JPH06107558A - Agent for prevention and treatment of anaphylactic shock - Google Patents

Abstract

PURPOSE:To provide an agent for preventing and treating anaphylactic shock, blocking the anaphylactic reaction at its origin and useful for clinical treatment. CONSTITUTION:The anaphylactic shock preventing and treating agent contains a peptide capable of bonding with human IgE as an active component. Concretely, the peptide is a high-affinity immunoglobulin E receptor alpha-chain or a soluble fragment of a high-affinity globulin E receptor alpha-chain capable of bonding with human IgE.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel use of a peptide capable of binding to human IgE. More specifically, the present invention relates to a prophylactic / therapeutic agent for anaphylactic shock, which comprises a high-affinity immunoglobulin E receptor α chain (FcεRIα) and a high-affinity immunoglobulin E receptor α chain soluble fragment (sFcεRIα) capable of binding to human IgE. .

[0002]

2. Description of the Related Art Anaphylactic shock is a system in which chemical mediators such as histamine and leukotriene and various enzymes are released from mast cells and basophils by the invasion of an exogenous factor, which affects the whole body. Suddenly causing serious disorders of the respiratory system and circulatory system (dyspnea, arrhythmia, myocardial infarction, etc.), and if proper treatment is delayed, death often occurs in a very short time.

Factors that cause anaphylactic shock include drugs such as penicillin and insulin, desensitizing allergens such as mites and fungi, dietary allergens such as eggs and peanuts, iodine contrast agents, and local opiates.

[0004] Currently, for anaphylactic shock, adrenaline administration, steroid administration, etc. are used as drug therapy, and it is said that the prognosis is good if the initial treatment is properly performed, but they are all symptomatic. It is just a therapy, and prevention of anaphylactic shock is emphasized. However, there is no definite preventive method, and at most, it is very vague to prevent the entry of highly dangerous factors into the body.

Therefore, in patients requiring drugs such as antibiotics such as penicillin, and in the case of using an iodine contrast agent in X-ray diagnosis, it should be administered while recognizing the risk of anaphylactic shock. The reality is unavoidable, and strong preventive measures are eagerly awaited.

[0006]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a clinically useful prophylactic / therapeutic agent for anaphylactic shock which blocks the anaphylactic reaction at its root.

[0007]

Means for Solving the Problems As a result of intensive studies to achieve the above objects, the present inventors have found that a peptide capable of binding to human IgE was administered before and / or after administration of a drug or a contrast agent. It was found that administration at the time of administration can suppress the onset of anaphylactic shock, and that administration of a peptide capable of binding to human IgE even after the occurrence of anaphylactic shock improves the symptoms, Completed the invention.

The present invention has been completed based on such new findings, and relates to an agent for preventing and / or treating anaphylactic shock, which uses a peptide capable of binding to human IgE.

In the present invention, the peptide capable of binding to human IgE means a peptide which specifically binds to human IgE and inhibits its function. Specifically, high affinity immunoglobulin E receptor α chain (FcεRIα) and human I
Examples include peptides containing a high-affinity immunoglobulin E receptor α chain soluble fragment (sFcεRIα) capable of binding to gE.

These are the high-affinity immunoglobulin E receptor (specifically, the high-affinity immunoglobulin E receptor that binds to the Fc portion of IgE with high affinity on the cell membrane of mast cells and basophils, which is the origin of the expression mechanism of anaphylactic reaction. Ig to FcεRI)
It blocks the binding of E and thus blocks the anaphylactic reaction at its source.

FcεRI is a tetrameric glycoprotein consisting of α chain, β chain, and two γ-chain subunits that are disulfide-bonded, and α is required for high affinity binding to IgE.
It has been shown that only the extracellular region of the chain is involved [Blank. U et al., J. Biol. Chem., 266 , 2639.
(1991)].

FcεRIα is the α chain of FcεRI, and sFcεRIα corresponds to the extracellular region of the α chain.

The peptide capable of binding to human IgE used in the present invention need only include at least the extracellular region of the α chain, that is, sFcεRIα.
There is no problem using RIα or FcεRI itself. Further, a mutant artificially modified by a genetic engineering method or a DDS derivative such as a conjugate of albumin may be used as long as it is capable of binding to human IgE, and particularly sFcεRIα. Is preferred. Further, these are preferably of human origin.

SFcεRIα can be produced by a cell culture method or a genetic engineering method. Blank. U et al. Have succeeded in expressing sFcεRIα using CHO cells lacking the dihydrofolate reductase (DHFR) gene (above-mentioned document).

The present inventors have also (1) added sFcεR in the upstream of which a promoter capable of controlling the expression in animal cells was added.
Animal cells transformed with a plasmid incorporating the Iα gene and the DHFR gene to which the urokinase (UK) promoter has been added, (2) sFcε with a promoter capable of controlling expression in animal cells added to the upstream portion
An animal cell transformed with the plasmid carrying the DNA encoding the RIα gene and the plasmid carrying the DNA encoding the DHFR having the UK promoter added to the upstream thereof at the same time, (3) above (1) or ( SFcεRIα by culturing the animal cell of 2)
Has established a mass production method (see Reference Example below).

The sFcεRIα may be produced by a genetic engineering method in addition to the above-mentioned method using animal cells and Escherichia coli or yeast.

The peptide capable of binding to human IgE used in the present invention is, for example, dissolved in physiological saline for injection or distilled water for injection and administered parenterally, preferably intravenously.

When used as a prophylactic agent, the drug or the contrast medium may be administered several days before, for example, four days before, about once or twice a day. The dose is preferably twice or more the amount of IgE to the exogenous factor. When used as a therapeutic agent, it is desirable to administer a large amount as early as possible after the onset, preferably 10 times or more the amount of IgE with respect to an exogenous factor.

[0019]

INDUSTRIAL APPLICABILITY The present invention provides a clinically useful prophylactic / therapeutic agent for anaphylactic shock that blocks the anaphylactic reaction at its root.

[0020]

EXAMPLES Reference examples to explain the present invention in more detail,
Experimental examples and examples are given, but the present invention is not limited thereto.

Reference Example Production of human sFcεRIα [I] Construction of FcεRI / pMT1 [Promoter capable of controlling expression in animal cells (SV4
0) was added to the upstream region to construct a plasmid constructed by incorporating a DNA encoding human sFcεRIα] Bla
nk et al. [Blank. U et al., J. Biol. Chem. 266,
2639 (1991)], the plasmid pko [Doren, KV
et al., J. Viral, 50, 606, (1984)] on human FcεR.
A plasmid FcεRI / pMT1 (FIG. 1) in which the leader sequence of Iα-human sFcεRIα gene was integrated was constructed. The DN encoding the human sFcεRIα gene
The A sequence and the deduced amino acid sequence are Nuclei
c Acids Research, Volume 16 Number 8, 3584 (1988)
Is disclosed in.

[II] Construction of pTT06 [Construction of a plasmid constructed by incorporating a DNA encoding DHFR having a UK promoter added at the upstream part] Biosci. Biotech. Biochem., 56 (4), 600-604, 1
According to 992, UK promoter, DHFR cDNA, SV4
A plasmid pTT06 containing the 0 poly A addition signal was constructed (see Figure 2).

[III] Introduction into animal cells and establishment of high-producing cells (i) Materials; -Plasmid DNA FcεRI / pMT1: a plasmid in which the leader sequence of human FcεRIα-human sFcεRIα gene is incorporated into pko. pTT06: a plasmid consisting of a DHFR gene expression unit controlled by a UK promoter (upstream about 800 bp 5'regulatory region including the transcription start point of urokinase gene). Cell: CHO DXB-11 cell line (DHFR deficient strain) G. Urlaub et al., Proc, Natl. Acad. Sci. USA, 77 ,
It was prepared and propagated by the method described in 4216-4220 (1980).・ Methotrexate (MTX); (+) Amet manufactured by Sigma
Hopterin was dissolved in 0.14 M NaCl, 0.02 M HEPES (Nacalai Tesque) to prepare a 2 mM stock solution. This was added to the medium to a desired concentration and used.

(Ii) Method DNA Assay and Assay of Transfectant Supernatant DHFR-deficient CHO DXB-11 cells passaged with α modified Eagle MEM, 10% FCS were treated with trypsin (0.25%) from the dish. It was peeled off and suspended in Hanks solution to give 10 ⁷ cells / ml. 0.5 ml of this suspension was added to 5 × 10 ⁶ cells of plasmid DNA (1 μg of pTT06 and 40 μg of Fcε).
RI / pMT1) was simultaneously introduced by electroporation. After culturing with α modified Eagle MEM (without ribonucleic acid / deoxyribonucleic acid) and 10% FCS, the resulting colonies were picked up and expanded sequentially, and the human sFcεRIα activity in the culture supernatant of these transfectants was determined as follows. Measurement was performed according to the method described in 1 above, and the strain in which high activity was observed was subjected to DNA amplification by MTX.

Quantification of human sFcεRIα The concentration of human sFcεRIα was measured by the inhibition of binding of ¹²⁵ I-labeled mouse IgE to FcεRI present on rat basophil cell line. The materials and methods are shown below. (Material) ・ Cells: Rat basophil cell line RBL-2H3 contains 10%
Cultured in Eagle MEM containing FCS, 100 U / ml penicillin and 100 μg / ml streptomycin.・ IgE; anti-dinitrophenyl mouse monoclonal I
gE (Hi-DNP-E-26-82) or anti-trinitrophenyl (TNP) mouse monoclonal IgE was used. Anti-T
NP mouse monoclonal IgE was purified from the culture supernatant of mouse hybridoma IGELb4 (ATCC No. TIB141) or mouse ascites. -IgE iodine label; Bio-Rad Enzymobeads
The method was used. 50 μl of 0.2M phosphate buffer (pH 7.2), 10 μl of mouse monoclonal IgE (2.14 mg / ml), Enzy
mobeads reagent 50 μl, Na ¹²⁵ I 25 μl (95MBq), 1%
Mix 25 μl of β-D glucose solution at room temperature for 15-20
Let react for minutes. 150 μl of 10 mg / ml tyrosine, 10%
After mixing with phosphate buffered saline containing glycerin and 0.1% xylene cyanol, the protein was recovered by gel filtration using a PD-10 column (Pharmacia). (Method) IgE binding assay; (1) CHO cells were modified with 10% FCS α modified Eagle ME
Since it is maintained in M (without ribonucleic acid / deoxyribonucleic acid), a sample diluted with this culture solution was prepared (125-25
0 μl). (2) Dulbecco's phosphate buffered saline containing 3% bovine serum albumin and 0.1% NaN ₃ (PBS / 3% BS
¹²⁵ I-labeled IgE diluted in A) was added to the sample of (1). The total volume is 500 μl and the concentration of ¹²⁵ I-labeled IgE is 100 ng / ml. The mixture was incubated at room temperature for 3-6 hours. (3) RBL-2H suspended in PBS / 3% BSA
50 μl of 3 cells (3-8 × 10 ⁷ / l) was added, and 1-
Incubated for 2 hours. Further, in the unlabeled IgE-added group for investigating non-specific adsorption of labeled IgE to cells, 150 μl of RBL-2H3 cell suspension and 15 μl of RBL-2H3 cell suspension were prepared in advance.
μl of 2.1 mg / ml unlabeled IgE was mixed and 55 μl of this mixture was added to the mixture of (2). (4) The cells were precipitated by centrifugation at 1000 rpm for 5 minutes, and the supernatant was discarded. (5) After washing the cells once with PBS / 3% BSA, the bound radioactivity was measured with a gamma counter. (6) 10% FCS-added α modified Eagle MEM (without ribonucleic acid / deoxyribonucleic acid) as radioactivity without additives
The binding inhibition rate was measured according to the following formula 1 by using cpm when adding.

[0026]

[Equation 1]

The human sFcεRIα producing strain obtained by amplification of the transgene by MTX was added to 5-10 × 10 5
Selective medium 8-10 containing 10 nM MTX at ⁵ cells / dish
Planted in a 10 cm dish containing ml. If the medium is changed about every 3 days and the culture is continued for 2 to 4 weeks, a sufficient number of 10 nM MTX-resistant cells can be obtained.
Concentrated medium was subcultured. Thus starting from 10nM MTX concentration, 50nM, 100nM, 200nM, 500nM, 1μM, 2μ
Gene amplification was performed by successively increasing the MTX concentration in the order of M, 4 μM and 10 μM.

Cloning of Human sFcεRIα Producing Strains Some of the human sFcεRIα producing strains for which MTX was amplified were cloned by the limiting dilution method. That is, MTX resistant cells at each concentration were cultured in α modified Eagle MEM (without ribonucleic acid / deoxyribonucleic acid) containing 10% FCS and 2 to 10 μM MTX, and seeded in a 96-well plate. Cells were recovered from the wells in which proliferation was observed, and the cells were sequentially expanded to a 24-well plate and further to a 10 cm dish, and the amount of human sFcεRIα produced in the supernatant at the stage of confluence was examined.

[IV] Purification of human sFcεRIα 983 ml of the culture supernatant of a clonal cell line that produced relatively high levels of human sFcεRIα contained 1/20 volume of 1 mM Tris, pH8.
0 and 10 mM benzamidine, 10 mM ethylenediaminetetraacetic acid, 0.02% sodium azide were added, and the sample filtered with a 0.22 μm filter was used as an IgE Sepharose 4B column [mouse hybridoma IGELb4 (ATCC No. TIB141) anti-trituration purified from the culture supernatant. Nitrophenyl IgE 10 mg is 0.
Activated in 1M sodium bicarbonate CH Sepharose (Pharma
cia) bound to 1 g]. After washing the column with PBS containing 0.02% sodium azide, the protein bound to the column was washed with 0.2M acetic acid, 0.2M sodium chloride, and 0.1M sodium chloride.
Elution with 02% sodium azide, pH 2.8. The purified human sFcεRIα obtained was dialyzed against 10 mM ammonium bicarbonate, and 200 μl was dried with SpeedVac to obtain SDS.
When examined by electrophoresis, a clear band was detected at a molecular weight of around 50 kDa under both reducing and non-reducing conditions by Coomassie blue staining, and no protein other than this band was detected. This protein had an activity of inhibiting the binding of radiolabeled IgE to basophil cells (RBL-2H3 cells; supra) in a radioimmunoassay. Such purified human F
The concentration of cεRIα is determined by the Bradford method [Bradford, MM, An
al. Biochem. 72, 248 (1976)].

Example 1 Injection Preparation 100 mg of purified human sFcεRIα and 100 mg of glucose were dissolved in distilled water for injection to give a human sFcεRIα chain concentration of 2 mg.
It was prepared so as to be / ml. This was filtered through a 0.45 μm membrane filter, and the filtrate was aseptically dispensed into a 5 ml vial, filled with nitrogen gas and then sealed to give an intravenous injection.

Test Example 1 Effect on anaphylactic shock of active sensitized rats Method of Levine et al. [Levine, BB et al., Int. Arch. Al
lergy Appl. Immunol., 39, 156 (1979)]. 10 μg of DNP-Asris was dissolved in 2 mg Al (OH) ₃ gel / ml and intraperitoneally administered to ICR rats for sensitization. After 4 weeks of sensitization, the human sFcεRI prepared in Example 1 above was used.
The α injection was intravenously administered twice at a rate of 100 μg / kg / day. 24 hours after administration, DNP-ovalbumin (10 mg / kg) was intravenously administered to induce anaphylactic shock,
The number of lethal animals within 24 hours was measured (n = 5). As a result, 4 animals died in the control group, whereas human sFc
There were 0 εRIα-administered group.

Test Example 2 Effect on anaphylactic shock of passively sensitized rats M. Pittman, FG Germuth, _JR ., Soc. Exptl. Biol. Me
It was carried out by the following method with reference to d. 87: 425 (1954).
B. pertussis vaccine (2 × 10 ⁹ ) was intraperitoneally administered to female ICR mice (8 weeks old). Two days later, anti-TNP IgE antibody (2
μg; IGELb4, ATCC141) was intraperitoneally administered. Ig above
24 hours after E sensitization, human sFcεRIα (~ 5 mg / kg)
Was administered intravenously. 72 hours after the above IgE sensitization, that is, 48 hours after the administration of human sFcεRIα, TNP-HS
Antigen challenge with A (50 μg) was performed by intravenous administration. The test was carried out with a total number of mice of 15 to 20 per group.
The test was performed on four groups, each of which had a different purchase time. The survival rate of the mouse was calculated by the following formula 2.

[0033]

[Equation 2]

As a result, the survival rate 60 minutes after the antigen challenge was improved by the administration of human sFcεRIα (see FIG. 3).

[Brief description of drawings]

FIG. 1 shows the structure of plasmid FcεRI / pMT1.

FIG. 2 shows the steps for preparing the plasmid pTT06.

Figure 3: Human sFcε 60 minutes after antigen challenge.
It is a figure showing the relation between the dose of RIα, and the survival rate.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Internal reference number for FI Technical indication C12P 21/00 C 8214-4B (72) Inventor Minoru Hirama 2-25 Otani, Otani, Hirakata, Osaka No. 1 Incorporated company Midori Cross Central Research Institute (72) Inventor Yasushi Okumura 1-14-9 Matsunami, Chuo-ku, Chiba-shi, Chiba Prefecture

Claims (5)

[Claims] 1. A prophylactic / therapeutic agent for anaphylactic shock, which comprises a peptide capable of binding to human IgE as an active ingredient. 2. The preventive / therapeutic agent for anaphylactic shock according to claim 1, wherein the peptide capable of binding to human IgE is a peptide containing a high-affinity immunoglobulin E receptor α chain. 3. The prophylactic / therapeutic agent for anaphylactic shock according to claim 2, wherein the high affinity immunoglobulin E receptor α chain is of human origin. 4. The preventive / therapeutic agent for anaphylactic shock according to claim 1, wherein the peptide capable of binding to human IgE is a peptide containing a soluble fragment of a high affinity immunoglobulin E receptor α chain. 5. The prophylactic / therapeutic agent for anaphylactic shock according to claim 4, wherein the soluble fragment of the high-affinity immunoglobulin E receptor α chain is of human origin.