U.S. patent application number 09/752510 was filed with the patent office on 2001-08-30 for protein which induces interferon-gamma production by immunocompetent cell.
This patent application is currently assigned to KABUSHIKI KAISHA HAYASHIBARA SEIBUTSU KAGAKU KENKYUJO. Invention is credited to Akita, Kenji, Fujii, Mitsukiyo, Kurimoto, Masashi, Nukada, Yoshiyuki, Tanimoto, Tadao.
Application Number | 20010018212 09/752510 |
Document ID | / |
Family ID | 27299437 |
Filed Date | 2001-08-30 |
United States Patent
Application |
20010018212 |
Kind Code |
A1 |
Akita, Kenji ; et
al. |
August 30, 2001 |
Protein which induces interferon-gamma production by
immunocompetent cell
Abstract
A protein of human cell origin, which induces the IFN-.gamma.
production by immunocompetent cells and has the amino acid sequence
of SEQ ID NO:1 near at the N-terminus. It can be produced from
human cells such as lymphoblasts, lymphocytes, monoblasts,
monocytes, myeloblasts, myelocytes, granulocytes and macrophages,
and used for preventing and/or treating IFN-.gamma. susceptive
diseases.
Inventors: |
Akita, Kenji; (Okayama,
JP) ; Nukada, Yoshiyuki; (Okayama, JP) ;
Fujii, Mitsukiyo; (Okayama, JP) ; Tanimoto,
Tadao; (Okayama, JP) ; Kurimoto, Masashi;
(Okayama, JP) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.
SUITE 300
624 NINTH STREET, N.W.
WASHINGTON
DC
20001-5303
US
|
Assignee: |
KABUSHIKI KAISHA HAYASHIBARA
SEIBUTSU KAGAKU KENKYUJO
2-3, 1-chome, Shimoishii, Okayama-shi, Okayama
Okayama-shi
JP
|
Family ID: |
27299437 |
Appl. No.: |
09/752510 |
Filed: |
January 3, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09752510 |
Jan 3, 2001 |
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08832198 |
Apr 8, 1997 |
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6242255 |
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08832198 |
Apr 8, 1997 |
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08721018 |
Sep 26, 1996 |
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Current U.S.
Class: |
435/366 ;
424/85.2; 530/351 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 37/08 20180101; A61P 31/12 20180101; A61K 38/00 20130101; A61P
31/04 20180101; C07K 14/52 20130101; A61P 43/00 20180101; A61P
37/00 20180101 |
Class at
Publication: |
435/366 ;
424/85.2; 530/351 |
International
Class: |
A61K 038/20; C07K
014/54; C12N 005/08 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 26, 1995 |
JP |
270725/1995 |
Feb 29, 1996 |
JP |
67434/1996 |
Sep 20, 1996 |
JP |
REF. NO. 10050403 |
Claims
What is claimed is:
1. A purified protein which induces interferon-.gamma. production
by an immunocompetent human cell, wherein said protein has a
molecular weight of 14,000-24,000 daltons on sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of a
reducing agent, and is obtainable by separation from human cells by
the process of transplanting human hematopoietic cells to a
non-human warm-blooded animal, propagating the cells in the
non-human warm-blooded animal while allowing the cells to receive
the body fluid of the animal, collecting and disrupting the
propagated cells, collecting protein from the resulting mixture,
and separating the 14,000-24,000 dalton protein therefrom which has
the property of inducing interferon-.gamma. production by an
immunocompetent human cell.
2. A pharmaceutical composition comprising the purified protein of
claim 1 as an active ingredient and a pharmaceutically acceptable
carrier.
3. The pharmaceutical composition of claim 2, further comprising a
stabilizer.
4. The pharmaceutical composition of claim 3, wherein said
stabilizer is selected from the group consisting of albumin,
gelatin, and a combination thereof.
5. The pharmaceutical composition of claim 2, wherein said purified
protein is present in a range of 0.000001-100 w/w % on a dry solid
basis.
6. The pharmaceutical composition of claim 2, further comprising
interleukin 2.
7. The pharmaceutical composition of claim 2, further comprising
interleukin 3.
8. The pharmaceutical composition of claim 2, further comprising
interleukin 12.
9. A method for treating atopic diseases, tumors, viral diseases,
infectious diseases, or immunopathies, comprising administering to
a patient in need thereof an effective amount of the protein of
claim 1.
10. A method for treating malignant tumor, comprising: contacting
mononuclear cells and lymphocytes, isolated from peripheral blood
of a patient with malignant tumor, with a combination of the
protein of claim 1 and interleukin 2; culturing the mononuclear
cells and lymphocytes, collecting NK cells or LAK cells; and
introducing the collected NK cells or LAK cells into the patient to
exhibit cytotoxicity on malignant tumor cells.
11. A method for treating malignant tumors, comprising
administering an effective amount of the protein of claim 1 to a
subject in need thereof prior to or at the same time as the
administration of interleukin 2.
12. A method for treating leukopenia or thrombocytopenia,
comprising administering an effective amount of the protein of
claim 1 and interleukin 3 to a subject in need thereof.
13. A method for enhancing the cytotoxicity of NK cells, comprising
contacting NK cells with the protein of claim 1 to enhance the
cytotoxicity of NK cells.
14. The method of claim 13, wherein said NK cells are also
contacted with interleukin 2.
15. A method for inducing the formation of LAK cells, comprising
contacting LAK cell-containing lymphocytes with the protein of
claim 1 to induce the formation of LAK cells.
16. The method of claim 15, wherein said lymphocytes are also
contacted with interleukin 2.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a novel protein which
induces the interferon-.gamma. (hereinafter abbreviated as
"IFN-.gamma.") production by immunocompetent cells.
[0003] 2. Description of the Prior Art
[0004] It is known that IFN-.gamma. is a protein which has
antiviral-, antioncotic- and immunoregulatory-activities and is
produced by immunocompetent cells that are stimulated with antigens
or mitogens. Because of these biological activities, IFN-.gamma.
has been expected for use as an antitumor agent since it was
discovered, and studied energetically on clinical trials as a
therapeutic agent for malignant tumors in general including brain
tumors. IFN-.gamma. preparations commercially available now are
roughly classified into two groups, i.e. one group of natural
IFN-.gamma.s produced by immunocompetent cells and another group of
recombinant IFN-.gamma.s produced by transformants obtained by
introducing DNAs which encode natural IFN-.gamma.s into
microorganisms of the species Escherichia coli. In the above
clinical trials, one of these two groups of IFN-.gamma.s is
administered to patients as an "exogenous IFN-.gamma.".
[0005] Among these IFN-.gamma.s, natural IFN-.gamma.s are usually
produced by culturing established immunocompetent cell lines in
nutrient culture media admixed with IFN-.gamma. inducers to produce
IFN-.gamma.s, and purifying the produced IFN-.gamma.s from the
resulting cultures. It is known that IFN-.gamma. inducers greatly
influences on the IFN-.gamma. yield, the facility of IFN-.gamma.
purification, and the safety of final IFN-.gamma. preparations.
Generally, mitogens such as concanavalin A (Con A), lentil lectin,
pokeweed lectin, endotoxin and lipopolysaccharides can be used as
IFN-.gamma. inducers. However, these mitogens have the following
problems: (i) their molecules and qualities vary and change
depending on their origins and purification methods, and (ii)
preparations with a constant IFN-.gamma. inducibility are not
readily prepared in a satisfactory yield. In addition, most of
these mitogens might induce unfavorable side effects when
administered to living bodies, and some of them might cause
toxicity, so that it is substantially difficult to induce
IFN-.gamma. production by directly administering IFN-.gamma.
inducers to the living bodies.
SUMMARY OF THE INVENTION
[0006] The present invention was made based on a novel protein
which induces the interferon-.gamma. production by immunocompetent
cells. During the study of cytokines produced by mammalian cells,
the present inventors noticed that the existence of a substance
which induces IFN-.gamma. production in mouse liver cells which had
been treated with a lipopolysaccharide and inactivated whole cells
of Corynebacterium. They isolated the substance by many
purification methods using column chromatography as a main
technique and studied the properties and features, and have found
that the reality is a protein having the following physicochemical
properties:
[0007] (1) Molecular weight
[0008] 19,000.+-.5,000 daltons on sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE);
[0009] (2) Isoelectric point (pI)
[0010] pI of 4.8.+-.1.0 on chromatofocusing;
[0011] (3) Partial amino acid sequence
[0012] Having the partial amino acid sequences of SEQ ID NOS:8 and
9; and
[0013] (4) Biological activity
[0014] Inducing the IFN-.gamma. production by immunocompetent
cells.
[0015] The data concluded that the substance is novel because no
protein with these physicochemical properties is known. The present
inventors continued studying on mouse liver cells and have
succeeded to isolate a DNA which encodes the protein. The inventors
decoded the DNA and have found that it consists of 471 base pairs
and encodes the amino acid sequence of SEQ ID NO:10 (where the
symbol "Xaa" means "methionine" or "threonine").
[0016] Based on these findings, the present inventors further
studied on human liver cells to obtain a DNA which encodes another
novel substance that induces the IFN-.gamma. production by
immunocompetent cells. They revealed that the reality is a
polypeptide, then decoded the DNA and found that it has the amino
acid sequence of SEQ ID NO:6 (where the symbol "Xaa" is
"isoleucine" or "threonine"). They introduced the DNA into
Escherichia coli to express the polypeptide and to produce it in
the resulting culture in a satisfactorily high yield. These
findings were disclosed in Japanese Patent Laid-Open Nos. 27,189/96
and 193,098/96, applied by the present applicant. In Japanese
Patent Application No. 78,357/95 applied by the applicant, the
polypeptide is disclosed as an agent for susceptive diseases.
Although biologically active proteins which are administered to
humans after mixed with pharmaceuticals should be generally human
cell origin, no human cell which produces such a polypeptide is
reported.
[0017] In view of the foregoing, the object of the present
invention is to provide a protein of human cell origin, which
induces the IFN-.gamma. production by immunocompetent cells.
[0018] The another object of the present invention is to provide a
process for producing the protein.
[0019] The further object of the present invention is to provide
the use of the protein as an agent for susceptive diseases.
[0020] The first object of the present invention is attained by a
protein of human cell origin which induces the IFN-.gamma.
production by immunocompetent cells and has the amino acid sequence
of SEQ ID NO:1.
[0021] The second object of the present invention is attained by a
process for producing the protein by propagating human cells which
produce the protein, and collecting the protein from the propagated
cells.
[0022] The third object of the present invention is attained by an
agent for susceptive diseases, which contains the protein as an
effective ingredient.
BRIEF EXPLANATION OF THE ACCOMPANYING DRAWING
[0023] FIG. 1 is a peptide map of the present protein.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The protein according to the present invention induces the
IFN-.gamma. production by immunocompetent cells when allowed to act
on the cells alone or together with an appropriate cofactor.
[0025] The protein is derived from human cells, and it can be
readily prepared by the present process using human cells.
[0026] The agent for susceptive diseases according to the present
invention induces the IFN-.gamma. production by immunocompetent
cells in the human body when administered to humans, and exerts
positive effects in the treatment and prevention of IFN-.gamma.
susceptive diseases. When the protein augments the cytotoxicity of
killer cells or induces the formation of killer cells, it exerts
positive effects on inveterate diseases including malignant
tumors.
[0027] The preferred embodiments according to the present invention
will be described hereinafter. The wording "protein" as referred to
in the present invention means polypeptides and glycoproteins in
general which induce the IFN-.gamma. production by immunocompetent
cells and have the amino acid sequence of SEQ ID NO:1. Depending on
the types and propagation conditions of human cells, the protein
has the amino acid sequences of SEQ ID NOS:1 and 3 near at the N-
and C-termini, respectively, and occasionally has the amino acid
sequence of SEQ ID NO:6, as a complete amino acid sequence,
including the amino acid sequences of SEQ ID NOS:4 and 5 as an
internal fragment (where the symbol "Xaa" means "isoleucine" or
"threonine"). The protein is detected as a protein band at a
position corresponding to a molecular weight of 14,000-24,000
daltons, usually, 1,000-19,500 daltons when determined on sodium
dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in
the presence of a reducing agent. Depending on the types and
propagating conditions of human cells, one or more amino acids may
be added to the above N- and/or C-termini of SEQ ID NOS:1 and 3 or
one or more amino acids in the N- and/or C-termini may be defected.
Any protein can be used in the present invention as long as it is
derived from a human cell, as well as having either of these amino
acid sequences and inducing the IFN-.gamma. production when acting
on immunocompetent cells alone or together with an appropriate
cofactor.
[0028] These proteins can be produced by the present process using
human cells. Usually, the human cells used in the present invention
include cell lines derived from human hematopoietic cells such as
lymphoblasts, lymphocytes, monoblasts, monocytes, myeloblasts,
myelocytes, granulocytes and macrophages. Examples of these cell
lines are lymphomas and leukemias such as myelocytic leukemia,
promyelocytic leukemia, adult T-cell leukemia, and hairy cell
leukemia, specifically, HBL-38 cell, HL-60 cell (ATCC CCL240),
K-562 (ATCC CCL243), KG-1 cell (ATCC CCL246), Mo cell (ATCC
CRL8066), THP-1 cell (ATCC TIB202), and U-937 cell (ATCC CRL1593)
as reported by Jun MINOWADA in "Cancer Review", Vol.10, pp.1-18
(1988), and A-253 cell (ATCC HTB41), an epidermoid carcinoma,
submaxillary gland, human. Mutants of these cell lines can be also
used in the present invention. Because these cell lines readily
proliferate and more produce the present protein, they can be
advantageously used in the present invention. Especially,
epidermoid carcinoma cell lines such as A-253 cell, and human
myelomonocytic cell lines such as HBL-38 cell, HL-60 cell, KG-1
cell, THP-1 cell, and U-937 cell have an extremely high
productivity of the present protein and are most satisfactorily
used in the present invention.
[0029] In the present process, the above human cells are first
allowed to propagate, then the present protein is collected from
the propagated cells. The method used to propagate these human
cells in the present invention is not specifically restricted, and
any conventional in vivo or in vitro propagation method can be
used. The in vivo propagation method means a method to propagate
cells using nutrient culture media, which comprises suspending
human cells in RPMI 1640 medium, MEM medium and DEM medium, which
are used conventionally to propagate animal cells in this field,
supplemented with 0.3-30 w/v % of fetal bovine serum to give a cell
density of about 1.times.10.sup.4-1.times.10.sup.7 cells/ml,
preferably, about 1.times.10.sup.5-1.times.10.sup.6 cells/ml, and
culturing these cells at a temperature of 36-38.degree. C.,
preferably, a temperature of about 37.degree. C. and at a pH of
7-8, preferably, a pH of 7.2-7.4, for about 1-7 days while
replacing these media with fresh ones. Thereafter, the propagated
cells were separated from the cultures to obtain the objective
protein. Depending on the types and culture conditions of human
cells, some cells extracellularly excrete the present protein while
culturing. When coexisted in culture media inducers such as
mitogens and/or IFN-.gamma.s which induce the production of the
present protein by the human cells, most of or all of the protein
may be produced extracellularly. In this case, the protein can be
collected from the culture supernatants.
[0030] The in vivo propagation method for human cells using
warm-blooded animals excluding human comprises injecting to
suppress the immunoreaction of the animals antilymphocyte
antibodies derived from rabbits into rodents such as new born mice,
nude mice, rats, nude rats, guinea pigs, and hamsters, injecting
subcutaneously or intraperitoneally about
1.times.10.sup.5-1.times.10.sup.8 cells/animal of the human cells
into the animals or placing the human cells in diffusion chambers
embedded in or out of the animals'0 body while allowing the
animals' body fluid to circulate in the chambers, and feeding the
animals by conventional methods for about 2-10 weeks. During the
feeding, the human cells propagate while receiving the animals'
body fluid. The propagated human cells are collected in the form of
a tumor mass, ascites or cell suspension. If necessary, the
objective protein is collected after suspending and washing these
human cells in and with an appropriate solvent. The in vivo
propagation method has a merit that as compared with the in vitro
propagation method it yields the present protein at a less labor
cost and time and in a satisfactorily high yield. The in vivo
propagation method is disclosed, for example, in Japanese Patent
Publication No. 54,158/81.
[0031] To collect the present protein from the propagated cells,
these cells are disrupted by ultrasonic before or after separating
the objective protein from the cultures, homogenizing, freezing and
thawing, or by soaking these cells in considerably-low osmotic
solvents, then the protein is collected from the resulting cell
debris or from a mixture of cell debris and culture supernatant. To
collect the protein from the cell debris or the mixture, the cell
debris or the mixture can be subjected directly or after incubation
at about 37.degree. C. for 1-24 hours to the following conventional
methods for purifying biologically active substances in this field:
salting out, dialysis, filtration, concentration, separatory
sedimentation, gel filtration chromatography, ion-exchange
chromatography, hydrophobic chromatography, adsorption
chromatography, affinity chromatography, chromatofocusing, gel
electrophoresis and/or isoelectrophoresis. Two or more of these
conventional methods can be selectively used in combination. The
collected protein can be concentrated and/or lyophilized into a
liquid or solid form to meet to final use. The monoclonal antibody
as disclosed in Japanese Patent Application No. 58,240/95 applied
by the present applicant is advantageously used to purify the
present protein. Immunoaffinity chromatography using the monoclonal
antibody yields the highest possible purity of the protein at the
lowest cost and labor.
[0032] As is described above, the protein according to the present
invention has a property of inducing the IFN-.gamma. production by
immunocompetent cells. Thus it can be satisfactorily used as an
inducer for IFN-.gamma. production by cell culture methods and used
in the treatment and prevention of IFN-.gamma. susceptive diseases
including viral diseases such as AIDS and condyloma acuminatum;
malignant tumors such as malignant nephroma, granuloma, mycosis
fungoides, and brain tumor; and immunopathies such as articular
rheumatism and allergosis.
[0033] The present protein is usually added to nutrient culture
media for IFN-.gamma. production by culturing immunocompetent cells
or administering to humans to treat and/or prevent IFN-.gamma.
susceptive diseases. In the former case, leukocytes separated from
mammalian peripheral blood and established cell lines of
immunocompetent cells such as HBL-38 cell, Mo cell (ATCC CRL8066),
Jurkat cell (ATCC CRL8163), HuT78 cell (ATCC TIB161), EL4 cell
(ATCC TIB39), L12-R4 cell, and mutants thereof are suspended in
culture media containing about 0.1-1,000 ng/ml of the present
protein, preferably, about 1-100 ng/ml of the protein. If
necessary, these cells are cultured in nutrient culture media
supplemented with T-cell stimulants such as mitogen, interleukin 2,
and anti-CD3 antibody for about 1-100 hours in conventional manner
while replacing the culture media with fresh ones. From the
resulting cultures the present protein can be collected by one or
more conventional methods used to purify IFN-.gamma. such as
salting out, dialysis, filtration, concentration, separatory
sedimentation, gel filtration chromatography, ion-exchange
chromatography, hydrophobic chromatography, adsorption
chromatography, affinity chromatography, chromatofocusing, gel
electrophoresis and isoelectrophoresis.
[0034] Because the present protein induces the IFN-.gamma.
production by human immunocompetent cells, agents for susceptive
diseases containing the protein as an effective ingredient
stimulate the human immunocompetent cells to produce IFN-.gamma. by
administering to humans, and exert positive effects on the
treatment and/or the prevention of IFN-.gamma. susceptive diseases.
Killer cells participate in the treatment and/or the prevention of
susceptive diseases when the present protein induces the
IFN-.gamma. production by immunocompetent cells, accelerates the
cytotoxicity of killer cells such as cytotoxic T-cells and
lymphokine activating killer cells including NK- and LAK-cells, and
induces the formation of killer cells similarly as the proteins in
the later described Experiments and Examples. The wording
"susceptive diseases" as referred to in the present invention means
diseases in general including IFN-.gamma. susceptive diseases,
which can be treated and/or prevented by IFN-.gamma.s and/or killer
cells: For example, viral diseases such as hepatitis, herpes,
condyloma acuminatum, and AIDS; infectious diseases such as
candidiasis, malaria, cryptococcosis, and Yersinia; malignant solid
tumors such as malignant tumor, mycosis fungoides, and chronic
granulomatous disease; hematopoietic malignant tumors such as adult
T-cell leukemia, chronic myelocytic leukemia, and malignant tumor;
and immunopathies such as allergosis and rheumatism. When used with
interleukin 3, the present protein positively effects on the
complete cure or the remission of leukopenia and thrombocytopenia
induced by radio- and chemotherapies to treat leukemia, myeloma,
and malignant tumors.
[0035] The present agent for susceptive diseases is widely used in
the treatment and/or the prevention of the above susceptive
diseases as an antitumor agent, antiviral agent, antiseptic,
immunotherapeutic agent, platelet-increasing agent, or
leukocyte-increasing agent. Depending on the type of agent and the
symptom of susceptive diseases to be treated, the present agent is
generally processed into a liquid, paste or solid form which
contains 0.000001-100 w/w %, preferably, 0.0001-0.1 w/w % of the
protein, on a dry solid basis (d.s.b.).
[0036] The present agent can be used intact or processed into
compositions by mixing with physiologically-acceptable carriers,
adjuvants, excipients, diluents and/or stabilizers, and, if
necessary, further mixing with one or more other
biologically-active substances such as interferon-.alpha.,
interferon-.beta., interleukin 2, interleukin 3, interleukin 12,
TNF-.alpha., TNF-.beta., carboquone, cyclophosphamide, aclarubicin,
thiotepa, busulfan, ancitabine, cytarabine, 5-fluorouracil,
5-fluoro-1-(tetrahydro-2-furyl)uracil, methotrexate, actinomycin D,
chromomycin A.sub.3, daunorubicin, doxorubicin, bleomycin,
mitomycin C, vincristine, vinblastine, L-asparaginase, radio gold
colloidal, Krestin.RTM. picibanil, lentinan, and Maruyama vaccine.
Among these combinations, a combination of the present protein and
interleukin 2 is especially useful because interleukin 2 acts as a
cofactor for the protein when the protein induces the IFN-.gamma.
production by immunocompetent cells. Another combination of the
protein and a natural or recombinant human interleukin 2 induces a
relatively high level of IFN-.gamma. production with only a small
amount of the protein which does not substantially induce the
IFN-.gamma. production by immunocompetent cells. While a
combination of the protein and interleukin 12 induces a greater
level of IFN-.gamma. production which could not be readily attained
by them each. Because the present protein increases the activity of
interleukin 12 to inhibit the production of immunoglobulin E
antibody in the human body, the protein is advantageously used as
an agent for immunopathies such as atopic diseases including atopic
asthma, atopic bronchial asthma, hay fever, allergic rhinitis,
atopic dermatitis, angioedema, and atopic digestive system's
disorder. Occasionally a relatively small amount of interleukin 12
exists in humans. In this case, a sole administration of the
protein to humans can attain the desired effect.
[0037] The form of the present agent for susceptive diseases
includes those in a unit dose form which means a physically
formulated medicament suitable for administration and contains the
protein in an amount from 1/40 to several folds, i.e. up to 4 folds
of a dosage. Examples of these are injections, liquids, powders,
granules, tablets, capsules, sublinguals, ophthalmic solutions,
nasal drops, and suppositories.
[0038] The present agent can be orally or parenterally administered
to patients, and as described below it can be used to activate
antitumor cells in vitro. In both administrations, the agent exerts
a satisfactory effect in the treatment and/or the prevention of
susceptive diseases. Varied depending on the types of susceptive
diseases and the symptoms of patients before and after the
administration, the agent is orally administered to them or
parenterally administered to their intradermal- and
subcutaneous-tissues, muscles, and veins at a dose of about 0.1
.mu.g to 50 mg per shot, preferably, about one .mu.g to one mg per
shot, 1-4 times/day or 1-5 times/week, for one day to one year.
[0039] The present agent can be also used in so called "antitumor
immunotherapy" using interleukin 2. Generally, the antitumor
immunotherapy is roughly classified into (i) a method for directly
administering interleukin 2 to patients with malignant tumors, and
(ii) a method for introducing antitumor cells which are previously
activated in vitro by interleukin 2, i.e. an adoptive
immunotherapy. The present protein significantly enhances the above
immunotherapeutic effect by interleukin 2 when used in combination.
In the method (i), the protein is administered to patients in an
amount of about 0.1 .mu.g/shot/adult to one mg/shot/adult at 1-10
times before the administration of interleukin 2 or at the same
time. The dose of interleukin 2 is generally about 10,000-1,000,000
units/shot/adult, though it varies depending on the types of
malignant tumors, patients' symptoms, and the dose of the present
protein. In the method (ii), mononuclear cells and lymphocytes,
collected from patients with malignant tumors, are cultured in the
presence of interleukin 2 and about 0.1 ng to one .mu.g of the
protein per 1.times.10.sup.6 cells of the blood cells. After
culturing for a prescribed period of time, NK cells or LAK cells
are collected from the culture and introduced into the same
patients. Diseases which can be treated by the present antitumor
immunotherapy are, for example, hematopoietic malignant tumors such
as leukemia and malignant lymphoma, and solid malignant tumors such
as colonic cancer, rectal cancer, large intestinal cancer, gastric
cancer, thyroid carcinoma, cancer of the tongue, bladder carcinoma,
choriocarcinoma, hepatoma, prostatic cancer, carcinoma uteri,
laryngeal, lung cancer, breast cancer, malignant melanoma, Kaposi's
sarcoma, cerebral tumor, neuroblastoma, tumor of the ovary,
testicular tumor, osteosarcoma, cancer of the pancreas, renal
cancer, hypernephroma, and hemangioendothelioma.
[0040] The following experiments explain the present protein:
EXPERIMENT 1
[0041] Preparation of protein
[0042] New born hamsters were suppressed their immunoreaction in
conventional manner by injecting a rabbit antiserum to hamster
antithymus into the hamsters, transplanted to their dorsal
subcutaneous tissues with about 5.times.10.sup.5 cells/hamster of
THP-1 cells (ATCC TIB202), a myelomonocytic cell line of a human
acute monocytic leukemia, and fed for 3 weeks in conventional
manner. Tumor masses formed in their subcutaneous tissues, about 15
g weight per hamster, were extracted, dispersed in conventional
manner in physiological saline, and washed with phosphate buffered
saline (hereinafter abbreviated as "PBS").
[0043] The propagated cells thus obtained were washed with 10-fold
volumes of cold 20 mM Hepes buffer (pH 7.4) containing 10 mM
potassium chloride, 1.5 mM magnesium chloride, and 0.1 mM disodium
ethylenediaminetetraacetat- e, allowed to stand in 3-fold volumes
of a fresh preparation of the same buffer under ice-chilled
conditions, freezed at -80.degree. C., and thawed to disrupt the
cells. The disrupted cells were centrifuged to obtain a supernatant
which was then fed to a column packed with "DEAE-SEPHAROSE", a gel
for ion-exchange column chromatography commercialized by Pharmacia
LKB Biotechnology AB, Uppsala, Sweden, which had been previously
equilibrated with 10 mM phosphate buffer (pH 6.6), followed by
washing the column with 10 mM phosphate buffer (pH 6.6), feeding to
the column with a gradient buffer of sodium chloride which
increases stepwisely from 0 M to 0.5 M in 10 mM phosphate buffer
(pH 6.6), and collecting a fraction eluted at about 0.2 M sodium
chloride.
[0044] The fraction was dialyzed against 10 mM phosphate buffer (pH
6.8) and fed to a column packed with "DEAE 5PW", a gel for
ion-exchange chromatography commercialized by Tosoh Corporation,
Tokyo, Japan, followed by feeding to the column a gradient buffer
of sodium chloride which increases stepwisely from 0 M to 0.5 M in
10 mM phosphate buffer (pH 6.8), and collecting fractions eluted at
about 0.2-0.3 M sodium chloride.
[0045] The resulting fractions were pooled, then dialyzed against
PBS, fed to a plastic cylindrical column packed with a gel for
immunoaffinity chromatography using a monoclonal antibody which had
been prepared according to the method as disclosed in Japanese
Patent Application No. 58,240/95 applied by the present applicant,
and washed with PBS. The column was fed with 100 mM glycine-HCl
buffer (pH 2.5) to collect from the eluate fractions containing a
protein which induces the IFN-.gamma. production by immunocompetent
cells. These fractions were pooled, dialyzed against sterile
distilled water, concentrated with a membrane filter, and
lyophilized to obtain a purified solid protein in a yield of about
50 ng per hamster.
EXPERIMENT 2
[0046] Molecular weight
[0047] In accordance with the method reported by U. K. Laemmli in
Nature, Vol.227, pp.680-685 (1970), a purified protein prepared by
the method in Experiment 1 was electrophoresed on a sodium dodecyl
sulfate polyacrylamide gel (SDS-PAGE) in the presence of 2 w/v %
dithiothreitol, resulting in a main protein band with an
IFN-.gamma. inducibility at a position corresponding to about
18,000-19,500 daltons. The marker proteins used in this experiment
were bovine serum albumin (MW=67,000 daltons), ovalbumin (MW=45,000
daltons), carbonic anhydrase (MW=30,000 daltons), soy bean trypsin
inhibitor (MW=20,100 daltons), and .alpha.-lactalbumin (MW=14,400
daltons).
EXPERIMENT 3
[0048] Amino acid sequence and peptide mapping near at the
N-terminus
EXPERIMENT 3-1
[0049] Amino acid sequence near at the N-terminus
[0050] The purified protein in Experiment 1 was analyzed on "MODEL
473A", a protein sequencer commercialized by Perkin-Elmer Corp.,
Instrument Div., Norwalk, USA, and revealed that it has the amino
acid sequence of SEQ ID NO:1, particularly, SEQ ID NO:2.
EXPERIMENT 3-2
[0051] Peptide mapping
[0052] A purified protein obtained by the method in Experiment 1
was dissolved in an adequate amount of sterile distilled water, and
the solution was fed to a column packed with "ASAHIPAK.RTM. C4P-50
4E", a gel for high-performance liquid chromatography (HPLC)
commercialized by Showa Denko, K.K., Tokyo, Japan, which had been
previously equilibrated with 0.1 v/v % aqueous trifluoroacetic acid
solution, followed by washing the column with 0.1 v/v % aqueous
trifluoroacetic acid solution and feeding to the column a linear
gradient solution of acetonitrile increasing from 0 v/v % to 90 v/v
% in a mixture solution of trifluoroacetic acid and acetonitrile at
a flow rate of 60 ml/hour. Fractions containing a protein which
induces the IFN-.gamma. production by immunocompetent cells were
collected from the eluted fractions, pooled, neutralized with 1 M
aqueous tris solution (pH 11.2), and concentrated in conventional
manner. To 50 mM Tris-HCl buffer (pH 8.5), dissolving an adequate
amount of clostripain commercialized by Sigma Chemical Company, St.
Louis, MO., USA, was added the protein in an amount of about 50
folds of the clostripain by molar ratio while removing
acetonitrile, and the resulting mixture was allowed to react at a
pH of 8-9 and at 37.degree. C. for 12 hours to obtain a reaction
mixture containing fragments of the protein.
[0053] The reaction mixture was fed to a column packed with
"ODS-120T", a gel for HPLC commercialized by Tosoh Corporation,
Tokyo, Japan, which had been previously equilibrated with 0.1 v/v %
aqueous trifluoroacetic acid solution, followed by washing the
column with 0.1 v/v % aqueous trifluoroacetic acid solution and
feeding to the column a linear gradient solution of acetonitrile
increasing from 0 v/v % to 70 v/v % in a mixture solution of
trifluoroacetic acid, acetonitrile and water where the
concentration of trifluoroacetic acid was 0.09 v/v % at a flow rate
of 30 ml/hour while monitoring the absorption level of the peptide,
i.e. the concentration of the peptide, at a wave length of 214 nm.
FIG. 1 is the resulting peptide map.
[0054] In FIG. 1, peptide fragments eluted at about 59, 62 and 68
min after initiating the elution are respectively named peptide
fragments 1, 2 and 3. These peptide fragments were separatory
collected and analyzed for amino acid sequence on "MODEL 473A", a
protein sequencer commercialized by Perkin-Elmer Corp., Instrument
Div., Norwalk, USA, in conventional manner. As a result, it was
revealed that the peptide fragments 1 and 2 have the amino acid
sequences of SEQ ID NOS:3 and 7, respectively, while the peptide
fragment 3 has those of SEQ ID NOS:4 and 5. The comparison of these
amino acid sequences with the one of SEQ ID NO:6 revealed that the
peptide fragments 1 to 3 correspond to the positions 148-157, 1-13
and 45-58 or 80-96 in the amino acid sequence of SEQ ID NO:6,
respectively. These results confirmed that the peptide fragments 1
and 2 correspond to the C- and N-terminal fragments of the protein
used for analysis, and the peptide fragment 3 corresponds to an
internal fragment of the protein.
[0055] It is concluded that the purified protein obtained by the
method in Experiment 1 contains the amino acid sequence of SEQ ID
NO:6 when totally evaluating these results, the fact as revealed in
Experiment 2 that the purified protein has a main protein band at a
position corresponding to a molecular weight of about 18,000-19,500
daltons on SDS-PAGE, and the fact that the purified protein is
calculated to have a molecular weight of 18,199 daltons from the
amino acid sequence of SEQ ID NO:6.
EXPERIMENT 4
[0056] Biological activity
EXPERIMENT 4-1
[0057] IFN-.gamma. production by immunocompetent cell
[0058] Blood was sampled from a healthy volunteer by a heparinized
syringe and diluted by 2-fold with serum free RPMI 1640 medium (pH
7.4). The diluted blood was overlaid on a ficoll commercialized by
Pharmacia LKB Biotechnology AB, Uppsala, Sweden, followed by
centrifugation to collect lymphocytes. These lymphocytes were
washed with RPMI 1640 medium (pH 7.4) supplemented with 10 v/v %
fetal bovine serum and suspended in a fresh preparation of the same
medium to give a cell density of 5.times.10.sup.6 cells/ml. The
cell suspension was distributed to a 96-well microplate in a volume
of 0.15 ml/well.
[0059] A purified protein obtained by the method in Experiment 1
was diluted with RPMI 1640 (pH 7.4) supplemented with 10 v/v %
fetal bovine serum, and the dilution was distributed to the
microplate in a volume of 0.05 ml/well. To the microplate was added
a fresh preparation of the same buffer either with or without 2.5
.mu.g/ml Con A or 50 units/ml of a recombinant human interleukin 2
in a volume of 0.05 ml/well, and the microplate was incubated at
37.degree. C. for 24 hours in a 5 v/v % CO.sub.2 incubator. After
completion of the culture, 0.1 ml of a culture supernatant was
sampled from each well and assayed for IFN-.gamma. activity by
conventional enzyme immunosorbent assay (EIA). As a control, a
system free of the purified protein was provided and treated
similarly as above. The results were in Table 1 where the
IFN-.gamma. content was assayed and expressed in terms of
international unit (IU) with respect to "Gg23-901-530", an
international standard for IFN-.gamma. obtained from the National
Institute for Health, Bethesda, Md., USA.
1 TABLE 1 IFN-.gamma. yield (IU/ml) Protein concentration Protein
Protein (ng/ml) Protein plus Con A plus interleukin 2 0 <0.5
<2 <0.5 0.32 <0.5 6 .+-. 2 2 .+-. 1 1.6 10 .+-. 2 70 .+-.
20 60 .+-. 20 8 140 .+-. 10 490 .+-. 80 570 .+-. 30 40 180 .+-. 20
620 .+-. 10 880 .+-. 50 200 260 .+-. 20 800 .+-. 20 1500 .+-. 400
Note: In the table, the wording "protein" means the present
protein.
[0060] The results in Table 1 show that lymphocytes as an
immunocompetent cell produced IFN-.gamma. by the action of the
present protein. As is evident from the results, the IFN-.gamma.
production is increased in the presence of interleukin 2 or Con A
as a cofactor.
EXPERIMENT 4-2
[0061] Increase of cytotoxicity by NK cell
[0062] Blood was sampled from a healthy volunteer by a heparinized
syringe and diluted with PBS by 2-fold. The dilution was overlaid
on a ficoll, and the resultant was centrifuged to obtain a high
density layer of lymphocytes. The lymphocytes were suspended in
RPMI 1640 medium (pH 7.2) containing 10 .mu.g/ml kanamycin,
5.times.10.sup.-5 M 2-mercaptoethanol and 10 v/v fetal bovine
serum, and the suspension was distributed to a 12-well microplate
in a volume of 0.5 ml/well. A purified protein obtained by the
method in Experiment 1 was appropriately diluted with a fresh
preparation of the same buffer, and the dilution was distributed to
the microplate in a volume of 1.5 ml/well, followed by adding to
the microplate 0.5 ml/well of a fresh preparation of the same
buffer either with or without 50 units/ml of a recombinant human
interleukin 2, incubating the microplate at 37.degree. C. for 24
hours in a 5 v/v % CO.sub.2 incubator, and washing the resultant
cells with PBS to obtain cultured lymphocytes containing NK cells
as an effector cell. 1.times.10.sup.4 cells/well aliquots of K-562
cells (ATCC CCL243), derived from human chronic myelocytic leukemia
as a NK-cell susceptive target cell, which had been labelled with
.sup.51Cr in conventional manner, were distributed to a 96-well
microplate, and mixed with the above NK cells in a ratio of 2.5:1,
5:1 or 10:1 (=(effector cells):(target cells)). The microplate was
incubated at 37.degree. C. for 4 hours in a 5 v/v % CO.sub.2 or,
followed by counting the radio activity of each supernatant to
count the dead target cells. In each system, the percentage (%) of
the dead target cells with respect to the target cells used in this
experiment was calculated for evaluating cytotoxicity. The results
were in Table 2.
2TABLE 2 Protein Cytotoxicity concentration Effector cells/Target
cells (pM) Concentration of interleukin 2 2.5/1 5/1 10/1 0 0 19 36
59 0 10 28 44 61 0.5 0 22 41 63 0.5 10 31 54 69 5 0 28 49 66 5 10
36 58 71 50 0 29 53 67 50 10 42 62 72 500 0 33 56 84 500 10 57 78
96 Note: In the table, the symbol "pM" means 10.sup.-12 M, and the
wording "protein" means the present protein.
[0063] The results in Table 2 show that the protein according to
the present invention has a property of enhancing the cytotoxicity
by NK cells. As is evident from the results, the cytotoxicity is
more enhanced by the coexisting interleukin 2.
EXPERIMENT 4-3
[0064] Induction of LAK cell formation
[0065] 1.times.10.sup.4 cells/well aliquots of Raji cell (ATCC
CCL86), a human Burkitt's lymphoma as an NK-cell non-susceptive
target cell labelled with .sup.51Cr in conventional manner were
distributed to a 96-well microplate, and mixed with a cell
suspension of the target cells and cultured lymphocytes containing
LAK cells as an effector cell, prepared similarly by the method in
Experiment 4-2 except for culturing 72 hours, in a ratio of 5:1,
10:1 or 20:1 (=(effector cells):(target cells)), followed by
incubating the microplate at 37.degree. C. for 4 hours in a 5 v/v %
CO.sub.2 incubator and counting the radio activity of each
supernatant in conventional manner. Thereafter, the cytotoxicity
(%) was calculated similarly as in Experiment 4-2. The results were
in Table 3.
3TABLE 3 Protein Cytotoxicity concentration Effector cells/Target
cells (pM) Concentration of interleukin 2 5/1 10/1 20/1 0 0 12 23
31 0 10 14 25 35 0.5 0 14 24 34 0.5 10 18 32 42 5 0 16 26 37 5 10
21 36 50 50 0 22 41 49 50 10 26 52 56 500 0 27 44 61 500 10 33 59
72 Note: In the table, the symbol "pM" means 10.sup.-12 M, and the
wording "proteint" means the present protein.
[0066] The results in Table 3 show that the present protein has a
property of inducing the LAK-cell formation. As is evident from
these results, this induction is more enhanced by the coexisting
interleukin 2.
EXPERIMENT 5
[0067] Acute toxicity test
[0068] A purified protein obtained by the method in Experiment 1
was injected percutaneously, orally or intraperitoneally into
8-week-old mice in conventional manner. As a result, the LD.sub.50
of the protein was about one mg/kg mouse or higher independent of
these administration routes. This evidences that the present
protein is safe to incorporate into medicaments which are
administrable to humans.
[0069] It is well known that IFN-.gamma. deeply relates to the
inhibition of bacterial infection and the propagation of malignant
tumors, the regulation of human biophylaxis through the
immunoregulatory function, and to the inhibition of immunoglobulin
E antibody's production. As is described above, IFN-.gamma. is now
commercially available and used as an agent for human susceptive
diseases, and the diseases to be treated, dose, administration, and
safety are almost revealed. It is described in "Cytokines in Cancer
Therapy", edited by Frances R. Balkwill, translated by Yoshihiko
WATANABE, published by Tokyo-Kagaku-Dojin, Tokyo, Japan (1991) that
treatments using killer cells such as NK- and LAK-cells are used as
an antitumor immunotherapy and applied to human diseases, and
reported that most of them exert a satisfactory therapeutic effect.
Recently focussed is the relationship between the therapeutic
effect and the augmentation of killer cells' cytotoxicity or the
induction of killer cells' formation using cytokines. For example,
T. Fujioka et al. reported in "British Journal of Urology", Vol.73,
No. 1, pp.23-31 (1994) that interleukin 2 strongly induced the
formation of LAK cells in an antitumor immunotherapy using LAK
cells and interleukin 2, and exerted a satisfactory effect on the
metastasis of human cancer without substantially inducing serious
toxicity and side effects.
[0070] Thus it is revealed that IFN-.gamma. and killer cells
closely relate to the treatment and the prevention of human
diseases for complete cure and remission. Under these backgrounds
as shown in the results in Experiments 4 and 5, the fact that the
present protein induces the IFN-.gamma. production by
immunocompetent cells, enhances the NK cells' cytotoxicity, and
induces the LAK cells' formation indicates that the present agent
containing the protein can be administered to humans over a
relatively long period of time and exerts a satisfactory
therapeutic effect on the treatment and the prevention of
IFN-.gamma. and/or killer cell related diseases without
substantially inducing serious side effects.
[0071] The following Examples explain the preferred embodiments of
the present invention in more detail. Examples A-1 to A-8 are the
preferred embodiments of the preparation of the present protein,
and Examples B-1 to B-6 are the preferred embodiments of the
present agent for susceptive diseases:
EXAMPLE A-1
[0072] Preparation of protein
[0073] New born hamsters were suppressed their immunoreaction in
conventional manner by injecting a rabbit antiserum to hamster
antithymus into the hamsters, transplanted to their dorsal
subcutaneous tissues with about 5.times.10.sup.5 cells/hamster of
THP-1 cells (ATCC TIB202), a myelomonocytic cell line of a human
acute leukemia, and fed for 3 weeks in conventional manner. Tumor
masses, about 15 g weight each, subcutaneously formed in each
hamster were extracted, suspended in physiological saline in
conventional manner, and washed with PBS.
[0074] In accordance with the method by Matthew J. Kostura et al.
in "Proceedings of the National Academy of Sciences of the United
States of America", Vol.86, pp.5,227-5,231 (1989), the suspended
cells were washed with 10-fold volumes of cold 20 mM Hepes buffer
(pH 7.4) containing 10 mM potassium chloride, 1.5 mM magnesium
chloride, 0. 1 mM disodium ethylenediaminetetraacetate, allowed to
stand in 3-fold volumes of a fresh preparation of the same buffer,
allowed to stand for 20 min under ice-chilled conditions,
lyophilized at -80.degree. C., and thawed to disrupt cells. The
disrupted cells were centrifuged, and the supernatant was fed to a
column packed with "DEAE-SEPHAROSE", a gel for ion-exchange
chromatography commercialized by Pharmacia LKB Biotechnology AB,
Uppsala, Sweden, followed by washing the column with 10 mM
phosphate buffer (pH 6.6), fed with a gradient buffer of sodium
chloride increasing stepwisely from 0 M to 0.5 M, and collecting
fractions eluted at about 0.2 M sodium chloride.
[0075] The fractions were pooled, dialyzed against 10 mM phosphate
buffer (pH 6.8), fed to a column packed with "DEAE 5PW", a gel for
ion-exchange chromatography commercialized by Tosoh Corporation,
Tokyo, Japan, which had been previously equilibrated with 10 mM
phosphate buffer (pH 6.8), fed with a linear gradient buffer of
sodium chloride increasing from 0 M to 0.5 M in 10 mM phosphate
buffer (pH 6.8), and collected fractions eluted at about 0.2-0.3 M
sodium chloride.
[0076] The resulting fractions were pooled and dialyzed against
PBS. The dialyzed inner solution was fed to a cylindrical plastic
column prepared by first packing a gel for immunoaffinity
chromatography of a monoclonal antibody, which had been prepared
according to the method disclosed in Japanese Patent Application
No. 58,240/95 applied by the present applicant, then washing with
PBS. One hundred mM glycine-HCl buffer (pH 2.5) was fed to the
column to effect fractionation, followed by collecting fractions
containing a protein which induces the IFN-.gamma. production by
immunocompetent cells from the eluate, dialyzing the fractions
against sterile distilled water, concentrating the dialyzed inner
solution with a membrane filter, and lyophilizing the concentrate
to obtain a solid purified protein. The yield was about 50 ng per
hamster.
EXAMPLE A-2
[0077] Preparation of protein
[0078] New born nude mice were injected into their dorsal
subcutaneous tissues with about 1.times.10.sup.6 cells/nude mouse
of KG-1 cells (ATCC CCL246), a myelomonocytic cell line derived
from human acute myelomonocytic leukemia, and fed for 4 weeks in
conventional manner. Tumor masses, about 20 g weight each, formed
subcutaneously in each nude mouse were extracted and dispersed in
physiological saline in conventional manner. The cells were washed
and disrupted similarly as in Example A-1, and the resulting
mixture was purified to obtain a purified protein which induces the
IFN-.gamma. production by immunocompetent cells in a yield of about
20 ng per nude mouse.
[0079] A portion of the purified protein was analyzed for amino
acid sequence in accordance with the method in Experiments 2-4,
revealing that the protein has the partial amino acid sequence of
SEQ ID NO:1 near at the N-terminus and a similar molecular weight
and biological activity as the protein in Experiment 1.
EXAMPLE A-3
[0080] Preparation of protein
[0081] HL-60 cells (ATCC CCL240), a myelomonocytic cell line
derived from human acute promyelocytic leukemia, were suspended in
RPMI 1640 (pH 7.4) placed in an about 10-ml plastic cylindrical
diffusion chamber in which was installed a membrane filter with a
diameter of 0.5 .mu.m, then the chamber was intraperitoneally
embedded in an aged rat. The rat was fed for 4 weeks in
conventional manner, then the chamber was removed. The propagated
cells in the chamber were collected, washed with physiological
saline, and disrupted similarly as in Example A-1, followed by
purifying the resulting mixture to obtain a purified protein which
induces the IFN-.gamma. production by immunocompetent cells. The
yield was about 20 ng per rat.
[0082] A portion of the purified protein was analyzed for amino
acid sequence in accordance with the method in Experiments 2-4,
revealing that the protein has the partial amino acid sequence of
SEQ ID NO:1 near at the N-terminus and has a similar molecular
weight and biological activity to the protein in Experiment 1.
EXAMPLE A-4
[0083] Preparation of protein
[0084] THP-1 cells (ATCC TIB202), a myelomonocytic cell line
derived from human acute monocytic leukemia, were suspended in RPMI
1640 medium (pH 7.2) supplemented with 10 v/v % fetal bovine serum
to give a cell density of about 3.times.10.sup.5 cells/ml, and
cultured at 37.degree. C. for 3 weeks in a 10 v/v % CO.sub.2
incubator while replacing the medium with a fresh one. The
propagated cells were separated from the resulting culture, washed
with physiological saline, and disrupted similarly as in Example
A-1, followed by purifying the resulting mixture to obtain a
purified protein which induces the IFN-.gamma. production in a
yield of about 10 ng per litter of the culture.
[0085] A portion of the purified protein was analyzed for amino
acid sequence in accordance with the method in Experiments 2-4,
revealing that the protein has the partial amino acid sequence of
SEQ ID NO:1 near at the N-terminus and has a similar molecular
weight and biological activity to the protein in Experiment 1.
EXAMPLE A-5
[0086] Preparation of protein
[0087] New born hamsters were immunosuppressed by injecting a
rabbit antithymus serum in conventional manner, injected to the
dosal subcutaneous tissues with about 5.times.10.sup.5 cells/head
of A-253 cells (ATCC HTB41), an epidermoid carcinoma, submaxillary
gland, human, and fed for 3 weeks in usual manner. Thereafter, the
tumor masses formed subcutaneously, about 10 g weight in each
hamster, were extracted, dispersed in physiological saline, and
washed with PBS.
[0088] The propagated cells thus obtained were washed with 20 mM
Hepes buffer (pH 7.4) containing 10 mM potassium chloride, 1.5 mM
magnesium chloride, and 0.1 mM disodium
ethylenediaminetetraacetate, suspended in a fresh preparation of
the same buffer to give a cell density of about 2.times.10.sup.7
cells/ml, disrupted by a homogenizer, and centrifuged to remove
cell debris to obtain a supernatant, followed by concentrating the
supernatant by a membrane for ultrafiltration to obtain a cell
extract containing a protein which induces the interferon-.gamma.
production by immunocompetent cells. The extract was purified
similarly as the method in Example A-1, concentrated, and
lyophilized to obtain a solid purified protein in a yield of about
3 .mu.g of per hamster.
[0089] The purified protein was sampled and analyzed in accordance
with the methods in Examples 2-4 revealing that it has the amino
acid sequence of SEQ ID NO:1 nearness to the N-terminus and has a
similar molecular weight and biological activities to those of the
protein in Experiment 1.
EXAMPLE A-6
[0090] Preparation of protein
[0091] A seed culture of A-253 cell was inoculated into RPMI 1640
medium (pH 7.4) supplemented with 10 v/v % fetal calf serum and
cultured in conventional manner at 37.degree. C. until forming a
monolayer of cells. Thereafter, the cells were detached from the
surface of the culture vessel used by using "TRYPSIN-EDTA", a
trypsin commercialized by Gibuco BRL, NY, USA, and washed with PBS.
In accordance with the method in Example A-1, the cells were
disrupted, and the disrupted cells were purified and centrifuged to
obtain a supernatant which was then incubated at 37.degree. C. for
6 hours, purified, concentrated, and lyophilized to obtain a solid
purified protein which induces the IFN-.gamma. production by
immunocompetent cells in a yield of about one .mu.g per 10.sup.7
cells.
[0092] The supernatant was sampled and analyzed in accordance with
the method in Experiments 2-4 revealing that it has the amino acid
sequence of SEQ ID NO:1 near at the N-terminus and has a similar
molecular weight and biological activities to those of the protein
in Experiment 1.
EXAMPLE A-7
[0093] Preparation of protein
[0094] A seed culture of A-253 cell was inoculated into RPMI 1640
medium (pH 7.4) supplemented with 10 v/v % fetal calf serum and
cultured in conventional manner at 37.degree. C. until forming a
monolayer of cells. Thereafter, the culture medium was replaced
with a serum-free RPMI 1640 medium (pH 7.4) supplemented with 10
IU/ml of a natural IFN-.gamma. derived from KG-1 cell as an
IFN-.gamma. inducer, and incubated at 37.degree. C. for 48 hours.
The culture was centrifuged to obtain a supernatant which was then
purified by the method in Example A-1, concentrated, and
lyophilized to obtain a solid purified protein which induces the
IFN-.gamma. production by immunocompetent cells in a yield of about
5 ng per 10.sup.7 cells.
[0095] The supernatant was sampled and analyzed in accordance with
the method in Experiments 2-4 revealing that it has the amino acid
sequence of SEQ ID NO:1 nearness to the N-terminus and has a
similar molecular weight and biological activities to those of the
protein in Experiment 1.
EXAMPLE A-8
[0096] Preparation of protein
[0097] A purified protein obtained by the method in Example A-1 was
dissolved in an adequate amount of sterile distilled water, and the
solution was fed to a column packed with "ASAHIPAK.RTM. C4P-50 4E",
a gel for high-performance liquid chromatography commercialized by
Showa Denko K.K., Tokyo, Japan, which had been previously
equilibrated with 0.1 v/v % aqueous trifluoroacetic acid, followed
by washing the column with 0.1 v/v % aqueous trifluoroacetic acid
and feeding to the column a linear gradient solution of
acetonitrile increasing from 0 v/v % to 90 v/v % in a mixture
solution of trifluoroacetic acid and acetonitrile at a flow rate of
60 ml/hour. Fractions containing a protein which induces the
IFN-.gamma. production by immunocompetent cells were collected from
the eluted fractions, pooled, neutralized with 1 M aqueous tris
solution (pH 11.2), and concentrated in conventional manner,
followed by removing acetonitrile from the resulting concentrate to
obtain a concentrated protein with a purity of at least 95% in a
yield of about 10% by weight with respect to the material protein,
d.s.b.
[0098] In accordance with the method in Experiment 2, the
concentrated protein was sampled and analyzed for molecular weight,
resulting in a single protein band, which induces an IFN-.gamma.
production, at a position corresponding to a molecular weight of
18,400.+-.1,000 daltons. Another fresh sample was analyzed for
amino acid sequence in accordance with the method in Experiments 3
and 4, revealing that it has the amino acid sequence of SEQ ID NO:3
and the one of SEQ ID NO:1 near at the N-terminus, more
particularly, the one of SEQ ID NO:7, and further it has the amino
acid sequence of SEQ ID NOS:4 and 5 as an internal fragment and
exhibited a similar biological activity to the protein of
Experiment 1 even when concentrated into a relatively high
level.
EXAMPLE B-1
[0099] Liquid
[0100] A purified protein obtained by the method in Example A-1 was
dissolved in physiological saline containing one w/v % human serum
albumin as a stabilizer, followed by sterilely filtering the
solution to obtain a liquid.
[0101] The product with a satisfactory stability can be used as an
injection, collunarium or nebula to treat and/or prevent susceptive
diseases such as malignant tumors, viral diseases, bacterial
infections, and immunopathies.
EXAMPLE B-2
[0102] Dried injection
[0103] A purified protein obtained by the method in Example A-2 was
dissolved in physiological saline containing one w/v % of a
purified gelatin as a stabilizer, and the solution was sterilely
filtered in conventional manner. The sterile solution was
distributed to vials by one ml and lyophilized, then the vials were
cap sealed.
[0104] The product with a satisfactory stability can be used as an
injection, collunarium or nebula to treat and/or prevent susceptive
diseases such as malignant tumors, viral diseases, bacterial
infections, and immunopathies.
EXAMPLE B-3
[0105] Dry injection
[0106] A solid pharmaceutical was prepared similarly as in Example
B-2 except for using a purified protein obtained by the method in
Example A-5 and "TREHAOSE", a crystalline trehalose powder
commercialized by Hayashibara Co., Ltd., Okayama, Japan, as a
stabilizer.
[0107] The product with a satisfactorily stability can be
advantageously used as a dry injection for treating and/or
preventing malignant tumors, viral diseases, bacterial infections,
and immunophathies.
EXAMPLE B-4
[0108] Ointment
[0109] "HI-BIS-WAKO 104", a carboxyvinylpolymer commercialized by
Wako Pure Chemicals, Tokyo, Japan, and "TREHAOSE", a crystalline
trehalose powder commercialized by Hayashibara Co., Ltd., Okayama,
Japan, were dissolved in sterile distilled water in respective
amounts of 1.4 w/w % and 2.0 w/w %, and the solution was mixed to
homogeneity with a purified protein obtained by the method in
Example A-3, then adjusted to pH 7.2 to obtain a paste containing
about one mg of a purified protein per g of the paste.
[0110] The product with a satisfactory spreadability and stability
can be used as an injection, collunarium or nebula to treat and/or
prevent susceptive diseases such as malignant tumors, viral
diseases, bacterial infections, and immunopathies.
EXAMPLE B-5
[0111] Tablet
[0112] A purified protein obtained by the method in Example A-4 and
"LUMIN (1-1'-1"-triheptyl-11-chinolyl(4).4.4'
-penthamethinchynocyanine-1-1"-dij- odide)" as a cell activator
were mixed to homogeneity with "FINETOSE.RTM.", an anhydrous
crystalline .alpha.-maltose powder commercialized by Hayashibara
Co., Ltd., Okayama, Japan, and the mixture was tabletted in
conventional manner to obtain tablets, about 200 mg weight each,
containing the purified protein and LUMIN in an amount of one mg
each.
[0113] The product with a satisfactory swallowability, stability
and cell-activating activity can be used as an injection,
collunarium or nebula to treat and/or prevent susceptive diseases
such as malignant tumors, viral diseases, microbism, and
immunopathies.
EXAMPLE B-6
[0114] Agent for adoptive immunotherapy
[0115] Human monocytes were separated from peripheral blood of a
patient with malignant lymphoma, suspended in RPMI 1640 medium (pH
7.2), which had been supplemented with 10 v/v % human AB serum and
preheated at 37.degree. C., to give a cell density of about
1.times.10.sup.6 cells/ml, mixed with about 10 ng/ml of a purified
protein obtained by the method in Example A-1 and about 100
units/ml of a recombinant human interleukin 2, and incubated at
37.degree. C. for one week, followed by centrifugally collecting
LAK cells.
[0116] The LAK cells exerted a strong cytotoxicity on lymphoma
cells when introduced into the patient, and the therapeutic effect
is significantly higher than that of the conventional adoptive
immunotherapy using interleukin 2 alone. Cytotoxic T-cells,
obtained by treating a patient's tumor tissue invasive lymphocyte
instead of the patient's monocytes, showed a similar effect as in
the LAK cells when reintroduced into the patient. The agent for
adoptive immunotherapy can be suitably applied to solid tumors such
as malignant nephroma, malignant melanoma, large intestinal cancer,
and lung cancer.
[0117] As is described above, the present invention was made based
on a novel protein which induces the IFN-.gamma. production by
immunocompetent cells and a discovery of human cells which produce
the protein. The protein with a partly revealed amino acid sequence
stably induces the IFN-.gamma. production by immunocompetent cells.
Therefore, the protein can be used widely as an IFN-.gamma. inducer
for IFN-.gamma. production by culturing cells, and a therapeutic
and/or prophylactic agent for IFN-.gamma. susceptive diseases such
as viral diseases, malignant tumors, and immunopathies which are
susceptible to IFN-.gamma.. The present agent for susceptive
diseases which contains the protein as an effective ingredient
exerts an outstanding effect on the treatment of inveterate
diseases such as malignant tumors.
[0118] Because the protein has a strong IFN-.gamma. production
inducibility and has a relatively low toxicity, it induces
generally a desired level of IFN-.gamma. production with only a
small amount and does not substantially cause serious side effects
even when administered to patients at a relatively high dose.
Therefore, the protein is advantageous in that it quickly induces a
desired level of IFN-.gamma. production without strictly
controlling the dose. Especially, the present protein of human cell
origin is advantageous in that it less causes side effects and less
induces antibodies when administered to humans in the form of a
pharmaceutical composition as compared with artificially produced
polypeptides by the recombinant techniques.
[0119] The present protein having these satisfactory properties can
be produced in a desired amount by the present process using human
cells.
[0120] Thus the present invention with these significant functions
and effects is a significant invention which greatly contributes to
this field.
[0121] While there has been described what is at present considered
to be the preferred embodiments of the invention, it will be
understood the various modifications may be made therein, and it is
intended to cover in the appended claims all such modifications as
fall within the true spirit and scope of the invention.
* * * * *