JP4197545B2 - Specific cell removal material - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a cell removal material having a protein and / or peptide having affinity for a cell surface on the surface of a porous material. The cell removing material of the present invention is used for removing and collecting specific cells from a cell population such as blood.
[0002]
[Prior art]
In recent years, selective treatment of leukemia, etc. for the purpose of treatment of autoimmune diseases such as systemic lupus erythematosus, malignant rheumatoid arthritis, multiple sclerosis, ulcerative colitis, Crohn's disease, leukemia, cancer, etc. or immunosuppression before transplantation The removal technology has progressed.
Thus, materials with fixed peptides, proteins, synthetic products, etc. have been developed for the purpose of selectively separating and removing cells. In particular, a technique for specifically removing blood cells using a material in which a protein such as an antibody or a peptide is immobilized has been advanced.
Proteins and peptides are compounds in which amino acids are linked by peptide bonds, and amino acids are electrolytes that share an acidic carboxyl group and a basic amino group. The three-dimensional structure of proteins and peptides is held by weak non-covalent bonds such as hydrogen bonds, hydrophobic bonds and van der Waals forces, in addition to covalent bonds such as peptide bonds and -SS-bonds. It is an extremely unstable substance. Therefore, denaturation that destroys the three-dimensional structure inherent to proteins and peptides due to physical, chemical, and biological factors is extremely likely to occur. In particular, in an aqueous solution, it is general that the intrinsic function is not exhibited just by leaving it at room temperature for about one day. Furthermore, long-term storage is impossible even by refrigerated storage. As described above, proteins and peptides have problems in terms of stability. Therefore, in order to maintain the function, it is necessary to specially devise and keep the three-dimensional structure stable. There has never been a method for stably maintaining the three-dimensional structure of a protein and / or peptide in a solution in a cell removal material having a protein and / or peptide having affinity for the cell surface on the surface of the porous material, and stabilizing the protein. There is a strong demand for a method of holding the substrate.
[0003]
[Problems to be solved by the invention]
In view of the problem of instability of the protein and / or peptide, an object of the present invention is to provide a specific leukocyte-removing material that stably retains the three-dimensional structure of the protein and / or peptide.
[0004]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventors have found that a nonionic surfactant containing a polyoxyethylene chain together with a protein and / or peptide having an affinity for the cell surface exists on the surface of the porous material. As a result, it was surprisingly found that the three-dimensional structure of the protein and / or peptide can be stably maintained, and the effect of maintaining the cell specificity is greatly improved. That is, the present invention is a cell removal material having a protein and / or peptide having affinity for the cell surface on the surface of the porous material, and has a nonionic surfactant containing a polyoxyethylene chain on the surface of the cell removal material. It is a specific cell removal material characterized by this.
[0005]
The porous material referred to in the present invention refers to a porous material having a pore through which cells can pass and a surface with which cells can contact. The shape preferably has a large surface area so that the cells can contact the liquid layer at a high frequency. Examples of preferable shapes include fiber structures such as nonwoven fabric, fiber, cotton, thread, bundle, cocoon, and woven fabric, polymer porous bodies such as sponge, beads, and gel. Shape. In particular, when leukocytes are removed from blood components, woven fabrics and non-woven fabrics are more preferable in terms of removal efficiency. Among these, nonwoven fabrics are most preferable in terms of structural stability, ease of control, and a large surface area.
[0006]
The protein and / or peptide having affinity for the cell surface in the present invention is a substance having affinity specifically or selectively for cells. Generally, proteins or peptides that interact with cell surface antigens are used. Preferably, a peptide having about 3 to 50 amino acids, an antibody or the like due to its high affinity. More preferably, a monoclonal antibody or a peptide comprising a part thereof is preferably used because of its high affinity with cells. Examples of antibodies by their functions include anti-CD4, anti-CD8, anti-CD3, anti-CD2, anti-CD1a, anti-CD1b, anti-CD5, anti-CD3R, anti-CD6, anti-CD7, anti-CD9, anti-CD10, anti-CDlla, anti-CD18, Anti-CD19, anti-CD20, anti-CD21, anti-CD22, anti-CD23, anti-CD24, anti-CD37, anti-CD40, anti-CD72, anti-CD77, anti-CD16, anti-CD32, anti-CD33, anti-CD34, anti-CD35, anti-CD64, anti-CD65 , Anti-CDw65, anti-CD66b, anti-CD66e, anti-CD89, anti-CDw90, anti-CD56, anti-CD57, anti-CD94, anti-CD105, anti-CD106, anti-CD46, anti-CD31, anti-CDw36, anti-CD41, anti-CD42a, anti-CD42b, anti-CD42b CD63, anti-CD11a, anti-CD11b, anti-CD11c, anti-CD18, CD29, anti-CD44, anti-CD48, anti-CD49a, anti-CD49b, anti-CD49c, anti-CD49d, anti-CD49e, anti-CD49f, anti-CD50, anti-CD51, anti-CD54, anti-CD58, anti-CD61, anti-CD62E, anti-CD62L, anti-CD62P , Anti-CD103, anti-CD26, anti-CD30, anti-CD69, anti-CD70, anti-CD71, anti-CD95, anti-CD25, anti-CD117, anti-CD122, anti-CDw124, anti-CD126, anti-CD127, anti-IL-2R, etc. However, it is not limited to these. Furthermore, these antibodies can be used alone, but a plurality may be fixed. Furthermore, peptides comprising a part of these antibodies can also be used favorably.
[0007]
In the specific cell removal material of the present invention, the protein and / or peptide may be directly bonded to the surface of the porous material, or may be bonded via an active group. As the binding method at the time of fixation, any reaction can be used satisfactorily as long as the activity of the protein and / or peptide can be maintained. For example, substitution reaction, condensation reaction, ring-opening reaction, etc. can be used. it can. The active group may be any known functional group as long as it has a structure capable of binding a protein and / or peptide. For example, an α-acetamino halogen group in which the surface of a porous material is activated using N-hydroxymethyl haloacetamide such as N-hydroxymethyl iodoacetamide, N-hydroxymethyl bromoacetamide, N-hydroxymethyl chloroacetamide, etc. , Α activated porous material surface using N-hydroxymethyldihalopropionamide, etc., α activated porous material surface using β-propionaminohalogen group, N-hydroxymethyldihaloacetamide etc. , Α-acetaminodihalogen group, N-hydroxymethyltrihaloacetamide and the like activated the surface of the porous material, using α, α, α-acetaminotrihalogen group, etc. Epoxy with activated porous material using epichlorohydrin, etc. And a xyl group. Other examples include, but are not limited to, isocyanate groups, isothiocyanate groups, amino groups, carboxyl groups, hydroxyl groups, vinyl groups, and bromocyan active groups. In particular, an active group using a haloacetoaminoalkane agent is preferably used from the viewpoint that the activity of the protein and / or peptide can be maintained and the reaction can be performed under mild conditions.
[0008]
The protein stabilizer referred to in the present invention is present in the vicinity of a protein and / or peptide having affinity for the cell surface of the porous material surface, and keeps the protein and / or peptide stable at room temperature or low temperature for a long time. It is a material that can be used. As the protein stabilizer, a surfactant sharing a hydrophobic group and a hydrophilic group is preferably used. Having a hydrophobic group adsorbs to the surface of the porous material or the hydrophobic portion of the protein and / or peptide, and having a hydrophilic group makes the surface of the material hydrophilic to stably hold the protein and / or peptide. Conceivable. It has been found that a surfactant which is materially stable and electrically neutral is preferred. By being electrically neutral, the material can be stabilized without unnecessary charge. Nonionic surfactants are more preferred because they are more materially stable than zwitterionic surfactants, and nonionic surfactants having a structure containing a polyoxyethylene chain in the hydrophilic portion are most preferred.
The basic structure of a surfactant is a compound that shares a hydrophilic part and a hydrophobic part in one molecule. When the surfactant is dissolved or dispersed in water, the surfactant is roughly classified according to the ionicity exhibited when a part of the hydrophobic portion is dissociated.
Examples of nonionic surfactants containing a polyoxyethylene structure include polyoxyethylene sorbitan ester, polyoxyethylene sorbitol ester, polyoxyethylene isooctyl phenyl ether, polyoxyethylene dodecyl ether, polyoxyethylene polystyryl. Examples thereof include, but are not limited to, phenyl ether, polyoxyethylene polyoxypropylene glycol, polyoxyethylene glycerin ester, polyoxyethylene alkylamine, and the like. Most preferably, polyoxyethylene sorbitan monolaurate belonging to polyoxyethylene sorbitan ester, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan trioleate, etc. Can be mentioned.
However, as an exception, surfactants having a membrane protein solubilizing action, for example, non-ionic octyl β-glucoside, octyl β-D-glucopyranosai, zwitterionic 3-cholamidopropyldimethylammonio-1- Propanesulfonic acid, 3-3-cholamidopropyldimethylammonio-2-hydroxy-1-propanesulfonic acid and the like are not preferable.
[0009]
The specific cell removal material of the present invention is not only as it is, but is filled in a container having at least an inlet and an outlet, and is effective for specific removal or recovery of cells and extracorporeal circulation for the purpose of cell-specific removal. Used. These specific cell removal materials can be used effectively for specific separation, adsorption, removal or the like of each fractional cell such as leukocytes, lymphocytes, monocytes, granulocytes and the like. Furthermore, it can be used favorably for specific removal and recovery of plasma components.
The state of cells in the present invention includes not only whole blood but also blood treated with platelet concentrate, erythrocyte concentrate, etc. in addition to lymphocyte suspension, mononuclear cell suspension, bone marrow fluid, buffy coat, etc. . Furthermore, it can be favorably used for peripheral blood or the like to which hematopoietic factors such as extracorporeal circulation and G-CSF are administered. Moreover, an anticoagulant, a blood preservation solution, etc. can be added to these as required and used satisfactorily.
Although the Example of this invention is shown below with a comparative example, this invention is not limited by these.
[0010]
[Example 1]
To a glass flask, 0.6 g of N-hydroxymethyl iodoacetamide, 5.7 mL of concentrated sulfuric acid and 7.2 mL of nitrobenzene were added and dissolved by stirring, and 0.04 g of paraformaldehyde was further added and stirred in an ice bath. To this was added 0.3 g of a nonwoven fabric made of polypropylene (average fiber diameter 3.3 μm) and reacted for 4 hours. After reacting for 4 hours, the nonwoven fabric was taken out, washed with ethanol and pure water, and vacuum-dried to obtain an activated nonwoven fabric.
An anti-human CD4 monoclonal antibody solution (hereinafter referred to as anti-human) in which 4 sheets of this activated non-woven fabric cut into a circle having a diameter of 0.68 cm are dissolved in a phosphate buffered saline solution (hereinafter referred to as PBS (−)) not containing calcium or magnesium. It was immersed in 400 μL (abbreviated as CD4 solution) (16 μg / 400 μL) for 2.5 hours, fixed with a monoclonal antibody (hereinafter abbreviated as antibody), and washed with PBS (−) after 2.5 hours. Next, a 0.2% polyoxyethylene sorbitan monolaurate / PBS (-) solution (hereinafter abbreviated as Tween 20 solution) was added and fixed for 2.5 hours, washed with PBS (-), and then CD4 positive cells (hereinafter CD4). A specific cell removal material that specifically removes (+) (abbreviated as cell) was obtained.
A specific cell removing device was prepared by filling 4 sheets of the above specific cell removing material (filling density 0.1 g / cm ³ ) into a 1 mL container having an inlet and an outlet, and filling with PBS (−) as a filling solution. .
ACD-A-added human fresh blood (5 mL) (blood: ACD-A = 8: 1) was fed at a flow rate of 1 mL / min with a syringe pump and flowed from the column inlet. The treated liquid was recovered from the column outlet. Furthermore, the remaining blood was collected by applying pressure with air.
The removal rate of CD4 (+) cells at this time was measured with a flow cytometer (COULTER EPICS ELITE ESP ASSY NO. 660556078), and the removal rate of CD4 (+) cells determined from the prevalence of cells before and after treatment was 96. %Met. At this time, the removal rate of CD8 (+) cells was 33%, the removal rate of B lymphocytes was 40%, and the removal rate of platelets was 15%, and CD4 (+) cells could be specifically removed.
[0011]
[Example 2]
The specific cell remover prepared in the same manner as in Example 1 was heated at 50 ° C. for 1 hour and then washed with PBS (−).
ACD-A-added human fresh blood (5 mL) (blood: ACD-A = 8: 1) was fed at a flow rate of 1 mL / min with a syringe pump and flowed from the column inlet. The treated liquid was recovered from the column outlet. Furthermore, the remaining blood was collected by applying pressure with air.
The removal rate of CD4 (+) cells at this time was measured with a flow cytometer (COULTER EPICS ELITE ESP ASSY NO. 660556078), and the removal rate of CD4 (+) cells determined from the prevalence of cells before and after treatment was 96. %Met. At this time, the removal rate of CD8 (+) cells was 33%, the removal rate of B lymphocytes was 27%, and the removal rate of platelets was 10%, and CD4 (+) cells could be specifically removed.
[0012]
[Comparative Example 1]
A specific cell removal material was prepared in the same manner as in Example 1 except that a PBS (−) solution was used instead of the 0.2% Tween 20 solution, and the same experimental operation as in Example 2 was performed. At this time, the removal rate of CD4 (+) cells was 23%. At this time, the removal rate of CD8 (+) cells was 10%, the removal rate of B lymphocytes was 10%, and the removal rate of platelets was 30%. It was considered that the protein was denatured by heating.
[0013]
[Example 3]
A specific cell remover was prepared in exactly the same manner as in Example 1 except that 1% chitosan / PBS (-) (hereinafter abbreviated as chitosan solution) was used instead of PBS (-) as the filling solution. The specific cell remover was sterilized by irradiation with γ-rays of 50 kGy, heated at 50 ° C. for 1 hour, and washed with PBS (−).
ACD-A-added human fresh blood (5 mL) (blood: ACD-A = 8: 1) was fed at a flow rate of 1 mL / min with a syringe pump and flowed from the column inlet. The treated liquid was recovered from the column outlet. Furthermore, the remaining blood was collected by applying pressure with air.
The removal rate of CD4 (+) cells at this time was measured by a flow cytometer method, and the removal rate of CD4 (+) cells determined from the presence rate of cells before and after treatment was 97%. At this time, the removal rate of CD8 (+) cells was 24%, the removal rate of B lymphocytes was 30%, and the removal rate of platelets was 16%, and CD4 (+) cells could be specifically removed.
[0014]
[Comparative Example 2]
A specific cell removal material was prepared in the same manner as in Example 1 except that a chitosan solution was used instead of the 0.2% Tween 20 solution, and a chitosan solution was used instead of using PBS (−) solution as a filling solution. A specific cell remover was prepared in the same manner as in Example 1 except that. Thereafter, the same experimental operation as in Example 3 was performed. At this time, the removal rate of CD4 (+) cells was 50%. At this time, the removal rate of CD8 (+) cells was 35%, the removal rate of B lymphocytes was 38%, and the removal rate of platelets was 30%.
[0015]
【The invention's effect】
The specific cell removal material of the present invention has a three-dimensional structure of a protein by retaining a protein stabilizer on the surface of the cell removal material having a protein and / or peptide having affinity for the cell surface on the porous material surface. It can be kept stable and the storage stability of the cell specificity of proteins and / or peptides is greatly improved. Furthermore, by combining with a sterilization protective agent, the affinity with the sterilization protective agent was increased, and the storage stability of the cell specificity was further improved.
The specific cell removal material of the present invention stably retains the protein and / or peptide structure for a long period of time even at room temperature and low temperature storage, so that it is practically possible to specifically remove target cells from a cell suspension such as blood. I came to be able to do it.

Claims (2)

A cell removing material having a protein and / or peptide having affinity for the cell surface on the surface of the porous material, wherein the cell removing material has a nonionic surfactant containing a polyoxyethylene chain on the surface. Specific cell removal material.   The specific cell removal material according to claim 1, wherein the protein and / or peptide having affinity for the cell surface is an antibody against a cell surface antigen and / or a part thereof.