JP3357139B2 - Adsorbent for anti-deoxyribonucleic acid antibody - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an adsorbent obtained by immobilizing a peptide on a carrier. The adsorbent provided by the present invention, which is obtained by immobilizing a peptide capable of binding to an anti-deoxyribonucleic acid antibody (hereinafter, referred to as an anti-DNA antibody) on a carrier, can be used for treating diseases involving the anti-DNA antibody. Useful for

[0002] Anti-DNA antibodies are autoantibodies mainly detected in the body fluid of patients with systemic lupus erythematosus (hereinafter referred to as SLE), which is an autoimmune disease. The vasculitis seen in SLE patients is due to the deposition of immune complexes formed by the reaction between anti-DNA antibodies and DNA on the blood vessel wall, and nephritis, which affects the prognosis of SLE patients, It is known that anti-DNA antibodies are caused by deposition directly on renal glomeruli. Therefore, removal of anti-DNA antibodies from body fluids such as blood and plasma is necessary for treating diseases involving anti-DNA antibodies such as SLE.

[0003]

2. Description of the Related Art As an adsorbent for an anti-DNA antibody, an immunoadsorbent obtained by immobilizing a hydrophobic compound on an insoluble carrier (see JP-A-57-122875) and DNA immobilized on a polymer carrier are used. Adsorbent (JP-A-61-226)
No. 059), an adsorbent obtained by immobilizing a compound having an anionic functional group on a water-insoluble porous material (Japanese Patent Application Laid-Open No. Sho.
No. 4-68272) is known.

[0004]

The above-mentioned JP-A-57-1
The adsorbent targeted by the immunoadsorbent described in Japanese Patent No. 22875 is an immunoglobulin and / or an immune complex, and an anti-DNA antibody is exemplified as the immunoglobulin. It does not have the ability to selectively adsorb and remove, and also adsorbs and removes immunoglobulins useful for the human body.

[0005] The DNA-immobilized adsorbent described in Japanese Patent Application Laid-Open No. 61-226059 uses DNA extracted from natural products as a raw material. If DNA elutes into the body fluid when used in contact with body fluids such as blood or plasma, it may form an immune complex with the anti-DNA antibody in the body fluid and worsen the medical condition There are drawbacks such as.

An adsorbent for an anti-DNA antibody described in Japanese Patent Application Laid-Open No. 64-68272 is a compound having an anionic functional group immobilized on a carrier, such as a peptide polyglutamic acid, polyaspartic acid, or an amino acid. It is described that glycine can be used, but the adsorbent on which the peptide or amino acid is immobilized does not have sufficient adsorption capacity, and it is desired to further improve the adsorption capacity.

[0007] As described above, in the conventionally known adsorbents for anti-DNA antibodies, from the viewpoints of adsorption specificity, safety, production cost, adsorption capacity, and the like, anti-DNA antibodies appear in body fluids. Not suitable for use in therapy.

[0008] It is an object of the present invention to selectively adsorb and remove anti-DNA antibodies without adsorbing and removing components useful for the human body from body fluids such as blood and plasma, and to adsorb and remove antibodies during sterilization and storage. It is an object of the present invention to provide a highly safe adsorbent that has a very small decrease in removal ability.

[0009]

According to the present invention, the above object is achieved by the following general formula (I): HXAYZ-(I) wherein A is a compound represented by the following formula: Ala-BC-Glu-Ile-Leu (wherein
B and C represent Trp, Tyr or Phe. Represents a peptide fragment consisting of 6 to 12 amino acid residues containing the amino acid sequence represented by X); X and Y each represent a single bond or consist of Asp, Glu, Arg, Lys and His Represents a peptide fragment consisting of 2 to 10 amino acid residues selected from the group or at least one amino acid residue selected from the group, the other being Asp, Glu, Arg, Lys and
2 to 10 amino acid residues selected from the group consisting of His or at least one amino acid residue selected from the group
Z represents a hydroxyl group or an amino group. Is achieved by providing an anti-DNA antibody adsorbent obtained by immobilizing the peptide represented by the formula (1) on a carrier.

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 Glu: L-glutamic acid residue Gln: L-glutamine 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 Trp: L-tryptophan residue Tyr : L-tyrosine residue

Further, in the present specification, the amino acid sequence of the peptide is described in a conventional manner such 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.

A in the general formula (I) is, as described above, a formula: Ala-BC-Glu-Ile-Leu (where B and C are
Represents Trp, Tyr or Phe. ) Represents a peptide fragment consisting of 6 to 12 amino acid residues having an amino acid sequence represented by the following as an essential component. Such a peptide fragment is a partial peptide fragment of protein A or an amino acid residue constituting the same. Those that have undergone homologous substitution are preferred. In addition, protein A is a Staphylococcus aureus (Staphyro
coccus aureus), whose primary structure has already been elucidated [Journal
Of Biological Chemistry (The Journal of
Biological Chemistry, Vol. 259, p. 1695 (1984)].

A in the general formula (I) is represented by the following formula (1): Ala Phe Tyr Glu Ile Leu (SEQ ID NO: 1);
Formula (2): Ala Trp Tyr Glu Ile Leu (SEQ ID NO:
2), Formula (3): Ala Tyr Tyr Glu Ile Leu (SEQ ID NO: 3), Formula (4): Ala Phe Trp Glu Ile Leu (SEQ ID NO: 4), Formula (5): Ala Phe Phe Glu Ile Leu (SEQ ID NO: 5), Formula (6): Gln Gln Asn Ala Phe Tyr Gl
u Ile Leu (SEQ ID NO: 6), Formula (7): Gln Gln Asn A
la Trp Tyr Glu Ile Leu (SEQ ID NO: 7), Formula (8):
GlnGln Asn Ala Tyr Tyr Glu Ile Leu (SEQ ID NO:
8), Formula (9): Gln Gln AsnAla Phe Trp Glu Ile Leu
(SEQ ID NO: 9), Formula (10): Gln Gln Asn Ala PheP
he Glu Ile Leu (SEQ ID NO: 10), Formula (11): Ala
Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu (SEQ ID NO:
11), Formula (12): Ala Trp Tyr Glu Ile Leu Asn Me
t Pro Asn Leu (SEQ ID NO: 12), Formula (13): Ala T
yr Tyr Glu Ile Leu Asn Met Pro Asn Leu (SEQ ID NO:
13), Formula (14): Ala Phe Trp GluIle Leu Asn Met
Pro Asn Leu (SEQ ID NO: 14) or Formula (15): Al
a PhePhe Glu Ile Leu Asn Met Pro A peptide fragment of the amino acid sequence represented by Asn Leu (SEQ ID NO: 15) is more preferred.

Since protein A is a physiologically active protein derived from Staphylococcus aureus, A in the general formula (I)
However, even if a partial peptide of protein A having the amino acid sequence represented by the formula (1) or a homologous substitution of amino acid residues constituting the partial peptide has 13 or more amino acid residues, In this case, toxicity and antigenicity to the human body are increased, and synthesis is complicated.

X and Y in the general formula (I) are defined as described above, but the addition of X and Y to A imparts hydrophilicity to A, and is therefore represented by the general formula (I) The peptide is efficiently immobilized on the carrier. Further, when an adsorbent in which the peptide represented by the general formula (I) is immobilized on a carrier is used in contact with a body fluid such as blood or plasma, even if the peptide is released and mixed into the body, Since the peptide is rendered hydrophilic by the addition of X and Y, it is easily excreted in urine, has low antigenicity to the human body, and is safe. When both X and Y are a single bond, or when any of X and Y is an amino acid residue or a peptide fragment different from those defined above, the peptide represented by the general formula (I) Adsorbent immobilized on a carrier is treated with anti-DNA
The ability to adsorb and remove antibodies may be significantly reduced.

The peptide fragments represented by X and Y in the general formula (I) include, for example, the following peptide fragments. -Asp-Asp-, -Glu-Glu-, -Lys-Lys-,-
His-His-, -Arg-Arg-, -Asp-Glu-, -Glu-Asp-, -Glu-Ly
s-, -Lys-Glu-, -His-Asp-, -Asp-His-, -His-Lys-, -L
ys-His-, -Arg-Lys-, -Lys-Arg-,-(Asp) ₅ -,-(Arg)
₅ -,-(Lys) ₅ -,-(Glu) ₅ -,-(His) _5- , -Lys-Glu-Glu-Asp
-, -Asp-Glu-His-Lys-,-(Asp) ₁₀ -,-(Arg) ₁₀ -,-(Lys)
₁₀ -,-(Glu) ₁₀ -,-(His) _10- , -Lys-Glu-His-Arg-Asp-Ly
s-Lys-Glu-, -Lys-Glu-Glu-Asp-Arg-Lys-Lys-His-

The peptide represented by the general formula (I) preferably has no more than about 20 amino acid residues from the viewpoints of ease of synthesis and low antigenicity to the human body.

The peptide represented by the general formula (I) is synthesized by a method usually used in peptide synthesis, for example, a liquid phase synthesis method such as a solid phase synthesis method, a stepwise extension method or a fragment condensation method. It is easy to operate by a solid phase synthesis method [for example, Journal of the American Chemical Society (Journalo).
f the American Chemical Society), Vol. 85, No. 2
149-2154 (1963); The Biochemical Society of Japan, edited by the Biochemical Society of Japan, "Chemistry of Proteins IV Chemical Modification and Peptide Synthesis", Nov. 15, 1977, issued by Tokyo Chemical Dojin, Ltd., Nos. 207-207. 495; edited by the Biochemical Society of Japan, “Seizoku Chemistry Experiment Course 2: Protein Chemistry (2)” (May 20, 1987, published by Tokyo Chemical Dojin, Ltd.), No. 6.
41 to 694].

In the production of the peptide represented by the general formula (I) by a solid phase synthesis method, for example, a polymer such as a styrene-divinylbenzene copolymer which is insoluble in a reaction solvent is added to the C-terminal of the peptide of interest. From the side toward the N-terminus, the corresponding amino acid is protected by protecting a functional group such as an α-amino group other than the α-carboxyl group of the amino acid, and then condensed and bonded. The peptide chain is extended by sequentially repeating the operation of removing the protecting group of the amino group forming a peptide bond such as an amino group to form a peptide chain corresponding to the target peptide, and The target peptide can be obtained by leaving from the union and removing the protecting group from the protected functional group. If necessary, the peptide can be further purified to obtain a highly pure peptide. It is effective that the peptide is purified by reversed-phase high-performance liquid chromatography.

The peptide represented by the general formula (I) is efficiently immobilized on a carrier, and the obtained adsorbent is more useful for the human body than body fluids such as blood and plasma of a disease patient involving an anti-DNA antibody. Anti-DNA antibodies can be selectively adsorbed and removed without adsorbing and removing components. General formula (I)
As a carrier used when immobilizing the peptide represented by, an amino group having a hydrophilic surface, and which can be used to form a covalent bond with the peptide, a carboxyl group, a hydroxyl group and the like Those having a reactive functional group are preferred. Further, the carrier is preferably insoluble in body fluids such as blood and plasma and porous. A porous carrier having a large effective surface area capable of adsorbing an anti-DNA antibody has an exclusion limit protein molecular weight in the range of about 10 ⁶ to 10 ⁹ or an average pore diameter in the range of about 50 to 1000 nm. It is preferred to use one. The carrier can be in any shape such as a particle, a fiber, a sheet, and a hollow fiber.

As such a carrier, for example, CM-Cellulofine CH (exclusion limit protein molecular weight: about 3 × 1
0 ^6, cellulosic carriers, such as Seikagaku Corporation sold), CM- Toyopearl 650C (exclusion limit protein molecular weight: 5 × 10 ^6, manufactured by Tosoh Corporation) Polyvinyl alcohol based carrier, such as, CM- Trisacryl M (CM-Tris
acryl M) [exclusion limit protein molecular weight: 1 × 10 ⁷ , Pharmacia-LKB (Sweden)
Polyacrylamide-based carrier such as Sepharose CL-4B (exclusion limit protein molecular weight: 2 × 10 ⁷ , Pharmacia-L, Sweden)
Organic carriers such as agarose-based carriers such as KB (Pharmacia-LKB), and CPG-10-1000 [exclusion limit protein molecular weight: 1 × 10 ⁸ , average pore size: 1]
00 nm, US Electro-Nucleonics (Electr
o-nucleonics), and inorganic carriers such as porous glass.

The immobilization of the peptide represented by the general formula (I) on a carrier is carried out according to a method generally employed when immobilizing a peptide or protein on a carrier. As an immobilization method, for example, a carboxyl group of a carrier is reacted with N-hydroxysuccinimide to convert it into a succinimideoxycarbonyl group, and the peptide represented by the general formula (I) is converted to an amino group. (Active ester method), the carboxyl group or the amino group of the peptide represented by the general formula (I) is condensed to the amino group or the carboxyl group of the carrier in the presence of a condensing reagent such as dicyclohexylcarbodiimide. A method of reacting (condensation method), a method of crosslinking a carrier and a peptide represented by the general formula (I) using a compound having two or more functional groups such as glutaraldehyde (carrier crosslinking method), and the like. . Above all, the immobilization method using the active ester method can immobilize the peptide represented by the general formula (I) on a carrier without substantially reducing the binding ability between the peptide and the anti-DNA antibody. preferable.

The amount of the peptide represented by the general formula (I) to be immobilized on the carrier is usually about 3 × so that the resulting adsorbent can effectively adsorb a significant amount of the anti-DNA antibody. 1
It is preferably at least 0 ^-8 mol / g (carrier). In order for the peptide represented by the general formula (I) immobilized on the carrier to be effectively used for adsorption of the anti-DNA antibody, about 1x
More preferably, it is in the range of 10 ^{−7 to} 5 × 10 ⁻⁵ mol / g (carrier).

The removal of the anti-DNA antibody is performed by contacting an adsorbent obtained by immobilizing the peptide represented by the general formula (I) on a carrier with a body fluid such as blood or plasma containing the anti-DNA antibody. This is performed by adsorbing an anti-DNA antibody on an adsorbent. For example, the adsorbent is used by filling the column. The column used for this purpose has an inlet part and an outlet part which can be easily connected to the blood circuit, and is made of polyester or the like between the inlet part and the adsorbent layer and between the outlet part and the adsorbent layer. It is desirable to have a filter made of a material.

Examples of the material of the column include polyethylene, polypropylene, polycarbonate, polyester, and polymethyl methacrylate. Of these, polypropylene and polycarbonate columns are particularly preferred in that they can be subjected to a sterilization treatment such as autoclave sterilization or γ-ray sterilization.

[0026]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to the examples.

Synthesis Example 1 A peptide represented by the formula (16): Lys Lys Ala Phe Tyr Glu Ile Leu (SEQ ID NO: 16) was synthesized by an automatic peptide synthesizer [Applied Biosystems, USA
430 (Model 430A) manufactured by ystems). Styrene-divinylbenzene copolymer having 4-hydroxymethylphenoxymethyl group at a ratio of 0.85 mmol / g (resin) [constituent ratio (molar ratio of styrene and divinylbenzene): 99: 1]
0.29 of a granular resin (HMP resin manufactured by Applied Biosystems, USA)
g, according to a series of operations shown in Table 1, from the C-terminal side to the N-terminal side of the target peptide, in the corresponding order, L-alanine, L-glutamic acid, L-isoleucine, L-leucine, L-Lysine, L-phenylalanine and L-tyrosine were coupled. In the condensation reaction, the above amino acids are 9-fluorenylmethoxycarbonyl-L-alanine, 9-fluorenylmethoxycarbonyl-L-glutamic acid-γ-t-butyl ester, and 9-fluorenylmethoxycarbonyl-L-, respectively.
Isoleucine, 9-fluorenylmethoxycarbonyl-
L-leucine, N ↑ α-9-fluorenylmethoxycarbonyl-N ↑ ε-t-butyloxycarbonyl-L-lysine, 9-fluorenylmethoxycarbonyl-L-phenylalanine and 9-fluorenylmethoxycarbonyl- It was used as Ot-butyl-L-tyrosine, and their amount was about 2 times the molar amount of the substrate.

[0028]

[Table 1]

After the completion of the reaction procedure for all amino acids, the obtained resin was washed sequentially with dichloromethane and methanol on a glass filter, and then dried under vacuum to obtain 600 mg of a dried resin. In a vial, 600 mg of dry resin and 1 trifluoroacetic acid
0 ml, 0.5 ml of water, 0.5 ml of thioanisole, 0.25 ml of ethanedithiol and 0.75 g of phenol were mixed. After standing at room temperature for 20 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 dried under vacuum, extracted with a 2N aqueous solution of acetic acid, and the extract was freeze-dried to obtain a peptide.

The obtained peptide was subjected to high performance liquid chromatography for analysis [column: column packed with octadecylated silica gel having a particle size of 5 μm (inner diameter: 4.6 mm, length: 100 m)
m, TSKgel ODS-80TMCTR manufactured by Tosoh Corporation); mobile phase:
Mixed solvent of acetonitrile and water containing 0.05% by volume of trifluoroacetic acid (the concentration of acetonitrile was gradually changed from 5% by volume to 50% by volume in 30 minutes); flow rate: 1 ml / min; detection Method: absorbance at a wavelength of 210 nm] showed a single sharp peak at 16.2 minutes. The molecular weight of the peptide determined by FAB (fast atom bombardment) mass spectrum was 1010 (theoretical value: 1011.18).

Synthesis Examples 2 to 29 Peptides shown in Tables 2 to 6 were obtained by performing solid phase synthesis of peptides in the same manner as in Synthesis Example 1. However, in Synthesis Examples 10 and 11, a resin for an amide peptide (available from Kokusan Chemical Co., Ltd.) was used as the resin for the solid phase. In the condensation reaction, L-arginine, L-aspartic acid, L-
Asparagine, L-glutamine, L-histidine, L-
Methionine, L-proline and L-tryptophan are each 9-fluorenylmethoxycarbonyl-N
-4-methoxy-2,3,6-trimethylbenzenesulfonyl-L-arginine, 9-fluorenylmethoxycarbonyl-L-aspartic acid-β-t-butyl ester, 9-fluorenylmethoxycarbonyl-L-asparagine , 9-fluorenylmethoxycarbonyl-L-glutamine, 9-fluorenylmethoxycarbonyl-N ^im
-Trityl-L-histidine, 9-fluorenylmethoxycarbonyl-L-methionine, 9-fluorenylmethoxycarbonyl-L-proline and 9-fluorenylmethoxycarbonyl-L-tryptophan.

[0032]

[Table 2]

[0033]

[Table 3]

[0034]

[Table 4]

[0035]

[Table 5]

[0036]

[Table 6]

When the obtained peptides were subjected to analytical reverse-phase high performance liquid chromatography, all showed a single peak. Table 7 shows the molecular weights of these peptides determined by the FAB method mass spectrum.

[0038]

[Table 7]

Example 1 In 50 ml of anhydrous dioxane (a commercially available dioxane distilled in the presence of metallic sodium), 10 g of cellulose particles (CM Cellulofine CL, manufactured by Chisso Corp.) were suspended, and the suspension was obtained. 0.5 g of N-hydroxysuccinimide and dicyclohexylcarbodiimide were added to the suspension.
0 g was added and the mixture was shaken and stirred at room temperature overnight. The resulting mixture was treated with 0.02 mol / l phosphate buffer (pH
Washed in 7.4) and suction-filtered. The particles obtained were mixed with 20 ml of a 0.02 mol / l phosphate buffer (pH 7.4) containing 20 mg of the peptide obtained in Synthesis Example 1,
The mixture was stirred at 4 ° C. overnight. The obtained mixture was subjected to suction filtration, and the filtrate was subjected to reverse phase high performance liquid chromatography for analysis, but no unreacted peptide remained (an immobilization rate of the peptide on the carrier: about 100).
%). Thus, about 10 g of an adsorbent on which 20 mg of the peptide obtained in Synthesis Example 1 was immobilized was obtained.

Example 2 In Example 1, 10 g of polyvinyl alcohol particles (CM-Toyopearl 650C manufactured by Tosoh Corporation) were used instead of 10 g of the cellulose particles, and the peptide obtained in Synthesis Example 1 was used instead of the peptide obtained in Synthesis Example 1. 20 mg of the obtained peptide
Except for using the same method, about 10 g of polyvinyl alcohol particles having 18.8 mg of the peptide obtained in Synthesis Example 2 immobilized thereon (immobilization ratio of peptide on carrier:
About 94%).

Example 3 Porous glass particles [CPG-10-100 manufactured by Electro-nucleonics, USA]
0] of 10 g was reacted with 100 ml of a toluene solution containing 5 ml of γ-aminopropyltriethoxysilane while heating under reflux for 24 hours. The resulting mixture was washed with anhydrous dioxane and filtered by suction. The obtained particles are suspended in 100 ml of anhydrous dioxane, and 3 g of succinic anhydride is added to the suspension.
Was added and the mixture was stirred overnight at room temperature. The resulting mixture was washed with anhydrous dioxane and filtered by suction. The obtained particles were suspended in 50 ml of anhydrous dioxane, and 0.5 g of N-hydroxysuccinimide and 1.0 g of dicyclohexylcarbodiimide were added to the suspension, and the mixture was stirred at room temperature overnight. 0.02 mol / l of the resulting mixture
Was washed with a phosphate buffer (pH 7.4) and filtered with suction. The obtained particles were combined with the peptide obtained in Synthesis Example 3 by 20.
mg of a 0.02 mol / l phosphate buffer (pH 7.
4) Mix with 20 ml and stir the mixture at 4 ° C. overnight. The obtained mixture was subjected to suction filtration to obtain about 10 g of an adsorbent on which 20 mg of the peptide obtained in Synthesis Example 3 was immobilized (immobilization rate of the peptide on the carrier: about 100%).

Examples 4 to 24, Comparative Examples 1 to 5 Except that 20 mg of the peptides shown in Tables 2 to 6 were used, the adsorbent on which the peptide was immobilized was prepared in the same manner as in any of Examples 1 to 3. Got each. Table 8 shows the peptide used, the particulate carrier, and the immobilization rate of the peptide on the carrier.

[0043]

[Table 8]

From Table 8, it is clear that in the case of a peptide in which both X and Y are single bonds in the general formula (I) as in Comparative Examples 1 to 5, the immobilization rate on the carrier is low. .

Test Example 1 Example 1 was applied to 3 ml of plasma of an SLE patient showing a high anti-double-chain deoxyribonucleic acid antibody (hereinafter referred to as anti-dsDNA antibody).
1g of the adsorbent obtained in any one of Examples 1 to 24 or an untreated particulate carrier [cellulose particles: Chisso Corporation]
CM-Cellulofine, polyvinyl alcohol particles: Tosoh Corporation CM-Toyopearl 650C, porous glass particles: U.S.A. Electro-nucleonics (CPG-10-1000)
1 g was added and suspended at 37 ° C. for 2 hours. The obtained suspension was centrifuged to obtain a supernatant. The concentrations of globulin and albumin in the obtained supernatant were measured using an A / G test kit (A / GB
Test Wako, manufactured by Wako Pure Chemical Industries, Ltd.)
The antibody A concentration was determined by the method of Suzuki et al. [SRL Takarakan, Vol.
Table (1984)], and the adsorption removal rate was calculated from the following equation (1).

[0046]

(Equation 1)

For comparison, an experiment similar to the above was carried out using an adsorbent having polyglutamic acid, polyaspartic acid or glycine immobilized on a carrier instead of the adsorbent obtained in Examples 1 to 24. Table 9 together with the results of
Shown in Note that the polyglutamic acid-immobilized adsorbent and the polyaspartic acid-immobilized adsorbent were synthesized using the same method as in Synthesis Example 1 and prepared using polyglutamic acid (Glu Glu Glu Glu Gl
u Glu Glu Glu Glu Glu Glu Glu, molecular weight determined by FAB method mass spectrum: 1567) and polyaspartic acid (Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp
Asp Asp, molecular weight determined by FAB method mass spectrum: 1400) prepared in the same manner as in Example 1 except for using 20 mg (immobilization ratio of polyglutamic acid on carrier: about 95%, Aspartic acid immobilization rate: about 93%), and the glycine-immobilized adsorbent was prepared in the same manner as in Example 1 except that 20 mg of glycine (manufactured by Kyowa Hakko Kogyo Co., Ltd.) was used (carrier). (Immobilization rate of glycine on top: about 80%) was used.

[0048]

[Table 9]

Test Example 2 In Test Example 1, except that SLE patient plasma having a high anti-single-chain deoxyribonucleic acid antibody (hereinafter referred to as anti-ssDNA antibody) was used instead of using SLE patient plasma having a high anti-dsDNA antibody. Perform a suspension treatment of plasma in a similar manner,
Albumin, globulin, anti-ssDNA in the obtained supernatant
Antibody concentration [SRL Takarakan, Vol. 8, p. 24 (1984)
Table 1) shows the results of calculating the adsorption removal rate.
0 is shown.

[0050]

[Table 10]

From Tables 9 and 10, it can be seen that by using the adsorbent of the present invention, it is possible to selectively remove anti-D with little adsorption and removal of albumin and globulin useful for the human body.
It is clear that the NA antibody can be removed by adsorption.

Test Example 3 In Test Example 1, Examples 5, 14, 15, 16, 1
7, 18, 19, 22, plasma suspension treatment was carried out in the same manner except that the adsorbent obtained in Comparative Examples 1 to 5 and the adsorbent subjected to autoclave sterilization were used. Table 11 shows the results of measuring the concentrations of albumin, globulin, and anti-dsDNA antibody and calculating the adsorption removal rate.
In addition, as an adsorbent subjected to autoclave sterilization treatment, 1 g of the adsorbent on which the peptide was immobilized was added with 0.1 g of sodium chloride.
The suspension was used in 5 ml of a 0.02 mol / l phosphate buffer (pH 7.4) containing 15 mol / l and heat-treated at 121 ° C. for 20 minutes under pressure in an autoclave sterilizer.

[0053]

[Table 11]

From these results, it was found that the adsorbent in which the peptide in which both X and Y are a single bond in the general formula (I), such as the adsorbents of Comparative Examples 1 to 5, was immobilized by autoclave sterilization. It is clear that the adsorbent of the present invention sufficiently retains the ability to adsorb and remove anti-DNA antibodies even after autoclaving.

[0055]

Industrial Applicability The adsorbent of the present invention can selectively adsorb and remove anti-DNA antibodies without adsorbing and removing components useful for the human body from body fluids. Useful for the treatment of

[0056]

[Sequence list] SEQ ID NO: 1 Sequence length: 6 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 2 Sequence length: 6 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 3 Sequence length: 6 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 4 Sequence length: 6 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 5 Sequence length: 6 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 6 Sequence length: 9 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 7 Sequence length: 9 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 8 Sequence length: 9 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 9 Sequence length: 9 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 10 Sequence length: 9 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 11 Sequence length: 11 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 12 Sequence length: 11 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 13 Sequence length: 11 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 14 Sequence length: 11 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 15 Sequence length: 11 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 16 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 17 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 18 Sequence length: 7 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 19 Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 20 Sequence length: 16 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Lys Lys Lys Lys Lys Ala Phe Tyr Glu Ile Leu Asp Asp Asp Asp Asp Asp Asp 1 5 10 15

SEQ ID NO: 21 Sequence length: 13 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Glu Glu Glu Glu Glu Ala Phe Tyr Glu Ile Leu Glu Lys 1 5 10

SEQ ID NO: 22 Sequence length: 11 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 23 Sequence length: 9 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 24 Sequence length: 11 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 25 Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 26 Sequence length: 10 Sequence type: amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 27 Sequence length: 17 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Ala Phe Tyr Glu Ile Leu 1 5 10 15 Glu

SEQ ID NO: 28 Sequence length: 11 Sequence type: amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 29 Sequence length: 11 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 30 Sequence length: 10 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 31 Sequence length: 13 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Lys Lys Ala Phe Tyr Glu Ile Leu Asn
Met Pro Asn Leu 15
10

SEQ ID NO: 32 Sequence length: 13 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence Ala Phe Tyr Glu Ile Leu Asn Met Pro
Asn Leu Asp Asp 15
10

SEQ ID NO: 33 Sequence length: 12 Sequence type: amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 34 Sequence length: 8 Sequence type: amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 35 Sequence length: 8 Sequence type: amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 36 Sequence length: 7 Sequence type: amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 37 Sequence length: 8 Sequence type: amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 38 Sequence length: 8 Sequence type: amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 39 Sequence length: 7 Sequence type: amino acid Topology: Linear Sequence type: Peptide

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-6-192290 (JP, A) JP-A-3-32680 (JP, A) JP-A-3-58937 (JP, A) JP-A-6-192 105909 (JP, A) JP-A-64-68272 (JP, A) JP-A-61-226059 (JP, A) JP-A-57-122875 (JP, A) JP-A-59-186558 (JP, A) JP-A-62-41667 (JP, A) JP-A-57-192560 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) A61M 1/36 500 A61K 38/00 ABA C07K 7 / 06

Claims (1)

(57) [Claims] (1) A compound represented by the general formula: H—X—A—Y—Z (wherein A is a compound represented by the formula: Ala-BC—Glu-Ile-Leu (wherein
B and C represent Trp, Tyr or Phe. Represents a peptide fragment consisting of 6 to 12 amino acid residues containing the amino acid sequence represented by X); X and Y each represent a single bond or consist of Asp, Glu, Arg, Lys and His Represents a peptide fragment consisting of 2 to 10 amino acid residues selected from the group or at least one amino acid residue selected from the group, the other being Asp, Glu, Arg, Lys and
2 to 10 amino acid residues selected from the group consisting of His or at least one amino acid residue selected from the group
Z represents a hydroxyl group or an amino group. ] An adsorbent for an anti-deoxyribonucleic acid antibody obtained by immobilizing the peptide represented by formula