JPS63203626A - Immunosuppression agent - Google Patents

Abstract

PURPOSE:To obtain an immunosuppression agent containing secretor IL-2 receptor as an active ingredient. CONSTITUTION:An immunosuppression agent containing secretor IL-2 receptor [a receptor having properties specifically combining with IL-2, soluble in an aqueous medium and having such properties as to be produced by a transformed strain and secreted into a culture medium when DNA sequence coding the IL-2 receptor is carried on a proper vector plasmid and introduced into a proper host in order to transform, e.g. a receptor having 40-43kd molecular weight, pI5.2-4.4 and the following amino acid sequence (N-terminated)] as an active ingredient. The agent is effective to remedy and prevention for an autoimmune disease, organ, rejection to bone marrow transplantation, etc., and can continuously be used without side effect. Intravenous injection is preferably used, though there are injection and drops in the formulation. The agent is preferably administered in an amount of 1-50ml/kg/day using the receptor having concentration of 5-250mg/ml.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to an immunosuppressant useful for the treatment and prevention of autoimmune diseases, organ transplant rejection, and the like.

[Conventional technology]

In recent years, immunotherapy has become important as a means that can be clinically applied to patients with immune diseases including various infectious diseases, cancer, immunodeficiency diseases, autoimmune diseases, organ transplant rejection, and the like. This has led to attempts to develop therapeutic and preventive agents for many immune diseases.

In the immune system, each immunocompetent cell possesses a variety of functions, and in recent years, many factors, ie, lymphokines, that stimulate or suppress activation or differentiation induction between cells have been discovered. Lurinhokine was previously produced in small amounts by lymphocytes, etc., but recently several products have been discovered that can be mass-produced using genetic engineering techniques.
Furthermore, using purified products, their effects on immunocompetent cells have been demonstrated, and the mechanism of action between cells has gradually been elucidated.

For example, interleukin 2 (hereinafter referred to as rrt, -2°) is produced by helper hypolymph cells, and killer T
It has been shown to be involved in the proliferation and differentiation of lymphoid cells, and its effects on other cells, including inducing differentiation of NK (natinal killer cells), LAK (lymphokine activated cells), and B lymphocytes, are currently being elucidated. It's arrived. As a result, in vivo animal experiments and clinical applications using IL-2 are currently being put into practical use.

On the other hand, it has been suggested that diseases such as autoimmune diseases and allergies may be caused by an abnormal balance of these lymphokines acting on immunocompetent cells. Furthermore, in organ and bone marrow transplants, the host-versus-graft reaction (H
ost versus Graftreaction
: HV G reaction) or transplant versus host reaction (Gr
aft vs host reaction:
It has been shown that killer T cells activated by lymphokines, mainly IL-2, are deeply involved in the onset of G V H reactions.

In this way, in autoimmune diseases, organ transplant rejection, etc., the immune reactions that are triggered in the body are shown as responses that damage the body itself, so activation of immunocompetent cells in these reactions, It is necessary to specifically and effectively suppress functional expression.

Immunosuppressive therapies that have been used clinically for autoimmune diseases and organ transplants include administration of DNA synthesis inhibitors such as methotrexate, azathioprine, 6-mercaptopurine (6-MP), and endoxan, steroids, etc. X-ray irradiation has been the mainstream, but problems have long been pointed out regarding the sustained use of this drug due to the low graft survival rate and side effects.

However, in recent years, cyclosphorin A (csA
) has dramatically improved the survival rate of organ transplants. It has already been revealed that the mechanism of action of CS A is through specific inhibition of IL-2 production from helper T cells; No clear conclusion has yet been reached regarding the effect on expression.

However, despite this dramatic improvement in the survival rate of grafts, it has been reported that C9A has the effect of inducing severe renal failure, and when using CsA, the administration schedule must be adjusted. It has been raised that there is a need to pay sufficient attention to the dosage and duration of administration. Therefore, the development of safer immunosuppressants is strongly desired.

[Problem that the invention seeks to solve]

Therefore, an object of the present invention is to provide an immunosuppressant that has an effective immunosuppressive effect for the treatment and prevention of autoimmune diseases, organ transplant rejection, etc., and can be used continuously without side effects. That's true.

[Means for solving problems]

Under these circumstances, as a result of intensive research, the present inventors discovered a concept that had not been realized until now, that is, the property of IL-2 to bind to the IL-2 receptor with high specificity. IL-2 plays a central role in immune response regulation.
We attempted to specifically and artificially control the functional expression of As a result, they discovered that the secreted IL-2 receptor specifically and effectively suppresses the functional expression of IL-2, and that it can be effectively used as an immunosuppressant, leading to the completion of the present invention. That is, the present invention is an immunosuppressant containing a secreted IL-2 receptor as an active ingredient.

The secreted IL-2 receptor used in the present invention is I
A suitable vector plasmid carries a DNA sequence that has the property of specifically binding to L-2, is soluble in an aqueous medium, and codes for this secreted IL-2 receptor, and is introduced into a suitable host. A substance that has the property of being produced by a transformed strain and secreted into the culture medium when transformed. More specifically, at least the polypeptide portion starting from the 220th (Val) position counting from the N-terminal amino acid (GJu) of the IL-2 receptor was deleted. “Extrace-11ula”
2 of the IL-2 receptor.
The polypeptide portion from positions 21 to 238 has thick hydrophobicity, and when produced by an IL-2 receptor producing strain, it is captured in the cell membrane, so it is called the “cell membrane region (tr)”.
ansmembrane domain)”
Also, the part that exists inside the cell downstream from this is the “intracellular domain (cytoplasmic domain)”.
”. The secreted IL-2 receptor of the present invention is
From the IL-2 receptor to at least the “cell membrane region” and “
(1) By removing the "intracellular region", the protein is made soluble in aqueous medium while retaining the ability to bind to L-2.

Specific examples of such secreted IL-2 receptors include those having the following properties.

(a) Molecular weight = 40 to 43Kd et al.) p15.2 to 4.4 (c) Amino acid sequence (N-terminus) Glu-Leu-Cys-Asp-Asp-Asp-P
ro-Pro-Glu-11e-Pro-His-Al
a-Thr-Phe-Lys-Ala-Met-AIa
-Tyr-Lys-G 1u-G ly-Thr-Me
t-Leu-Asn-Cys-G 1u-Cys-L
ys-Arg-Gly-Phe-^rg-Arg-1
1e-Lys-Ser-Gly-3er-Leu-Ty
r-Me t-Leu-Cys-T h r-G 1
y-As n-3e r-3er-H1s-3e r-
3e r-Trp-Asp-^5n-Gln-Cys-
Gln-Cys-Thr-Ser-5er-Ala-T
h r-A rg-Asn-Th r-Th r-Ly
s-G In-Va 1-Th r-Pro-G 1n
-P r o-G 1u-G 1u-G 1 n-Ly
s-G lu-Arg-Lys-Th r-Th r-
G lu-Met-G 1 n-3ern-3er-P
ro-In-Pro-Va 1-Asp-G In-A
1 a-5er-Leu-Pro-GI y-Hi
s-Cys-Arg-G 1u-P ro-Pro-P
ro-T r p-G 1u-As n-G 1u-
A 1a-Th r-G 1u-Ar g-11e-T
y r-tl 1s-Phe-Va 1-Va 1-G
1y-G In-Me t-Va 1-Tyr-Ty
r-G 1n-Cys-Va ]-G ] n-G
] y-T y r-Arg-A 1a-Leu-Hi
s-Arg-G 1y-Pro-^1 a-G 1 u
-3er-Va 1-Cys-Lys-Met-Th
r-H1s-G 1y-Lys-Th r-Arg-T
r p-Th r-G In-Pro-G In-L
eu-11e-Cys-Th r-G 1y-G lu
-Met-G 1 u-Th r-Ser-G In-
Phe-P ro-G 1 y-G 1u-G 1u-
Lys-Pro-Gln-A 1a-3er-Pr
o-G 1u-G 1y-Ar g-Pro-G I
u-5er-G 1 u-Th r-3e r-Cys
-Leu-Va 1-Thr-Thr-Thr-As
p-Phe-G 1 n-11e-G ] n-Th
r-G lu-Me t-A 1a-A 1a-Th
r-Met-Glu-Thr The above-mentioned secreted IL-2 receptor is patent application No. 1048/1986.
It can be produced by the method described in No. 86 specification.

The method will be explained below.

The above-mentioned secreted IL-2 receptor has a gene encoding the following polypeptide (hereinafter referred to as rIL-2R gene) located downstream of the promoter sequence so that it can be expressed, and can proliferate within the host cell. Vector DNA
It can be produced by culturing cells or microorganisms belonging to the genus Escherichia.

X-Glu-Leu-Cys-Asp-Asp-Asp
-Pro-Pro-Glu-[1e-GAG CTCT
GT GACGAT GACCCG CCA GAG
ATCG I u-G I y-Th r-Met-L
eu-As n-Cys-G l u-Cys-L y
s-Arg-GAA GGA ACCATG TTG
AACTGT GAA TGCAAG AGAG I
y-Phe-Arg-Arg-11e-Lys-3er
-Gly-3er-Leu-Tyr-GGT
TTCCGCAGA Ar8 AAA AGCGG
G TCA CTCT TTrp-Asp-Asn
-GIn-Cys-GI n-Cys-Thr-5er
-3er-Ala-TGG GAC8 ACCAA
TGT CAA TGCACA AGCTCT
GCCX is Met or a signal peptide having Met at the N-terminus. Note that the above base sequence is an example of a gene encoding the above polypeptide.

In order to place the IL-2R gene downstream of the promoter sequence so that it can be expressed, in the case of L cells, a vector that can be expressed within this cell, such as a plasmid pK.
CRH2 (Nature, 3 0 7.
The rL-2R gene may be placed downstream of the SV40 early gene promoter of 604-608 (1984)). In the case of L cells, the start codon is M e
It is better to distribute power by using t and a signal peptide as exemplified below as X.

Specifically, pKCR-Tac-2・A plasmid (Na
ture, 311, 631-635 (1984))
5 at the AatII restriction enzyme site of the IL-2R cDNA of
The synthetic compound shown in Figure 1 with phosphorylated ' end N A (a
), a stop codon TGA is placed immediately after the threonine codon of the IL-2R gene. In this way, pKCRIL2RBTMLESS can be constructed in which the start codon ATG, the gene encoding the signal peptide, and the gene encoding the secretory IL-2 receptor are placed downstream of the SV40 early gene promoter (first
(see figure).

In the case of a microorganism belonging to the genus Escherichia, the IL-2R gene is placed further downstream of the SD sequence downstream of the promoter of the expression vector of the microorganism. For example, SD
Place the sequence and then the IL-2R gene.

A specific example using the trp promoter in Escherichia coli is shown below. pDR720 plasmid (Ge
ne, 20.231 (1982)) with the restriction enzyme sa
It is cut with lI and 3maI to obtain a cut linear plasmid. The above pKCRIL2RBTMLE
The SS plasmid was cleaved with the restriction enzyme HindIII to obtain a fragment containing the secreted IL 2R cDNA, which was further cleaved with the restriction enzyme Fok I, treated with DNA polymerase (Klenow fragment), and digested with the restriction enzyme Sac I.
Cut with. The largest fragment is recovered from this reaction. This recovered fragment and synthetic RNA with phosphorylated 5' end (
5) (see Figure 2) and the pDR720 plasmid cut with the above restriction enzymes sal I and Sma I as DNA! Bind with J gauze (Figure 2).

Thus, pDRI has the SD sequence, start codon ATG, and IL-2R gene arranged downstream of the trp promoter.
L2RBTMLESS plasmid can be constructed.

To transform a host cell using a vector DNA containing the IL-2R gene, there are commonly used transformation methods as shown below. When the host cell is an L cell, the DNA
methods include infecting the plasmid as a calcium phosphate precipitate, microinjection, encasing the plasmid in red blood cells or liposomes, treating cells with reagents such as lysophosphatidylcholine, or using viral vectors. .

When the host cell is a microorganism of the genus Escherichia, competent cells capable of taking up the DNA can be transformed by the well-known calcium chloride method after collecting the cells in the logarithmic growth phase. M g Cf! in the transformation reaction solution.
Transformation efficiency will be improved if 2 or Rb(l) is present. It is also possible to prepare host cell protoplasts and then transform them.

Mouse L transformed with IL-2R gene inserted
A method for producing secreted IL-2 receptor protein from cells may be according to a commonly used method for culturing adherent cells. That is, Falcon 3024 under 5% carbon dioxide gas aeration.
．． 3028 flask (Falcon Labware,
Div, Becton, Dickinson and
It can be cultured using tissue culture flasks such as Co. Also, roller flasks such as the Falcon 3027 can be used. The medium may be a synthetic medium commonly used in tissue culture.

For example, Dulbecco's modified Eagle's medium (DMEM), R
Examples include PM11640 medium. When actually using these media, 10% fetal bovine serum albumin, 100%
Unit/nil! of penicillin, 100 μg/ml of streptomycin, and 2 g/β of Na HCO3. The amount of secreted IL-2 receptor protein produced by culturing using the mouse L cells changes over time. The following method may be used to efficiently produce secreted IL-2 receptor protein.

That is, DMEM medium containing 10% Fe2 (10
0U/rnl penicillin, 100μg/mf! Streptomycin, 2 g/f! Contains NaHCOs)
Start culturing at a cell density of lXl0'/rnl using
After 4 to 5 days, when the cells become dense, collect the culture supernatant,
Furthermore, fresh medium is added and culture is continued for 3 to 4 days.

After the culture is completed, the medium is collected and the amount of secreted IL-2 receptor protein produced is quantified, and it is found that approximately the same amount of secreted IL-2 receptor and ter protein are produced in the first and second half of the culture.

From the culture supernatant of the mouse L cells obtained in this way, the secreted IL-2 receptor can be detected using a column immobilized with IL-2, such as IL-2 Sepharose 4B or IL-2
- Can be purified by affinity chromatography using Affi-gel. For even higher purity separation and purification, use an ODS column (Octa decyl 5il
This may be carried out using reverse phase HPLC (high performance liquid chromatography) using ane).

The thus obtained IL-2R protein lacking the cell membrane and intracellular region, that is, the secreted IL-2 receptor, has the following properties: 1) Molecular weight; 43-40 Kd 2) Isoelectric point; 5.2-4. 4, and has binding ability with IL-2 and reactivity with anti-IL-2R antibody (anti-Tac).

Next, it will be explained that it is possible to specifically suppress various immune responses mediated by IL-2 using secretory IL-2 receptors.

, IL-2 maintains the proliferation of TIJ lymphocytes, CTL cells (
cytotoxic T Lyo+phocyte C
It has been reported that it has a variety of biological activities, such as inducing differentiation into e11 (cytotoxic T lymphocytes), activating NK cells, activating LAK cells, and inducing differentiation and proliferation of B cells.

The present inventors used the IL-2-dependent mouse T cell line CTLL-
2 proliferation, activation of memory CTL by IL-2, secondary mixed culture reaction of mouse tile lymphocytes, blastogenesis reaction of mouse lymphocytes by ConA, primary CT from mouse tile cells
induction of lymphocyte proliferation by IL-1 in the coexistence of PHA (phytohemagglutinin) (PHA-IL
l co-mitogenic activity),
Examining the inhibitory effect on secretory IL-2 receptors in systems such as activation of mouse NK cells by IL-2, PHA-induced blastogenesis of human tonsil lymphocytes, and primary mixed culture reaction of human tonsil lymphocytes. did. It is known that IL-2 plays an important function in both systems, but by coexisting with secreted IL-2 receptors, each reaction is significantly suppressed in each system. I received it. This means that in each reaction system, endogenous IL-2 or secreted IL-2 receptors in addition to exogenous IL-2 specifically bind to the membrane of IL-2-responsive cells. This indicates that it inhibits binding to the IL-2 receptor. This means that secreted IL-
2 receptors cause HVG reactions, which are a problem during organ transplants.
Alternatively, it is possible to specifically suppress the onset of GVH reaction,
In other words, this shows that it is useful as a therapeutic agent for immune diseases such as organ transplant rejection and certain autoimmune diseases.

Next, the test method and results that confirmed the immunosuppressive effect of secreted IL-2 receptor will be explained.

(1) Growth suppressive effect of IL-2-dependent cell line CTLL by secreted IL-2 receptor.

50 μl of IL-2 serially diluted 2:200.
20.2μg/- of secreted IL-2 receptor, 50p
H was reacted at 37° C. for 30 minutes in a 96-well microplate manufactured by Corning. Next, the mouse T cell line CT was maintained and subcultured in the presence of rat IL-2.
Wash LL cells thoroughly with medium, add 4 x 10' cells (100 μl) per well, and incubate at 37°C, 5% C for 20 hours.
○2 Culture was performed during aeration. 20 hours after starting the companion meal, 0
．． Add 5 μCi/well of 3H-thymidine (50 μm),
After another 4 hours, the cells were harvested and the 3H incorporated into the cells was
-The radioactivity of thymidine was measured. As shown in FIG. 3, at a final concentration of secreted IL-2 receptor of 50 μg/-, CTLL cell proliferation was clearly suppressed compared to the control. In addition, IL-3 dependent mouse cell line FDC-P
It showed no effect on cell proliferation.

(2) Memory CTL by secreted IL-2 receptor
Activation Suppressing Effect Since it was recognized that the secretory IL-2 receptor suppresses the proliferation of CTLL, the present inventors next demonstrated that the secretory IL-2 receptor suppresses the proliferation of memory CTLs by IL-2.
We investigated whether it is possible to suppress the activation of IL-2, that is, the biological activity of IL-2 as a differentiation factor. Memo IJ-CT L cells, C3H/HeN tile cells as responding cells, and X-ray irradiated DBA/21g as stimulator cells! ! ! MLC (mixed lymphocyte culture) was performed using the cells, and viable cells after 11 days were used. Secretory rL-2 receptor was added in the presence of various concentrations of IL-2, cultured for 3 days in a Corning 96-well microplate, and 51Cr release assay was performed using P815@tumor cells as targets. Ta.

200 u/rnf! When adding a concentration of IL-2, P
The injury rate (%) of 815 tumor cells increased from 11.9% to 65% compared to the case without addition, indicating that memory CTLs were clearly activated by IL-2. 200 u/ml ~ 100 u/mj! The injury rate almost plateaus in the presence of IL-2 of 100 u/
In the presence of IL-2 below m1, the injury rate decreases depending on the IL-2 concentration. When secreted IL-2 receptor coexists in such a system, the concentration is 50 μg/ml! A significant inhibition of memory CTL activation was observed at a concentration of .

Comparing the injury rates in the presence of 200 u/mN IL-2 with and without the addition of secreted IL-2 receptors, they were 28.9% and 65.2%, respectively;
It can be seen that the addition of the secreted IL-2 receptor clearly reduces the injury rate. Also, 100 u/rnl
In the presence of IL-2, 50 μg/mI! The injury rate was 62.4% due to the coexistence of secreted IL-2 receptors.
This decreases from 13.5% to 13.5%. This 13.5% figure is ■
This corresponds to the injury rate without the addition of L-2. This means that
This shows that most of the externally added IL-2 was neutralized.

(3) Human tonsil lymphocyte mixed culture reaction using secreted IL-2 receptor (Mixed Lymphocyt
e Reaction: MLR) Suppressive effect on human major histocompatibility antigen (Human lymphocytosis)
teAntigen: HLA After tonsils obtained from two humans with different A) types were ground and separated into individual cells, red blood cells were removed by discontinuous density gradient centrifugation (Ficoll treatment). Responding cells are 4x10
Stimulated cells were seeded in a 96-well microplate manufactured by Corning Co., Ltd., so that the number of stimulator cells was 5. OOOr a d's X-
Irradiation was performed to stop DNA synthesis, and the response cell/stimulator cell (R/S) ratio was 4/2.4/1.410.5.41.
Each hole was seeded at 0. After 5 days of culture in 5% CO2 aeration, 1.0 μCi/well of 3H-
Thymidine was added and cultured, and the cells were collected. As is clear from FIG. 5, the addition of secreted IL-2 receptor significantly inhibited the uptake of 3H-thymidine.

This indicates that added secreted IL-2 receptors can also neutralize IL-2 produced in vitro.

The immunosuppressive agent of the present invention is suitably used as an injection or a drip. For example, a secreted IL-2 receptor that does not contain bacteria or pyrodiene and is in a freeze-dried state is dissolved in distilled water for injection in the Japanese Pharmacopoeia before use. Once it has been confirmed that the drug has completely dissolved, it is administered to patients suffering from autoimmune diseases, or who are scheduled to undergo organ transplantation or bone marrow transplant surgery, or who have completed surgery. The preferred route of administration is intravenous injection, and the concentration and dosage vary depending on each patient and the patient's situation, but it is 5 to 250 mg/mj! with a concentration of 1 to 50
It is appropriate to administer about mg/kg/day.

The immunosuppressive agent of the present invention is formulated according to methods commonly used for protein preparations.

That is, a solution of secreted IL-2 receptor containing excipients such as gelatin and human serum albumin, and stabilizers such as glutathione and various amino acids is poured into a vial exclusively used for injection preparations and freeze-dried. All of these operations must be performed under aseptic conditions.

Next, reference examples and formulation examples will be shown to further specifically explain the present invention.

Reference Example 1 As shown in FIG. 1, (1) Plasmid pKCR-TaC-2・A (Nature,
311.631-635 (1984)) was partially digested with the restriction enzyme AatI to obtain approximately 6.5 kilobases (
Fragments of the linear plasmid (kb) were separated and recovered by agarose gel electrophoresis. The amount of the recovered fragment was 1 μg. The fragment was digested with the restriction enzyme BamHI, and 0.35 μg of a 5.4 Kb fragment and 0.07 μg of a 1.1 kb fragment were separated and recovered by agarose electrophoresis.

5′GAGATCTCACGT” was synthesized using an automatic DNA synthesizer (Model 380A manufactured by Applied Biosystems) and subjected to reverse-phase HPL.
After purification at C, 5
' side was phosphorylated. 5.4kb fragment and 1. lk
0.04 pmol of each of the b fragments and 0.16 pmol of oligonucleotide having the above 12 sequences were incubated in a 10 μl reaction solution at 15°C for 1 hour, then diluted to 100 μl, and further incubated at 15°C for 12 hours. A! It was bound with J gauze.

(2) Using 10 μl of this reaction solution, Escherichia coli HBIOI was transformed by a conventional method to obtain about 250 ampicillin-resistant strains. Randomly select 12 plasmids from among these, extract their plasmid DNA, and confirm that ■ the size of the plasmid is approximately 6.5 kb, ■ the restriction enzyme Aat
I [The restriction enzyme Bgl II derived from 012 oligonucleotides has one cleavage site.
Five transformants were obtained that were transformed with a plasmid that satisfied the following three requirements: the cleavage site was present at the restriction enzyme Aat II cleavage site. The nucleotide sequence of the plasmid in the transformed strain was determined using the dideoxynucleotide chain method (Pr
oc, Natl, Acad, Sci.

It was confirmed that it had the base sequence of pKCRIL2RBTMLESS in Figure 1.

(3) From the transformed strain obtained in this way, pKCRIL2
The RBT MLEss plasmid was purified by cesium chloride equilibrium centrifugation, and the purified plasmid was infected into mouse L cells (tk-mutant strain) by the following method. That is, Dulbecco's modified Eagle's medium containing 10% tallow serum (D
IX10' cells suspended in MEM)10- were placed in a Falcon 3003 strainer and cultured at 37°C in a 5% carbon dioxide gas incubator for 20 hours. After that, use the same medium for 10mj! The medium was exchanged and cultured under the same conditions for 4 hours. After culturing, solution A (50mM Hepes-28
0mM NaCj! -1,5mM sodium phosphate buffer, pH 7,22) 0.5- and solution B (2M Ca
Cj! 2 10 μg I) K CRI R2
HBTMLESS-1MgpBR322-herpes
Tk) 0.5m1. was added and cultured for 12 hours at 37°C in a 5% carbon dioxide gas incubator. TBS
(0,137M NaCl-0,05M KCl
The plate was washed with 5.6mM Naz HPOHPO4-25O Lis-HCl buffer (pH 7,5) and treated with a TBS solution containing 2.5% glycerol for 3 minutes. Immediately after treatment, wash with TBS and add 10ml of DMEM containing 10% tallow serum.
l! was added and cultured for 2 days at 37°C in a 5% carbon dioxide gas incubator. After that, HAT medium (13,61℃
1g/I! Hyboxanthin, 3.88 tng/l thymidine, 0, 176 mg/l aminopterin and 10
Change the medium to DMEM (containing 10% tallow baby serum), and
The cells were cultured for 2 days in a 5% carbon dioxide gas incubator at °C. This medium exchange was performed every two days thereafter. On the 14th day, 12 colonies appeared per petri dish. Each colony that appeared in this way was transferred to a 96-well microplate and cultured in a 5% carbon dioxide gas incubator at 37°C, and when it became dense, it was cultured for an additional 2 days.

The secreted IL-2 receptor in the cultured fluid of each colony was identified by the method described below. As a result, secretory type I
L-2 receptor producing cells BTMLESS-J and B
TMLESS-Q was obtained.

AT expressing numerous IL-2 receptors on its membrane surface
L-derived cell line MT-1 cells can be killed by treatment with anti-IL-2R monoclonal antibody (anti-Tac antibody) and complement. Therefore, the secreted IL-2 receptor-containing specimen is reacted with the antibody, and then MT1 cells are added and further treated with complement. If a secreted IL-2 receptor is present in this specimen, it will bind to antibodies and reduce the amount of antibodies that react with MT-1 cells, so that MT-1 cells will not die even when treated with complement.

Due to this principle, a very small amount (approximately 0.1 ng) of secreted IL
-2 receptor can be identified.

The specific method for identifying the secreted IL-2 receptor is to first incubate the standard lOμ! in a 96-well V-bottom microplate. Or 10 μl of this diluted with RPMI 1640 medium containing 10% tallow serum and 1, 6 dissolved in the same medium.
Add 10 μl of ng anti-Tac antibody solution (mix and incubate at 0°C for 3
Leave for 0 minutes. and 5X105 suspended in the above medium.
Add 20 µl of /- MT1 cells, mix and leave at 0°C for 30 minutes. Centrifuge at 800 rpm for 5 minutes, discard the supernatant, and remove complement (rabbit newborn serum diluted 15 times with RPMI 1640 medium containing 10% tallow serum) 2
Add 0 μl, mix and leave at 37°C for 30 minutes. Then, 20 μl of a PBS solution containing 0.5% trypan blue is added, and the number of dead cells among 200 cells is counted.

(4) The secreted IL-2 receptor thus produced can be stored frozen at -20°C, and its properties remain unchanged even if it is concentrated by ultrafiltration (for example, Centricon 10). Furthermore, it is possible to bind to the IL-2-Sephadex 4B (IL-2 column). In other words, 100 μβ of the sample concentrated 10 times with Centricon 10 can be combined with the IL-2-Sephadex 4B (IL-2 column).
-2 was adsorbed onto a column (containing 200Mg), and 0.1M
When eluted with sodium citrate buffer (pH 3,0), the secreted IL-2 receptor is eluted, so this eluted fraction is immediately neutralized in the IM) IJ space. The secreted IL-2 receptor obtained by
-1 cells inhibited the killing effect of anti-Tac antibody and complement.

Reference Example 2 The culture supernatant of the mouse L cells containing the secreted IL-2 receptor protein was concentrated 10 times using Amicon hollow fiber and separated from other contaminant proteins by IL-2-Sepharose 4B column chromatography. That is, the mouse L
Cells were grown in DMEM medium containing 10% FC5 (100 U/m
l Penicillin, 100μg/mI! Streptomycin, 2g/! , N a HC03) 7Flucon 3027
Culture was carried out using a 0-Ra bottle to obtain culture supernatant 15A. This supernatant was concentrated 10 times using Amicon hollow fiber (molecular weight: 10,000), bound at 1500mm, and made into a volume of 1ml.
L-2 Sepharose 4B column (I/rnl of gel)
L-21.9 mg was combined). After sample development,
10mI! The column was thoroughly washed with PBS, and further washed with distilled water in an amount equal to the column volume.

The secreted IL-2 receptor protein was eluted from the IL-2 Sepharose 4B column with 0.1 M acetic acid (pH 3,1). With this procedure, the number of secreted IL-2 receptors was 15,000.
It was purified approximately 1,000 times, and the recovery rate was 75%. Furthermore, the elution fraction of the IL-2 Sepharose 4B column was analyzed using ODS.
It was purified to high purity by reverse phase HPLC using a column (Yamamura Kagaku, 4 mm x 60 mn+). 4 rnl of the IL-2 Sepharoise 4B column elution fraction was equilibrated with 0.1% trifluoroacetic acid (pH 2,0). Add the adsorbed protein to the DS column at a flow rate of 0.5 me/min.
Elution was performed with a linear gradient of 0% acetonitrile.

Secreted IL-2 receptor was eluted with 38-40% acetonitrile. By the above purification method, culture supernatant 1
Finally, we were able to separate and purify the secreted IL-2 receptor molecule 35,000 times more than 51 with a recovery rate of 50%.

[Molecular weight of secreted IL-2 receptor] The molecular weight of the secreted IL-2 receptor was examined by HPLC followed by secreted IL-2 di-2 receptor label AGE. The sample was added to a 10% polyacrylamide gel containing 0.1% SDS and electrophoresed. As a result, a single band was obtained at a position corresponding to a molecular weight of 40 Kd to 43 Kd. In addition, as standard proteins,
Bovine serum albumin, ovalbumin, chymotrypsinogen, and soybean trypsin inhibitor were used.

[pI] Purified secreted IL-2 di-2 receptor marker pI was measured. 2 5mM Bis-Tris-1mino
dia-cptic Ac1d (pH 7.0)
MonoP column (Pharmacia
Fine Chemicals, Sweden) and Polyvar t7y-74-Iminodia
It was eluted with cetic^cid (pH 4.0). It was revealed that the secreted IL-2 receptor was eluted in the pH range of 5.2 to 4.4 and had an isoelectric point of 5.2 to 4.4.

[N-terminal structure of secreted IL-2 receptor molecule] In order to determine the amino acid sequence of secreted IL-2 receptor, purified secreted IL-2 receptor was analyzed using a protein sequencer (Applied 8iosystemCo,
) was introduced. The method for determining the amino acid sequence is described in J. Biol
, Chem, 193. 265-275 (1951)
The method described in was followed.

It has been revealed that the secreted IL-2 receptor protein consists of a molecule having the N-terminal structure shown below.

Glu-Leu-Cys-Asp-Asp-Asp-P
The single ro-Pro-Glu-11e structure completely matched the amino acid structure predicted from the base sequence of the IL-2R gene.

Formulation Example 100 mg of secreted IL-2 receptor dissolved in 10 mj2 of physiological saline was placed in a vial and lyophilized.

When using, add 10ml of distilled water and use as an injection or drip.

〔Effect of the invention〕

The immunosuppressive agent of the present invention is extremely effective for the treatment and prevention of autoimmune diseases and organ/bone marrow transplant rejection, and unlike conventional immunosuppressive agents, it has no side effects and can be used continuously. can.

[Brief explanation of the drawing]

Figure 1 shows the plasmid pKC, which contains the gene encoding the secreted IL-2 receptor and can be expressed in L cells.
It is a drawing showing the construction process of RIL2R BTMLESS. FIG. 2 is a diagram showing the construction steps of plasmid pDRIL2R BTMLESS, which contains a gene encoding secreted IL-2 receptor and is expressible in Escherichia coli. Figure 3 shows IL-2 by secreted IL-2 receptor.
It is a graph showing the growth inhibitory effect of dependent cell line CTLL. Figure 4 shows memo IJ by secreted IL-2 receptor.
- It is a drawing showing the effect of suppressing CTL activation. FIG. 5 is a graph showing the suppressive effect of the secreted 1L-2 receptor on the mixed culture reaction of human tonsil lymphocytes. Figure 3? 2.5 Ll/ml (Final concentration) l L-2 dilution ratio Figure 4 200 100 50 25 12.5 0 Figure 5

Claims (2)

[Claims] (1) An immunosuppressant containing secreted IL-2 receptor as an active ingredient. (2) The immunosuppressant according to claim (1), wherein the secreted IL-2 receptor has the following properties. (a) Molecular weight: 40 to 43Kd (b) pI5.2 to 4.4 (c) Amino acid sequence (N-terminus) [There is an amino acid sequence. ]