JPH1112298A - Ige-fc fragment-binding peptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject peptide which has a specific amino acid sequence, histamine release-suppressing property, and IgE-FC fragment-binding property, suppresses crisis of allergy I, and is useful to prevent/treat pollenosis, atopic dermatitis, and so on. SOLUTION: This peptide, which has an amino acid sequence shown by the formula, is a new IgE-FC fragment-binding peptide having histamine release- suppressing property, suppresses release of histamine from mastocytes and the like, prevents crises of allergy I, and is useful to prevent/treat pollenosis, atopic dermatitis, urticaria, and so on, is obtained by estimating an amino acid sequence of the binding site between IgE and the Fc receptor by the screening of homology box between IgE and the high affinity-type receptor with its Fc region for the rate of concordance for the sense-antisense pair between arbitrary several amino acid sequences using a computer software, followed by chemical synthesis according to the well-known peptide synthesis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a peptide for use in medical preparations for preventing the onset of type I allergy, and more particularly to IgE which suppresses the release of histamine from mast cells and the like.
-Fc fragment binding peptides.

[0002]

2. Description of the Related Art Type I allergies such as hay fever, atopic dermatitis and urticaria are caused by sensitized immunoglobulin (Ig).
E) that the Fc region of the basophil or mast cell
It is known that mast cells and basophils activated by binding to the gE-Fc fragment produce mediators such as histamine and release these mediators.

In recent years, basophils or mast cells have a high affinity type (FcεR1) or a low affinity type (FcεR2) as a receptor for the Fc region of IgE, and the former FcεR1 is an IgE antibody. It has been found that a degranulation (release of granulocytes containing histamine, etc.) after the antigen is bound to the liposome is induced.

[0004] More recently, the structure of FcεR1 has been identified, and soluble FcεR1 obtained by producing the extracellular domain thereof by genetic engineering techniques can be used to obtain IgE and FcεR1.
There is also a report that it inhibits binding to cεR1 and suppresses type I allergy (Chise Ra, International Immunolo
gy, vol5, No.1, pp.47-54.1993 or Japan Clinical No. 54
Vol. 2, February 1996).

[0005]

However, despite the subsequent studies, the amino acid sequence of the extracellular domain of the α chain, which is the binding site for IgE in FcεR1, has not been specifically identified, and is similar to that of FcεR1. No short peptide having a binding property to an IgE-Fc fragment and a function of suppressing type I allergy has been obtained by any method of isolation or synthesis.

As described above, in the prior art, the Fc of IgE
As a receptor for the region, the entire extracellular domain of the α chain of FcεR1 must be used. In this case, many peptides (peptides) are unnecessary for a peptide for a medical preparation aimed at suppressing the function of IgE. There is a problem that mass production is difficult.

[0007] Further, such a polypeptide having a function of suppressing type I allergy has a problem that it cannot be produced efficiently by a known chemical synthesis method because the number of constituent amino acids is large and an effective region is not specified. There are points.

[0008] Accordingly, an object of the present invention is to solve the above-mentioned problems, and has an IgE-Fc fragment binding property having a specific amino acid sequence having a function of inhibiting the type I allergy and having a binding property to the Fc region of IgE. Is to provide a short peptide of Another object of the present invention is to provide a peptide having a binding property to the Fc region of IgE which can be efficiently produced by chemical synthesis.

[0009]

In order to solve the above-mentioned problems, the present invention provides an IgE-Fc fragment-binding peptide having the following amino acid sequence and inhibiting histamine release. Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp or an IgE-Fc fragment-binding peptide having a histamine release inhibitory property consisting of a hexaploid of a peptide sequence in which Lys is bonded to the C-terminal of the above amino acid sequence. is there.

The Ig of the present invention configured as described above
The E-Fc fragment-binding peptide consists of a short peptide of 11 specific amino acid sequences, which is not FcεR1 itself despite having binding to the Fc region of IgE. Is considered to be unable to bind to basophils or mast cells, thereby suppressing the release function of histamine and the like.

Further, such an IgE-Fc fragment-binding peptide having histamine release inhibitory properties includes 1
Since it is a short peptide having one defined amino acid or a peptide having a specific sequence of 16-fold thereof, it can be efficiently produced by a well-known chemical synthesis method or the like.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION A peptide having a predetermined amino acid sequence according to the present invention can be produced by a known peptide preparation method such as a chemical synthesis method as described above. When a peptide is synthesized by solid phase synthesis, the amino acid at the C-terminus of the peptide to be synthesized is immobilized on an insoluble support such as cross-linked polystyrene, and a commercially available synthesizer controlled by a microcomputer using the amino acid at the C-terminus as a starting point. N-α-t-butoxycarbonylated amino acid (B
oc-amino acid) or N-α-9-fluorenylmethoxycarbonylated amino acid (Fmoc-amino acid) is appropriately removed with a protecting group to extend the required peptide chain in order. Incidentally, as a manufacturer or a sales company of the peptide synthesizer, there are Pharmacia Biotech, PerkinElmer Japan, Aloka, and Shimadzu.

[0013] A peptide consisting of the above-mentioned predetermined amino acid sequence was discovered by estimating the binding site (short peptide) to IgE in FcεR1. For this purpose, one of the hypotheses about protein interaction, sense- This is performed based on the antisense theory.

That is, the sense-antisense theory is
A protein encoding a complementary mRNA and a base mRNA
Has a strong interaction with the protein encoded by NA,
This is the folding or ligand-receptor interaction within a protein molecule.
epter interaction).

[0015] Two short amino acid sequences dense in such a sense-antisense pair (two different amino acid sequences within the same molecule).
Between two molecules. The part in parentheses) is called a homology box, and its biological activity has been biochemically verified.

When a homology box is searched between IgE and FcεR1, the identity ratio of a sense-antisense pair is searched between any number of amino acid sequences using computer software, and the binding of FcεR1 to IgE is determined. The partial amino acid sequence was predicted. For example, when 10 out of 10 amino acids form a sense-antisense pair, the identity is 80%.

Specifically, the following four Fcε-derived sites showed a high identity rate of a sense-antisense pair with IgE. (U61) Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp (U62) Lys Val Ser leu Asn Pro Pro Trp Asn Arg (U75) Gly Gln Pro Leu Phe Leu Arg Cys His Gly (U74) Trp Leu Leu Leu Gln Ala Ser Ala Glu Val A peptide having four or more sequences was synthesized, and an experiment was carried out on a peptide modified in the form of MAP (multiple antigen peptide) having lysine bound to the C-terminus of U61 as follows.

[0018]

【Example】

[Example 1] The amino acid sequence was changed to Ser by a chemical synthesis method (using BOP as a condensing agent in a solid phase synthesis method by the t-Boc method) using an automatic peptide synthesizer (manufactured by Biosystems: 431A). Gly Thr Tyr Tyr Cys Thr Gl
A peptide consisting of y Lys Val Trp was prepared and purified by high performance liquid chromatography (HPLC) using a reversed phase column.

The HPLC was performed by Wako Pure Chemical Industries, Ltd. packed with _C18 silica gel having a particle size of 5 μm, having an inner diameter of 4.6 mm and a length of 25 mm.
Using a 0 mm column (Wakosil-II, 5C18 AR), the peptide was eluted under the following conditions: the initial solvent was 0.1% tetrafluoroacetic acid (TFA) in 5% acetonitrile (C
H ₃ CN), and the mixture was eluted at a flow rate of 1.5 ml / min for 30 minutes with a gradient of acetonitrile concentration ranging from 5 to 65% over time.

The HPLC detected the peak of the absorbance using an ultraviolet detector having a wavelength of 220 nm, and the results are shown in FIG. The peak report of FIG. 1 is as follows. Peak number Detection time (min) Area Height Concentration 1 13.7 79908 941 1.8 2 14.0 4184632 365716 96.1 3 15.4 89015 7324 2.0.

Example 2 The amino acid sequence of Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp was determined in exactly the same manner as in Example 1 by a chemical synthesis method using an automatic peptide synthesizer.
And a diploid was obtained by coupling Lys to the C-terminus, and then Lys was newly added to further link the diploid-linked Lys. The modified peptide is an octaploid and then a hexaploid, which is separated from the solid support (resin) with hydrogen fluoride (HF), and is a modified structure in which the short peptide of Example 1 is branched.
A hexaploid MAP (Multiple antigen peptide) was produced. The branched sequence of this MAP is shown in FIG.

The method for producing the MAP (Example 2) is described in Biochemistry, Vol. 85, pp. 5409-5413 (19).
James (issued in August 1988)
P. TAM).

The obtained peptide was subjected to high performance liquid chromatography (HPL) using a reversed phase column in the same manner as in Example 1.
Purified by C).

Comparative Example 1 A peptide having an amino acid sequence of Lys Val Serleu Asn Pro Pro Trp Asn Arg was prepared by a chemical synthesis method using an automatic peptide synthesizer in the same manner as in Example 1, Purification was performed by high performance liquid chromatography (HPLC) using a column under exactly the same conditions as in Example 1.

The HPLC detected the absorbance peak using an ultraviolet detector having a wavelength of 220 nm, and the results are shown in FIG. The peak report of FIG. 3 is as follows. Peak number Detection time (min) Area Height Concentration 1 14.2 17181 2031 0.42 15.4 4091313 129073 99.6.

Comparative Example 2 The amino acid sequence was determined to be Gly by a chemical synthesis method using the same peptide synthesizer as in Example 1.
A peptide consisting of Gln Pro Leu Phe Leu Arg Cys His Gly was prepared, and the same operation as in Example 2 was performed to prepare a 16-ploid having Lys bonded to the C-terminal of the above amino acid sequence. Then, the obtained peptide was purified by high performance liquid chromatography (HPLC) using a reversed phase column.

Comparative Example 3 The amino acid sequence was determined to be Trp Le by a chemical synthesis method using the same peptide synthesizer as in Example 1.
A peptide consisting of u Leu Leu Gln Ala Ser Ala Glu Val was prepared, and the same operation as in Example 2 was further performed to prepare a 16-ploid in which Lys was bonded to the C-terminal of the above amino acid sequence. Then, the obtained peptide was purified by high performance liquid chromatography (HPLC) using a reversed phase column.

The following test was conducted to examine the histamine release inhibitory properties of the obtained peptides. The peptide having the predetermined amino acid sequence in Examples 1 and 2 or Comparative Examples 1 to 3 was diluted with a phosphate buffer at pH 7.4 to a final concentration of 5 mg / ml and left at room temperature for 3 hours. Used in.

[Histamine Release Inhibition Test] At 37 ° C., each type of peptide corresponding to Examples 1 and 2 and Comparative Examples 1 to 3 (the amino acid sequence is shown in Table 1) is tested. Was. That is, heparin blood (heparin blood of a burnt epidermis mite-specific antibody holder) as a specimen was placed in a test tube in an amount of 1 ml at a time, and Examples 1 and 2 and Comparative Examples 1 to 3 were placed.
Was diluted with a phosphate buffer of pH 7.4 and dissolved and adjusted to 50, 100 or 500 μg / ml, and 100 μl was added. For a blank test (peptide concentration 0), 100 μl of a phosphate buffer of pH 7.4 was added. Was used.

Then, 100 μl of an antigen of Dermatophagoides Pteronyssinus (manufactured by Sanko Junyaku Co., Ltd .: Arastat) was added to each test tube, and left at 37 ° C. for 1 hour, and then centrifuged at 4 ° C. and 3000 rpm. The histamine concentration (ng / ml) of the supernatant was determined by HPL.
The analysis was performed by the C fluorescence method, and the results are shown in Table 2. The control of heparin blood in healthy subjects was 6.8 ng / ml in the histamine concentration when only the allergen was added, and the concentration of histamine in the heparin blood of the burn epidermis mite-specific antibody carrier was 5 ng / ml in the condition where only the peptide was added. .

[0031]

[Table 1]

[0032]

[Table 2]

As is clear from the results shown in Table 2, the peptides of Comparative Examples 1 to 3 were used at 50, 100 or 500 μg / m 2.
The histamine concentration in allergen-containing heparin blood added at a concentration adjusted to 1 showed no change or rather a tendency to increase, as compared to those without the peptide.

On the other hand, the peptide of Example 1 was prepared at various concentrations (5
Allergen-containing heparin blood added at 0, 100, or 500 μg / ml) was lower than the histamine concentration in the case of no peptide addition (concentration 0), particularly 50 μg.
/ Ml peptide suppressed histamine release most.

The peptide of Comparative Example 2 was used in an amount of 100 μg /
Allergen-containing heparin blood added at a concentration of 500 μg / ml or 500 μg / ml is lower than the histamine concentration in the absence of peptide (concentration 0).
The μg / ml peptide suppressed histamine release most.

By the way, the histamine value was examined when the peptide of Example 2 (U73) was added at a concentration of 50, 100, or 500 μg / ml to heparin blood of a healthy person or a burner epidermis mite-specific antibody carrier. Is shown in FIG. In FIG. 4, the symbol ● represents a heparin blood sample of a healthy subject, the symbol ◎ represents a heparin blood sample of a burn epidermis mite-specific antibody carrier, and the symbol ○ represents a heparin blood control of a burn epidermis mite-specific antibody carrier (no peptide added). Is shown.

As is clear from the results of FIG. 4, the IgE-Fc of Example 2 was used for a sample of heparin blood of a healthy subject.
50, 100,
Even when added at each concentration of 500 μg / ml, no change corresponding to the concentration was observed. The amount of histamine release in the blood of the antibody carrier was suppressed in a concentration-dependent manner.
It can be seen that the peptide of Example 1 accurately exerts a histamine release inhibitory action.

Example 2 (MAP 16 of Example 1)
Is difficult to be degraded by proteases in human blood, binds to basophils in blood, suppresses the binding of the added allergen to the IgE complex, and suppresses histamine release.

[0039]

As described above, the peptide of the present invention is a short peptide having a predetermined amino acid sequence or a peptide having a predetermined amino acid sequence of 16-fold thereof, and binds to the Fc region of IgE. And has the advantage of being a novel IgE-Fc fragment-binding peptide that inhibits the release of histamine from mast cells and the like.

Further, the IgE-Fc fragment-binding peptide of the present invention has the above advantages and also has the advantage that it can be efficiently produced by chemical synthesis.

[0041]

[Sequence list]

SEQ ID NO: 1 Sequence length: 11 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence SEQ ID NO: 2 Sequence length: 10 Sequence type: amino acid Topology: linear Sequence type: peptide sequence SEQ ID NO: 3 Sequence length: 10 Sequence type: amino acid Topology: linear Sequence type: peptide sequence SEQ ID NO: 4 Sequence length: 10 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence

[Brief description of the drawings]

FIG. 1 is a chart showing a measurement chart of HPLC of Example 1.

FIG. 3 is a chart showing the amino acid sequence of Example 2 which is a branched peptide.

FIG. 3 is a chart showing an HPLC measurement chart of Comparative Example 1.

FIG. 4 is a chart showing the relationship between the histamine concentration and the peptide concentration of Example 2.

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[Procedure amendment]

[Submission date] October 7, 1997

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] Brief description of drawings

[Correction method] Change

[Correction contents]

[Brief description of the drawings]

FIG. 1 is a chart showing a measurement chart of HPLC of Example 1.

FIG. 2 is a chart showing the amino acid sequence of Example 2 which is a branched peptide.

FIG. 3 is a chart showing an HPLC measurement chart of Comparative Example 1.

FIG. 4 is a chart showing the relationship between the histamine concentration and the peptide concentration of Example 2.

Continued on the front page (71) Applicant 596141952 Higashi Narumi 137-1, Shiomi-cho, Showa-ku, Nagoya-shi, Aichi Prefecture (72) Inventor Carlos Adriel del Carpio Inventor Yasuki Kojima 20-1 Tawara, Tawara-cho, Atsumi-gun, Aichi Pref.

Claims (2)

[Claims] 1. An IgE-Fc fragment-binding peptide having the following amino acid sequence and inhibiting histamine release. Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp 2. An IgE-Fc fragment-binding peptide having a histamine release inhibitory property, comprising a hexaploid of a peptide sequence in which Lys is bonded to the C-terminus of the following amino acid sequence. Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp