JPH07309893A - Anti-interepidermal antibody-binding peptide and adsorbent - Google Patents

Abstract

PURPOSE:To obtain the subject peptide having ability to specifically binding to an anti-interepidermal antibody, and capable of giving an adsorbent with the peptide immobilized on a carrier, which is useful for treating the diseases involving the antiinterepidermal antibody. CONSTITUTION:This peptide contains an amino acid sequence consisting of consecutive 5 to 50 amino acid residues among the amino acid sequence expressed by the formula (1) : Val-Gly-Ile-Asp-Gln-Pro-Pro-Phe-Gly-Ile-Phe-Val- Val-Asp-Lys-Asn-Thr-Gly-Asp-Ile-Asn-Ile-Thr-Ala-Ile-Val-Asp-Arg-Glu-Glu. The other objective adsorbent is obtained by immobilizing this peptide on a carrier.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a peptide and an adsorbent obtained by immobilizing the peptide on a carrier. Since the peptide provided by the present invention has the ability to specifically bind to the anti-epidermal intercellular antibody, the adsorbent obtained by immobilizing the peptide on a carrier involves the anti-epidermal intercellular antibody. It is useful in the treatment of existing diseases. Anti-epidermal intercellular antibodies are mainly detected in body fluids of patients with autoimmune diseases such as pemphigus. It is presumed that the anti-epidermal intercellular antibody is deposited between epidermal cells to cause blistering and is closely related to the cause or progression of pemphigus. Therefore, removal of anti-epidermal intercellular antibodies from body fluids such as blood and plasma is useful in treating autoimmune diseases such as pemphigus.

[0002]

As an adsorbent for autoimmune diseases, an immunoadsorbent obtained by immobilizing protein A on a silica matrix (see JP-A-62-242628), a hydrophobic compound immobilized on an insoluble carrier. There are known immunoadsorbents (see JP-A-57-122875).

[0003]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention
The protein A-immobilized immunoadsorbent described in Japanese Patent No. 42628 adsorbs and removes immunoglobulins and immune complexes containing autoantibodies from body fluids such as blood and plasma.
It does not have the ability to selectively adsorb and remove only autoantibodies, and it also adsorbs and removes immunoglobulin useful to the human body. In addition, Protein A is a bioactive protein derived from Staphylococcus aureus, and it is costly to produce. Depending on the handling conditions during immobilization on a carrier, sterilization, and storage after immobilization, bioactivity may be inactivated. If the protein A elutes when used in contact with body fluids such as blood and plasma, the eluted protein A
Has the drawback that it may cause serious symptoms to the human body. Similarly, JP-A-57-122875
The immunoadsorbents described in JP-A No. 1994-52, which are obtained by immobilizing a hydrophobic compound on an insoluble carrier, do not have the ability to selectively adsorb and remove only autoantibodies, and they also contain immunoglobulins useful for the human body. It will be removed by adsorption.

As described above, conventionally known immunoadsorbents do not have the ability to selectively adsorb and remove only anti-epidermal intercellular antibodies, and autoimmune caused by anti-epidermal intercellular antibodies. No adsorbent useful for treating diseases has been obtained.

One object of the present invention is to provide a peptide which has the ability to specifically bind to an anti-epidermal intercellular antibody, but does not bind to a component useful for the human body.
Another object of the present invention is to provide an adsorbent capable of selectively adsorbing and removing the anti-epidermal intercellular antibody from body fluids such as blood and plasma.

[0006]

According to the present invention, the above object is to provide an amino acid sequence represented by the following formula (1), which is composed of 5 or more consecutive amino acid residues. And a total number of constituent amino acid residues of 50 or less, and another object by providing an adsorbent comprising the peptide immobilized on a carrier. To be done.

Formula (1): Val Gly Ile Asp Gln Pro Pro
Phe Gly Ile Phe Val Val Asp Lys Asn Thr Gly Asp Il
e Asn Ile Thr Ala Ile Val Asp Arg Glu Glu (SEQ ID NO: 1)

In the present specification, various amino acid residues are described by the following abbreviations. Ala: L-alanine residue Arg: L-arginine residue Asp: L-aspartic acid residue Asn: L-asparagine residue Cys: L-cysteine residue Glu: L-glutamic acid residue Gln: L-glutamine residue Gly: Glycine residue His: L-histidine residue Ile: L-isoleucine residue Leu: L-leucine residue Lys: L-lysine residue Met: L-methionine residue Phe: L-phenylalanine residue Pro: L -Proline residue Ser: L-serine residue Thr: L-threonine residue Trp: L-tryptophan residue Tyr: L-tyrosine residue Val: L-valine residue Further, in the present specification, a peptide is used according to a conventional method. The amino acid sequence of is described so that the N-terminal amino acid residue is located on the left side and the C-terminal amino acid residue is located on the right side.

The peptide of the present invention may contain only one amino acid sequence composed of 5 or more consecutive amino acid residues among the amino acid sequences represented by the above formula (1). Also, it may contain a plurality of one or more kinds of amino acid sequences. Furthermore, the side chains of the constituent amino acid residues may be chemically modified, or the side chains may be replaced with homologous amino acids. Of the amino acid sequence represented by the above formula (1), a peptide containing an amino acid sequence composed of 4 or less consecutive amino acid residues is:
It is not preferable because the ability to specifically bind to the anti-epidermal antibody is low. In the peptide of the present invention, the total number of constituent amino acid residues is 50 or less, preferably 40 or less, 30
Less than or equal to the number is more preferable. Total number of constituent amino acid residues is 50
When it exceeds the number, the antigenicity to the human body becomes high,
Further, the synthesis becomes complicated, which is not preferable.

Further, when the peptide has a high solubility in an aqueous solvent, the reactivity with the anti-epidermal intercellular antibody and the amount of immobilization on the carrier are improved.
Those having 1 to 10 amino acid residues selected from the group consisting of p, Glu, Lys, His and Arg are preferable. A preferred amino acid sequence of this peptide terminal portion is exemplified below. Asp Asp, Glu Glu, Lys Lys, His His, Arg Arg, A
sp Glu, Glu Asp, GluLys, Lys Glu, His Asp, Asp Hi
s, His Lys, Lys His, Arg Lys, Lys Arg, AspAsp Asp
Asp Asp, Arg Arg Arg Arg Arg, Lys Lys Lys Lys Ly
s, Glu Glu GluGlu Glu, His His His His, Lys Gl
u Glu Asp, Asp Glu His Lys, Asp AspAsp Asp Asp Asp
Asp Asp Asp Asp, Arg Arg Arg Arg Arg Arg Arg Arg
Arg Arg, Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys,
Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu, His His H
is His His His His His His, Lys Glu His Arg As
pLys Lys Glu, Lys Glu Glu Asp Arg Lys Lys His

The peptide of the present invention has the formula (2): Va
l Gly Ile Asp Gln Pro Pro Phe Gly Ile Phe Val Val
Asp Lys Asn Thr Gly Asp (SEQ ID NO: 2), Formula (3): LysLys Val Gly Ile Asp Gln Pro Pro Phe Gly
Ile Phe Val Val Asp Lys Asn ThrGly Asp (SEQ ID NO: 3), Formula (4): Asp Lys Asn Thr Gly Asp Ile As
n IleThr Ala Ile Val Asp Arg Glu Glu (SEQ ID NO:
The amino acid sequence represented by 4) is preferred.

The peptide of the present invention is synthesized by a method commonly used in peptide synthesis, for example, a solid phase synthesis method or a liquid phase synthesis method such as a stepwise extension method or a fragment condensation method. Is easy to operate [for example, the Journal of the American Chemical Society (Journal of the America
Chemical Society), Volume 85, 2149-215
P. 4 (1963); "Biochemistry Experiment Course 1 Protein Chemistry IV Chemical Modification and Peptide Synthesis", edited by the Japanese Biochemical Society (November 15, 1972, published by Tokyo Kagaku Dojin), pp. 207-495. ; Biochemical Society of Japan, "Seikagaku Chemistry Laboratory 2 Protein Chemistry (2)" (May 20, 1987)
Sun, Tokyo Kagaku Doujinshi, pp. 641-694].

Production of the peptide of the present invention by the solid phase synthesis method is carried out, for example, on a polymer such as a styrene-divinylbenzene copolymer which is insoluble in a reaction solvent in the direction from the C-terminal side to the N-terminal side of the target peptide. Toward the corresponding amino acid other than the α-carboxyl group possessed by the amino acid
-Sequentially repeating an operation of condensing and binding after protecting a functional group such as an amino group and an operation of removing a protecting group carried by an amino group forming a peptide bond such as an α-amino group in the bound amino acid. By extending the peptide chain to form a peptide chain corresponding to the desired peptide, then removing the peptide chain from the polymer, and removing the protecting group from the protected functional group. A peptide can be obtained. If necessary, a highly pure product can be obtained by further purifying this peptide. It is effective to purify the peptide by reverse phase high performance liquid chromatography.

The adsorbent obtained by immobilizing the peptide of the present invention on a carrier selectively selects an anti-epidermal cell antibody in a body fluid such as blood or plasma of a patient with a disease in which an anti-epidermal cell antibody is involved. Can be removed by adsorption. The carrier used when immobilizing the peptide of the present invention has a hydrophilic surface and can be used to form a covalent bond with the peptide, such as a reaction of an amino group, a carboxyl group or a hydroxyl group. Those having a functional group of sexuality are preferable. The carrier is preferably insoluble in body fluids such as blood and plasma and porous. As a porous carrier with a wide effective surface area capable of adsorbing the anti-epidermal intercellular antibody,
It is preferred to use those having an exclusion limit protein molecular weight in the range of about 10 ⁶ to 10 ⁹ or an average pore size in the range of about 50 to 1000 nm. The carrier may have any shape such as a particle shape, a fiber shape, a sheet shape, and a hollow fiber shape.

Examples of such carriers include cellulosic carriers such as CM-Cellulofine C-500 (molecular weight of exclusion limit protein: about 1.5 × 10 ⁶ , sold by Seikagaku Corporation), CM-Toyopearl 650C ( Exclusion limit protein molecular weight: 5 × 10 ⁶ , polyvinyl alcohol type carrier such as Tosoh Corporation, CM-Trisacryl M
(CM-Trisacryl M) [Exclusion limit protein molecular weight:
1 × 10 ⁷ , Sweden Pharmacia-LKB (Pha
rmacia-LKB), etc.], a polyacrylamide carrier, Sepharose CL-4B (Sepharose CL-4B)
Organic carrier such as agarose carrier such as [Exclusion limit protein molecular weight: 2 × 10 ⁷ , manufactured by Pharmacia-LKB, Sweden], and CPG
-10-1000 [Exclusion limit protein molecular weight: 1 × 1
0 ^8, an average pore diameter: 100 nm, USA Electro - Nucleonics Nix (Electro-nucleonics) Co. Ltd.] include inorganic carriers such as porous glass, such as.

Immobilization of the peptide of the present invention on a carrier is
Generally, it is performed according to the method adopted when immobilizing a peptide or protein on a carrier. As the immobilization method, for example, the carboxyl group of the carrier is converted to N
By converting it to a succinimidooxycarbonyl group by reacting with hydroxysuccinimide, and reacting the peptide at the amino group portion (active ester method), or dicyclohexylcarbodiimide for the amino group or carboxyl group of the carrier. A method in which a carboxyl group or an amino group of a peptide is subjected to a condensation reaction in the presence of a condensation reagent (condensation method), and a method in which a carrier and a peptide are crosslinked with a compound having two or more functional groups such as glutaraldehyde (carrier Crosslinking method) and the like. Among them, the immobilization method by the active ester method is preferable because the peptide can be immobilized on the carrier with almost no decrease in the binding ability between the peptide and the anti-epidermal intercellular antibody.

The amount of the peptide of the present invention immobilized on the carrier is usually about 3 × 10 ^-8 mol so that the resulting adsorbent can effectively adsorb a significant amount of anti-epidermal intercellular antibody. / G (carrier) or more, and in order for the peptide of the present invention immobilized on the carrier to be effectively utilized for adsorption of anti-epidermal intercellular antibody, it is about 1 × 10 ^{−7 to} 5 ×. 10 ^-5
More preferably, it is within the range of mol / g (carrier).

The anti-epidermal intercellular antibody can be removed by contacting the adsorbent obtained by immobilizing the peptide of the present invention on a carrier with a body fluid such as blood or plasma containing the anti-epidermal intercellular antibody. It is carried out by adsorbing the anti-epidermal intercellular antibody to the. For example, the adsorbent is used by packing it in a column. The column used for this purpose has an inlet portion and an outlet portion that are easily connected to the blood circuit, and is made of polyester or the like between the inlet portion and the adsorbent layer and between the outlet portion and the adsorbent layer, respectively. It is desirable to have a filter made of material.

Examples of the material of the column include polyethylene, polypropylene, polycarbonate, polyester, polymethylmethacrylate and the like. Of these, polypropylene and polycarbonate columns are particularly preferable because they can be subjected to sterilization treatment such as autoclave sterilization and γ-ray sterilization.

[0020]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to the examples. In the condensation reaction of peptide synthesis in Examples, L-aspartic acid, L-asparagine, L-glutamine, glycine, L-isoleucine, L-lysine, L-phenylalanine, L-proline, L-threonine, L- Valine, L
The amino acids of -alanine, L-arginine and L-glutamic acid are respectively 9-fluorenylmethoxycarbonyl-L-aspartic acid-β-t-butyl ester,
9-fluorenylmethoxycarbonyl-L-asparagine, 9-fluorenylmethoxycarbonyl-L-glutamine, 9-fluorenylmethoxycarbonylglycine,
9-fluorenylmethoxycarbonyl-L-isoleucine, N ↑ α-9-fluorenylmethoxycarbonyl-N
↑ ε-t-butyloxycarbonyl-L-lysine, 9-
Fluorenylmethoxycarbonyl-L-phenylalanine, 9-fluorenylmethoxycarbonyl-L-proline, 9-fluorenylmethoxycarbonyl-Ot-butyl-L-threonine, 9-fluorenylmethoxycarbonyl-L- Valine, 9-fluorenylmethoxycarbonyl-L-alanine, 9-fluorenylmethoxycarbonyl-N-4-methoxy-2,3,6-trimethylbenzenesulfonyl-L-arginine and 9-fluorenylmethoxycarbonyl- It was used as L-glutamic acid-γ-t-butyl ester, and the amount used was about twice the molar amount of the substrate.

Example 1 Formula (2): Val Gly Ile Asp Gln Pro Pro Phe Gly Ile
Phe Val Val Asp Lys Asn Thr Gly Asp (SEQ ID NO:
The peptide represented by 2) is converted into an automatic peptide synthesizer [Applied Biosystems (US).
s) Model 430A (Model 430A)] was used for the solid phase synthesis. Granular resin composed of a styrene-divinylbenzene copolymer [constituent ratio of styrene and divinylbenzene (molar ratio): 99: 1] having a 4-hydroxymethylphenoxymethyl group at a ratio of 0.85 mmol / g (resin) [ Applied Biosystems (Ap
0.29 g of HMP resin manufactured by Plied Biosystems) was used, and amino acids were bound in the corresponding order from the C-terminal side to the N-terminal direction of the target peptide according to the series of operations shown in Table 1. .

[0022]

[Table 1]

After completion of the reaction procedure for all amino acids, the obtained resin was washed successively with dichloromethane and methanol on a glass filter, and then vacuum dried to obtain 600 mg of a dried resin.
In a vial, 600 mg of dry resin, 10 ml of trifluoroacetic acid, 0.5 ml of water, 0.5 m of thioanisole
1, 0.25 ml of ethanedithiol and 0.75 g of phenol were mixed. After standing at room temperature for 2 hours, the mixture was filtered with a glass filter, diethyl ether was added to the filtrate, and the mixture was centrifuged to obtain a white precipitate. The obtained precipitate was vacuum dried, extracted with 2N aqueous acetic acid solution, and the extract was freeze-dried to obtain a peptide.
The obtained peptide was analyzed by high performance liquid chromatography [column: column packed with octadecylated silica gel having a particle size of 5 μm (inner diameter: 4.6 mm, length: 100 mm, TSKgel ODS-80TMCTR manufactured by Tosoh Corp.); A mixed solvent of acetonitrile and water containing 0.05% by volume of fluoroacetic acid (concentration of acetonitrile is 5% by volume in 30 minutes).
To 50% by volume. ); Flow rate: 1 ml / min; Detection method: absorbance at a wavelength of 210 nm], and a single sharp peak was shown at 21.9 minutes. The molecular weight of the peptide determined by FAB (Fast Atom Bombardment) mass spectrum was 2018 (theoretical value 2018.22).

Example 2 In the same manner as in Example 1, the formula (3): Lys Lys Val Gly was used.
Ile Asp Gln Pro ProPhe Gly Ile Phe Val Val Asp Lys
Solid phase synthesis of the peptide represented by Asn Thr Gly Asp (SEQ ID NO: 3) was performed. The resulting peptide was subjected to analytical reverse phase high performance liquid chromatography and showed a single peak at 19.9 minutes. The molecular weight of the peptide determined by FAB (Fast Atom Bombardment) mass spectrum was 22.
It was 75 (theoretical value was 2274.56).

Example 3 In the same manner as in Example 1, Formula (4): Asp Lys Asn Thr
Gly Asp Ile Asn IleThr Ala Ile Val Asp Arg Glu Glu
Solid-phase synthesis of the peptide represented by (SEQ ID NO: 4) was performed. Subjecting the resulting peptide to reverse phase high performance liquid chromatography for analysis revealed a single peak at 18.1 minutes. The molecular weight of the peptide determined by FAB (Fast Atom Bombardment) mass spectrum is 1903 (theoretical value is 1
903.02).

Example 4 10 g of cellulose particles (manufactured by Chisso Corporation, CM Cellulofine) were suspended in 50 ml of anhydrous dioxane (commercial dioxane distilled in the presence of sodium metal) to obtain a suspension. 0.5 g of N-hydroxysuccinimide and 1.0 of dicyclohexylcarbodiimide were added to the liquid.
g was added and the mixture was shaken and stirred overnight at room temperature. The resulting mixture was added with 0.02 mol / l of phosphate buffer (pH 7.
It was washed with 4) and suction filtered. The particles obtained are mixed with 20 ml of 0.02 mol / l phosphate buffer (pH 7.4) containing 20 mg of the peptide obtained in Example 2,
The mixture was stirred overnight at 4 ° C. The obtained mixture was suction filtered, and the filtrate was subjected to analytical reverse phase high performance liquid chromatography, but no unreacted peptide remained was observed (immobilization rate of peptide on the carrier: about 100).
%). Thus, about 10 g of the adsorbent having 20 mg of the peptide obtained in Example 2 immobilized thereon was obtained.

Test Example 1 Pemphigus patient serum was diluted 10-fold with a neutral phosphate buffer, and the peptides obtained in Example 1 or Example 3 were diluted to final concentrations of 1, 5, 10, 15, 20, respectively. It was added at 50 μg / ml. After mixing at 37 ° C for 1 hour, the mixture was left standing at 4 ° C overnight, centrifuged (10000 xg, 1 hour), and the resulting supernatant was diluted with neutral phosphate buffer solution at 20, 40, 8 respectively.
Dilute 0, 160, 320, 640 and 1280 times,
It was mixed with epidermal cells separated from a normal person. After reacting for 1 hour and washing with a neutral phosphate buffer, fluorescence-labeled anti-human Ig
The G antibody was added, the reaction was continued for 1 hour, and the sample was washed with a neutral phosphate buffer solution to give a sample. The sample was observed by the fluorescent antibody method, and the upper limit dilution ratio at which IgG deposition was observed between epidermal cells was defined as the antibody titer. According to this evaluation method, the anti-epidermal cell antibody bound to the peptide does not deposit between the epidermal cells, so the higher the proportion of the anti-epidermal cell antibody bound to the peptide, the lower the antibody titer. The results are shown in Table 2.
From this result, it is clear that the peptide of the present invention has high reactivity with the anti-epidermal intercellular antibody in the serum of pemphigus patients.

[0028]

[Table 2]

Test Example 2 To 800 μl of pemphigus patient plasma, 200 mg of the adsorbent obtained in Example 4 or 200 mg of an untreated particulate carrier [cellulose particles: CM-Cellulofine] manufactured by Chisso Corporation as a control was added. Suspended at 37 ° C for 2 hours. The obtained suspension was centrifuged to obtain a supernatant. The antibody titer in the obtained supernatant was measured by the same method as in Test Example 1. In this evaluation method, the lower the antibody titer, the more the anti-epidermal intercellular antibody is adsorbed and removed. The antibody titer of the plasma treated with the adsorbent obtained in Example 4 was 320 times, and that of the control plasma was 640 times. Further, the globulin and albumin concentrations in the supernatant were measured using an A / G test kit (A / GB Test Wako, manufactured by Wako Pure Chemical Industries, Ltd.), and the adsorption removal rate of globulin and albumin was set to 1 As a result, the plasma treated with the adsorbent obtained in Example 4 had a globulin adsorption removal rate of 2% and an albumin adsorption removal rate of 3%.

[0030]

[Equation 1]

As described above, it is clear that the use of the adsorbent of the present invention enables selective adsorption and removal of the anti-epidermal intercellular antibody with almost no adsorption and removal of albumin and globulin useful for the human body.

[0032]

INDUSTRIAL APPLICABILITY According to the present invention, a peptide having the ability to specifically bind to an anti-epidermal intercellular antibody is provided. The adsorbent having the peptide immobilized on a carrier can selectively remove anti-epidermal intercellular antibody from body fluid without adsorbing and removing components useful for the human body from body fluid. It is useful for treating diseases involving epidermal cell-cell antibodies.

[0033]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 30 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Val Gly Ile Asp Gln Pro Pro Phe Gly Ile Phe Val Val Asp Lys Asn 1 5 10 15 Thr Gly Asp Ile Asn Ile Thr Ala Ile Val Asp Arg Glu Glu 20 25 30

SEQ ID NO: 2 Sequence length: 19 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Val Gly Ile Asp Gln Pro Pro Phe Gly Ile Phe Val Val Asp Lys Asn 1 5 10 15 Thr Gly Asp

SEQ ID NO: 3 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Lys Lys Val Gly Ile Asp Gln Pro Pro Phe Gly Ile Phe Val Val Asp 1 5 10 15 Lys Asn Thr Gly Asp 20

SEQ ID NO: 4 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Asp Lys Asn Thr Gly Asp Ile Asn Ile Thr Ala Ile Val Asp Arg Glu 1 5 10 15 Glu

Claims (2)

[Claims] 1. An amino acid sequence represented by the following formula (1), which contains an amino acid sequence composed of 5 or more consecutive amino acid residues and has a total number of constituent amino acid residues of 50 or less. Is a peptide. Formula (1): Val Gly Ile Asp Gln Pro Pro Phe Gly Ile
Phe Val Val Asp Lys Asn Thr Gly Asp Ile Asn Ile Th
r Ala Ile Val Asp Arg Glu Glu (SEQ ID NO: 1) 2. An adsorbent obtained by immobilizing the peptide according to claim 1 on a carrier.