JPH09299478A - Method for eliminating anti-cardiolipin beta 2 glycoprotein i conjugated antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for eliminating efficiently anti-cardiolipin β2 glycoprotein I conjugated antibody from a humor contg. anti-cardiolipin β2 glycoprotein I conjugated antibody. SOLUTION: A method for eliminating efficiently anti-cardiolipin β2 glycoprotein I conjugated antibody from a humor contg. anti-cardiolipin β2 glycoprotein I conjugated antibody consists of a process wherein β2 glycoprotein I is added in the above described humor and a process wherein the above described β2 glycoprotein I-added humor is brought into contact with a solid substance with an anionic functional group.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for efficiently removing an anti-cardiolipin β2 glycoprotein I complex antibody from a body fluid.

[0002]

2. Description of the Related Art Antiphospholipid antibody syndrome (hereinafter "APS")
Is an autoimmune disease in which an antiphospholipid antibody such as an anticardiolipin antibody appears in the blood of a patient, and the main symptoms are repeated arteriovenous thrombosis, habitual miscarriage, thrombocytopenia and the like. APS is systemic lupus erythematosus (hereinafter "SL
Most commonly found in patients with collagen disease such as "E"),
It is widely recognized in patients who do not necessarily meet the diagnostic criteria for SLE, pulmonary hypertension of unknown cause, young-onset myocardial infarction, and recurrent miscarriage.

In recent years, anti-cardiolipin antibodies found in APS include anti-cardiolipin antibodies (hereinafter referred to as "anti-CL antibodies") that directly bind to cardiolipin (hereinafter referred to as "CL"), CL and β2 glycoprotein I (hereinafter referred to as "β2").
Anti-cardiolipin β2 glycoprotein I complex antibody (hereinafter “anti-CLβ”) that binds to a complex with GPI ”.
2GPI-complex antibody ”) has been demonstrated (E. Matsuura et al., Lanc.
et 386, p. 177 (1990)), now
Among them, the anti-CLβ2GPI complex antibody is considered to be closely related to the symptoms of APS.

As a mechanism of expression of APS, β having a function of suppressing the activity of coagulation factors, particularly prothrombinase
Anti-CLβ2GPI complex antibody binds to 2GPI,
It has been suggested that inhibition of the function of 2GPI may promote the coagulation system and promote the tendency of thrombus formation. Therefore, it is considered that reducing the anti-CLβ2GPI complex antibody in blood is effective for the treatment of APS.

Anticoagulant therapy has been used as a treatment method for APS, but like other autoimmune diseases, drug therapy such as steroids, immunosuppressants, immunomodulators, anti-inflammatory agents, and plasma exchange. Blood purification therapy such as immunoadsorption has also been tried.

[0006]

However, steroids are most commonly used in drug therapy, but steroids tend to cause side effects even when administered in small amounts. In the treatment of APS, a large amount of steroids and long-term administration are often performed, which may cause more serious side effects, and the large number of side effects makes it impossible to administer a sufficient amount to bring about a therapeutic effect. Sometimes. Simple plasma exchange requires a large amount of healthy human plasma,
In addition to being expensive, there is also the risk of transmission through the plasma.

As a therapeutic method for overcoming these drawbacks, adsorption therapy has been developed which selectively removes a causative agent with almost no loss of active ingredients in body fluids. For example, an adsorption method using an adsorption column using a dextran sulfate-immobilized cellulose gel as an adsorbent can be mentioned. In this method, although anti-CL antibody can be removed efficiently, anti-C antibody can be removed.
In the removal of the Lβ2GPI complex antibody, there were large individual differences, and in some cases, a sufficient removal effect was not always obtained.

In view of the above, the present invention is directed to anti-CLβ2GPI.
It is an object of the present invention to provide a method for efficiently removing anti-CLβ2GPI complex antibody from a body fluid containing the complex antibody.

[0009]

The present invention provides anti-CLβ2G
From the body fluid containing the PI complex antibody, anti-CLβ2GPI
A method for removing a complex antibody, which comprises adding β2G to the body fluid
A method for removing an anti-CLβ2GPI complex antibody, which comprises a step of adding PI and a step of bringing the β2GPI-added body fluid into contact with a solid substance having an anionic functional group.
Hereinafter, the present invention will be described in detail.

The present invention is a method for removing an anti-CLβ2GPI complex antibody from a body fluid containing the anti-CLβ2GPI complex antibody. The body fluid means blood, plasma, serum, ascites fluid, lymph fluid, synovial fluid, cerebrospinal fluid, fractionated components obtained from these, other biologically derived liquid components, and the like. The body fluid containing the anti-CLβ2GPI complex antibody is, for example, plasma of a patient in which the blood concentration of the anti-CLβ2GPI complex antibody is not decreased by the adsorption therapy using the dextran sulfate-immobilized cellulose as an adsorbent. Can be used.

In the present specification, "β2GPI" is also generally called apolipoprotein H and has a molecular weight of 5
It is 0 kD, has an isoelectric point of 6.5 to 7.0, and is a phospholipid having a negative charge such as cardiolipin, phosphatidylserine, phosphatidylinositol, phosphatidic acid, and phosphatidylglycerol; a protein having binding properties to heparin and the like.

In the first step of the present invention, the above anti-CL is used.
The above-mentioned β2GPI is added to the body fluid containing the β2GPI complex antibody.
Is added. As a method of adding the β2GPI,
For example, a method of adding the body fluid to a suitable container and then adding the body fluid, a method of continuously adding the body fluid into a circuit for extracorporeal circulation, and the like can be mentioned.

The amount of β2GPI added is preferably 25 μg or more per 1 ml of body fluid. When it is less than 25 μg, the amount of anti-CLβ2GPI complex antibody adsorbed hardly increases.

In the second step of the present invention, the above β2G is used.
The PI-added body fluid is contacted with a solid material having anionic functional groups. The anionic functional group in the present invention means pH
Means a functional group that is negatively charged near neutral. The anionic functional group is not particularly limited, and examples thereof include a carboxyl group, a sulfonic acid group, a sulfone group, a sulfate ester group, a silanol group, a phosphate ester group, and a phenolic hydroxyl group.

In the present invention, the anionic functional group is preferably at least one selected from the group consisting of a sulfuric acid ester group, a sulfonic acid group, a carboxyl group and a phosphoric acid ester group.

The solid substance in the present invention means a substance which is solid at room temperature and normal pressure and is insoluble in water. The solid substance may have a solid surface covered with a hydrophilic substance. Examples of the solid substance include granular substances, plate-shaped substances, film-shaped substances, fibrous substances, and hollow fiber-shaped substances, but the shape is not particularly limited.

When the solid substance is packed in a column and used, it must be able to form a gap through which cells contained in the body fluid can sufficiently pass, and when the solid substance is granular, the average particle size is small. The diameter is preferably 5 to 1000 μm, more preferably 25 to 1000 μm, and further preferably 50 to 600 μm. Of these,
A narrow particle size distribution is preferable because it does not cause an increase in pressure loss. When the body fluid is blood, the average particle size of the solid substance is preferably 200 μm or more.

When the solid substance is fibrous and hollow fiber, the inner diameter is preferably 1 μm or more. If it is less than 1 μm, it will not pass sufficiently when cells are contained in the body fluid. More preferably 2 to 500 μm
And more preferably 5 to 200 μm.

In the present invention, the solid substance preferably has a porous structure having a large number of pores of an appropriate size. Examples of the solid substance having a porous structure include, for example,
A solid substance having a space (macropore) formed by agglomeration of microspheres when the base polymer matrix forms one spherical particle by agglomeration of microspheres, and one microsphere forming the base polymer matrix A solid substance having pores formed between cores and agglomerates of cores, present when a copolymer having a three-dimensional structure (polymer network) is swollen in an organic solvent with affinity Examples thereof include a solid substance having micropores (micropores).

The pore diameter of the solid substance having the above-mentioned porous structure can be expressed by using the exclusion limit molecular weight. In the present specification, the “exclusion limit molecular weight” means the molecular weight of the molecule having the smallest molecular weight among the molecules that cannot enter (exclude) in the pores in gel permeation chromatography (Hiroyuki Hatano, Toshihiko Hanai, Experiments). High performance liquid chromatography, chemistry coterie).

The above exclusion limit molecular weight varies depending on the target compound and is generally well known for globular proteins, dextran, polyethylene glycol and the like, and globular proteins which are considered to be most similar to the anti-CLβ2GPI complex antibody ( It is suitable to use the value obtained by using (including virus), and the exclusion limit molecular weight is 100,000 to 60 as the pore size of the solid substance having the above-mentioned porous structure.
It is preferably, 000,000.

The anti-CLβ2GPI complex antibody is Ig
It is composed of immunoglobulins such as G and IgM (immunoglobulin M) and is a macromolecule having a molecular weight of 160,000 to 900,000. Therefore, in order to efficiently adsorb it, at least anti-C
It is necessary for the Lβ2GPI complex antibody to have a surface pore size that allows it to easily penetrate into the solid substance. If the exclusion limit molecular weight is less than 100,000, the anti-CLβ2GPI complex antibody cannot penetrate into the solid substance, so the amount of adsorption is not sufficient, and if it exceeds 60 million, adsorption of components other than the anti-CLβ2GPI complex antibody, that is, non- The specific adsorption increases and the selectivity remarkably decreases, and the surface area decreases substantially, and the adsorption amount decreases. More preferably, it is 400,000 to 200,000 from the viewpoint of being more selective and having a larger adsorption capacity.
It is 0,000.

The porous structure of the solid substance in the present invention is preferably wholly porous rather than surface porous.
The pore volume of the solid substance is preferably such that the adsorption capacity is large. Therefore, for example, the pore volume into which spherical proteins having a molecular weight of about 160,000 can penetrate is preferably 20% or more.

In order to know the porous structure, mercury porosimetry, gas adsorption method, etc. can be used for the pore distribution measurement. These measurement methods measure the pore distribution in a dry state, In many cases, the shape of the solid substance changes due to drying, which is not applicable in many cases.
Since it is not suitable for expressing the pore volume into which many globular proteins can enter, in order to know the porous structure of the solid substance, the solid substance is packed in a column in the same manner as in the measurement of the exclusion limit molecular weight. , The volume (V0) at which this protein elutes when a protein solution having a molecular weight above the exclusion limit is injected and the solution is passed through
Similarly, a globular protein having a molecular weight of 160,000 was similarly injected, and the outflow volume (VX) at the time of passing was measured, and "(VX-V0) / (V
-V0) "(V: column volume) is suitable.

The surface of the solid substance in the present invention is preferably smooth. A rough surface is not preferable because the adherence of autologous blood cells and platelets is increased when blood cells are contained in the body fluid. The solid substance may be coated with a polymer such as a polymer of hydroxyethyl methacrylate.

The method for obtaining the solid substance having an anionic functional group used in the present invention is not particularly limited, and for example, a compound having an anionic functional group or a functional group which can be easily converted into an anionic functional group is used. Examples include a method of forming an adsorbent by polymerization or copolymerization used as a monomer or a cross-linking agent (method (1)), a method of fixing a compound having an anionic functional group to a solid substance (method (2)), and the like. .

In the present invention, the solid substance having an anionic functional group is preferably a water-insoluble solid substance having a porous structure to which the compound having an anionic functional group is immobilized.

The monomer having an anionic functional group in the above method (1) is not particularly limited, and examples thereof include acrylic acid, methacrylic acid and styrene sulfonic acid. The monomer having a functional group that can be easily converted into an anionic functional group is not particularly limited,
For example, acrylic acid ester, methacrylic acid ester and the like can be mentioned, and these can be easily converted into an anionic functional group by a reaction such as alkali saponification.

The solid substance having an anionic functional group can be obtained by polymerizing the monomer having an anionic functional group or by copolymerizing two or more kinds of monomers, and the monomer having an anionic functional group and styrene or chlorostyrene. Can be obtained by a method of copolymerizing with a monovinyl monomer such as, and a method of copolymerizing the monomer having an anionic functional group with a polyvinyl monomer such as divinylbenzene or trivinylbenzene as a crosslinking agent.

As the above method (2), a method of directly introducing an anionic functional group itself into a solid substance or the like, or a compound having a moiety other than an anionic functional group in the molecule and having an anionic functional group is used as a solid substance. And the like.

The method for directly introducing the above-mentioned anionic functional group itself into a solid substance is not particularly limited, and for example, a sulfuric acid ester group or a sulfonic acid group is directly reacted by reacting the solid substance with a reagent such as chlorosulfonic acid or concentrated sulfuric acid. Examples thereof include a method of introducing a group and the like.

As a method for introducing a compound having a moiety other than an anionic functional group in the molecule and having an anionic functional group, a known method can be used, but the storage and stability of the adsorptive property can be used. Since it is important that the compound having an anionic functional group is not eliminated for maintenance,
A covalent bond method capable of firmly fixing is preferable. in this case,
The compound having a portion other than the anionic functional group in the molecule and having an anionic functional group is more preferably a compound having a functional group used for bonding to a solid substance, in addition to the anionic functional group. preferable.

The functional group used for binding to the solid substance is not particularly limited, and examples thereof include amino group, amide group, carboxyl group, acid anhydride group, succinimide group,
Examples thereof include a hydroxyl group, a thiol group, an aldehyde group, a halogen group, an epoxy group, a silanol group and a tresyl group.

Examples of the compound having an anionic functional group which has a functional group other than the anionic functional group used for binding to the solid substance in the molecule include, for example, diols, triols, sugars and polysaccharides. Partial sulfuric acid esterification products of polyhydric alcoholic compounds such as These are preferably used because they have both an anionic functional group and a functional group used for immobilization.

In this case, the compound having an anionic functional group is preferably a polyanionic compound having a plurality of anionic functional groups in the molecule. Above all,
Since many anionic functional groups can be easily introduced, those having a molecular weight of 1,000 or more are preferable, and those having a molecular weight of 3,000 or more are more preferable. The upper limit of the molecular weight is not particularly limited, but from the practical viewpoint, it is 100
10,000 is preferred. The anionic functional group contained in the polyanionic compound may be one type or two or more types.

The polyanionic compound is not particularly limited, and examples thereof include polyacrylic acid, polyvinyl sulfuric acid,
Synthetic polyanionic compounds such as polyvinyl sulfonic acid, polystyrene sulfonic acid, polyglutamic acid, polyaspartic acid, polymethacrylic acid, polyphosphoric acid, styrene-maleic acid copolymers; synthetic acidic polysaccharides such as dextran sulfate and carboxymethyl cellulose; chondroitin sulfate -Derived acidic mucopolysaccharides having sulfate ester groups such as dermatan sulfate and keratan sulfate; biological-derived acidic mucopolysaccharides having N-sulfonic acid groups or sulfate ester groups such as heparin and heparan sulfate; chondroitin and phosphomannan Examples thereof include polysaccharides having an anionic functional group derived from a living body; nucleic acids derived from a living body such as deoxyribonucleic acid and ribonucleic acid.

Of these, it is preferable to use a synthetic compound rather than a biologically-derived compound as it is because a highly pure substance can be obtained at a low cost and the introduction amount of an anionic functional group can be controlled. Synthetic polyanionic compounds such as polyacrylic acid, polyvinyl sulfuric acid, polyvinyl sulfonic acid, polystyrene sulfonic acid, polyglutamic acid, polyaspartic acid, polymethacrylic acid, polyphosphoric acid, styrene-maleic acid copolymers; dextran sulfate, carboxymethyl cellulose, etc. Synthetic acidic polysaccharides are preferably used.

When the polyanionic compound is immobilized on the solid substance, the solid substance having the anionic functional group may be one type or two or more types.

In this case, it is preferable that the above solid substance also has a functional group used for binding. The functional group used for the above bonding is not particularly limited, and for example,
Amino group, amide group, carboxyl group, acid anhydride group, succinimide group, hydroxyl group, thiol group, aldehyde group,
A halogen group, an epoxy group, a silanol group, a tresyl group, etc. can be mentioned.

The solid substance having a functional group used for the above binding is not particularly limited, and examples thereof include inorganic solid substances such as glass and silica gel; polysaccharides such as cellulose, chitin, chitosan, agarose, dextran and their derivatives. And solid polymers such as polyvinyl alcohol, polyacrylamide, polyacrylic acid, polymethacrylic acid, derivatives thereof, and synthetic polymers such as copolymers thereof. The solid substance having a functional group used for the above-mentioned bonding may be activated by a method such as a cyanogen halide method, an epichlorohydrin method, a bisepoxide method, a bromoacetyl bromide method or the like.

The solid substance having an anionic functional group used in the present invention preferably has an anionic functional group in an amount of 100 nmol to 10 mmol per unit volume (1 ml) of the solid substance. When it is less than 100 nmol,
There is no effect of the anionic functional group, and if it exceeds 10 mmol, nonspecific adsorption may occur. More preferably 1 μmo
1 to 5 mmol, more preferably 5 μmol to 3
mmol.

In the present invention, as a method for bringing the β2GPI-added body fluid into contact with the solid substance having the anionic functional group, for example, the β2GPI-added body fluid is stored in a bag or the like, and the β2GPI-added body fluid having the anionic functional group is stored. A method for obtaining a body fluid from which the anti-CLβ2GPI complex antibody has been removed by filtering the solid substance having an anionic functional group after adsorbing and removing the anti-CLβ2GPI complex antibody by contacting with a solid substance, and an inlet and an outlet of the body fluid And a solid substance having an anionic functional group, which does not pass a solid substance having an anionic functional group at its outlet, is filled with the solid substance having an anionic functional group, and the β2G
Examples include a method of flowing a PI-added body fluid. Either method may be used, but the latter method is easy to operate, and by incorporating it in an extracorporeal circulation circuit, it is possible to efficiently and online perform the above-mentioned anti-CL from a patient's body fluid, especially blood.
It is preferable because it is possible to remove the β2GPI complex antibody.

In the extracorporeal circulation circuit, only the method of the present invention can be performed, but it can also be used in combination with another extracorporeal circulation treatment system. Examples of the system that can be used in combination include, for example, an artificial dialysis circuit,
It can also be used in combination with dialysis therapy.

In the method for removing anti-CLβ2GPI complex antibody of the present invention, the adsorption amount of the anti-CLβ2GPI complex antibody in the body fluid to the solid substance having the anionic functional group is
Since it increases depending on the concentration of the β2GPI contained in the body fluid, the β2GPI is added to the body fluid containing the anti-CLβ2GPI complex antibody, and then contacted with the solid substance having the anionic functional group. Thus, the anti-CLβ2GPI complex antibody in the body fluid can be efficiently adsorbed and removed.

[0045]

BEST MODE FOR CARRYING OUT THE INVENTION FIG. 1 shows the anti-CLβ2GP of the present invention.
Anti-CLβ2, which is an embodiment of a method for removing an I-complex antibody
The schematic cross-sectional view of the GPI complex antibody removal device is shown. In FIG. 1, 1 is a body fluid inlet, 2 is a body fluid outlet,
3 is a solid substance having an anionic functional group, 4 and 5
Represents a filter or mesh that can pass components contained in body fluids and fluids but not solid substances having anionic functional groups, 6 represents a column, and 7 represents a container. The filter 4 on the inlet side of the body fluid may or may not be present.

In the anti-CLβ2GPI complex antibody removing apparatus, the method of adding the β2GPI to the body fluid is not particularly limited, but the continuous injection method at the entrance side of the anti-CLβ2GPI complex antibody removing apparatus is simple and preferable. .

[0047]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Production Example 1 Porous cellulose gel (CK-A22, manufactured by Chisso Corp., exclusion limit molecular weight of spherical protein: 20 million, particle size: 45 to 10)
5 μm, cross-linked gel) 100 ml, 20% NaOH 40
g, 120 g of heptane, and 10 drops of the nonionic surfactant Tween 20 (manufactured by Kao Atlas). 40 ℃
After stirring for 2 hours, add 50 g of epichlorohydrin and add 2
After stirring for a time, the gel was washed with water and filtered to obtain an epoxidized cellulose gel.

To 5 ml of this epoxidized cellulose gel,
4 g of dextran sodium sulfate (molecular weight: about 5000, sulfur content: 15%) and 5 ml of water were added, and the mixture was adjusted to pH 9 and permeated at 45 ° C. for 16 hours. Then, the gel is filtered off, washed in this order with a 2M aqueous sodium chloride solution, a 0.5M aqueous sodium chloride solution and water, and 0.5% monoethanolamine aqueous solution is added and shaken to seal unreacted epoxy groups A cellulose gel adsorbent having dextran sulfate sodium immobilized thereon was obtained. The amount of anionic functional groups introduced by the immobilized dextran sulfate is 16 μm per 1 ml of the adsorbent.
It was ol.

Example 1 Using physiological saline (Otsuka Pharmaceutical Co., Ltd.), β2GPI (Yamasa Shoyu Co., Ltd.) was adjusted to 400, 200, 100, 50, 25 and 12.5 μg / ml concentrations in 5 steps. 0.25 ml of each of the adsorbents obtained in Production Example 1 was weighed into a polypropylene centrifuge tube, and each β2GPI solution and anti-CLβ were measured.
After adding 1 ml of a solution in which serum containing the 2GPI complex antibody was mixed at a ratio of 1: 1 and shaking at 37 ° C. for 2 hours,
The anti-CLβ2GPI complex antibody titer of the supernatant was measured. Anti-C
The Lβ2GPI complex antibody titer was measured using an anti-CLβ2GPI kit “Yamasa” (manufactured by Yamasa Shoyu Co., Ltd.). N =
The measurement results of 3 were averaged.

The evaluation results show that anti-CLβ2G was obtained when Subrad A liquid (manufactured by Fuso Pharmaceutical Co., Ltd.) was used instead of the adsorbent.
The ratio (%) of the antibody titer at the concentration of each β2GPI solution to the PI complex antibody titer is shown in Table 1. From Table 1, it can be seen that the proportion of the anti-CLβ2GPI complex antibody titer decreases as the concentration of β2GPI added increases.

[0052]

[Table 1]

[0053]

INDUSTRIAL APPLICABILITY According to the present invention, the anti-CLβ2GPI complex antibody can be efficiently removed from the body fluid containing the anti-CLβ2GPI complex antibody.

[Brief description of drawings]

FIG. 1 is a schematic cross-sectional view of an anti-CLβ2GPI complex antibody removing apparatus according to an embodiment of the present invention.

[Explanation of symbols]

 1 Inlet 2 Outlet 3 Adsorbent 4 Filter 5 Filter 7 Container

Claims (4)

[Claims] 1. From a body fluid containing an anti-cardiolipin β2 glycoprotein I complex antibody, anti-cardiolipin β
A method for removing a 2 glycoprotein I complex antibody, comprising the steps of adding β2 glycoprotein I to the body fluid and contacting the β2 glycoprotein I-added body fluid with a solid substance having an anionic functional group. A method of removing an anti-cardiolipin β2 glycoprotein I complex antibody, which comprises: 2. The anionic functional group is a sulfate ester group,
The method for removing an anti-cardiolipin β2 glycoprotein I complex antibody according to claim 1, which is at least one selected from the group consisting of a sulfonic acid group, a carboxyl group, and a phosphoric acid ester group. 3. A solid substance having an anionic functional group,
The compound having an anionic functional group is immobilized on a solid substance which is water-insoluble and has a porous structure.
Or the anticardiolipin β2 glycoprotein I described in 2 above
Method for removing complex antibody. 4. The method for removing anti-cardiolipin β2 glycoprotein I complex antibody according to claim 3, wherein the compound having an anionic functional group is a polyanionic compound.