JPH06263795A - Peptide and adsorbent comprising the same immobilized on carrier - Google Patents

Abstract

PURPOSE:To obtain a peptide usable for treating, etc., chronic articular rheumatism, having a specific amino acid sequence without binding to immunoglobulin in spite of its ability to bind to an immune complex, useful as an adsorbent for the immune complex by immobilizing thereof on a carrier. CONSTITUTION:The peptide is expressed by formula I [A is peptide composed of 6-12 amino acid residues containing the amino acid sequence expressed by formula II (B and C are Trp, Tyr, Phe); X and Y are single bond, Asp, Gln, Lys, His, etc.; Z is OH or amino] and is obtained by using a granular resin of a styrene-divinylbenzene copolymer having 4-hydroxymethylphenoxymethyl group as a solid-phase support, successively binding protected amino acids in the direction from the C-terminal of the peptide to the N-terminal thereof, synthesizing a peptide chain according to a solid-phase synthetic method for the peptide, treating the resultant peptide with trifluoroacetic acid, etc., and then purifying the prepared peptide by high-performance liquid chromatography. This peptide has the ability to bind to an immune complex and is useful as an adsorbent, etc. for the immune complex.

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 an ability to bind to an immune complex, an adsorbent obtained by immobilizing the peptide on a carrier is useful for treating a disease in which the immune complex is involved. Is.

Immune complexes are detected in body fluids of patients with autoimmune diseases such as rheumatoid arthritis and Guillain-Barre syndrome, and patients with diseases such as malignant tumors, chronic infections and allergies. It is presumed that immune complexes deposit in tissues and cause damage, influence the immune system by various mechanisms, and have a close relationship with the cause or progression of the above diseases. Therefore, the removal of immune complexes from body fluids such as blood and plasma is necessary to treat the above diseases.

[0003]

As an adsorbent for immune complexes, an immunoadsorbent obtained by immobilizing protein A on a silica matrix (see JP-A-62-242628) and a hydrophobic compound immobilized on an insoluble carrier. Immunoadsorbents (see JP-A-57-122875) and immunoadsorbents (see Japanese Patent Publication No. 2-501985) in which a protein A analog (polypeptide) is immobilized on an insoluble carrier. There is. Protein A is a physiologically active protein derived from Staphyrococcus aureus, and its primary structure has already been clarified [Journal of Biological Chemistry (The
Journal of Biological Chemistry), Volume 259, 1
Pp. 695 (1984)].

[0004]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention
The protein A-immobilized immunoadsorbent described in Japanese Patent No. 42628 is 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 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.

The immunoadsorbent described in JP-A-57-122875, in which a hydrophobic compound is immobilized on an insoluble carrier, can be produced at a lower cost than the protein A-immobilized adsorbent described above and can be removed by adsorption. Although its ability is stable, it also does not have the ability to selectively adsorb and remove immune complexes, and it also adsorbs and removes immunoglobulin useful to the human body.

[0006] An immunoadsorbent substance described in Japanese Patent Publication No. 2-501985, which is immobilized with a protein A analog (polypeptide), selectively adsorbs and removes an immune complex from a body fluid such as blood or plasma. Useful for
Since the protein A analog is prepared by the recombinant DNA manipulation technique, there is a drawback that the product cost is high. Further, as the Protein A analog, one consisting of 29 to 51 amino acid residues is described, but when it is eluted into the body fluid such as blood or plasma when contacted with the body fluid, it shows antigenicity or toxicity, May cause undesired effects on the human body.

[0007] As described above, in the conventionally known immunoadsorbent, from the viewpoint of adsorption specificity, safety, manufacturing cost, etc.
It is not suitable for use in the treatment of emerging diseases of immune complexes.

[0008] One object of the present invention is to provide peptides which have the ability to bind immune complexes but which do not bind components useful to the human body. Another object of the present invention is that the immune complex can be selectively adsorbed and removed from body fluids such as blood and plasma, and the adsorption and removal capacity during sterilization and storage is extremely low, and the safety is high. To provide a high adsorbent.

[0009]

According to the present invention, the above object is achieved by the following general formula (I) H-X-A-Y-Z ... (I) [wherein A is a formula: Ala-BC-Glu-Ile-Leu (in the formula,
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 the formula; X and Y each represent a single bond or consist of Asp, Glu, Arg, Lys and His. 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
Represents a peptide fragment consisting of 1; Z represents a hydroxyl group or an amino group. ] It is achieved by providing the peptide represented by the above, and also it is achieved by providing the adsorbent of the immune complex formed by immobilizing the peptide 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 a peptide is described according to a conventional method 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 represented by the formula: Ala-BC-Glu-Ile-Leu (wherein B and C are
Represents Trp, Tyr or Phe. ) Represents a peptide fragment consisting of 6 to 12 amino acid residues, which has an amino acid sequence represented by the formula (1) as an essential component. Those that have undergone homologous substitution are preferred. Examples of the peptide fragment of A include 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 Ty
r Tyr Glu IleLeu (SEQ ID NO: 3), Formula (4): Ala Ph
e Trp Glu Ile Leu (SEQ ID NO: 4), Formula (5): Ala P
he Phe Glu Ile Leu (SEQ ID NO: 5), formula (6): Gln
Gln Asn Ala Phe Tyr Glu Ile Leu (SEQ ID NO: 6),
Formula (7): Gln Gln Asn Ala Trp Tyr Glu Ile Leu (SEQ ID NO: 7) Formula (8): Gln Gln Asn Ala Tyr Tyr Gl
uIle Leu (SEQ ID NO: 8), Formula (9): Gln Gln Asn Al
a Phe Trp Glu Ile Leu (SEQ ID NO: 9), formula (1
0): Gln Gln Asn Ala Phe Phe Glu Ile Leu (SEQ ID NO: 10), Formula (11): Ala Phe Tyr Glu Ile Leu As
n Met Pro Asn Leu (SEQ ID NO: 11), Formula (12): A
la Trp Tyr Glu Ile Leu Asn Met Pro Asn Leu (SEQ ID NO: 12), Formula (13): Ala Tyr Tyr Glu Ile Leu As
n Met Pro AsnLeu (SEQ ID NO: 13), Formula (14): Al
a Phe Trp Glu Ile Leu Asn Met ProAsn Leu (SEQ ID NO: 14) or Formula (15): Ala Phe Phe Glu Ile Leu
A peptide fragment of the amino acid sequence represented by u Asn Met Pro Asn Leu (SEQ ID NO: 15) is more preferable. Since protein A is a physiologically active protein derived from Staphylococcus aureus, a partial peptide of protein A having the amino acid sequence represented by formula (1) or a homologous substitution in the amino acid residues constituting it However, when the number of amino acid residues is 13 or more, toxicity and antigenicity to the human body become high, and synthesis becomes complicated.

X and Y in the general formula (I) are defined as described above, but by adding X and Y to A, hydrophilicity is imparted to A, and therefore, they are represented by the general formula (I). The peptide can be efficiently immobilized on the carrier. Further, when an adsorbent having a peptide represented by the general formula (I) immobilized on a carrier is used in contact with a body fluid such as blood or plasma, even if the peptide is liberated and contaminated in the body, Since the peptide is rendered hydrophilic by the addition of X and Y, it is easily excreted in urine, and is safe with low antigenicity to the human body. A peptide of the general formula (I) when both X and Y are single bonds or when either X and Y are amino acid residues or peptide fragments different from those defined above. The adsorbent in which is immobilized on a carrier may have a significantly reduced ability to adsorb and remove the immune complex due to sterilization.

Examples of the peptide fragment represented by X and Y in the general formula (I) include the following peptide fragments. -Asp-Asp-, -Glu-Glu-, -Lys-Lys-, -His-His-, -Arg-A
rg-, -Asp-Glu-, -Glu-Asp-, -Glu-Lys-, -Lys-Glu-,-
His-Asp-, -Asp-His-, -His-Lys-, -Lys-His-, -Arg-Ly
s-, -Lys-Arg-,-(Asp) ₅ -,-(Arg) ₅ -,-(Lys) ₅ -,-(Gl
u) ₅ -,-(His) _5- , -Lys-Glu-Glu-Asp-, -Asp-Glu-His-Ly
s-,-(Asp) ₁₀ -,-(Arg) ₁₀ -,-(Lys) ₁₀ -,-(Glu) ₁₀ -,-
(His) _10- , -Lys-Glu-His-Arg-Asp-Lys-Lys-Glu-, -Lys-
Glu-Glu-Asp-Arg-Lys-Lys-His-

The peptide represented by the general formula (I) preferably has about 20 or less amino acid residues from the viewpoints of easiness of synthesis and low antigenicity to human body.

The peptide represented by the general formula (I) is synthesized by a method usually 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. However, it is easy to operate by solid-phase synthesis [for example, the Journal of the American Chemical Society (Journa
l of the American Chemical Society), Vol. 85, pp. 2149-2154 (1963); "Biochemistry Experimental Course 1 Chemistry IV of Protein Chemistry and Chemical Synthesis and Peptide Synthesis" (November 15, 1972) edited by The Biochemical Society of Japan. , Tokyo Kagaku Doujinshi, pp. 207-495; edited by The Biochemical Society of Japan, "Seikagaku Chemistry Laboratory Course 2 Protein Chemistry (2)" (May 20, 1987, Tokyo Kagaku Doujinshi) ), 6th
41-694].

The peptide represented by the general formula (I) can be produced by the 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 desired peptide. From the side toward the N-terminal direction, the corresponding amino acid is protected by a functional group such as an α-amino group other than the α-carboxyl group possessed by the amino acid and then condensed and bonded, and α in the bonded amino acid -The peptide chain is extended by sequentially repeating the operation of removing the protective group of the amino group that forms a peptide bond such as an amino group to form a peptide chain corresponding to the target peptide, and then the peptide chain is The target peptide can be obtained by removing the protective group from the functional group that is protected from the combined group. 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 peptide represented by the general formula (I) is efficiently immobilized on a carrier, and the obtained adsorbent is an immune complex in a body fluid such as blood or plasma of a diseased patient in which the immune complex is involved. Can be selectively adsorbed and removed. The carrier used when immobilizing the peptide represented by the general formula (I) is an amino group or 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 and a hydroxyl group are preferable. The carrier is preferably insoluble in body fluids such as blood and plasma and porous. The porous carrier having a large effective surface area capable of adsorbing the immune complex has an exclusion limit protein molecular weight in the range of about 10 ⁶ to 10 ⁹ or an average pore size of about 50 to 1000.
It is preferred to use those in the range of 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 a carrier include CM-cellulofine CH (molecular weight of exclusion limit protein: 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-Tr
isacryl M) [Exclusion limit protein molecular weight: 1 x 1
0 ^7, Sweden Pharmacia -LKB (Pharmacia
-LKB), etc., and agarose-based carrier such as Sepharose CL-4B (Sepharose CL-4B) [Exclusion limit protein molecular weight: 2 × 10 ⁷ , manufactured by Pharmacia-LKB, Sweden] Organic carrier such as carrier, and CPG-10-
An inorganic carrier such as a porous glass such as 1000 [exclusion limit protein molecular weight: 1 × 10 ⁸ , average pore size: 100 nm, manufactured by US Electro-nucleonics] can be used.

The immobilization of the peptide represented by the general formula (I) on the carrier is generally carried out according to the method adopted when immobilizing the peptide or protein on the carrier. As the immobilization method, for example, the carboxyl group of the carrier is reacted with N-hydroxysuccinimide to convert into a succinimidooxycarbonyl group, and the peptide represented by the general formula (I) is converted into an amino group. In the presence of a condensation reagent such as dicyclohexylcarbodiimide to the amino group or carboxyl group of the carrier in the presence of a condensation reagent such as dicyclohexylcarbodiimide. Examples thereof include a reaction method (condensation method) and a method of crosslinking the carrier and the peptide represented by the general formula (I) with a compound having two or more functional groups such as glutaraldehyde (carrier crosslinking method). . In particular, the immobilization method by the active ester method can immobilize the peptide represented by the general formula (I) on a carrier with almost no decrease in the binding ability between the peptide and the immune complex. preferable.

The amount of the peptide represented by the general formula (I) immobilized on the carrier is usually about 3 × so that the resulting adsorbent can effectively adsorb a significant amount of the immune complex. 10
^-8 mol / g (carrier) or more is preferable, and in order to effectively utilize the peptide represented by the general formula (I) immobilized on the carrier for adsorption of the immune complex, it is about 1 × 10
^It is more preferably within the range of ^{−7 to} 5 × 10 ⁻⁵ mol / g (carrier).

The immune complex is removed by using an adsorbent obtained by immobilizing the peptide represented by the general formula (I) on a carrier.
It is carried out by bringing the immune complex into contact with a body fluid such as blood or plasma containing the immune complex to adsorb the immune complex. 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.

[0024]

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

EXAMPLE 1 Formula (16): Lys Lys Ala Phe Tyr Glu Ile Leu (SEQ ID NO: 16) was used as an automatic peptide synthesizer [Applied Biosystems (US).
The model 430A (Model 430A) manufactured by ystems) was used for the solid phase synthesis. Styrene-divinylbenzene copolymer having 4-hydroxymethylphenoxymethyl group at a ratio of 0.85 mmol / g (resin) [Structural ratio of styrene and divinylbenzene (molar ratio): 99: 1]
0.29 of granular resin [HMP resin manufactured by Applied Biosystems, USA]
g, according to the series of operations shown in Table 1 from the C-terminal side to the N-terminal direction 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 attached. In the condensation reaction, the above amino acids are respectively 9-fluorenylmethoxycarbonyl-L-alanine, 9-fluorenylmethoxycarbonyl-L-glutamic acid-γ-t-butyl ester, 9-fluorenylmethoxycarbonyl-L-
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 the amount used was about twice the molar amount of the substrate.

[0026]

[Table 1]

After the reaction procedure for all amino acids was completed, 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. 600 mg dry resin and 1 trifluoroacetic acid in a vial
0 ml, water 0.5 ml, thioanisole 0.5 ml, ethanedithiol 0.25 ml and phenol 0.75 g were mixed. After standing at room temperature for 20 hours, the mixture was filtered through 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 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 (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], a single sharp peak was shown at 16.2 minutes. The molecular weight of the peptide determined by FAB (Fast Atom Bombardment) mass spectrum was 1010 (theoretical value 1011.18).

Examples 2 to 24, Comparative Examples 1 to 5 By carrying out solid-phase peptide synthesis in the same manner as in Example 1, the peptides shown in Tables 2 to 6 were obtained. However, as the resin for the solid phase, in Examples 10 and 11, a resin for amide peptide (sold by Kokusan Kagaku Co.) was used. 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
Used as -trityl-L-histidine, 9-fluorenylmethoxycarbonyl-L-methionine, 9-fluorenylmethoxycarbonyl-L-proline and 9-fluorenylmethoxycarbonyl-L-tryptophan.

[0030]

[Table 2]

[0031]

[Table 3]

[0032]

[Table 4]

[0033]

[Table 5]

[0034]

[Table 6]

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

[0036]

[Table 7]

Example 25 10 g of cellulose particles (CM Cellulofine CL, manufactured by Chisso Corp.) were suspended in 50 ml of anhydrous dioxane (commercial dioxane distilled in the presence of metallic sodium). 0.5 g of N-hydroxysuccinimide and dicyclohexylcarbodiimide 1.
0 g was added and the mixture was shaken and stirred overnight at room temperature. The resulting mixture was added with 0.02 mol / l phosphate buffer (pH
It was washed with 7.4) 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 1,
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 1 immobilized thereon was obtained.

Example 26 In Example 25, 10 g of polyvinyl alcohol particles (CM-Toyopearl 650C manufactured by Tosoh Corp.) were used in place of 10 g of the cellulose particles, and the peptide obtained in Example 1 was used in place of Example 2. 20 peptides obtained
By the same method except that mg was used, about 10 g of polyvinyl alcohol particles having 18.8 mg of the peptide obtained in Example 2 immobilized thereon were obtained (immobilization rate of the peptide on the carrier was about 94%).

Example 27 Porous glass particles [CPG-10-100 manufactured by Electro-nucleonics, USA
0] was reacted in 100 ml of a toluene solution containing 5 ml of γ-aminopropyltriethoxysilane under heating and reflux for 24 hours. The resulting mixture was washed with anhydrous dioxane and suction filtered. The obtained particles were suspended in 100 ml of anhydrous dioxane, and 3 g of succinic anhydride was added to this suspension.
Was added and the mixture was stirred at room temperature overnight. The resulting mixture was washed with anhydrous dioxane and suction filtered. The particles obtained were suspended in 50 ml of anhydrous dioxane, 0.5 g of N-hydroxysuccinimide and 1.0 g of dicyclohexylcarbodiimide were added to this suspension, and the mixture was stirred overnight at room temperature. 0.02 mol / l of the resulting mixture
It was washed with a phosphate buffer solution (pH 7.4), and filtered with suction. The particles obtained were converted to the peptide obtained in Example 3 by 20 times.
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 filtered by suction to obtain about 10 g of the adsorbent having 20 mg of the peptide obtained in Example 3 immobilized thereon (immobilization rate of peptide on carrier: about 100%).

Examples 28-48, Comparative Examples 6-10 Peptide-immobilized adsorbents were prepared in the same manner as in Examples 25-27 except that 20 mg of the peptides shown in Tables 2-6 were used. Got each. Table 8 shows the peptides and the particulate carriers used, and the immobilization ratio of the peptides on the carriers.

[0041]

[Table 8]

From Table 8, it is clear that in the case of peptides of the general formula (I) in which both X and Y are single bonds as in Comparative Examples 6 to 10, the immobilization rate on the carrier is low. .

Test Example 1 1 g of the adsorbent obtained in Examples 25 to 48 in 3 ml of plasma of a patient with rheumatoid arthritis, or an untreated particulate carrier as a control [cellulose particles: CM manufactured by Chisso Co., Ltd.
-Cellulofine, polyvinyl alcohol particles: Tosoh Corporation CM-Toyopearl 650C, porous glass particles: US Electro-nuclearonics (Electro-nu)
cleonics) CPG-10-1000] 1 g,
Suspended at 37 ° C for 2 hours. The obtained suspension was centrifuged to obtain a supernatant. The globulin and albumin concentrations in the obtained supernatant were measured using an A / G test kit (A / GB Test Wako, manufactured by Wako Pure Chemical Industries, Ltd.), and the immune complex concentration was measured using an immune complex detection reagent kit [Immune complex]. Body (C1q-Ig
G) "Kuraray", Immunomedics
Table 9 shows the results obtained by calculating the adsorption removal rate by using the following equation (1).

[0044]

[Equation 1]

For comparison, instead of the adsorbents obtained in Examples 25 to 48, an adsorbent having protein A immobilized on a carrier and an adsorbent having tryptophan as a hydrophobic compound immobilized on the carrier were used. The results of the same experiment as above are shown together in Table 9. The protein A-immobilized adsorbent was prepared in the same manner as in Example 25 except that 20 mg of protein A [manufactured by Pierce Chemical Company] was used (immobilization of protein A on a carrier. Example 2 except that the tryptophan-immobilized adsorbent used was 20 mg of tryptophan (manufactured by Kyowa Hakko Kogyo Co., Ltd.).
What was prepared by the same method as in Example 5 (immobilization rate of tryptophan on the carrier: about 80%) was used.

[0046]

[Table 9]

From Table 9, the use of the adsorbent of the present invention
It is clear that the immune complex can be selectively adsorbed and removed with almost no adsorption of albumin and globulin, which are useful for the human body.

Test Example 2 In Test Example 1, Examples 29, 38, 39, 40, 4
1, 42, 43, 46, the adsorbents obtained in Comparative Examples 6 to 10 and the autoclave-sterilized adsorbents described above were used for suspension of plasma in the same manner as described above. Table 10 shows the results of measuring the concentration of albumin, globulin, and immune complex and calculating the adsorption removal rate. In addition, as the adsorbent subjected to the autoclave sterilization treatment, 1 g of the peptide-immobilized adsorbent was added with 0.1 g of sodium chloride.
The suspension was suspended in 5 ml of a 0.02 mol / l phosphate buffer solution (pH 7.4) containing 5 mol / l, and the suspension was heat-treated under pressure in an autoclave sterilizer at 121 ° C. for 20 minutes.

[0049]

[Table 10]

From the above results, it can be seen that the adsorbents to which the peptides of the general formula (I) in which both X and Y are single bonds are immobilized, such as the adsorbents of Comparative Examples 6 to 10, were subjected to autoclave sterilization to obtain an immune complex. It is clear that the adsorbent of the present invention markedly decreases the adsorbent-removal ability of the immune complex even after the autoclave sterilization treatment.

[0051]

INDUSTRIAL APPLICABILITY According to the present invention, a peptide having the ability to bind to an immune complex is provided. The adsorbent having the peptide immobilized on a carrier can selectively adsorb and remove the immune complex from the body fluid without adsorbing and removing a component useful for the human body from the body fluid. It is useful for the treatment of diseases associated with.

[0052]

[Sequence Listing] 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 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 1 5 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 1 5 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

Claims (2)

[Claims] 1. A general formula: H-X-A-Y-Z, wherein A is a formula: Ala-B-C-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 the formula; X and Y each represent a single bond or consist of Asp, Glu, Arg, Lys and His. 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
Represents a peptide fragment consisting of 1; Z represents a hydroxyl group or an amino group. ] The peptide represented by these. 2. An adsorbent for an immune complex obtained by immobilizing the peptide according to claim 1 on a carrier.