JPH06105909A - Antiplatelet antibody adsorbent material - Google Patents

Abstract

PURPOSE:To provide the method of removing antiplatelet antibodies by using a peptide contg. a specific amino acid arrangement or an org. compd. prepared by modifying the peptide and bonding the peptide or the compd. to the antiplatelet antibodies and the antiplatelet antibody adsorbent material formed by immobilizing the peptide or the org. compd. to a water insoluble carrier. CONSTITUTION:This antiplatelet antibody adsorbent material is prepd. by immobilizing at least one kind among the peptide of the amino acid arrangement having bondability with the antiplatelet antibodies or the amino acid of 1 to 60 pieces of both ends or one-side ends of the peptide or the modified peptide bonded with the side chains expressed by formula -A-(CH2)n-B- (A, B are N or CO, n is 1 to 20 integer) to the water insoluble carrier.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention utilizes an antiplatelet antibody, which is an autoantibody produced in blood for some reason, by using a peptide containing a specific amino acid sequence or an organic compound containing the peptide region. The present invention relates to a method for removing an antiplatelet antibody, which comprises binding with an antiplatelet antibody, and an antiplatelet antibody adsorbent characterized by immobilizing the peptide or an organic compound on a carrier.
The present invention is particularly useful in the treatment of diseases such as idiopathic thrombocytopenic purpura (hereinafter referred to as ITP) that causes various symptoms such as bleeding and purpura due to the decrease in the number of platelets due to the presence of antiplatelet antibodies. It is useful.

[0002]

ITP is a type of autoimmune disease characterized by shortened platelet lifespan and increased antiplatelet antibody, which is a platelet-binding immunoglobulin. In the bone marrow, the number of megakaryocytes is normal or increased. Immune thrombocytopenia that does not show findings suggesting the presence of a disease does not have a causative disease such as collagen disease, lymphoproliferative disease, or drug allergy.

ITP is divided into an acute type and a chronic type. The acute form is more common in children, and bleeding symptoms are severe but relatively easy to cure. Antecedent infections such as upper respiratory tract infection and viral infection are often found 3 weeks before the onset. Since thrombocytopenia is observed in the convalescent stage of infection, there is a strong possibility of an immune complex disease in which the formed immune complex binds to the platelet membrane Fc receptor and platelets are nonspecifically destroyed. On the other hand, the chronic type has a weak bleeding symptom but persists for a long period of time, and it is considered to be an autoimmune disease due to antiplatelet antibody, which is common among adults, especially in women in their 20s.

Although there are individual differences in the symptoms observed due to the decrease in the number of platelets due to the antiplatelet antibody, in general, bleeding symptoms are not generally observed when the number of platelets is 50,000 / μl or more,
At 30,000 to 50,000 cells / μl, easy bleeding during trauma, ecchymosis, 10,000 to 30,000 cells / μl
Then, the appearance of purpura on the exposed area, frequent menses, 10000 /
Hemouria, irregular genital bleeding, epistaxis, and gingival bleeding are observed at μl or less, and intracranial bleeding and gastrointestinal bleeding are often the direct causes of death.

In the treatment of ITP, administration of a corticosteroid agent, removal of the spleen, administration of an immunosuppressive agent and the like are applied depending on the degree of the symptoms. However, there are many cases where the platelet count is not effectively recovered by such drug therapy or splenectomy. Mechanisms for the development of various autoimmune diseases including ITP have not been fully elucidated, and the absolute treatment method for such patients has not been established at present, and measures are urgently needed. There is.

As a method for treating such autoimmune diseases,
Various attempts have been made to adsorb and remove autoantibodies by extracorporeal circulation of blood using an immunoadsorption column. For example, application of a column in which protein A having a binding property with immunoglobulin is immobilized on a carrier is also under study. However, protein A is not very useful because it is expensive, has poor adsorption specificity, and has side effects. In addition, various immobilization columns for antibodies against the substance to be removed have been studied, but safety, storage stability problems, immobilization that allows antibodies to react effectively, and sterilization methods are limited. There are many problems in using it as medical equipment for reasons.

[0007]

DISCLOSURE OF THE INVENTION The present invention recognizes anti-platelet antibodies in blood and has binding properties for the purpose of enabling effective cure of ITP for which an absolute therapeutic method has not been established. A relatively low molecular weight peptide or modified peptide is used for binding and removal. Further, the present invention provides an adsorbent in which the above-mentioned peptide or modified peptide is immobilized on a water-insoluble carrier, and is intended to be applied to blood purification therapy such as extracorporeal circulation using a column packed with the adsorbent.

[0008]

Means for Solving the Problems The present invention, which has achieved the above object, has as its gist the removal of antiplatelet antibodies with a peptide or modified peptide containing an amino acid sequence which can serve as an antigenic determinant of antiplatelet antibodies. . An anti-platelet antibody adsorbent is obtained by immobilizing the above peptide or modified peptide on a water-insoluble carrier, and blood purification treatment by extracorporeal circulation or the like can be performed using a column packed with this.

In general, antiplatelet antibody in ITP patients is a complex of glycoproteins present on the surface of platelet membrane G
It is known that it is often an antibody against P2b / 3a or GP1b / 9. The peptide or modified peptide in the present invention means one having an amino acid sequence determined by predicting and screening a region that can be an antigenic determinant from the primary structure of these glycoproteins by various methods, or the obtained peptide is 1 to up to 60 other amino acids or -NH (CH _{2) n}
It is a modified peptide in which the side chain represented by CO- (n is an integer of 1 to 20) is bound by a peptide bond.

GP3a is a glycoprotein consisting of a single polypeptide having a molecular weight of about 105 kilodalton, and its cDNA has already been cloned (J. Biol.
Chem. , 262, 3939, 1987). It is composed of 762 amino acid residues and 26 at the N-terminal.
There is a signal peptide consisting of amino acid residues. The extracellular portion has 689 amino acids, the transmembrane domain has 29 amino acids, and the cytoplasm has 41 amino acids. 460 to 62
33 to 38 in the region consisting of the 7th amino acid residue
There are four cysteine-rich domain repeats consisting of 4 amino acids.

GP2b has a molecular weight of about 140 kilodaltons and is composed of an H chain of about 125 kilodaltons and an L chain of about 25 kilodaltons, which are linked by SS bonds. It consists of 1039 amino acid residues and its sequence has been determined (J. Biol. Chem., 2
62, 8476, 1987). There is a signal peptide consisting of 871 in the H chain, 137 in the L chain and 30 amino acid residues at the N-terminal part. Transmembrane domain is L
It is present in the chain and is composed of 26 hydrophobic amino acids.

GP3a and GP2b form a complex in bone marrow megakaryocytes or their progenitor cells in the bone marrow. GP2b and GP3a form a 1: 1 complex by a non-covalent bond on the surface of the platelet membrane. 55 in GP2b
It is presumed that the portion of the 8th to 747th amino acid residue and the portion of the 114th to 303rd amino acid residue in GP3a are associated (medical history,
160, 681, 1992). Latent receptors for adhesive proteins such as fibrinogen are present on this molecule. GP2b / 3a is the most abundant glycoprotein on the surface of platelet membranes.

GP1b / 9 is GP1b α chain, β chain, GP
Von Willebr composed of 3 subunits of 9 and inactivated platelets in subendothelial tissue
It is considered to be important as a molecule receptor that recognizes and factor and a molecule that controls the very early stage of platelet thrombus formation. The α chain and the β chain are linked by SS bonds, and the molecular weights are 140 kilodalton and 24 kilodalton, respectively. GP9 is 17 to 18 kilodaltons and is considered to be non-covalently bound to GP1b (Medical Ayumi, 160, 677, 1992). The amino acid sequence of each of them was determined, and the number of α chains was 610 (Proc. Natl. Acad. Sci. USA, 8
4,5615, 1987), 181 β chains (Pro
c. Natl. Acad. Sci. USA, 85, 21
35, 1988) and 160 GP9 (Proc. Na.
tl. Acad. Sci. USA, 86, 6773, 1
989).

As an example of the most abundant peptide having immunogenicity against the antiplatelet antibody for each of the above glycoproteins, GP2b / 3a, which is the most abundant peptide, corresponds to the 68th to 78th Pro position in the amino acid sequence of GP3a. -Leu-Ser-Asp-Lys-Gly-
Ser-Gly-Asp-Ser-Ser, 349th to 364th Gly-Lys-Ile-Arg-Se.
r-Lys-Val-Glu-Leu-Glu-Val
-Arg-Asp-Leu-Pro-Glu, 475th to 490th Glu-Glu-Asp-Tyr-A
rg-Pro-Ser-Gln-Gln-Asp-Gl
u-Cys-Ser-Pro-Arg-Glu, 619
To 623rd Lys-Phe-Asp-Arg-
Glu, 741-744th Thr-Ala-A
Five regions of sn-Asn are regions confirmed by enzyme immunoassay (hereinafter referred to as ELISA method). All of these regions are regions having relatively strong hydrophilicity and are presumed to be regions existing on the GP3a molecule surface. For GP2b, Glu-Thr
-Arg-Asn-Val-Gly-Ser (106th to 112th), Glu-Lys-Thr-Glu-
Glu-Ala-Glu-Lys-Thr (148th to 156th), Glu-Pro-Glu-Gln-P
Three regions of ro-Ser-Arg-Leu (896th to 903th) are immunogenic to the antiplatelet antibody. These are also highly hydrophilic regions and are presumed to exist on the surface of the molecule. Also with respect to GP1b / 9, several peptides have been confirmed to have immunogenicity against antiplatelet antibodies.

The above-mentioned region is considered to be the minimum required region for binding with antiplatelet antibody. When the antiplatelet antibody is actually removed using such a peptide, it is necessary to reproduce the state close to the reaction mode with the antiplatelet antibody present in blood. Further, it is often necessary to consider the stability of the structure when it is present in blood, or to increase the solubility in an aqueous solvent when it is immobilized on a carrier.

Therefore, one or both end regions of the above-mentioned peptide region is extended according to the amino acid sequence of each protein, or several amino acids such as lysine for increasing water solubility are added to the end. It is preferable to bind or to bind an amino acid such as cysteine to the terminal to facilitate immobilization on the carrier. The total number of amino acid residues in the peptide or modified peptide actually used is preferably 60 or less, but more preferably 30 or less considering the difficulty of synthesis, cost, stability and the like. Furthermore, considering the ease of formation of higher-order structures required to express immunogenicity, etc., 10 to 2
It can be said that up to 5 are optimal.

In consideration of the immobilization reaction and stability,
For example, it is also preferable to use an organic compound obtained by modifying the above-mentioned peptide by amidating the N-terminal or introducing an appropriate functional group at the terminal. In addition, multiple peptides
_{(CH 2) n -B- (A} , B represents NH or CO)
It is possible to further enhance the binding to the antiplatelet antibody by using a side chain as shown in (4). In this case, the value of n is preferably 1 to 20, and more preferably 3 to 10.

The method of peptide synthesis is not particularly limited, but it is easier to apply the solid phase synthesis method than the liquid phase synthesis method. In this case, an amino acid corresponding to the C-terminal of the peptide to be synthesized is bound to a support that is insoluble in an organic solvent, and corresponding amino acids with functional groups such as α-amino groups other than α-carboxyl groups protected in the N-terminal direction are sequentially condensed. After binding by reaction, the peptide chain is extended by alternately repeating the reaction of removing the protecting group after binding.

After the desired peptide is obtained, the peptide chain is cleaved from the support and deprotected. Hydrogen fluoride is often used for this, but in terms of safety and ease of handling, trifluoromethanesulfonic acid (hereinafter TFM) is used.
It is suitable to use SA). After deprotection by reacting with thioanisole and 1,2-ethanediol in TFMSA, trifluoroacetic acid (hereinafter referred to as TFA)
The peptide is recovered by cleavage from the support. The crude peptide is obtained by freeze-drying this.

The above crude peptide is a high performance liquid chromatography (hereinafter referred to as HPLC) using a reversed phase column.
The product is collected and purified. The HPLC conditions are usually optimized based on the system used for protein purification. A fraction corresponding to the obtained chromatographic peak is collected and freeze-dried. Molecular weight analysis of the obtained purified peptide fraction by mass spectrometry,
Identification is performed by amino acid composition analysis or amino acid sequence analysis.

The water-insoluble carrier used for immobilizing the above-mentioned peptide or organic compound is not particularly limited, but when used for blood purification, the complement system or coagulation system upon contact with blood components It is preferable to use a porous carrier of cellulose, polystyrene, polyvinyl alcohol, polyacrylic acid ester or a derivative thereof because it is necessary to consider the influence on the above. The average pore size is preferably 20 to 3000 nm,
It is more preferably 30 to 1000 nm. The carrier may be in the form of beads, fibers, or membranes (including hollow fibers).

Further, at the time of introducing the ligand, it is more preferable to immobilize it via a hydrophilic spacer for the purpose of improving adsorption efficiency by reducing steric hindrance and suppressing non-specific adsorption. As the hydrophilic spacer, it is preferable to use, for example, a polyalkylene oxide derivative whose both ends are substituted with a carboxyl group, an amino group, an aldehyde group, an epoxy group or the like, and the degree of polymerization thereof is preferably 10 to 1000, and 100 to 100 Those of 500 are more preferable.

The peptides and organic compounds used as the above-mentioned ligands are relatively stable and low-molecular weight substances, and their immobilization reaction conditions are less restricted as compared with the case of immobilizing proteins such as enzymes and antibodies. That is also one of the advantageous factors. Therefore, the immobilization method is not particularly limited, but an immobilization method such as a Schiff base reaction, an epoxy reaction, a condensation reaction using a carbodiimide reagent, etc. often used when immobilizing an enzyme or an antibody is applied. It is preferable.

[0024]

EXAMPLES The method of introducing the peptide-based substance into the water-insoluble carrier in the present invention is not particularly limited as long as it is based on the above-mentioned one. The present invention will be described below with reference to examples.

Example 1 Lys-Lys-Pro-
Leu-Ser-Asp-Lys-Gly-Ser-G
Immobilization of ly-Asp-Ser-Ser to Cellulose (1) Synthesis of Peptide Ly in which two lysine residues are bound to the N-terminal of the 68th to 78th region having immunogenicity in GP3a
s-Lys-Pro-Leu-Ser-Asp-Lys
The peptide having the sequence of -Gly-Ser-Gly-Asp-Ser-Ser was synthesized by a solid phase synthesis method using a peptide synthesizer Model 430A (manufactured by Applied Biosystems). PAM serine (t-Boc-), which is a support having C-terminal serine bound thereto.
L-Ser (Bzl)) 0.5 mmol (manufactured by Applied Biosystems) was used to repeat the deprotection reaction and the condensation reaction in the order of the N-terminal in accordance with the synthetic program published in the above peptide synthesizer to repeat the peptide chain. Extended. That is, the protecting group t-butoxycarbonyl group was removed with TFA and dichloromethane (hereinafter referred to as DCC), washed with dichloromethane (hereinafter referred to as DCM), neutralized with diisopropylethylamine and DCC, and then dimethylformamide (hereinafter referred to as The procedure of washing with DMF), the condensation reaction with DMF, and washing with DCM was repeated. Amino acid is t-
Boc-L-Lys (Cl-Z), t-Boc-L-S
er (Bzl), t-Boc-L-Asp (OBz
l), t-Boc-L-Gly, t-Boc-L-Le
_{u · H 2 O, t-} Boc-L-Pro ( both Applied Biosystems) was used cartridges 2.0mmol of.

(2) Deprotection group, cleavage of peptide chain 1 m of thioanisole was added to 1 g of the support after completion of the above reaction.
After 0.5 ml of 1,1,2-ethanediol was added and stirred for 10 minutes, 10 ml of TFA was added while cooling with ice water and stirred for 10 minutes. Further, 1 ml of TFMSA was added and the mixture was stirred at room temperature for 30 minutes. Pre-cooled diethyl ether is added to this until it does not appear, and the mixture is stirred, filtered while washing with diethyl ether using a medium-pore glass filter, and TFA is added to dissolve the peptide in ether. Collected. The peptide in ether was filtered through a fine-hole glass filter, the peptide on the glass filter was dissolved in 2N acetic acid, and freeze-dried to obtain a crude peptide.

(3) Purification of Peptide The above crude peptide was dissolved again in 2N acetic acid to give a mixture of 0.
The solution filtered with a 2 μm membrane filter is HPL
Subjected to C. For HPLC, Model 130A system (manufactured by Applied Biosystems) was used, and for the column, reverse phase Aquapore Prep-10, C8 (manufactured by Applied Biosystems) was used. As the mobile phase, water containing 0.1% TFA was used as solution A, and 70% acetonitrile / water (v / v) containing 0.1% TFA was used as solution B.
Elution was performed with a linear concentration gradient from solution to solution B. The chromatographic peak was almost single, and the corresponding fractions were collected. The fractionation was repeated several times, and this was freeze-dried to obtain a purified peptide. The obtained peptide is BIOION
It was confirmed by analysis with a 20 mass analyzer (manufactured by Applied Biosystems) that the target peptide was obtained.

(4) Immunogenicity of peptide The immunogenicity of the obtained peptide was confirmed by hemagglutination. Alsever solution-preserved sheep erythrocytes (manufactured by Sedalene) in PBS at 2.5% concentration (v / v)
It was suspended in (pH 7.2). 0.0 to 10 ml of suspension
A 05% tannic acid solution was added, and the mixture was stirred at 37 ° C. for 15 minutes for reaction, washed with the above PBS by centrifugation at 1500 rpm for 10 minutes, and then resuspended in 10 ml of physiological saline. On the other hand, the purified peptide was dissolved in 0.15M phosphate buffer (pH 6.4) so as to have a concentration of 2 mg / ml, and was mixed with the suspension in an equivalent amount. Stir at 37 ° C for 15 minutes, wash with the above PBS twice, and then use the same PB again.
Suspended in S. 0.01M on 96-well microplate
Add 25 μl of EDTA-added veronal buffer,
Add an equal amount of serum from ITP patients (5 types), mix, and mix 2.
Make serial dilutions in series. The above-mentioned red blood cell suspension was added drop by drop and mixed, and after reacting at 37 ° C. for 2 hours, the aggregating property was confirmed. The results are as shown in Table 1, and the maximum dilution ratio in which agglutination was confirmed was displayed.

[0029]

[Table 1] In Table 1, N. D. Indicates that it did not react.

(5) Immobilization of Peptide on Cellulose The above peptide was immobilized on porous cellulose. Cellulofine series GCL-1 for cellulose
000 m (manufactured by Chisso) was used. Dissolve 3.5 g of sodium periodate in 300 ml of 1N sulfuric acid to give GCL-1.
000 m100 g was added and reacted by stirring at room temperature for 20 hours. The reaction product was recovered and thoroughly washed to obtain [aldehyde]-[cellulose]. The aldehyde content was determined by the oxime method and was 0.61 meq / g.

Next, 15 g of a polyalkylene oxide having a molecular weight of 5000 and having amino groups at both ends (hereinafter referred to as PEO amine) was dissolved in 300 ml of a carbonate buffer having a pH of 9.5, and the above [aldehyde]-[cellulose] was added. The reaction was carried out by stirring at room temperature for 24 hours. The reaction product is collected, washed thoroughly, and then [PEO amine]-[cellulose].
Got Amino group content is measured by potentiometric titration with hydrochloric acid
Using TITE 101 (made by Hiranuma Sangyo), 0.5
It was 4 meq / g.

The above [PEO amine]-[cellulose] was suspended in 300 ml of a carbonate buffer solution having a pH of 9.5, 30 ml of a 25% glutaraldehyde solution was added, and the mixture was reacted at room temperature for 24 hours with stirring. The reaction product was recovered and thoroughly washed to obtain [PEO aldehyde]-[cellulose].
The aldehyde content was determined by the oxime method to be 0.4.
It was 9 meq / g.

On the other hand, 25 mg of the above purified peptide was added to pH 9.
It was dissolved in 50 ml of the carbonate buffer solution of No. 5, 5 g of [PEO aldehyde]-[cellulose] was added, and the mixture was reacted by stirring at room temperature for 24 hours. The reaction product was recovered and thoroughly washed to obtain [peptide]-[cellulose]. The peptide immobilization rate was calculated by quantifying the nitrogen content in the reaction residual liquid by the micro Kjeldahl method, and 85% was immobilized. PH of the above [peptide]-[cellulose]
The suspension was suspended in 50 ml of a carbonate buffer solution of 9.0, 1 g of sodium borohydride was added, and the mixture was reacted by stirring at room temperature for 20 hours. The reaction product was recovered and thoroughly washed to obtain the target adsorbent.

(6) Evaluation of Adsorption Performance of Peptide-Immobilized Adsorption Material The adsorption performance of the antiplatelet antibody was evaluated using the above-mentioned serum of the ITP patient in the adsorption material. 1 ml of the above adsorbent
Serum of the above ITP patients (5 types) collected in a vial
3 ml was added and incubated at 37 ° C. for 30 minutes with shaking. The suspension was centrifuged at 3000 rpm for 10 minutes, and the amount of antiplatelet antibody in the supernatant was quantified by the ELISA method (Acta. Haemat., 66, 25.
1, 1981). The results are shown in Table 2, and the adsorption rate [%] calculated from the value of the serum before incubation was shown. The nonspecific adsorption of albumin was also quantified using Albumin B-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). The results are shown in Table 3, and the adsorption rate [%] was shown as in Table 2.

[0035]

[Table 2]

[0036]

[Table 3]

<Example 2> Glu-Glu-Asp-
Tyr-Arg-Pro-Ser-Gln-Gln-A
sp-Glu-Cys-Ser-Pro-Arg-Gl
Immobilization of u on cellulose Glu-Glu-Asp-Tyr-Arg-Pro corresponding to the 475th to 490th regions in GP3a.
-Ser-Gln-Gln-Asp-Glu-Cys-
Regarding the synthesis of the peptide having the sequence of Ser-Pro-Arg-Glu, PAM glutamic acid (t-Boc-
L-Glu (OBzl)) (manufactured by Applied Biosystems) and t-Boc-L-Arg (Tos), t.
-Boc-L-Pro, t-Boc-L-Ser (Bz
l), t-Boc-L -Cys (4-CH 3 OBz
l), t-Boc-L-Glu (OBzl), t-Bo
c-L-Asp (OBzl), t-Boc-L-Gl
It carried out like Example 1 using n, t-Boc-L-Tyr (Br-Z) (all are the Applied Biosystems company make). Deprotection group of the support after synthesis,
Cleavage of the peptide chain, purification by reverse phase HPLC and identification were also carried out in the same manner as in Example 1.

The immunogenicity of the obtained purified peptide was determined in the same manner as in Example 1. The results are shown in Table 1, and the maximum dilution ratio in which agglutination was confirmed was displayed. The peptide was also immobilized on cellulose in the same manner as in Example 1. That is, the above purified peptide 25
5 mg of [PEO aldehyde]-[cellulose] obtained in Example 1 was dissolved in 50 ml of carbonate buffer having a pH of 9.5.
Was added, and the mixture was reacted by stirring at room temperature for 24 hours. The reaction product was recovered and thoroughly washed to obtain [peptide]-[cellulose]. The peptide immobilization rate was determined in the same manner as in Example 1, and 88% was immobilized. The above [peptide]-[cellulose] was added to a carbonate buffer solution having a pH of 9.0 at 50 m.
1 g of sodium borohydride was added to the suspension, and the mixture was reacted by stirring at room temperature for 20 hours. The reaction product was recovered and thoroughly washed to obtain the target adsorbent. The performance evaluation of the obtained adsorbent was performed in the same manner as in Example 1. That is, the adsorption performance of antiplatelet antibody and albumin in the sera of 5 ITP patients was evaluated. The respective adsorption rates are shown in Tables 2 and 3.

Example 3 Arg-Ala-Lys-
Trp-Asp-Thr-Ala-Asn-Asn-P
Immobilization of ro-Leu-Tyr-Lys-Glu on cellulose: Thr-A at positions 741 to 744 in GP3a
Arg-Ala-Lys-Trp-Asp-Thr in which both ends of the sequence of la-Asn-Asn are extended by 5 residues each.
-Ala-Asn-Asn-Pro-Leu-Tyr-
Regarding the synthesis of the peptide having the Lys-Glu sequence, PAM glutamic acid (t-Boc-L-Glu (OB
zl)) (manufactured by Applied Biosystems) and t
-Boc-L-Lys (Cl-Z), t-Boc-L-
Tyr (Br-Z), t-Boc-L-Leu.H
₂ O, t-Boc-L-Pro, t-Boc-L-As
n, t-Boc-L-Ala, t-Boc-L-Thr
(Bzl), t-Boc-L-Asp (OBzl), t
-Boc-L-Trp (CHO), t-Boc-LA
rg (Tos) (all manufactured by Applied Biosystems) was used in the same manner as in Example 1. For the deprotecting group of the support after the synthesis, t-Bo is added in the sequence.
Since it contains c-L-Trp (CHO), 1g first
1,2-ethanediol 0.2 was added to the support after the synthesis of
ml, m-cresol 0.8 ml, dimethyl sulfide 3 ml, TFA 5 ml, TFMSA 1 ml were added, and the mixture was stirred for 3 hours while cooling with ice water to react. The reaction product was filtered through a glass filter (medium hole) while being washed with diethyl ether that had been cooled beforehand, and then the deprotecting group reaction carried out in Example 1 was carried out. Hereinafter, the peptide chain cleavage, purification by reverse phase HPLC and identification were carried out in the same manner as in Example 1.

The immunogenicity of the obtained purified peptide was determined in the same manner as in Example 1. The results are shown in Table 1, and the maximum dilution ratio in which agglutination was confirmed was displayed. The peptide was also immobilized on cellulose in the same manner as in Example 1. That is, the above purified peptide 25
5 mg of [PEO aldehyde]-[cellulose] obtained in Example 1 was dissolved in 50 ml of carbonate buffer having a pH of 9.5.
Was added, and the mixture was reacted by stirring at room temperature for 24 hours. The reaction product was recovered and thoroughly washed to obtain [peptide]-[cellulose]. The peptide immobilization rate was determined in the same manner as in Example 1, and 84% was immobilized. The above [peptide]-[cellulose] was added to a carbonate buffer solution having a pH of 9.0 at 50 m.
1 g of sodium borohydride was added to the suspension, and the mixture was reacted by stirring at room temperature for 20 hours. The reaction product was recovered and thoroughly washed to obtain the target adsorbent. The performance evaluation of the obtained adsorbent was performed in the same manner as in Example 1. That is, the adsorption performance of antiplatelet antibody and albumin in the sera of 5 ITP patients was evaluated. The respective adsorption rates are shown in Tables 2 and 3.

Example 4 Arg-Ala-Lys-
Trp-Asp-Thr-Ala-Asn-Asn-P
Immobilization of ro-Leu-Tyr-Lys-Glu on cellulose Arg-Ala-Lys-Tr obtained in Example 3
p-Asp-Thr-Ala-Asn-Asn-Pro
-Leu-Tyr-Lys-Glu hexamethylenediamine _{(H 2 N (CH 2)} 6 NH 2) via an immobilized were examined in the same manner as in Example 1. 100 g of hexamethylenediamine was dissolved in 50 ml of a carbonate buffer solution of pH 9.5, 5 g of [PEO aldehyde]-[cellulose] obtained in Example 1 was added, and the mixture was stirred at room temperature for 24 hours for reaction. The reaction product was recovered, washed thoroughly, suspended in 50 ml of a carbonate buffer having a pH of 9.0, added with 1 g of sodium borohydride, and reacted by stirring at room temperature for 20 hours.
The reaction product was recovered and thoroughly washed to obtain [PEO amine 2]-[cellulose]. Next, 25m of the above peptide
g was dissolved in 50 ml of a citrate buffer solution having a pH of 4.5 and then ice-cooled to obtain EDC which is a water-soluble carbodiimide reagent.
(1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide) (10 mg) was added, and the mixture was stirred for 30 minutes while cooling with ice. Further, the above [PEO amine 2]-[cellulose] was added and reacted at room temperature for 24 hours with stirring. The reaction product was recovered and thoroughly washed to obtain the target adsorbent. The immobilization rate of the peptide was determined in the same manner as in Example 1, and 79% was immobilized. The reaction product was recovered and thoroughly washed to obtain the target adsorbent. The performance evaluation of the obtained adsorbent was performed in the same manner as in Example 1. That is, the adsorption performance of antiplatelet antibody and albumin in the sera of 5 ITP patients was evaluated. The respective adsorption rates are shown in Tables 2 and 3.

[0042]

[Comparative example]

Comparative Example 1 Met-Asp-Leu-Ser-Ty
r-Ser-Met-Lys-Asp-Asp-Leu
Immobilization of Cellulose onto Cellulose Met-Asp-Leu-Ser-Tyr-Ser corresponding to the 118th to 128th regions in GP3a
For the synthesis of peptides having the sequence -Met-Lys-Asp-Asp-Leu, PAM leucine (t-B
oc-L-Leu) (manufactured by Applied Biosystems) and t-Boc-L-Asp (OBzl), t-.
Boc-L-Lys (Cl-Z), t-Boc-L-M
et, t-Boc-L-Ser (Bzl), t-Boc
-L-Tyr (Br-Z), (all manufactured by Applied Biosystems) was used and carried out in the same manner as in Example 1. This peptide region is also a relatively large hydrophilic region. Deprotection of the support after the synthesis, cleavage of the peptide chain, purification by reverse phase HPLC and identification were also carried out in the same manner as in Example 1.

The immunogenicity of the obtained purified peptide was also determined in the same manner as in Example 1. The results are shown in Table 1, and the maximum dilution ratio in which agglutination was confirmed was displayed. The peptide was also immobilized on cellulose in the same manner as in Example 1. That is, the above purified peptide 25
5 mg of [PEO aldehyde]-[cellulose] obtained in Example 1 was dissolved in 50 ml of carbonate buffer having a pH of 9.5.
Was added, and the mixture was reacted by stirring at room temperature for 24 hours. The reaction product was recovered and thoroughly washed to obtain [peptide]-[cellulose]. The peptide immobilization rate was determined in the same manner as in Example 1, and 85% was immobilized. The above [peptide]-[cellulose] was added to a carbonate buffer solution having a pH of 9.0 at 50 m.
1 g of sodium borohydride was added to the suspension, and the mixture was reacted by stirring at room temperature for 20 hours. The reaction product was recovered and thoroughly washed to obtain the target adsorbent. The performance evaluation of the obtained adsorbent was performed in the same manner as in Example 1. That is, the adsorption performance of antiplatelet antibody and albumin in the sera of 5 ITP patients was evaluated. The respective adsorption rates are shown in Tables 2 and 3.

<Comparative Example 2> Thr-Thr-Arg-
Thr-Asp-Thr-Cys-Met-Ser-S
Immobilization of er-Asn-Gly-Leu on cellulose Thr-Thr-Arg-Thr-Asp-Thr corresponding to the 561st to 673rd regions in GP3a.
-Cys-Met-Ser-Ser-Asn-Gly-
For the synthesis of peptides having the sequence of Leu, PAM
Leucine (t-Boc-L-Leu) (manufactured by Applied Biosystems) and t-Boc-L-Gly, t.
-Boc-L-Asn, t-Boc-L-Ser (Bz
l), t-Boc-L-Met, t-Boc-L-Cy.
_{s (4-CH 3 OBzl)} , t-Boc-L-Thr
(Bzl), t-Boc-L-Asp (OBzl), t
-Boc-L-Arg (Tos) (all manufactured by Applied Biosystems) was used in the same manner as in Example 1. This peptide region is also a relatively large hydrophilic region. Deprotection of the support after the synthesis, cleavage of the peptide chain, purification by reverse phase HPLC and identification were also carried out in the same manner as in Example 1.

The immunogenicity of the obtained purified peptide was determined in the same manner as in Example 1. The results are shown in Table 1, and the maximum dilution ratio in which agglutination was confirmed was displayed. The peptide was also immobilized on cellulose in the same manner as in Example 1. That is, the above purified peptide 25
5 mg of [PEO aldehyde]-[cellulose] obtained in Example 1 was dissolved in 50 ml of carbonate buffer having a pH of 9.5.
Was added, and the mixture was reacted by stirring at room temperature for 24 hours. The reaction product was recovered and thoroughly washed to obtain [peptide]-[cellulose]. The peptide immobilization rate was determined in the same manner as in Example 1, and 89% was immobilized. The above [peptide]-[cellulose] was added to a carbonate buffer solution having a pH of 9.0 at 50 m.
1 g of sodium borohydride was added to the suspension, and the mixture was reacted by stirring at room temperature for 20 hours. The reaction product was recovered and thoroughly washed to obtain the target adsorbent. The performance evaluation of the obtained adsorbent was performed in the same manner as in Example 1. That is, the adsorption performance of antiplatelet antibody and albumin in the sera of 5 ITP patients was evaluated. The respective adsorption rates are shown in Tables 2 and 3.

[0046]

INDUSTRIAL APPLICABILITY According to the present invention, antiplatelet antibodies in blood can be effectively bound, and the present invention can be applied to the treatment of patients with severe ITP. The blood purification therapy using the anti-platelet antibody adsorbent in the present invention is safe without side effects and is excellent in stability by storage. It is also advantageous as a medical device in that the activity as a ligand hardly decreases even when steam sterilization by autoclave treatment is performed.

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[Procedure amendment]

[Submission date] September 30, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 2

[Correction method] Change

[Correction content]

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Masakazu Tanaka 2-1-1 Katata, Otsu City, Shiga Prefecture Toyobo Co., Ltd.

Claims (2)

[Claims] 1. A glycoprotein forming a platelet membrane, GP3a, GP2b, GP1bα chain, GP1bβ.
At least one peptide present in either the chain or GP9 and consisting of 4 to 40 amino acid residues and having the ability to bind antiplatelet antibody is immobilized on a water-insoluble carrier An anti-platelet antibody adsorbent characterized by the following. 2. An amino acid, B- (A and B are NH or CO, n) at both ends or one end of the peptide according to claim 1.
Is an integer from 1 to 20), and at least one modified peptide having a total number of amino acid residues within 70 bound by a peptide bond is immobilized on a water-insoluble carrier. Antibody adsorbent.