JPH0624625B2 - Adsorbent for autoantibody-producing B cells and method for adsorbing and removing autoantibody-producing B cells - Google Patents

Description

TECHNICAL FIELD The present invention relates to an adsorbent for adsorbing and removing autoantibody-producing B cells from a body fluid, and a method for adsorbing and removing autoantibody-producing B cells using the adsorbent.

[Problems to be Solved by Conventional Techniques and Inventions] Living bodies are equipped with immunity to protect foreign substances invading from the outside.

Immunity includes innate immunity and adaptive immunity, and lymphocytes are responsible for the adaptive immunity.

T cells in lymphocytes directly attach to antigens and activate other cells, and B cells in lymphocytes secrete into proteins called antibodies and in blood and body fluids. Defending.

However, an immune response occurs not to the foreign substances from the outside but to the antigens of the cells and tissues of the self, and antibodies against the self (hereinafter referred to as autoantibodies) are generated or a complex formed by an antigen-antibody reaction ( Hereinafter, an immune abnormality called autoimmune disease may occur in which a large amount of (immune complex)) occurs.

Representative examples of autoimmune diseases and autoantibodies in the diseases are systemic lupus erythematosus (SL, respectively).
E) anti-double-stranded DNA antibody, anti-single-stranded DNA antibody, anti-Sm antibody; rheumatoid factor in chronic rheumatoid arthritis (hereinafter RA), anti-single-stranded DNA antibody; Sjogren's syndrome (hereinafter SjS) Anti-SS-A antibody, anti-SS in
-B antibody, anti-mitochondrial antibody; systemic sclerosis (hereinafter,
Anti-Sc 170 antibody in PSS), anti-single-stranded DNA antibody; anti-RN in mixed connective tissue disease (MCTD)
Examples include P antibody.

Therapeutic methods for these autoimmune diseases have been studied for a long time, and methods for removing autoantibodies and immunocomplexes formed from autoantibodies, which are considered to be causative agents, have been studied. That is, it is a method such as a plasma exchange method or an adsorption removal method of autoantibodies.

However, these conventional therapeutic methods aimed at removing the autoantibodies produced by the autoantibody-producing B cells in lymphocytes do not suppress or terminate the production of autoantibodies, and the pathogenic substance after the treatment. Since the autoantibody is produced, it cannot be said to be a sufficiently effective therapeutic method.

As a therapeutic method for solving the problems of the above method, a method for separating lymphocytes or B cells in lymphocytes has been studied, but in these methods, lymphocytes and B cells in lymphocytes are isolated.
There is a problem that the whole cell is separated.

[Means for Solving the Problems] In view of the present situation, the present inventors have selectively adsorbed and removed autoantibody-producing B cells involved in the production of a pathogen from a patient's body fluid as a method for suppressing the production of the pathogen. As a result of earnest studies to develop a method, it was found that, when a solid substance having an anionic functional group is brought into contact with a patient's body fluid, the autoantibody-producing B cells in B cells in lymphocytes are strongly adsorbed. Heading out, the present invention has been completed.

That is, the present invention provides a method for adsorbing and removing an autoantibody-producing B cell, which comprises contacting the adsorbent for an autoantibody-producing B cell made of a solid substance having an anionic functional group and a body fluid containing lymphocytes. Regarding

[Examples] The term "body fluid" as used herein refers to a liquid component derived from a living body, and specific examples thereof include blood, plasma, serum, ascites, lymph, joint fluid, and components obtained from these. Examples include image components.

The term “autoantibody-producing B cell” as used herein means a B cell in which the idiotype of the B cell surface immunoglobulin is the same as that of the autoantibody, its precursor cells, and plasma cells producing the autoantibody.

The solid substance in the present invention means that it is solid at room temperature and atmospheric pressure and is insoluble in water, and the surface of the solid substance has, for example, a protein that acts such as blocking blood cell damage that suppresses nonspecific adsorption. Water-soluble substances such as polysaccharides and water-soluble polymers may be present.

As long as the solid substance is a solid substance having an anionic functional group, its shape, particle size, surface state, material, etc. are not particularly limited, and the shape, particle size, etc. can be selected appropriately according to the method of use. You can use it.

Specific examples of the shape of the solid substance include a granular shape, a plate shape, a film shape, and a fibrous shape.

The particle size of the solid substance is not particularly limited, but when the solid substance is packed into a column and used, a gap is formed through which cells contained in the body fluid can sufficiently pass. Is preferred. For example, when the solid substance is in the form of particles, fine powder is not preferable, and it is preferable that the average particle size is about 200 μm or more. Further, if the particle size is too small or too large, It is preferable to use those removed, and those having an average particle diameter of 200 to 1000 μm and a narrow particle diameter distribution are preferable. When the solid substance is fibrous and hollow, the inner diameter is preferably 5 μm or more, and when the solid substance is clogged, the diameter is 1 μm.
It is preferably m or more.

Further, the surface state of the solid substance is not particularly limited, but if the surface is rough, non-specific adsorption increases and the selectivity tends to decrease, so that the surface is preferably smooth.

Further, as the material of the solid substance, for example, a cross-linked polyvinyl alcohol, a cross-linked polyacrylate, a synthetic polymer such as cross-linked polyacrylamide and crystalline cellulose, cross-linked cellulose, cross-linked agarose, an organic carrier comprising a polysaccharide such as cross-linked dextran, and further these Examples thereof include organic-organic composite carriers obtained by a combination of the above, and inorganic carriers such as glass and silica gel, but are not limited to these.

The anionic functional group in the present invention is a group containing a functional group which is negatively charged at around neutral pH, and may be any group as long as it has such a property.

Typical examples of such a functional group include, but are not limited to, a carboxyl group, a sulfonic acid group, a sulfuric acid ester group, a silanol group, a phosphoric acid ester group, a phenolic hydroxyl group, and the like.

The anionic functional group in the invention is 1 per functional group.
It may be a monoanionic functional group having one anionic group or a polyanionic functional group having a plurality of anionic groups. The polyanionic functional group is preferable in that it has a high affinity for autoantibody-producing B cells, and that many anionic groups can be easily introduced into a unit amount of the solid substance. Among them, a polyanionic functional group having a molecular weight of 1000 or more is more preferable from the viewpoints of affinity for autoantibody-producing B cells and introduction of many anionic groups. The polyanionic functional group may have one type or two or more types of anionic groups.

Further, the anionic functional group in the present invention may be composed of only one type of the above monoanionic functional group or polyanionic functional group, or may be composed of two or more types.

The anionic functional group is 1 per 1 mm ² of the surface area of the solid substance.
X10 ² to 1 x 10 ¹⁰ µmol is preferably contained, and particularly 1 x 10 ⁴ to 1 per 1 mm ² of the surface area of the solid substance.
It is preferable that the content of x10 ⁷ µmol is contained. If the content is too small, the effect of the anionic functional group becomes insufficient, and if it is too large, non-specific adsorption or cell damage tends to occur.

The adsorbent of the present invention comprises the solid substance having the anionic functional group.

The solid substance having an anionic functional group means that the solid substance itself may be composed of a material having an anionic functional group, or a solid substance composed of a material having no anionic functional group. An anionic functional group may be introduced by introducing a compound having an anionic functional group.

Representative examples of the compound for introducing the anionic functional group, preferably a polyanionic functional group into a solid substance, include, for example, polyacrylic acid, polyvinyl sulfuric acid, polyvinyl sulfonic acid, polyvinyl phosphoric acid, polystyrene sulfonic acid, polystyrene phosphoric acid. , Polyglutamic acid, polyaspartic acid, polymethacrylic acid, polyphosphoric acid, synthetic polyanion compounds such as styrene-maleic acid copolymer, heparin, dextran sulfate, chondroitin, chondroitin sulfate, polysaccharides having anionic groups such as phosphomannan, etc. However, the present invention is not limited to this.

Next, a method for producing the adsorbent of the present invention will be described.

One of the methods for producing the adsorbent of the present invention is a method of introducing an anionic functional group into a solid substance having no anionic functional group as described above. There are various methods for introducing an anionic functional group into a solid substance, and any method may be used. Typical examples of the method include the following methods.

(1) a method of forming an adsorbent by polymerization using a compound having an anionic functional group or a functional group that can be easily converted to an anionic functional group as a monomer or a crosslinking agent, (2) a compound having an anionic functional group Examples thereof include a method of immobilizing the compound having an anionic functional group on the solid substance by directly reacting the compound having an anionic functional group with the solid substance (3).

Representative examples of the anionic functional group used in the method (1) or a monomer or a cross-linking agent that can be easily converted into an anionic functional group include acrylic acid and its ester, methacrylic acid and its ester, and styrene sulfonic acid. However, it is not limited to these.

The method of (2), that is, the method of immobilizing the compound having an anionic functional group on the solid substance includes a method by physical adsorption, a method by ionic bond, a method by immobilization by covalent bond, and any method is used. But you can
Since it is important that the compound having an anionic functional group is not desorbed from the standpoint of storage stability and stability of the adsorbent, a covalent bond method capable of firmly fixing is desirable.

When the compound having an anionic functional group is immobilized by a covalent bond, the compound having an anionic functional group is preferably a polyfunctional compound having a functional group that can be used for immobilization in addition to the anionic functional group.

Representative examples of functional groups that can be used for immobilization include amino groups, amide groups, carboxyl groups, acid anhydride groups, succinylimide groups, hydroxyl groups, thiol groups, aldehyde groups, halogen groups, epoxy groups, and silanol groups. However, it is not limited to these.

For example, when a compound having a sulfate ester group is immobilized on a solid substance by a covalent bond, a representative example of the compound having a sulfate ester group is a sulfated compound of a compound having a hydroxyl group such as alcohol, sugar and glycol. , Among them, the partially sulfated ester of polyhydric alcohol, especially the sulfated ester of polysaccharide contains both a sulfate ester group and a functional group necessary for immobilization, and can be easily immobilized on a solid substance. , Particularly preferred.

Next, a representative example of the method of (3), that is, a method of immobilizing a compound having an anionic functional group in a solid substance by reacting a compound having an anionic functional group with a solid substance to introduce an anionic functional group As a method, there is a method of introducing a sulfate ester group into a solid substance having a hydroxyl group. In this case, the sulfate ester group can be directly introduced by reacting a solid substance having a hydroxyl group with chlorosulfonic acid, concentrated sulfuric acid or the like.

There are various methods for adsorbing and removing autoantibody-producing B cells from a body fluid using the adsorbent of the present invention. As a typical example, body fluid is taken out and stored in a bag or the like, and an adsorbent is mixed with this to adsorb and remove autoantibody-producing B cells, and then the adsorbent is filtered to remove autoantibody-producing B cells. There is a method of obtaining a body fluid, a method of filling a container equipped with a filter having an inlet and an outlet of the body fluid, through which the body fluid passes but does not pass an adsorbent, and the body fluid is caused to flow into the container. Either method may be used, but the latter method is easy to operate, and by incorporating it in the extracorporeal circulation circuit, it is possible to efficiently and online remove autoantibody-producing B cells from a patient's body fluid, especially blood. Is. The adsorbent of the present invention is suitable for this method.

The present invention relates to an adsorbent for adsorbing and removing autoantibody-producing B cells from a body fluid of a patient with autoimmune disease, and an adsorption-removing method using the adsorbent.
Among the cells, the effect is particularly remarkable on anti-DNA antibody-producing B cells that are the etiological agent of SLE.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples.

Example 1 Preparation of Adsorbent To about 700 g of glass beads (particle size 210 to 297 μm, manufactured by Toshiba Marottini Co., Ltd.) was added about 0.1 N nitric acid aqueous solution 500 ml,
The mixture was heated in a water bath at 70 to 80 ° C. for 3 hours and then washed with about 2 water to confirm that the liquid was neutral. Then, it was washed with about 300 ml of methanol and dried. To 200 g of the washed glass beads obtained, 80 g of toluene and 40 g of aminated silane coupling agent (A-1100 manufactured by Nippon Unicar Co., Ltd.) were added,
After reacting at 90 ° C. for 3.5 hours, the mixture was washed with about 150 ml of toluene and then with 200 ml of methanol and air-dried.

On the other hand, 3.0 g of dextran sulfate was dissolved in 20 ml of water, and 0.48 g of sodium hydroxide and sodium borohydride were dissolved.
A solution containing 90 mg was prepared. To this solution, 25 g of the aminated glass beads prepared above was added, reacted at room temperature for 2 hours, washed with water and methanol, and dried to prepare an adsorbent.

Preparation of lymphocyte suspension A heparinized SLE patient's peripheral blood was layered on the upper layer of Ficollback, and lymphocytes were separated by specific gravity centrifugation, and the lymphocyte fraction thoroughly washed with Hanks' buffer was added to 1 × 10 ⁷ ~ 5x
To a concentration of 10 ^{7 /} ml was adjusted with pH 7.20HEPES-HBSS buffer containing 0.1% bovine serum albumin.

Adsorption operation Adsorbent 1.00 g of polyvinyl chloride tube (φ3ID ×
85 mm) was filled with physiological saline (Japanese Pharmacopoeia, manufactured by Otsuka Pharmaceutical Co., Ltd.) and meshed on both ends (platelet adhesion measuring tube,
An adsorbent pressed by a medical shoinin machine Co., Ltd. was produced. Before passing the cell suspension through this adsorbent, rinsing was performed using the same buffer as the cell suspension. After that, the cell suspension was passed through at 25 ° C. at a flow rate of 0.4 ml / min.

Analytical method The lymphocyte adsorption rate was determined by sampling a part of the cell suspension and effluent that had not passed through the column and measuring the concentration with a Coulter counter.

The adsorption rate of B cells was determined by reacting a part of the cell suspension and effluent that had not passed through the column with FITC-labeled anti-human Igs antibody to cause B cells to fluoresce, and counted under a microscope to detect lymphocytes. It was determined from the ratio of B cells and the concentration of lymphocytes.

The following three methods are used to analyze the effect of removing anti-DNA antibody-producing B cells: (1) Detection of free surface immunoglobulins (2) Culture of purified B cells and immunization in the culture supernatant. Detection of globulins (3) Lymphocytes (B and T cells) were cultured and the immunoglobulin in the culture supernatant was detected.

In more detail about (1), referring to the cell surface globulin test method (clinical test, Vol.28 (No.4) p430 (1984)), the cell suspension not passed through the column and the column suspension The effluent was centrifuged (1000 rpm for 5 minutes), the buffer solution was replaced with PBS, and fetal bovine serum was added to PBS and W-MEM (Eagles MEM (manufactured by Nissui Pharmaceutical Co., Ltd.) to 2%. ), Adjust with W-MEM to 3 × 10 ⁶ / ml, and at 4 ℃
Stored for 48 hours. After centrifugation, lymphocytes were removed and free surface immunoglobulin was concentrated to give an immunoglobulin solution. The amount of anti-DNA antibody in these immunoglobulin solutions (obtained from the stock solution and the effluent) was quantified and calculated from the ratio.

(2) and (3) are B cell activator assay (Clinical test, Vol. 28.
(No. 7) p818). In the case of (2), B cells are separated and purified, and in the case of (3), the lymphocytes are left as they are, and the cell suspension not passed through the column and the effluent from the column are separated by 7%. Incubate in carbon dioxide incubator using 96 well microculture plate in RPMI 1640 medium with FCS, and after 4 days and / or 7
After a day, the supernatant was sampled and the anti-DNA antibody was quantified.

Analysis results Adsorption rate of lymphocytes 21% Adsorption rate of B cells 32% Adsorption rate of anti-DNA antibody-producing B cells 75% Example 2 Preparation of adsorbent Synthesis was performed in the same manner as in Example 1.

Preparation of lymphocyte suspension A heparinized SLE patient's peripheral blood is overlaid on the upper layer of ficoll back, lymphocytes are separated by specific gravity centrifugation, and the lymph-class fraction thoroughly washed with Hanks' buffer is added to 1 × 10 ⁷ ~ 5x
To a concentration of 10 ^{7 /} ml was adjusted with pH 7.20HEPES-HBSS buffer containing 1.0% bovine serum albumin.

Adsorption operation The same operation as in Example 1 was performed.

Analytical method The same as in Example 1.

Analysis results Adsorption rate of lymphocytes 18% Adsorption rate of B cells 28% Adsorption rate of anti-DNA antibody-producing B cells 65% Example 3 Preparation of adsorbent Synthesis was performed in the same manner as in Example 1.

Preparation of lymphocyte suspension A heparinized SLE patient's peripheral blood is overlaid on the upper layer of ficoll back, lymphocytes are separated by specific gravity centrifugation, and the lymph-class fraction thoroughly washed with Hanks' buffer is added to 1 × 10 ⁷ ~ 5x
The concentration was adjusted with pH 7.20 HEPES-HBSS buffer containing no bovine serum albumin so as to have a concentration of 10 ⁷ / ml.

Adsorption operation The same operation as in Example 1 was performed.

Analytical method The same as in Example 1.

Analysis results Adsorption rate of lymphocytes 29% Adsorption rate of B cells 45% Adsorption rate of anti-DNA antibody-producing B cells 85% [Effect of the invention] According to the present invention, the production of autoantibodies involved in the production of pathogenic substances from patient body fluids. B cells can be selectively adsorbed and removed.

Claims (5)

[Claims] 1. An adsorbent for autoantibody-producing B cells, which comprises a solid substance having an anionic functional group. 2. The adsorbent according to claim 1, wherein the anionic functional group is 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. 3. The adsorbent according to claim 1, wherein the anionic functional group is a polyanionic functional group having a plurality of anionic groups in the functional group. 4. The surface area 1 of the solid substance having an anionic functional group.
The adsorbent according to claim 1, which is present in an amount of 1 × 10 ⁴ to 1 × 10 ⁷ µmol per mm ² . 5. A method for adsorbing and removing autoantibody-producing B cells, which comprises bringing an adsorbent for autoantibody-producing B cells, which comprises a solid substance having an anionic functional group, into contact with a body fluid containing lymphocytes.