JP3329868B2 - Peptide and adsorbent obtained by immobilizing the peptide on a carrier - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

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 an ability to specifically bind to an anti-deoxyribonucleic acid antibody (hereinafter, referred to as an anti-DNA antibody) and / or an immune complex, the peptide is immobilized on a carrier. Such adsorbents are useful for treating diseases involving anti-DNA antibodies and / or immune complexes.

[0002] Anti-DNA antibodies and immune complexes are used in patients with autoimmune diseases such as systemic lupus erythematosus (hereinafter referred to as SLE), rheumatoid arthritis, Guillain-Barre syndrome, and diseases such as malignant tumors, chronic infections, and allergies. And is presumed to be closely related to the cause or progression of the disease. For example, vasculitis in SLE patients can be attributed to anti-DNA antibodies and D
Nephritis, which depends on the immune complex formed by the reaction with NA and deposits on the blood vessel wall and affects the prognosis of SLE patients, is due to the fact that in addition to the immune complex, anti-DNA antibodies are directly deposited on renal glomeruli. It is known to be caused by Therefore, anti-DNA from body fluids such as blood and plasma
Removal of antibodies and / or immune complexes is necessary to treat the above diseases.

[0003]

2. Description of the Related Art Conventionally, as an adsorbent for the above-mentioned diseases, an immunoadsorbent obtained by immobilizing protein A on a silica matrix (see Japanese Patent Application Laid-Open No. 62-242628), and a hydrophobic compound immobilized on an insoluble carrier. Adsorbent for immunoglobulins and / or immunocomplexes (JP-A-57-1)
No. 22875), an adsorbent obtained by immobilizing DNA on a polymer carrier (see Japanese Patent Application Laid-Open No. 61-226059), and an adsorbent obtained by immobilizing a compound having an anionic functional group on a water-insoluble porous material. (Japanese Unexamined Patent Publication No. 64-68272)
And JP-A-1-181875) are known.

[0004]

SUMMARY OF THE INVENTION The above-mentioned JP-A-62-2
No. 42628 discloses a protein A-immobilized immunoadsorbent for adsorbing and removing immunoglobulins and immune complexes from body fluids such as blood and plasma.
It does not have the ability to selectively adsorb and remove immune complexes, and also adsorbs and removes immunoglobulins that are useful for the human body. In addition, protein A is a bioactive protein derived from Staphylococcus aureus, which requires a high product cost, deactivates bioactivity depending on handling conditions during immobilization on a carrier, sterilization, and storage after immobilization. If protein A is eluted when used in contact with body fluids such as blood or plasma, eluted protein A
Have the drawback that they may cause severe symptoms to the human body.

It is described that the adsorbent for immunoglobulins and / or immunocomplexes described in JP-A-57-122875 can adsorb and remove anti-DNA antibodies and immunocomplexes at a high rate. However, the adsorbent is an anti-DNA
It does not have the ability to selectively adsorb and remove antibodies and immune complexes, and also has the disadvantage of adsorbing and removing immunoglobulins useful for the human body at a high rate.

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 JP-A-64-68272 and JP-A-1-181
No. 875 describes that the adsorbent for an immune complex can use peptide polyglutamic acid, polyaspartic acid, or amino acid glycine as a compound having an anionic functional group immobilized on a carrier. However, the adsorbent on which the peptide or amino acid is immobilized does not have sufficient adsorption capacity, and further improvement in adsorption capacity is desired.

[0008] As described above, conventionally known anti-DNA antibodies and / or adsorbents for immunoconjugates have the above-mentioned anti-DNA antibodies and / or immunocomplexes from the viewpoints of adsorption specificity, safety, production cost, adsorption capacity and the like. Or it is not suitable for use in the treatment of diseases in which immune complexes are present in body fluids.

[0009] One object of the present invention is to provide a peptide capable of specifically binding to an anti-DNA antibody and / or an immune complex. Another object of the present invention is to selectively adsorb and remove anti-DNA antibodies and / or immunocomplexes without adsorbing and removing components useful for the human body from body fluids such as blood and plasma, and sterilizing. An object of the present invention is to provide a highly safe adsorbent that has a very small decrease in adsorption removal ability during processing and storage.

[0010]

According to the present invention, the above object is achieved by the following general formula (I): H-X- (AB) n-YZ (I) A represents an amino acid residue selected from the group consisting of Trp and Phe or a peptide fragment consisting of two of at least one amino acid residue selected from the group;
X and Y each represent a single bond, or X or Y represents an amino acid residue selected from the group consisting of Asp, Glu, Arg, Lys and His; Or at least one selected from the group
Represents a peptide fragment consisting of 2 to 10 kinds of amino acid residues, the other being an amino acid residue selected from the group consisting of Asp, Glu, Arg, Lys and His or at least one amino acid residue selected from the group Represents a peptide fragment consisting of 2 to 10; Z represents a hydroxyl group or an amino group;
Represents an integer of 2 to 5. This is achieved by providing the peptide represented by the formula (1), and by providing an adsorbent obtained by immobilizing the peptide on a carrier.

In the present specification, various amino acid residues are described by the following abbreviations. Arg: L-arginine residue Asp: L-aspartic acid residue Glu: L-glutamic acid residue His: L-histidine residue Lys: L-lysine residue Phe: L-phenylalanine residue Trp: L-tryptophan residue

In the present specification, the amino acid sequence of a 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.

The peptide of the present invention is represented by the following general formula (II) (AB) n (II) (where A, B and n are the same as defined above). By containing a peptide fragment consisting of 15 amino acid residues, the ability to specifically bind to an anti-DNA antibody and / or an immune complex is exhibited. General formula (I
In I), the peptide fragment wherein n is an integer of 6 or more is
It is not preferable because synthesis becomes complicated and antigenicity to the living body becomes high. Further, a peptide fragment in which n is 1 is not preferable because its binding ability to an anti-DNA antibody and / or an immune complex is insufficient.

Examples of the peptide fragment represented by the general formula (II) include a peptide fragment represented by the following amino acid sequence. Formula (1): Trp Trp Phe Tr
p Trp Phe (SEQ ID NO: 1), Formula (2): Phe Phe Asp P
he Phe Asp Phe Phe Asp Phe Phe Asp (SEQ ID NO:
2), Formula (3): Phe Glu Phe Glu Phe Glu Phe Glu
(SEQ ID NO: 3), Formula (4): Trp Trp Asp Trp TrpAsp
Trp Trp Asp (SEQ ID NO: 4), Formula (5): Trp Trp Gl
u Trp Trp Glu TrpTrp Glu (SEQ ID NO: 5), Formula (6): Trp Phe Phe Trp Phe Phe (SEQ ID NO: 6),
Formula (7): Trp Phe Trp Phe Trp Phe (SEQ ID NO:
7), Formula (8): Phe AspPhe Asp Phe Asp Phe Asp Phe
Asp (SEQ ID NO: 8), Formula (9): Phe Glu Phe Glu Phe
Glu Phe Glu Phe Glu (SEQ ID NO: 9), Formula (10):
Trp Glu Trp GluTrp Glu Trp Glu Trp Glu (SEQ ID NO:
10), Formula (11): Trp Asp Trp AspTrp Asp Trp Asp
Trp Asp (SEQ ID NO: 11), Formula (12): Trp Phe As
p Trp Phe Asp Trp Phe Asp (SEQ ID NO: 12), Formula (13): Trp Phe Glu Trp Phe Glu Trp Phe Glu Trp
Phe Glu (SEQ ID NO: 13)

X and Y in the general formula (I) are defined as described above. By adding X and Y to the peptide fragment represented by the general formula (II), hydrophilicity is imparted to the peptide fragment. Therefore, the peptide represented by the general formula (I) 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 made hydrophilic by the addition of X and Y, it is easily excreted in urine, has low antigenicity to the living 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) The adsorbent having immobilized on a carrier may have a markedly reduced ability to adsorb and remove anti-DNA antibodies and / or immune complexes by sterilization.

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 -,-(Lys) ₁₀ -,-(Glu)
₁₀ -,-(His) ₁₀ -,-(Arg) _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) 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 target peptide. 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. Further purification of this peptide, if necessary, results in a higher purity. 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 used for the blood of a disease patient involving an anti-DNA antibody and / or an immune complex,
It is possible to selectively adsorb and remove anti-DNA antibodies and / or immune complexes without adsorbing and removing components useful for the human body from body fluids such as plasma. The carrier used for immobilizing the peptide represented by the general formula (I) includes an amino group, a carboxyl group, which has a hydrophilic surface and can be used to form a covalent bond with the peptide. Those having a reactive functional group such as a group or a hydroxyl group are preferred. Further, the carrier is preferably insoluble in body fluids such as blood and plasma and porous. The anti-DNA antibodies and / or immune complexes carrier effective surface area is wide porosity capable of adsorbing, or excluded limit protein molecular weight is in the range of about ^{106 to 109,} or an average pore diameter of about 50 It is preferred to use one that is in the range of 1000 nm. The carrier can be in any shape such as a particle, a fiber, a sheet, and a hollow fiber.

Examples of such a carrier include 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 performed 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 by the active ester method immobilizes the peptide represented by the general formula (I) on a carrier without substantially reducing the binding ability of the peptide to an anti-DNA antibody and / or an immune complex. It is preferable because it can be performed.

The amount of the peptide represented by the general formula (I) to be immobilized on the carrier is such that the resulting adsorbent can effectively adsorb a significant amount of an anti-DNA antibody and / or an immune complex. Is preferably about 3 × 10 ⁻⁸ mol / g (carrier) or more, and the peptide represented by the general formula (I) immobilized on the carrier is preferably an anti-DNA antibody and / or an immunoconjugate. To be effectively used for adsorption, about 1
More preferably, it is in the range of × 10 ^{−7 to} 5 × 10 ⁻⁵ mol / g (carrier).

In the removal of the anti-DNA antibody and / or the immune complex, the adsorbent obtained by immobilizing the peptide represented by the general formula (I) on a carrier contains an anti-DNA antibody and / or an immune complex. This is carried out by contacting the adsorbent with an anti-DNA antibody and / or an immune complex by bringing the adsorbent into contact with a bodily fluid such as blood or plasma. 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, polymethyl methacrylate and the like. 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.

[0025]

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

EXAMPLES 1-15, COMPARATIVE EXAMPLE 1 The peptides shown in Table 1 were converted to an automatic peptide synthesizer [Applied Biosystems, USA]
Using a model 430A (Model 430A). A granular resin comprising a styrene-divinylbenzene copolymer having a 4-hydroxymethylphenoxymethyl group at a ratio of 0.85 mmol / g (resin) [constituent ratio (molar ratio of styrene and divinylbenzene): 99: 1] [ US Applied Biosystems (Applie
d Biosystems) HMP resin] 0.29 g,
In accordance with a series of operations shown in Table 2, L-arginine, L-aspartic acid, L-glutamic acid, L-histidine, and L-histidine were used in the corresponding order from the C-terminal side to the N-terminal side of the target peptide. Lysine, L-phenylalanine and L-tryptophan were coupled. In the condensation reaction, each of the above amino acids is 9-fluorenylmethoxycarbonyl-N-4-methoxy-2,3,6
-Trimethylbenzenesulfonyl-L-arginine, 9
-Fluorenylmethoxycarbonyl-L-aspartic acid-β-t-butyl ester, 9-fluorenylmethoxycarbonyl-L-glutamic acid-γ-t-butyl ester, 9-fluorenylmethoxycarbonyl-N ^im -trityl -L-histidine, N ↑ α-9-fluorenylmethoxycarbonyl-N ↑ ε-t-butyloxycarbonyl-L-lysine, 9-fluorenylmethoxycarbonyl-L-phenylalanine and 9-fluorenylmethoxycarbonyl They were used as -L-tryptophan, and their amount used was about 2 times the molar amount of the substrate.

[0027]

[Table 1]

[0028]

[Table 2]

After the reaction operation for all amino acids was completed, the obtained resin was sequentially washed with dichloromethane and methanol on a glass filter, and then dried under vacuum to obtain a dried resin. In a vial, 600 mg of the dry resin was mixed with 10 ml of trifluoroacetic acid, 0.5 ml of water, 0.5 ml of thioanisole, 0.25 ml of ethanedithiol and 0.75 g of phenol. 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], all showed a single peak. Table 3 shows the theoretical values calculated from the amino acid sequences and the molecular weights of the peptides determined by FAB (fast atom bombardment) mass spectra for these peptides.

[0031]

[Table 3]

Examples 16 to 30, Comparative Example 2 The peptides obtained in Examples 1 to 15 and Comparative Example 1 were
An adsorbent was produced by immobilizing each on a carrier by the following method. [When Cellulose Particles are Used as a Carrier] Cellulose particles (Chisso Corporation) are added to 50 ml of anhydrous dioxane (a commercially available dioxane distilled in the presence of sodium metal).
10 g of CM Cellulofine CL) was added, and 0.5 g of N-hydroxysuccinimide and 1.0 g of dicyclohexylcarbodiimide were added to the obtained suspension, and the mixture was shaken and stirred at room temperature overnight. The resulting mixture was washed with 0.02 mol / l phosphate buffer (pH 7.4) and filtered by suction. The obtained particles were prepared in Examples 1, 2, and 4
-8, 10, 11, 13, 14 or 20 mg of the peptide obtained in Comparative Example 1 was mixed with 20 ml of a 0.02 mol / l phosphate buffer (pH 7.4) containing 4 mg of the peptide.
By stirring the mixture at ° C overnight, an adsorbent having the peptide immobilized thereon was obtained. Further, the obtained mixture was subjected to suction filtration, and the filtrate was subjected to reverse phase high performance liquid chromatography for analysis to determine the immobilization ratio of the peptide on the carrier.

[When polyvinyl alcohol particles are used as a carrier] In the above-mentioned method in which cellulose particles are used as a carrier, 10 g of polyvinyl alcohol particles (CM-Toyopearl 650C manufactured by Tosoh Corporation) are used instead of 10 g of cellulose particles. Except for using the same method,
An adsorbent on which the peptide obtained in Example 3 or 12 was immobilized was obtained.

[When porous glass particles are used as a carrier] Porous glass particles [CPG-10-1 manufactured by Electro-nucleonics, USA]
000] in 100 ml of a toluene solution containing 5 ml of γ-aminopropyltriethoxysilane under heating and refluxing for 24 hours. The resulting mixture was washed with anhydrous dioxane and filtered by suction. The obtained particles were suspended in 100 ml of anhydrous dioxane, 3 g of succinic anhydride was added to the suspension, and the mixture was stirred at room temperature overnight. 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. The resulting mixture was washed with 0.02 mol / l phosphate buffer (pH 7.4) and filtered by suction. The particles obtained are mixed with 20 ml of a 0.02 mol / l phosphate buffer (pH 7.4) containing 20 mg of the peptide obtained in Example 9 or 15, and the mixture is stirred at 4 ° C. overnight. As a result, an adsorbent on which the peptide was immobilized was obtained. Table 4 shows the peptides used, the particulate carrier, and the immobilization ratio of the peptide on the carrier.

[0035]

[Table 4]

From Table 4, as shown in Comparative Example 2, the compound represented by the general formula (I)
In the case of the peptide in which both X and Y are single bonds, it is clear that the immobilization rate on the carrier is low.

Test Example 1 1 g of the adsorbent obtained in Examples 16 to 30 or 3 g of the untreated particulate carrier [cellulose particles: CM manufactured by Chisso Co., Ltd.] was added to 3 ml of plasma of a patient with rheumatoid arthritis or a control.
-Cellulofine, polyvinyl alcohol particles: CM-Toyopearl 650C manufactured by Tosoh Corporation, porous glass particles: Electro-nucleonics (Electro-nu, USA)
cleonics) CPG-10-1000] 1 g,
Suspended at 37 ° C. for 2 hours. The obtained suspension was centrifuged to obtain a supernatant. Using an A / G test kit (A / GB Test Wako, manufactured by Wako Pure Chemical Industries, Ltd.), the concentration of immunoglobulin and albumin in the obtained supernatant was measured using an immunocomplex detection reagent kit [immunocomplex. Body (C1q-Ig
G) Kuraray, Immunomedics
The results are shown in Table 5 below.

[0038]

(Equation 1)

For comparison, an adsorbent obtained by immobilizing tryptophan, polyglutamic acid or polyaspartic acid on a carrier was used in place of the adsorbent obtained in Examples 16 to 30, and the same adsorbent as described above was used. Table 5 shows the results of the experiments. The tryptophan-immobilized adsorbent was prepared in the same manner as in Example 16 except that 20 mg of tryptophan (manufactured by Kyowa Hakko Kogyo Co., Ltd.) was used (an immobilization ratio of tryptophan on a carrier: about 80%). Using polyglutamic acid (Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu 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 Asp)
p, molecular weight determined by FAB method mass spectrum: 14
00) Except for using 20 mg, prepared in the same manner as in Example 16 (immobilization rate of polyglutamic acid on carrier: about 95%, immobilization rate of polyaspartic acid: about 9)
3%).

[0040]

[Table 5]

Test Example 2 A plasma suspension treatment was performed in the same manner as in Test Example 1 except that SLE patient plasma was used instead of rheumatoid arthritis patient plasma, and albumin and globulin in the resulting supernatant were obtained. Table 6 shows the results of measuring the concentration of the immune complex and calculating the adsorption removal rate.

[0042]

[Table 6]

From Tables 5 and 6, it is clear that the use of the adsorbent of the present invention enables selective adsorption and removal of immunocomplexes with little adsorption and removal of albumin and globulin useful for the human body.

Test Example 3 In Test Example 1, an anti-double-chain deoxyribonucleic acid antibody (hereinafter referred to as anti-dsD
Plasma suspension was performed in the same manner except that the plasma of an SLE patient showing a high level of NA antibody was used, and the concentrations of albumin, globulin, and anti-dsDNA antibody in the obtained supernatant were measured, and the adsorption was performed. Table 7 shows the result of calculating the removal rate.
The anti-dsDNA antibody concentration was determined by the method of Suzuki et al.
Vol. 24, p. 24 (1984)].

For comparison, an experiment similar to the above was carried out using an adsorbent in which polyglutamic acid, polyaspartic acid or glycine was immobilized on a carrier instead of the adsorbent obtained in Examples 16 to 30. Table 7 also shows the results of the above. The polyglutamic acid-immobilized adsorbent and the polyaspartic acid-immobilized adsorbent used were the same as those used in Test Examples 1 and 2, and the glycine-immobilized adsorbent was glycine (manufactured by Kyowa Hakko Kogyo) 20 mg A solution prepared in the same manner as in Example 16 except that glycine was used (an immobilization rate of glycine on a carrier: about 80%) was used.

[0046]

[Table 7]

Test Example 4 In Test Example 1, an anti-single-chain deoxyribonucleic acid antibody (hereinafter referred to as anti-s
Plasma suspension was performed in the same manner except that SLE patient plasma with a high level of sDNA antibody was used, and the concentrations of albumin, globulin, and anti-ssDNA antibody in the obtained supernatant [SRL Takara, No. 8 Vol., Page 24 (1984)], and the results of calculating the adsorption removal rate are shown in Table 8.

[0048]

[Table 8]

From Tables 7 and 8, it can be seen that the use of the adsorbent of the present invention allows the selective removal of anti-DN with little adsorption and removal of albumin and globulin useful for the human body.
It is clear that the antibody A can be adsorbed and removed.

Test Example 5 In the same manner as in Test Example 1, except that the adsorbent obtained in Examples 25, 26 and Comparative Example 2 and the above adsorbent subjected to autoclave sterilization were used, the plasma suspension treatment was carried out in the same manner. Performed, albumin, globulin in the resulting supernatant,
Table 9 shows the results of measuring the concentration of the immune complex and calculating the adsorption removal rate. As the adsorbent subjected to autoclave sterilization, 1 g of the adsorbent on which the peptide was immobilized was 0.02 mol / l containing 0.15 mol / l of sodium chloride.
Was suspended in 5 ml of a phosphate buffer (pH 7.4) and heat-treated at 121 ° C. for 20 minutes under pressure in an autoclave sterilizer.

[0051]

[Table 9]

From these results, it was found that the adsorbent obtained by immobilizing a peptide in which both X and Y are single bonds in the general formula (I), such as the adsorbent of Comparative Example 2, adsorbed the immune complex by autoclave sterilization. It is clear that the adsorbent of the present invention sufficiently retains the adsorbing and removing ability of the immune complex even after the autoclave sterilization treatment, while the removing ability is significantly reduced.

Test Example 6 In the same manner as in Test Example 3, except that the adsorbents obtained in Examples 25, 26 and Comparative Example 2 and the above-mentioned adsorbent subjected to autoclave sterilization were used, the plasma suspension treatment was carried out in the same manner. Performed, albumin, globulin in the resulting supernatant,
Table 10 shows the results of measuring the concentration of the anti-dsDNA antibody and calculating the adsorption removal rate.

[0054]

[Table 10]

From these results, it was found that the adsorbent obtained by immobilizing a peptide in which both X and Y are single bonds in the general formula (I), such as the adsorbent of Comparative Example 2, was adsorbed with an anti-DNA antibody by autoclave sterilization. It is clear that the adsorbent of the present invention sufficiently retains the anti-DNA antibody adsorption / removal ability even after the autoclave sterilization treatment, while the removal ability is significantly reduced.

[0056]

According to the present invention, an anti-DNA antibody and / or
Alternatively, a peptide having the ability to specifically bind to an immune complex is provided. The adsorbent in which the peptide is immobilized on a carrier can selectively adsorb and remove anti-DNA antibodies and / or immune complexes without adsorbing and removing components useful for the human body from body fluids, It is useful for treating diseases involving anti-DNA antibodies and / or immune complexes.

[0057]

[Sequence list]

SEQ ID NO: 1 Sequence length: 6 Sequence type: amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 2 Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 3 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 4 Sequence length: 9 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 5 Sequence length: 9 Sequence type: amino acid Topology: linear Sequence type: peptide

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

SEQ ID NO: 7 Sequence length: 6 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 8 Sequence length: 10 Sequence type: amino acid Topology: linear Sequence type: peptide

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

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

SEQ ID NO: 11 Sequence length: 10 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 12 Sequence length: 9 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 13 Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 14 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 15 Sequence length: 10 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 16 Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 17 Sequence length: 15 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Lys Lys Phe Phe Asp Phe Phe Asp Phe Phe Asp Phe Phe Asp Arg 1 5 10 15

SEQ ID NO: 18 Sequence length: 17 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Asp Asp Asp Asp Asp Phe Phe Asp Phe Phe Asp Phe Phe Asp Phe Phe 1 5 10 15 Asp

SEQ ID NO: 19 Sequence length: 15 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Asp Phe Phe Asp Phe Phe Asp Phe Phe Asp Phe Phe Asp Lys Lys 1 5 10 15

SEQ ID NO: 20 Sequence length: 14 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence His His His His His Phe Glu Phe Glu
Phe Glu Phe Glu Glu 1 5 10

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

SEQ ID NO: 22 Sequence length: 10 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 23 Sequence length: 11 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 24 Sequence length: 14 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Asp Trp Trp Asp Trp Trp Asp Trp Trp Asp Lys Asp Glu His 1 5 10

SEQ ID NO: 25 Sequence length: 13 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Lys Lys Trp Trp Asp Trp Trp Asp Trp Trp Asp Asp Glu 1510

SEQ ID NO: 26 Sequence length: 11 Sequence type: amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 27 Sequence length: 20 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Trp Trp Glu Trp Trp Glu 1510 15 Trp Trp Glu Asp 20

SEQ ID NO: 28 Sequence length: 16 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Asp Glu Trp Trp Glu Trp Trp Glu Trp Trp Glu His His His His 1 5 10 15

Claims (2)

(57) [Claims] 1. A compound represented by the general formula: H—X— (AB) n—YZ, wherein A is an amino acid residue selected from the group consisting of Trp and Phe, or at least one kind selected from the group Represents a peptide fragment consisting of two amino acid residues; B is Tr
X and Y each represent a single bond, or X or Y represents an amino acid residue selected from the group consisting of Asp, Glu, Arg, Lys and His; Or at least one selected from the group
Represents a peptide fragment consisting of 2 to 10 kinds of amino acid residues, the other being an amino acid residue selected from the group consisting of Asp, Glu, Arg, Lys and His or at least one amino acid residue selected from the group Represents a peptide fragment consisting of 2 to 10; Z represents a hydroxyl group or an amino group;
Represents an integer of 2 to 5. ). 2. An adsorbent obtained by immobilizing the peptide according to claim 1 on a carrier.