN-.alpha. Subtypes
[0098] According to the above experimental methods 2) and 3), the
supernatant of the subpool lot No. PS/199 (45 ml) and the
supernatant of the subpool lot No. PS/200 (45 ml) were fractionated
by the Water's .mu.Bondasphere (5.mu., C4, 300A) column
(19.phi..times.150 mm). There were ten major fractions: fr.4, fr.8,
fr.9, fr.11, fr.14, fr.18, fr.19, fr.21, fr.24, and fr.25.
[0099] Then, according to the above experimental method 4), RP-HPLC
RP-HPLC preparation was performed for each fraction using the
Bio-Rad's RP-304 (5.mu., C4, 300A) column to obtain 12 IFN-.alpha.
subtype fractions. As a result of purity analysis performed in
accordance with the method in 5) above for each fraction by the
reverse phase HPLC method using the RP-304 (5.mu., C4, 300A)
column, every sample exhibited a single peak confirming a high
purity.
[0100] Furthermore, according to the above experimental method 6),
purity analysis by the SDS-PAGE method was performed for each
fraction after reduction treatment, and every sample exhibited a
single band confirming the high purity. The molecular weight
obtained by the SDS-PAGE method is shown in Table 4 below.
4 TABLE 4 IFN-.alpha. subtype Molecular weight fr. No. kDa 4 26.8
8A 22.3 9 21.6 11A 22.6 11B 20.9 14 22.4 18A 21.2 18'B 22.2 19B1
20.9 21 28.2 24 23.0 25 22.6
2) Amino Terminal-Amino Acid Sequence Analysis of IFN-.alpha.
Subtypes
[0101] For 12 IFN-.alpha. subtypes isolated, the amino acid
sequence of the amino terminal was analyzed according to the above
experimental method 8) to obtain the following result.
[0102] The amino terminal-amino acid sequence of IFN-.alpha.
subtypes fr.8A and fr.9 coincided with the previously published
sequence of IFN-.alpha.2b.
[0103] The amino terminal-amino acid sequence of IFN-.alpha.
subtype fr.4 coincided with the previously published sequence of
IFN-.alpha.4.
[0104] The amino terminal-amino acid sequence of IFN-.alpha.
subtype fr.11A coincided with the previously published sequence of
IFN-.alpha.21.
[0105] The amino terminal-amino acid sequence of IFN-.alpha.
subtype fr.11B coincided with the previously published sequence of
IFN-.alpha.5.
[0106] The amino terminal-amino acid sequence of IFN-.alpha.
subtype fr.18A coincided with the previously published sequence of
IFN-.alpha.17.
[0107] The amino terminal-amino acid sequence of IFN-.alpha.
subtype fr.18'B coincided with the previously published sequence of
IFN-.alpha.7.
[0108] The amino terminal-amino acid sequence of IFN-.alpha.
subtype fr.19B1 coincided with the previously published sequence of
IFN-.alpha.17.
[0109] The amino terminal-amino acid sequence of IFN-.alpha.
subtype fr.21 coincided with the previously published sequence of
IFN-.alpha.8.
[0110] The amino terminal-amino acid sequence of IFN-.alpha.
subtypes fr.24 and fr.25 coincided with the previously published
sequence of IFN-.alpha.1.
[0111] The amino terminal-amino acid sequence of IFN-.alpha.
subtype fr.14 was similar to that of the previously published
IFN-.alpha.10a (=-.alpha.C) (Nature 1981 Mar. 5: 290, 20-26),
except that the sequence was novel in that the amino acid at
position 19 was Ala in stead of Gly. We have termed this novel
subtype IIIe. The amino terminal-amino acid sequence of the subtype
IIIe is shown in SEQ ID NO: 5.
3) Anti-Viral Activity of IFN-.alpha. Subtypes
[0112] According to the above experimental method 9), anti-viral
activity on Sindbis virus (SBV) was determined. The anti-viral
activity of each IFN subtype is shown in Table 5 below.
[0113] All IFN subtypes showed anti-viral activity and the
anti-viral activity of fr.8A (IFN-.alpha.2b), a major IFN subtype
of HLBI, was 1.67.times.10.sup.8 u/mg. The IFN-.alpha. subtype that
had the highest specific activity was fr.14 (IIIe), and its
anti-viral activity was 5.21.times.10.sup.8 u/mg which is higher
than that of any other conventionally known IFN-.alpha. subtypes.
On the other hand, the IFN-.alpha. subtype that had the lowest
specific activity was fr.24, and its anti-viral activity was
0.12.times.10.sup.8 u/mg. Thus, it was demonstrated that the novel
IFN subtype fr.14 (IIIe) had the highest anti-viral activity and
IFN subtype fr.24 whose amino terminal-amino acid sequence
coincides with that of IFN-.alpha.1 had the lowest anti-viral
activity.
5 TABLE 5 Classification based on the amino IFN-.alpha. subtype
terminal-amino acid Activity .times. fr. No sequence 10.sup.8 u/mg
4 IFN-.alpha.14 1.73 8A IFN-.alpha.2b 1.67 9 IFN-.alpha.2b 1.83 11A
IFN-.alpha.21 1.69 11B IFN-.alpha.5 1.44 14 (IIIe) 5.21 18A
IFN-.alpha.17 2.63 18'B IFN-.alpha.7 1.65 19B1 IFN-.alpha.17 2.57
21 IFN-.alpha.8 1.50 24 IFN-.alpha.1 0.12 25 IFN-.alpha.1 0.20
EXAMPLE 2
Analysis of the Primary Structure of the IFN-.alpha. Subtype
IIIe
[0114] The IFN-.alpha. subtype IIIe obtained in Example 1 is a
subtype that has a novel amino acid sequence and exhibits an
unprecedentedly high specific activity. In order to determine the
primary structure of said IIIe, the following experiment was
performed.
Experimental Method
1) Analysis of Primary Structure of the IFN-.alpha. Subtype
IIIe
[0115] As the method of analyzing primary structure, a method of
cleaving the Met residue with cyanogen bromide and a method of
cleaving the basic amino acid residue with trypsin were
employed.
The Method of Cleaving a Met Residue with Cyanogen Bromide
[0116] 30 .mu.g of the IFN-.alpha. subtype IIIe was dissolved in
200 .mu.l of 70% formic acid, to which 1 .mu.mole of cyanogen
bromide was added and allowed to stand at 24.degree. C. for 20
hours. After the disappearance of the raw material IIIe was
confirmed by a reverse phase HPLC using the PR-304 column, 9
volumes of water was added to stop the reaction and then
concentrated under reduced pressure by a speedvac concentrator
equipped with a NaOH trap.
[0117] To the cyanogen bromide-fragmented peptides, 0.2 ml of a 0.5
M Tris solution (pH 8.1) containing argon gas-displaced 6M
guanidine and 2 mM EDTA was added and dissolved. After adding 0.4
.mu.mole of DTT and displaced with argon gas, it was placed in the
dark and was reduced at 37.degree. C. for 3 hours. 0.8 .mu.mole of
monoiodo acetamide that was recrystalized was added, and alkylated
in the dark at 37.degree. C. for 1 hour. After passing the reaction
solution at a flow rate of 1 ml/min through the RP-304 (5.mu., C4,
300A) column (4.6 .phi..times.250 mm) manufactured by Bio-Rad
previously equilibrated with a 0.1% TFA solution, the column was
extensively washed by passing 15 ml of the 0.1% TFA solution at a
flow rate of 1 ml/min. Then, by a gradient method in which
acetonitrile concentration was increased, the constituent peptides
were eluted. The eluate was monitored at A220 nm. It was purified
under the following RP-HPLC condition.
RP-HPLC Condition
[0118] High performance liquid chromatography instrument: 5000 LC
manufactured by Varian
[0119] Column: RP-304 (5.mu., C4, 300A) column (4.6 .phi..times.250
mm) manufactured by Bio-Rad
[0120] Flow rate: 1 ml/min
[0121] Eluent A: 0.1% TFA, eluent B: 0.1% TFA-95% acetonitrile
[0122] Gradient elution condition: As described in Table 6
6 TABLE 6 Time: min 0 60 70 Solution B % 0 60 100
[0123] Detection: A220 nm, 1.28 aufs and A280 nm, 0.16 aufs
2) The Method of Cleaving with Trypsin and the Reductive
Carboxymethylation Method
[0124] 60 .mu.g of the subtype IIIe was dissolved in 200 .mu.l of a
0.2 M NaHCO.sub.3 (pH 8.3) solution, to which 2 .mu.g Of
TPCK-Trypsin was added and the mixture was allowed to stand at
37.degree. C. for 24 hours. To the trypsin-fragmented peptides,
0.05 ml of a 0.5 M Tris solution (pH 8.1) containing argon
gas-displaced 6M guanidine and 2 mM EDTA was added and dissolved.
After adding DTT at an amount of 50 times that of the Cys residue
and displaced with argon gas, it was placed in the dark and was
reduced at 37.degree. C. for 1 hour. Monoiodo acetamide that was
recrystalized was added at an amount of 100 times that of the Cys
residue, and alkylated in the dark at 37.degree. C. for 30 minutes.
After passing the reaction solution at a flow rate of 1 ml/min
through the RP-304 (5.mu., C4, 300A) column (4.6 .phi..times.250
mm) manufactured by Bio-Rad previously equilibrated with a 0.1% TFA
solution, the column was extensively washed by passing 15 ml of the
0.1% TFA solution at a flow rate of 1 ml/min. Then, by a gradient
method in which acetonitrile concentration was increased, the
constituent peptides were eluted. The eluate was monitored at A220
nm. It was purified under the following RP-HPLC condition.
RP-HPLC Condition
[0125] High performance liquid chromatography instrument: 5000 LC
manufactured by Varian
[0126] Column: RP-304 (5.mu., C4, 300A) column (4.6 .RTM..times.250
mm) manufactured by Bio-Rad
[0127] Flow rate: 1 ml/min
[0128] Eluent A: 0.1% TFA, eluent B: 0.1% TFA-95% acetonitrile
[0129] Gradient elution condition: As described in Table 7
7 TABLE 7 Time: min 0 10 40 60 Solution 0 25 50 100 B %
[0130] Detection: A220 nm, 0.08-0.16 aufs
3) Analysis of Amino Acid Composition
[0131] To 20-100 .mu.l of the fragmented peptide solution of the
purified IFN-.alpha. subtype IIIe, 1 nmol of Nle was added as an
internal standard, which was then concentrated under reduced
pressure in a speedvac concentrator. 0.2 ml of constant boiling
hydrochloric acid containing 0.1% thioglycolic acid was added, and
then sealed and hydrolyzed at 110.degree. C. for 24 hours. Using
the automatic amino acid analyzer model 835 manufactured by Hitachi
Seisakusho, the constituent amino acids that formed were analyzed
by the OPA hypo method, and amino sugars and Trp were analyzed by
the OPA method.
4) Sequencing of Amino Terminal-Amino Acids
[0132] The fragmented peptide solution of the purified IFN-.alpha.
subtype IIIe were subjected to automatic Edman degradation using
the gas-phase type protein sequencer model 477A manufactured by
Applied Biosystems, and the PTH-amino acids that formed were
identified using the PTH-amino acid analyzer model 120A
manufactured by Applied Biosystems.
Experimental Results and Discussion
1) Structural Analysis of Cyanogen Bromide-Fragmented Peptides
[0133] After 30 .mu.g of the IFN-.alpha. subtype IIIe was
fragmented with cyanogen bromide, the reductive-carboxymethylated
peptides were purified with the RP-304 (5.mu., C4, 300A) column
(4.6 .phi..times.250 mm) manufactured by Bio-Rad. Each peptide was
subjected to amino acid analysis, amino sugar analysis, and amino
acid sequencing. For peak fractions corresponding to SEQ ID NO:
21-60, SEQ ID NO: 61-106, SEQ ID NO: 113-149, and SEQ ID NO:
150-166, amino acid sequences were identified.
2) Structural Analysis of Trypsin-Fragmented Peptides
[0134] After 80 .mu.g of the IFN-.alpha. subtype IIIe was
fragmented with trypsin, the reductive-carboxymethylated peptide
was purified with the RP-304 column. Each peptide was subjected to
amino acid analysis, amino sugar analysis, and amino acid
sequencing. For peak fractions corresponding to SEQ ID NO: 1-12,
SEQ ID NO: 14-23, SEQ ID NO: 24-50, SEQ ID NO: 51-84, SEQ ID NO:
85-121, SEQ ID NO: 122-126, SEQ ID NO: 136-145, SEQ ID NO: 146-150
and SEQ ID NO: 151-160, amino acid sequences were identified.
[0135] As hereinabove described, the cyanogen bromide-fragmentation
and trypsin-fragmentation of the IFN-.alpha. subtype IIIe and the
following structural analysis of the constituent peptides confirmed
that the IFN-.alpha. subtype IIIe is comprised of 166 amino acid
residues. The identification result of all amino acid sequences is
shown in SEQ ID NO: 4. The primary structure of the subtype IIIe
was similar to that of the previously published IFN-.alpha.10a
(=-.alpha.C) (Nature 1981 Mar. 5: 290, 20-26), except that the
sequence was novel in that the amino acid at position 19 was Ala in
stead of Gly.
EXAMPLE 3
Gene Cloning of the IFN-.alpha. Subtype IIIe
[0136] The gene of the IFN-.alpha. subtype IIIe was specifically
amplified by PCR. As primers, U-10 (SEQ ID NO: 6) and L-10 (SEQ ID
NO: 7), sequences that are specific to the subtype IIIe, were used.
Since the IFN-.alpha. gene contained no introns, genomic DNA was
used as a template for PCR. The experimental method is described
below.
[0137] The genomic DNA of Namalwa cell was prepared using the DNA
Extraction kit (Stratagene) and was used as the template for PCR.
PCR was performed under the following condition using KOD DNA
polymerase (Toyobo), and primers U-10 and L-10. Thus, a reaction
mixture comprising 1 .mu.g of the template DNA, 0.5 .mu.g of each
primer, 1.times.KOD buffer, 1 mM MgCl.sub.2, 200 .mu.M of each
dNTP, and 2.5 U KOD was subjected to, after heating at 90.degree.
C. for 3 minutes, 30 cycles of PCR with each cycle comprising
95.degree. C. for 30 seconds, 68.degree. C. for 30 seconds, and
72.degree. C. for 90 seconds, and then cooled at 4.degree. C.
[0138] This PCR product was cloned into pUC18 vector to obtain a
recombinant plasmid of the IFN-.alpha. subtype IIIe.
[0139] Thereafter, using the ABI PRISM Dye Terminator Cycle
Sequencing Ready Reaction Kit (Perkin-Elmer) the above plasmid was
subjected to a dye-terminator reaction, and using the ABI PRISM 377
DNA Sequencer (Perkin-Elmer) the base sequence was analyzed. The
determined base sequence (567 bp) is shown in SEQ ID NO: 1, and the
amino acid sequence (189 amino acids) deduced from said base
sequence is shown in SEQ ID NO: 3. The sequence of the amino acid
sequence at position 24 and after of SEQ ID NO: 3 completely
coincided with the amino acid sequence (SEQ ID NO: 4) determined in
the above Example 2. The sequence at positions 1-23 of the amino
acid sequence as set forth in SEQ ID NO: 3 corresponds to the
signal sequence.
[0140] In the comparison of the base sequence of IIIe as set forth
in SEQ ID NO: 1 with the previously reported base sequence of
IFN-.alpha.10a, three bases (positions 66, 96, and 125) were
different.
[0141] As described above, for the novel IFN-.alpha. subtype IIIe,
a complete DNA sequence containing the signal sequence portion was
obtained.
Industrial Applicability
[0142] The present invention can provide a novel human IFN-.alpha.
and its derivative having an unprecedentedly high specific
activity, and a pharmaceutical composition comprising said
IFN-.alpha. and its derivative as active ingredient.
[0143] Sequence Listing Free Text
[0144] The amino acids at positions 1, 29, and 37 as set forth in
SEQ ID NO: 5 are unknown.
Sequence CWU 1
1
7 1 567 DNA Homo sapiens 1 atg gcc ctg tcc ttt tct tta ctt atg gcc
gtg ctg gtg ctc agc tac 48 Met Ala Leu Ser Phe Ser Leu Leu Met Ala
Val Leu Val Leu Ser Tyr 5 10 15 aaa tcc atc tgt tct cta ggc tgt gat
ctg cct cag acc cac agc ctg 96 Lys Ser Ile Cys Ser Leu Gly Cys Asp
Leu Pro Gln Thr His Ser Leu 20 25 30 ggt aat agg agg gcc ttg ata
ctc ctg gca caa atg gga aga atc tct 144 Gly Asn Arg Arg Ala Leu Ile
Leu Leu Ala Gln Met Gly Arg Ile Ser 35 40 45 cct ttc tcc tgc ctg
aag gac aga cat gat ttc cga atc ccc cag gag 192 Pro Phe Ser Cys Leu
Lys Asp Arg His Asp Phe Arg Ile Pro Gln Glu 50 55 60 gag ttt gat
ggc aac cag ttc cag aag gct caa gcc atc tct gtc ctc 240 Glu Phe Asp
Gly Asn Gln Phe Gln Lys Ala Gln Ala Ile Ser Val Leu 65 70 75 80 cat
gag atg atc cag cag acc ttc aat ctc ttc agc aca gag gac tca 288 His
Glu Met Ile Gln Gln Thr Phe Asn Leu Phe Ser Thr Glu Asp Ser 85 90
95 tct gct gct tgg gaa cag agc ctc cta gaa aaa ttt tcc act gaa ctt
336 Ser Ala Ala Trp Glu Gln Ser Leu Leu Glu Lys Phe Ser Thr Glu Leu
100 105 110 tac cag caa ctg aat gac ctg gaa gca tgt gtg ata cag gag
gtt ggg 384 Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Glu
Val Gly 115 120 125 gtg gaa gag act ccc ctg atg aat gag gac tcc atc
ctg gct gtg agg 432 Val Glu Glu Thr Pro Leu Met Asn Glu Asp Ser Ile
Leu Ala Val Arg 130 135 140 aaa tac ttc caa aga atc act ctt tat cta
ata gag agg aaa tac agc 480 Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu
Ile Glu Arg Lys Tyr Ser 145 150 155 160 cct tgt gcc tgg gag gtt gtc
aga gca gaa atc atg aga tcc ctc tcg 528 Pro Cys Ala Trp Glu Val Val
Arg Ala Glu Ile Met Arg Ser Leu Ser 165 170 175 ttt tca aca aac ttg
caa aaa aga tta agg agg aag gat 567 Phe Ser Thr Asn Leu Gln Lys Arg
Leu Arg Arg Lys Asp 180 185 2 498 DNA Homo sapiens 2 tgt gat ctg
cct cag acc cac agc ctg ggt aat agg agg gcc ttg ata 48 Cys Asp Leu
Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 5 10 15 ctc ctg
gca caa atg gga aga atc tct cct ttc tcc tgc ctg aag gac 96 Leu Leu
Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30
aga cat gat ttc cga atc ccc cag gag gag ttt gat ggc aac cag ttc 144
Arg His Asp Phe Arg Ile Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe 35
40 45 cag aag gct caa gcc atc tct gtc ctc cat gag atg atc cag cag
acc 192 Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln
Thr 50 55 60 ttc aat ctc ttc agc aca gag gac tca tct gct gct tgg
gaa cag agc 240 Phe Asn Leu Phe Ser Thr Glu Asp Ser Ser Ala Ala Trp
Glu Gln Ser 65 70 75 80 ctc cta gaa aaa ttt tcc act gaa ctt tac cag
caa ctg aat gac ctg 288 Leu Leu Glu Lys Phe Ser Thr Glu Leu Tyr Gln
Gln Leu Asn Asp Leu 85 90 95 gaa gca tgt gtg ata cag gag gtt ggg
gtg gaa gag act ccc ctg atg 336 Glu Ala Cys Val Ile Gln Glu Val Gly
Val Glu Glu Thr Pro Leu Met 100 105 110 aat gag gac tcc atc ctg gct
gtg agg aaa tac ttc caa aga atc act 384 Asn Glu Asp Ser Ile Leu Ala
Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 ctt tat cta ata gag
agg aaa tac agc cct tgt gcc tgg gag gtt gtc 432 Leu Tyr Leu Ile Glu
Arg Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 aga gca gaa
atc atg aga tcc ctc tcg ttt tca aca aac ttg caa aaa 480 Arg Ala Glu
Ile Met Arg Ser Leu Ser Phe Ser Thr Asn Leu Gln Lys 145 150 155 160
aga tta agg agg aag gat 498 Arg Leu Arg Arg Lys Asp 165 3 189 PRT
Homo sapiens 3 Met Ala Leu Ser Phe Ser Leu Leu Met Ala Val Leu Val
Leu Ser Tyr 5 10 15 Lys Ser Ile Cys Ser Leu Gly Cys Asp Leu Pro Gln
Thr His Ser Leu 20 25 30 Gly Asn Arg Arg Ala Leu Ile Leu Leu Ala
Gln Met Gly Arg Ile Ser 35 40 45 Pro Phe Ser Cys Leu Lys Asp Arg
His Asp Phe Arg Ile Pro Gln Glu 50 55 60 Glu Phe Asp Gly Asn Gln
Phe Gln Lys Ala Gln Ala Ile Ser Val Leu 65 70 75 80 His Glu Met Ile
Gln Gln Thr Phe Asn Leu Phe Ser Thr Glu Asp Ser 85 90 95 Ser Ala
Ala Trp Glu Gln Ser Leu Leu Glu Lys Phe Ser Thr Glu Leu 100 105 110
Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Glu Val Gly 115
120 125 Val Glu Glu Thr Pro Leu Met Asn Glu Asp Ser Ile Leu Ala Val
Arg 130 135 140 Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Ile Glu Arg
Lys Tyr Ser 145 150 155 160 Pro Cys Ala Trp Glu Val Val Arg Ala Glu
Ile Met Arg Ser Leu Ser 165 170 175 Phe Ser Thr Asn Leu Gln Lys Arg
Leu Arg Arg Lys Asp 180 185 4 166 PRT Homo sapiens 4 Cys Asp Leu
Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 5 10 15 Leu Leu
Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30
Arg His Asp Phe Arg Ile Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe 35
40 45 Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln
Thr 50 55 60 Phe Asn Leu Phe Ser Thr Glu Asp Ser Ser Ala Ala Trp
Glu Gln Ser 65 70 75 80 Leu Leu Glu Lys Phe Ser Thr Glu Leu Tyr Gln
Gln Leu Asn Asp Leu 85 90 95 Glu Ala Cys Val Ile Gln Glu Val Gly
Val Glu Glu Thr Pro Leu Met 100 105 110 Asn Glu Asp Ser Ile Leu Ala
Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Ile Glu
Arg Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu
Ile Met Arg Ser Leu Ser Phe Ser Thr Asn Leu Gln Lys 145 150 155 160
Arg Leu Arg Arg Lys Asp 165 5 40 PRT Homo sapiens MOD_RES (1)
Unknown amino acid 5 Xaa Asp Leu Pro Gln Thr His Ser Leu Gly Asn
Arg Arg Ala Leu Ile 5 10 15 Leu Leu Ala Gln Met Gly Arg Ile Ser Pro
Phe Ser Xaa Leu Lys Asp 20 25 30 Arg His Asp Phe Xaa Ile Pro Gln 35
40 6 30 DNA Homo sapiens 6 ataggatcca ggccgaagtt caaggttatc 30 7 34
DNA Homo sapiens 7 tacaagcttc aggatcattg ccatgttgaa ccag 34
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