WO2021106107A1

WO2021106107A1 - Material and column for collecting cells having recovery function of hypo-immune state

Info

Publication number: WO2021106107A1
Application number: PCT/JP2019/046383
Authority: WO
Inventors: 寺本　和雄; 一誠小笠原; 祐二上田
Original assignee: 国立大学法人滋賀医科大学
Priority date: 2019-11-27
Filing date: 2019-11-27
Publication date: 2021-06-03

Abstract

Disclosed are: an immunosuppressive cell-collecting material which includes a molded body having, as an aromatic substituent, a straight-chain aromatic polymer in which nonpolar side-chain amino acid residues are bonded at a ratio of 1-100 per 200 aromatic nuclei through a spacer having a length of 10 or more atoms; and an immunosuppressive cell-collecting column which is filled with said collecting material.

Description

Cell collecting material and cell collecting column having a function to recover hypoimmune state

The present invention relates to an immunosuppressive cell collecting material mainly used for recovery of a hypoimmune state in medical applications, and an immunosuppressive cell collecting column filled with the collecting material.

Prevention of death from cancer and infectious diseases is a major issue in modern medicine. The living body has an immune function, and in a healthy state, the immune function is properly maintained. However, if the function deteriorates too much, cancer development and intractable infectious diseases occur, and conversely, the immune function is promoted. Too much is thought to cause autoimmune disease. Along with the increase in life expectancy, the decline in immune function due to aging is a factor in the increase in cancer and severe infections. Immune-related proteins and leukocyte subclasses in the blood play important roles in maintaining the normal state of immune function.

In the case of cancer, it is known that the concentration of immunosuppressive proteins such as TGF-β and interleukin-10 is increased in the blood, and CD4 ⁺ CD25 ⁺ FoxP3 ⁺ are the cells that produce these. ^{Regulatory T cells (Non-Patent Document 1), CD4 +} and CD8 ⁺ regulatory T cells expressing latency-associated peptide (hereinafter abbreviated as LAP) on the cell surface (Non-patent) Documents 2 and 3), Gr1 ^high CD11b ^high myeloid-derived inhibitory cells (Non-Patent Document 4), regulatory B cells (Non-Patent Document 5), etc. are known, and it has been clarified in recent years that they suppress immunity. It has become.

In the case of infections caused by pathogenic viruses, gram-negative bacteria, gram-positive bacteria, etc., immunity is promoted in the early stage of infection, and interferon-gamma (hereinafter abbreviated as IFN-γ) and tumor necrosis factor-alpha (hereinafter abbreviated as IFN-γ) in the blood. Immune-activated lymphocytes, monocytes, etc. that produce (abbreviated as TNF-α) increase, contributing to the removal of pathogenic bacteria and the like. When the activation of activated lymphocytes becomes too strong, abnormal increases in blood interleukin-1β, interleukin-6, TNF-α, IFN-γ, etc. occur, which overlaps with the complement reaction. Abnormal vascular permeability and impaired blood coagulation can result in death. In addition, as a reaction to this immune activation, immunosuppressive cells increase from several days after infection, and the state of extremely reduced immune function continues for several days to several weeks. During this period, opportunistic infections such as Acinetobacter, enterococci, cytomegalovirus, herpes simplex virus, and Candida may be induced, resulting in death.

The most direct method for determining that a cell is immunosuppressive is to produce interleukin-10 and TGF-β, but the LAP molecule and T cell Ig and ITIM domain on the cell surface. It is also an important determination method that (hereinafter abbreviated as TIGIT), PD-1, Lag-3, Tim-3 and the like are expressed (Non-Patent Document 6).

Recently, immune checkpoint antibodies, which have appeared as therapeutic agents for cancers such as melanoma, have been used for severe infectious diseases because of the idea that death due to sepsis is caused by a decrease in immunity. There is concern that the reagents are expensive and that side effects such as the onset of autoimmune diseases may occur.

On the other hand, as another method for releasing immunosuppression, an extracorporeal circulation column for removing the causative substance and the inhibitory substance can be considered. As a conventional column for treating infectious diseases, there is an endotoxin adsorption column using polymyxin B as an adsorption ligand as a column for treating severe infectious diseases caused by gram-negative bacterial infection, which is clinically used. In addition, an enterotoxin adsorption column using a mannose-binding lectin as a ligand has been proposed for the treatment of Gram-positive bacterial infection. These are used to remove toxins. In addition, systemic inflammatory response syndrome is a serious symptom in which inflammatory cytokines induced by infectious diseases increase abnormally. Since one of the pathogenic substances is histone, a histone removal column has been proposed for treatment. (Patent Document 1). Although many methods have been proposed for removing the humoral pathogenic substance that causes the disease, it cannot be said that the therapeutic effect was sufficient.

Therefore, the present inventors have provided a method for directly removing immunosuppressive cells that produce a humoral pathogen as a more effective method. Until now, there was no effective adsorbent except for a column in which an antibody was immobilized on beads or the like as an adsorption ligand, but a LAP-positive cell selective adsorption column using a specific amino group as a ligand was found for the purpose of cancer treatment. We are studying (Patent Document 2). However, further performance improvement is required in order to improve the effectiveness of the column and expand the applicable range.

International Publication No. 2016/013540 International Publication No. 2012/133399

An object of the present invention is to provide an immunosuppressive cell collecting material having improved performance of selectively collecting immunosuppressive cells, and an immunosuppressive cell collecting column filled with the collecting material. And.

As a result of various studies on improving the performance of a medical column that selectively collects immunosuppressive cells in blood, the present inventors have conducted linear studies having a polyamine residue graft to which a non-polar side chain amino acid is bound. We have found that the above objectives can be achieved by using aromatic polymer fibers. In particular, it was found that the fibers of aminated polysulfone having leucine residues and isoleucine residues can remarkably and selectively collect immunosuppressive cells, and are significantly effective in saving the life of sepsis model rats. It is the first time in the world that such a therapeutic effect has been obtained for the treatment of sepsis with an extracorporeal circulation column of immunosuppressive cells.

The present invention has been completed by further studying based on these findings, and provides the following immunosuppressive cell collecting material and an immunosuppressive cell collecting column.

(I) Immunosuppressive cell collector
(I-1) A linear aromatic polymer in which non-polar side chain amino acid residues are bonded as aromatic substituents at a ratio of 1 to 100 per 200 aromatic nuclei via a spacer having a length of 10 atoms or more. An immunosuppressive cell collecting material consisting of a provided molded body.
(I-2) The non-polar side chain amino acid residue is at least one selected from the group consisting of leucine, isoleucine, norleucine, valine and linear peptides containing these, according to (I-1). Collection material.
(I-3) The collector according to (I-1) or (I-2), wherein the spacer is a linear polyamine.
(I-4) The collector according to any one of (I-1) to (I-3), wherein the linear aromatic polymer is polysulfone, polyetherimide, polyimide or a derivative thereof. ..
(I-5) The collecting material according to any one of (I-1) to (I-4), wherein the linear aromatic polymer is applied to the fiber.
(I-6) The collecting material according to any one of (I-1) to (I-5), wherein the immunosuppressive cell is a cell having a latency-associated protein on the cell surface.
(I-7) The collecting material according to any one of (I-1) to (I-5), wherein the immunosuppressive cell is a cell having TIGIT on the cell surface.
(I-8) The capture according to any one of (I-1) to (I-5), wherein the immunosuppressive cell is a cell that highly expresses a B cell antigen or a granulocyte antigen and a CD11b antigen. Collecting materials.

(II) Column for collecting immunosuppressive cells
(II-1) An immunosuppressive cell collection column filled with the collection material according to any one of (I-1) to (I-8).
(II-2) The column according to (II-1), which is for extracorporeal circulation.
(II-3) The column according to (II-1), which is for cell therapy.
(II-4) Any of (II-1) to (II-3) used for treatment of infectious diseases, treatment of burns, treatment of cancer, prevention of recurrence of cancer after surgery to remove cancer, or prevention of sepsis. The column according to item 1.
(II-5) A method for reducing the concentration of immunosuppressive cells in the patient's blood, in which the patient's blood is placed in vitro in the column according to any one of (II-1) to (II-3). A method that includes a step of circulating.
(II-6) Treatment of infectious diseases, treatment of burns, treatment of cancer, prevention of recurrence of cancer after surgery to remove cancer, or prevention of sepsis, any of (II-1) to (II-3) A method comprising the step of extracorporeally circulating the blood of a patient in need of these treatments or prophylaxis in the column according to paragraph 1.

The collection material and column of the present invention have significantly improved ability to selectively collect immunosuppressive cells from blood, and can reduce the concentration and increase immunity. Therefore, the collector and column of the present invention are expected to be applied to the treatment of severe infectious diseases and cancer.

Furthermore, since the collecting material of the present invention uses a non-proteinaceous material, it can be sterilized.

Hereinafter, embodiments of the present invention will be described in more detail.

Immunosuppressive Cell Collecting Material The immunosuppressive cell collecting material of the present invention has 1 to 1 per 200 aromatic nuclei via a spacer having a non-polar side chain amino acid residue of 10 atoms or more as an aromatic substituent. It is characterized by consisting of a molded product having a linear aromatic polymer bonded at a ratio of 100 pieces.

(Composition of collected material)
The immunosuppressive cells, which are the cells to be collected in the present invention, mean blood cells that directly or produce inhibitory cytokines such as interleukin 10 to suppress the functions of killer cells and helper T cells. ^{Specific examples include LAP +} CD4 ⁺ regulatory T cells, LAP ⁺ CD8 ⁺ regulatory T cells, LAP-positive B cells, monocytic cells and granulocyte cells, and TIGIT cells that have LAP on the cell surface. Examples include CD4 T cells, CD8 T cells and B cells on the surface, cells that highly express granulocyte antigen and CD11b (that is, cells that express Gr1 ^high CD11b ^high ), and cells that highly express B cell antigen. Can be done. The presence of these cells can be confirmed by flow cytometer analysis. The abundance of LAP ⁺ CD4 ⁺ regulatory T cells and LAP ⁺ CD8 ⁺ regulatory T cells in peripheral blood varies from individual organism to individual organism, but is generally 0.2% to 30% of each T cell subset. .. The LAP-positive rate for B cells and granulocytes is 1% to 60%, and that for monocytes is 50% to 100%.

In the present invention, "selectively capture" means that when blood is passed through a column filled with a collecting material, the abundance ratio of immunosuppressive cells in the passed blood is reduced as compared with that before the passage, and the captured cells. It means that the abundance ratio of immunosuppressive cells in the cells is higher than that before the passage.

High cell selectivity of the collecting material of the present invention is preferable for medical safety, but in order to improve this, it is generally better that the basicity of the amino group is low and the abundance density is low. good. Further, in the case of a method of directly treating blood and returning it to a living body such as extracorporeal circulation, it is preferable that the adsorptivity of plasma proteins such as albumin is as low as possible. In particular, it must be avoided for medical safety that the amino group of the collecting material stimulates the cells captured by the collecting material to release inflammatory cytokines and cause the adsorbed cells to cause necrosis. From these viewpoints, the amount of amino groups in the molded product of the present invention is 150 μmol or less in 1 g of the molded product, and the value obtained by subtracting the amount of carboxyl groups from the amount of amino groups is 110 μmol or less, more preferably. It is preferably 1 to 110 μmol.

The linear aromatic polymer in the present invention means an aromatic polymer in which the main chain of the polymer is linear and can be molded. Further, a polymer capable of withstanding the conditions of gamma ray sterilization and high pressure steam sterilization is particularly preferable. Solubility in an organic solvent is preferable because it can be applied to the surface of other molded products. In particular, having film-forming property is preferable in terms of safety because the mechanical stability of the processed product is increased and the probability of separation of fine particles from the molded product is reduced. Specific examples of linear aromatic polymers include polysulfone consisting of bisphenol A and diphenylsulfone-{(pC ₆ H ₄ ) -SO _2- (pC ₆ H ₄ ) -O- (pC ₆ H ₄ ) -C ( CH ₃ ) _2- (pC ₆ H ₄ ) -O} _n- , poly (p-phenylene ether sulfone)-{(pC ₆ H ₄ ) -SO _2- (pC ₆ H ₄ ) -O- (pC ₆ H) ₄ ) -O} _n -,-{(pC ₆ H ₄ )-SO _2- (pC ₆ H ₄ ) -O- (pC ₆ H ₄ ) -C (CF ₃ ) _2- (pC ₆ H ₄ )- Examples thereof include aromatic polysulfone polymers typified by O} _n- , polyetherimide, polyimide, and derivatives thereof. Among them, polysulfone is excellent as a medical material because it is inexpensive, has high workability, has high mechanical strength, is soluble in various kinds of organic solvents, and has the ability to form a tough film. It is particularly preferable because it is easy to introduce a functional group. Further, among the above polymers, those that dissolve in a non-chlorine type and low toxicity solvent such as tetrahydrofuran, dimethylsulfoxide, and N-methylpyrrolidone are particularly preferable in terms of preventing environmental pollution and maintaining occupational health. The molecular weight of the polymer is not particularly limited as long as it can be molded, but it is usually 10,000 to 5 million, particularly preferably 20,000 to 200,000 from the viewpoint of good moldability.

Preferred hydrophobic amino acids attached via spacers as aromatic substituents include leucine, isoleucine, norleucine, valine, methionine and linear peptides containing these, amino acids having a cyclic structure such as proline, tyrosine, phenylalanine and tryptophan. Examples thereof include linear peptides containing these. Among them, leucine, isoleucine, norleucine, valine and linear peptides containing these are preferable, and leucine, isoleucine and linear peptides containing these are more preferable. Specific preferable examples of the peptide containing leucine and isoleucine as constituents include glycyl leucine, glycyl glycyl leucine, leucine leucine, glycyl isoleucine and the like. The number of amino acid residues of the linear peptide is usually 2 to 10, preferably 2 to 4. The amino acid may be D-form, L-form or a mixture thereof, and is preferably L-form. These hydrophobic amino acids can be used alone or in combination of two or more.

The ratio of non-polar side chain amino acid residues bound to the linear aromatic polymer is usually 1 to 100, preferably 5 to 50, and more preferably 10 to 50 per 200 aromatic nuclei.

The spacer is a linear molecule with a length of 10 atoms or more, one end of which is bonded to the aromatic ring of the main chain of the aromatic polymer, and the other end of which is bonded to a hydrophobic amino acid. Examples of the spacer include linear polyamines. The length of the spacer is preferably 10 to 40 atoms, more preferably 10 to 20 atoms. The number of atoms here means the number of atoms in the main chain of the spacer, and examples of the atoms include carbon, nitrogen, and oxygen atoms. There are two types of binding directions for amino acids that bind to the spacer: a binding mode in which the amino acid is bonded from the amino group side and the carboxyl group remains at the end, and a bonding mode in which the amino group at the alpha position remains at the terminal by binding from the carboxyl group side. , The former is preferable because it is easy to manufacture. Since the spacer is manufactured based on the manufacturing process, it depends on the manufacturing method.

The shape of the molded body of the present invention is not particularly limited, and examples thereof include fibrous, non-woven fabric, film-like, hollow thread-like, powder-granular, and higher-order processed products thereof, and the shape is appropriately selected according to the intended use. .. Examples of the molded product of the present invention include a product obtained by molding the linear aromatic polymer itself, a product obtained by applying the linear aromatic polymer to another molded product, and the like.

Examples of the shape of the molded product include fibrous, non-woven fabric, film-like, hollow thread-like, powder-granular, and higher-order processed products thereof. Examples of the material of the molded product include polyester, polyamide, polyurethane and the like. Specific examples of polyester include polylactic acid, polyglycolic acid, polyethylene terephthalate, polybutylene terephthalate and the like. Specific examples of the polyamide include nylon-6, polyhexamethylene adipamide, and the like. The molecular weight of such a polymer is not particularly limited as long as it can be molded.

The collecting material of the present invention can selectively capture immunosuppressive cells from the extracellular fluid composed of various types of blood cells. Further, since the collecting material of the present invention does not contain a protein component, its function is not lost by sterilization operations such as high-pressure steam sterilization and radiation sterilization.

We do not want to be bound by any theory, but by inferring the mechanism of selective collection, LAP on LAP-positive T cells, which is one of the immunosuppressive cells, binds to a protein rich in polyleucine structure. The LAP molecule has an affinity for the amino group because the sugar chain in the LAP molecule has a phosphate group, and the stem polymer of the collector has an aromatic nucleus, and the aromatic nucleus Since it has a polyethyleneimine structure substituent having a polyleucine residue at the tip as a substituent, it is presumed that its chemical structure has bulkiness, hydrophobicity and polarity close to those of the polyleucine structure. Further, when the polyamine and the carboxyl group coexist and the carboxyl group is present at the ligand terminal, the carboxyl group forms an ion pair with the amino group inside in the graft molecule, so that the tip is curved inward. It is considered that the alkyl group such as leucine is exposed to the outside and easily interacts with the cell, so that the selective collection property is improved. In general, in order to selectively adsorb a specific cell, it is important to select a ligand having an affinity for the target cell, an appropriate ligand distribution, an appropriate cation / anion balance, and the like.

(Manufacturing method of collected material)
The simplest method for producing the immunosuppressive cell collecting material of the present invention is to synthesize a ligand compound having a spacer residue and to synthesize a linear aromatic such as a polysulfone having a reactive functional group such as a haloacetamide methyl group. There is a method of reacting with a polymer, or a method of reacting a linear aromatic polymer such as polysulfone having a spacer residue with a ligand compound. Further, a method of dissolving the obtained polymer in a solvent to form the molded product, a method of melt-molding the polymer, a method of surface-coating an existing molded product, or the like is adopted. When surface-coating an existing molded product, it can be coated with a linear aromatic polymer twice or more. As the ligand compound, one having the above-mentioned reactive functional group at one end and a hydrophobic amino acid residue at the other end is preferably adopted.

Examples of the reactive functional group include reactive functional groups such as chloromethyl group, haloacetamide methyl group, acid halide of carboxyl group, acid anhydride or ester, which have high reactivity and stability during processing. From the standpoint of balance, a chloroacetamide methyl group, an iodoacetamide methyl group and a bromoacetamide methyl group are particularly preferable.

As a method for synthesizing the ligand compound having the spacer residue, equimolar amounts of chloroacetyl-L-leucine, chloroacetyl-L-isoleucine, chloroacetyl-L-valine, and chloro in a solution of a linear polyamino compound such as diethylenetriamine are used. Haloacetyl amino acid derivatives such as acetyl-α-aminobutyric acid, chloroacetyl-L-norleucine, chloroacetyl-glycine leucine, chloroacetyl-alanyl leucine, chloroacetyl-L-leucine, or leucine and leucine glycine, glycine This can be achieved by adding a succinimide ester derivative of a peptide such as leucine at room temperature.

Further, as a method for producing a linear aromatic polymer having the spacer residue, a polysulfone having a reactive functional group such as a chloromethyl group, a haloacetamide methyl group, an acid halide of a carboxyl group, an acid anhydride or an ester is used. It can be achieved by adding a solution of a linear polyamino compound such as diethylenetriamine to a solution of a polymer such as or a molded product.

The linear polyamino compound is not particularly limited, and alkylene polyamine is preferable, and examples of the alkylene polyamine include butylene diamine, hexamethylenediamine, octamethylenediamine, dodecamethylenediamine, diethylenetriamine, triethylenetetramine, and tetraethylenepentamine. Can be mentioned.

As a method of coating the molded product with a linear aromatic polymer, a method of immersing the molded product in a polymer solution and then evaporating and removing the solvent is adopted. Specific examples are shown below, but the present invention is not limited to this.

First, 0.5-fold mol of N-hydroxymethyl-α-chloroacetamide is reacted with polysulfone to prepare chloroacetamide methylpolysulfone having a substitution rate of 50 mol%. Next, the polymer is dissolved in tetrahydrofuran to prepare a polymer solution, the polyethylene terephthalate nonwoven fabric is dipped in this solution, and then tetrahydrofuran is evaporated to obtain a polymer coated nonwoven fabric.

Next, the polymer-coated non-woven fabric was heated in a 50 mass% dimethylsulfoxide aqueous solution of 1 mass% diethylenetriamine at 40 ° C. for 1 hour, washed with 50 mass% dimethylsulfoxide water, and further of chloroacetyl-L-leucine. Immerse in 50 mass% dimethylsulfoxide aqueous solution and heat at 40 ° C. for 1 hour.

Then, this non-woven fabric is washed with 50% by mass dimethylsulfoxide water and water to obtain the immunosuppressive cell collecting material of the present invention. Here, if chloroacetyl chlorides of various ligands are used instead of chloroacetyl-L-leucine, a collecting material having various ligands can be obtained.

As described above, the coating amount of the linear aromatic polymer that coats the molded product is preferably 0.1 to 50% by mass, and more preferably 5 to 20% by mass with respect to the molded product.

Column for collecting immunosuppressive cells The column for collecting immunosuppressive cells of the present invention is characterized by being filled with the above-mentioned immunosuppressive cell collecting material.

When the collecting material of the present invention is in the form of a fibrous material or a non-woven fabric, the packing density of the collecting material of the present invention to be filled in the column is preferably 50 to 400 mg / cm ³ , particularly preferably 150 to 300 mg / cm. It is ^3. Within this range, the cell collection efficiency, selectivity, and pressure loss during fluid passage are in the appropriate range. When the pressure drop increases, the red blood cells hemolyze. In particular, hemolysis is contraindicated in extracorporeal circulation. Pressure drop has a positive correlation between filling density and blood flow rate. Since the blood flow rate must be at least 30 mL / min in order to complete extracorporeal circulation within 2 hours in humans, the pressure drop must be 100 mmHg or less to prevent erythrocyte hemolysis.

By using the column of the present invention as a column for extracorporeal circulation, immunosuppressive cells can be selectively collected from the blood and its concentration can be lowered. In addition, since leukocytes prepared from blood treated with the column of the present invention and leukocytes obtained by treating pre-isolated leukocytes with this column have improved antitumor activity, the column of the present invention is used for cell therapy for diseases such as cancer. Can also be used as a column for.

Examples of the container filled with the collecting material of the present invention include those made of glass, plastic, stainless steel, etc., and the size of the container is appropriately selected according to the purpose of use.

Since the column of the present invention can selectively collect immunosuppressive cells, it can be used for treating infectious diseases (particularly severe infections and opportunistic infections) caused by decreased immunity, and for bacterial infections for which antibiotics do not work. It is used for treatment, prevention of infections (in the case of severe trauma, surgery, burns, etc.), prevention of sepsis (after severe trauma), etc. It can also be used for the treatment of cancer and the prevention of recurrence of cancer after surgery to remove the cancer. When the column of the present invention is applied for the treatment of cancer, when used in combination with surgical therapy, radiotherapy, anticancer drug therapy, activated leukocyte therapy, vaccine therapy, etc., it helps to improve these therapeutic effects, especially to prevent metastasis and recurrence. Is also considered useful. The column of the present invention can be said to be a safe column because it can circulate extracorporeally even in a shock state.

The types of cancer to which the column of the present invention is applied include gastric cancer, colon cancer (rectal cancer, colon cancer), small bowel cancer, liver cancer, pancreatic cancer, lung cancer, pharyngeal cancer, esophageal cancer, renal cancer, biliary sac and bile duct cancer. Head and neck cancer, bladder cancer, prostate cancer, breast cancer, uterine cancer (cervical cancer, uterine body cancer), ovarian cancer, brain tumor, thoracic adenoma, leukemia, malignant lymphoma and the like can be mentioned.

Hereinafter, examples will be given to explain the present invention in more detail. However, the present invention is not limited to these examples and the like.

Unless otherwise specified, the measurement of the amount of amino groups, the analysis of cell surface antigens, the preparation of cancer-bearing rats, and the preparation of extracorporeal circulation of rats in this example were carried out by the following methods.

1. 1. Measurement of Picric Acid Adsorption Amount 0.1 g of a sample was immersed in 10 mL of a 0.1 M picric acid / 70% ethanol solution and gently shaken for 2 hours. Next, the sample was washed with a 70% ethanol solution and washed until the yellow color of the washing solution disappeared. Next, the sample was immersed in 10 to 50 mL of a 1% by mass diethylamine / 70% ethanol solution to elute picric acid. The picric acid concentration was determined from the absorbance at 320 nm, and the adsorption amount was divided by the accurate sample weight to obtain the picric acid adsorption amount per gram. This value corresponds to the amount of amino groups.

2. Analysis of cell surface antigens Cell surface antigens were analyzed using a multi-laser, multi-color flow cytometer CytoFlex S manufactured by Beckman Coulter. Multicolor measurement was performed with 7 to 9 colors. Antibodies for cell surface staining include fluorosane isothiocyanate (FITC) -labeled anti-rat granulocyte marker (HIS48) antibody and phycoerythrin-cocyanin 7 (PE-Cy7) -labeled anti-rat CD8a antibody from e-Bioscience. Biolegend's Peridinin Chlorofil Protein-Cyanin 5.5 (PerCP-Cy5.5) -labeled anti-rat CD45RA antibody, phycoerythrin (PE) -labeled anti-rat MHC-2 antibody, PE-labeled anti-rat LAP antibody, allophycocyanin (APC) Labeled anti-mouse LAP antibody, Alexa Fluor 700 labeled anti-rat CD45 antibody, allophycocyanin-cocyanin 7 (APC-Cy7) -labeled anti-rat CD4 antibody, APC-labeled anti-rat CD11b / c antibody, APC-labeled anti-mouse TIGIT antibody (Clone: 1G9) , And Becton Dickinson's V450-labeled anti-rat CD11b antibody, Brilliant Violet 605 (BV605) -labeled anti-rat CD3 antibody. Examples of antibody combinations include FITC-labeled anti-rat granulocyte marker (HIS48) antibody, PerCP-Cy5.5-labeled anti-rat CD45RA antibody, PE-labeled anti-mouse LAP antibody, PE-Cy7-labeled anti-rat CD8a antibody, and APC-labeled. Nine color combinations of anti-mouse TIGIT antibody, Alexa Fluor 700-labeled anti-rat CD45 antibody, APC-Cy7-labeled anti-rat CD4 antibody, V450-labeled anti-rat CD11b antibody, and BV605-labeled anti-rat CD3 antibody were used. For LAP and TIGIT, the corresponding mouse IgG was used as a comparative antibody.

100 μL of blood was collected in a 15 mL test tube, 100 μL of fax buffer containing 3% mouse serum (0.1% bovine serum albumin-containing commercial dalpecco phosphate buffer) containing fluorescent antibody dissolved was added, and then 30 at room temperature. Allowed to stand for minutes. Next, 1 mL of Becton Dickinson's dissolved blood was added to the cell pellet obtained by centrifugation, and the mixture was shaken for 20 minutes. After washing with fax buffer, it was dispersed in 200 μL of fax buffer and measured with a flow cytometer. 100,000 cells were collected and analyzed.

3. 3. Preparation of cancer-bearing rats (preparation of KDH-V cells)
4-Dimethylaminoazobenzene-induced liver cancer cell KDH-8 (Satoshi Yano, Hokkaido Medical Journal, Vol. 68, No. 5, 654-664 (1993)) in complete medium (RPMI1400 medium: fetal bovine serum 10% by volume, 2-mercaptoethanol 50 μg Subculture in / L, streptomycin 50 μg / mL, penicillin-G 50 units / mL), and suspended cells were collected from the passage and passaged in the above-mentioned complete medium to obtain KDH-V cells. Those with a viable cell rate of 95% or more were used.

(Preparation of cancer-bearing rats)
1 × 10 ⁵ cancer cells KDH-V were suspended in 0.5 mL PBS (-) and inoculated subcutaneously into the back of WKAH / Hkm rats (male, 8-16 weeks old) to prepare cancer-bearing rats. .. Twelve days after inoculation, the tumor diameter averaged 1 cm.

4. Preparation of septic rats Lipopolysaccharide (derived from Escherichia coli O111, phenol extract; Fujifilm Wako Junyaku Co., Ltd. 125-05201; hereinafter abbreviated as LPS) was dissolved in physiological saline to a concentration of 10 mg / mL, and the concentration was 0.22. It was sterilized and filtered through a μm film (MILLEX-GV filter unit) to obtain an LPS solution. Septic rats were prepared by intraperitoneally injecting 1 mL of LPS solution per kg of body weight into the abdominal cavity of WKAH / Hkm rats (male, 10-12 weeks old) 4 hours prior to extracorporeal circulation.

5. Extracorporeal circulation in rats (preparation of extracorporeal circulation column)
An extracorporeal circulation column was prepared by filling 0.3 g of a collecting material into a polypropylene cylindrical column having an inner diameter of 1 cm and an internal volume of 2 mL. After sterilization by passing 70% alcohol through the column and circuit, 40 mL of heparin-added physiological saline (5 units / mL) was flowed at a rate of 2 mL / min immediately before extracorporeal circulation for pretreatment.

(Extracorporeal circulation)
Three-kind mixed anesthetic (domitor 7.5 mg per 1 L of physiological saline, midazolam "sand" 400 mg, betorfar 500 mg; subcutaneous domitol 0.375 mg / kg rat body weight General anesthesia was performed by injection), the artery and vein of the left thigh were cannulated, blood was removed from the artery, passed through an extracorporeal circulation column using a microtube pump, and blood was returned to the vein. Extracorporeal circulation was performed for 30 minutes at a blood flow rate of 2 mL / min. Heparin was continuously administered at 100 units / hour using an infusion pump (Terufusion small syringe pump TE-361N; Terumo Corporation) during extracorporeal circulation. However, in the case of anesthesia of septic rats, 90% of the usual amount of anesthetic was used.

After the completion of extracorporeal circulation, a medetomidine antagonist (a solution containing 150 mg of antisedan per 1 L of physiological saline) was subcutaneously injected at a ratio of 0.75 mg of antisedan per 1 kg of rat body weight to awaken the patient.

[Example 1]
(Preparation of coating polymer 1)
A mixed solution of 20 mL of nitrobenzene and 40 mL of sulfuric acid is cooled to 0 ° C., and then 2.6 g (0.02 mol) of N-hydroxymethyl-2-chloroacetamide is added at a temperature of 0 to 10 ° C to dissolve the solution. It was added to a nitrobenzene solution of Udelpolysulfone P3500 (88.4 g: 0.2 mol / 1600 mL) with good stirring. Further, after stirring at 20 ° C. for 2 hours, the reaction mixture was placed in a large excess of cold methanol to precipitate the polymer. The precipitate was extracted with methanol until the odor of nitrobenzene disappeared and then dried to give 90.0 g of polymer. This polymer was dissolved in 2 L of dimethylacetamide and placed in a large excess of methanol for reprecipitation to give the coating polymer 1.

The polymer was dissolved in dimethylformamide, dimethylsulfoxide and tetrahydrofuran. The presence of the amide group was confirmed by absorption of ^{3290-3310, 1670, and 1528 cm -1} from the infrared absorption spectrum of the film prepared by dissolving this in chloroform and casting it on a glass plate. ^{The 1} H NMR spectrum of the deuterated chloroform solution was measured, and the peak derived from the methylene group hydrogen (2H) of the benzyl group of the amide methyl group with respect to the area of the peak (1.66 ppm; singlet) derived from the isopropyride group hydrogen (6H) of the polysulfone main chain. It was confirmed that the substitution rate was 10% from the area ratio of (4.22 ppm).

(Preparation of coating polymer 2)
A mixed solution of 130 mL of nitrobenzene and 270 mL of sulfuric acid is cooled to 0 ° C., and then 13.6 g (0.11 mol) of N-hydroxymethyl-2-chloroacetamide is added at a temperature of 0 to 10 ° C. to dissolve the solution. It was added to a nitrobenzene solution of Udelpolysulfone P3500 (44.2 g: 0.1 mol / 500 mL) with good stirring. Further, after stirring at 20 ° C. for 2 hours, the reaction mixture was placed in a large excess of cold methanol to precipitate the polymer. The precipitate was extracted with methanol until the odor of nitrobenzene disappeared and then dried to give 56.0 g of polymer. The polymer was dissolved in 1 L of dimethylformamide and placed in a large excess of methanol for reprecipitation.

Since part of the chloroacetamide methyl group was hydrolyzed to an aminomethyl group in this polymer, the following treatment was performed to convert it back to the chloroacetamide methyl group. That is, 30 g of the polymer was dissolved in 300 mL of N-methylpyrrolidone, 20 mL of chloroacetyl chloride was added dropwise on ice water and under stirring, and the mixture was reacted for 20 hours. The reaction mixture was placed in a large excess of cold methanol to precipitate the polymer and immersed in 2 L of water with 30 g of sodium hydrogen carbonate for 5 hours. This polymer was dissolved in dimethylformamide, placed in methanol, the polymer was precipitated again and vacuum dried to give 27 g of coating polymer 2.

The resulting polymer was dissolved in dimethylformamide, dimethylsulfoxide, methylene chloride and tetrahydrofuran. Elemental analysis: N: 2.6% Cl: 6.1%. The presence of the amide group was confirmed by absorption of ^{3290-3310, 1670, and 1528 cm -1} from the infrared absorption spectrum of the film prepared by dissolving this in chloroform and casting it on a glass plate. ^{The 1} H NMR spectrum of the deuterated chloroform solution was measured, and the peak derived from the methylene group hydrogen (2H) of the benzyl group of the amide methyl group with respect to the area of the peak (1.66 ppm; singlet) derived from the isopropyride group hydrogen (6H) of the polysulfone main chain. It was confirmed that the substitution rate was 100% from the area ratio of (4.22 ppm).

(Collecting material synthesis: Non-woven fabric cleaning treatment)
To remove foreign substances such as oils adhering to the surface of the fiber ^{, soak 88.2 g of polyethylene terephthalate fiber non-woven fabric (density 48 mg / cm 3} ; Japan Vilene Co., Ltd.) in 2 L of 0.5 mass% diethylenetriamine / dimethylsulfoxide solution. , 105 ° C. for 20 minutes. After washing with water, it was dried to obtain 87.2 g of washed non-woven fabric-1. The amount of picric acid adsorbed was 1 μmol / g.

<Preparation of Collecting Material-1 of the Present Invention>
(Collecting material synthesis: Polymer primary coating treatment)
6 g of the previously prepared coating polymer 1 is dissolved in 800 mL of tetrahydrofuran, 40 g of the previously prepared cleaning nonwoven fabric-1 is immersed in this solution, allowed to stand for 24 hours, and then rotated in a flask. Tetrahydrofuran was evaporated. This non-woven fabric was immersed in 2 L of 1 mass% diethylenetriamine / dimethylsulfoxide solution and heated at 40 ° C. for 60 minutes. After washing with water, it was dried to obtain 46 g of coated non-woven fabric-1.

(Collecting material synthesis: Secondary coating treatment of polymer)
1 g of the previously prepared coating polymer 2 is dissolved in 400 mL of dimethylsulfoxide, 40 g of the previously prepared coated nonwoven fabric-1 is immersed in this solution, and then the temperature is 25 ° C. while rotating in a flask. Dimethyl-sulfoxide was evaporated under vacuum to obtain a coated non-woven fabric-2.

(Ligand binding)
Add 1.0 g (4.8 mmol) of chloroacetyl-L-leucine to a solution consisting of 1 g (9.6 mmol) of diethylenetriamine, 200 mL of water and 200 mL of dimethylsulfoxide, stir at room temperature for 3 hours, and then 20 g of coated non-woven fabric. -2 was soaked and shaken in a water bath at 40 ° C. for 2 hours. The non-woven fabric is washed with water, extracted 6 times with warm water at 60 ° C., and then dried to use a diethylenetriamine derivative having a diethylenetriamine / acetyl-L-leucine bond ratio of 2 as a ligand (collecting material of the present invention-1) 22. got g. The amount of picric acid adsorbed on the non-woven fabric as a whole was 127 μmol / g. Assuming that the outermost layer of the non-woven fabric is completely covered with the coating polymer 2, ligands containing diethylenetriamine bind to 50 per 200 aromatic nuclei, half of which 25 contain acetyl-L-leucine. It will be combined.

<Preparation of Collecting Material-2 of the Present Invention>
(Ligand binding)
Add 2.0 g (10 mmol) of chloroacetyl-L-leucine to a solution consisting of 1 mL (9.6 mmol) of diethylenetriamine, 200 mL of water and 200 mL of dimethylsulfoxide, stir at room temperature for 3 hours, and then 20 g of coated non-woven fabric. -2 was soaked and shaken in a water bath at 40 ° C. for 2 hours. The non-woven fabric was washed with water, extracted 6 times with warm water at 60 ° C., and dried to obtain 21 g of a non-woven fabric having a diethylenetriamine / acetyl-L-leucine bond ratio of 1. The amount of picric acid adsorbed on the non-woven fabric as a whole was 143 μmol / g. Assuming that the outermost layer of the non-woven fabric is completely covered with the coating polymer 2, ligands containing diethylenetriamine are bound to 50 per 200 aromatic nuclei, all of which are bound to 1 acetyl-L-leucine. Will be there.

<Preparation of Collecting Material-3 of the Present Invention>
(Ligand binding)
Add 2.0 g (10 mmol) of chloroacetyl-L-isoleucine to a solution consisting of 2 mL (20 mmol) of diethylenetriamine, 200 mL of water and 200 mL of dimethylsulfoxide, stir at room temperature for 3 hours, and then 20 g of coated non-woven fabric. -2 was soaked and shaken in a water bath at 40 ° C. for 2 hours. The non-woven fabric is washed with water, extracted three times with warm water at 60 ° C., dried, and a non-woven fabric using a diethylenetriamine derivative having a diethylenetriamine / acetyl-L-isoleucine bond ratio of 2 as a ligand (collector of the present invention-3) 20. got g. The amount of picric acid adsorbed on the non-woven fabric as a whole was 120 μmol / g.

<Preparation of Collecting Material-4 of the Present Invention>
(Ligand binding)
Add 2.0 g (10 mmol) of chloroacetyl-DL-valine to a solution consisting of 2.1 g (20 mmol) of diethylenetriamine, 200 mL of water and 200 mL of dimethylsulfoxide, stir at room temperature for 3 hours, and then 20 g of coated non-woven fabric. -2 was soaked and shaken in a water bath at 40 ° C. for 2 hours. The non-woven fabric is washed with water, extracted three times with warm water at 60 ° C., dried, and the non-woven fabric using a diethylenetriamine derivative having a diethylenetriamine / acetyl-DL-valine bond ratio of 2 as a ligand (collector of the present invention-4) 21. got g. The amount of picric acid adsorbed on the non-woven fabric as a whole was 133 μmol / g.

[Example 2]
<Preparation of Collecting Material-5 of the Present Invention>
(Preparation of coating polymer 3)
A mixed solution of 150 mL of nitrobenzene and 100 mL of sulfuric acid is cooled to 0 ° C., and then 9.2 g (0.075 mol) of N-hydroxymethyl-2-chloroacetamide is added at a temperature of 0 to 10 ° C to dissolve the solution. Delpolysulfone P3500 was added to a nitrobenzene solution (60 g: 0.136 mol / 500 mL) with good stirring. Further, after stirring at 20 ° C. for 2 hours, the reaction mixture was placed in a large excess of cold methanol to precipitate the polymer. The precipitate was extracted with methanol until the odor of nitrobenzene disappeared and then dried to give 63.0 g of polymer. The polymer was dissolved in 1 L of dimethylformamide and placed in a large excess of methanol for reprecipitation and purification. 30 g of this polymer was dissolved in N-methylpyrrolidone, and 20 mL of chloroacetyl chloride was added dropwise on ice water and under stirring, and the mixture was reacted for 20 hours. The reaction mixture was placed in a large excess of cold methanol to precipitate the polymer and immersed in 2 L of water with 30 g of sodium hydrogen carbonate for 5 hours. After washing with water and drying, the polymer was dissolved in dimethylformamide, placed in methanol, the polymer was precipitated again and vacuum dried to give 29 g of coating polymer 3.

The resulting polymer was dissolved in dimethylformamide, dimethylsulfoxide, methylene chloride and tetrahydrofuran. ^{The 1} H NMR spectrum of the deuterated chloroform solution was measured, and the peak derived from the methylene group hydrogen (2H) of the benzyl group of the amide methyl group with respect to the area of the peak (1.66 ppm; singlet) derived from the isopropyride group hydrogen (6H) of the polysulfone main chain. It was confirmed that the substitution rate was 50% from the area ratio of (4.22 ppm).

(Collecting material synthesis: Polymer coating treatment)
Dissolve 2 g of the previously prepared coating polymer 3 in 400 mL of dimethyl sulfoxide, immerse 18 g of the previously prepared non-woven fabric-1 in this solution, and then rotate in a flask at 25 ° C. Dimethyl-sulfoxide was evaporated under vacuum to obtain a coated non-woven fabric-3.

(Ligand binding)
Add 0.5 g (2.6 mmol) of chloroacetyl-L-leucine to a solution consisting of 0.6 g (5.8 mmol) of diethylenetriamine, 200 mL of water and 200 mL of dimethylsulfoxide, stir at room temperature for 3 hours, and then 10 g of coated non-woven fabric. -3 was soaked and shaken in a water bath at 40 ° C. for 2 hours. The non-woven fabric was washed with water, extracted three times with warm water at 60 ° C., and dried to obtain 10 g of the collector of the present invention-5. The amount of picric acid adsorbed on the non-woven fabric as a whole was 110 μmol / g. Assuming that the outermost layer of the non-woven fabric is completely covered with the coating polymer 3, a ligand containing diethylenetriamine binds to 25 per 200 aromatic nuclei, and one acetyl-L-leucine binds to half of it. It will be.

[Preparation of comparative collection material]
(Ligand binding)
20 g of coated non-woven fabric-2 was immersed in a solution consisting of 4 mL of diethylenetriamine, 200 mL of water and 200 mL of dimethylsulfoxide, and shaken in a water bath at 40 ° C. for 2 hours. The non-woven fabric was washed with water, extracted 6 times with warm water at 60 ° C., and dried to obtain 20 g of a non-woven fabric having a ligand of diethylenetriamine (comparative collecting material-1). The amount of picric acid adsorbed was 108 μmol / g.

[Test Example 1] (Septicemia Treatment Experiment-1)
The septic rats were anesthetized, and 4 hours after the administration of LPS, extracorporeal circulation therapy was performed for 30 minutes using the column of the collection material-1 of the present invention, the column of the comparative collection material-1, and the empty column containing no collection material. .. In the case of a collector-filled column, 0.3 mL of blood was collected from the artery at the start and end of extracorporeal circulation, and surface antigen analysis of leukocytes was performed. The results are shown in Table 1. All 5 animals treated with the column packed with the collection material -1 of the present invention survived, but only 1 in 4 animals survived with the comparative collection material -1. In the case of 2 animals treated with an empty column and 5 animals without treatment, all died by the next day.

Table 2 shows the changes in the LAP-positive cell rate in ^{CD4 +} T cells in leukocytes at that time. The LAP positive rate decreased in the collection material of the present invention, but did not decrease in the column of the comparative collection material in many cases.

Similarly, Table 3 shows the rate of decrease in LAP-positive cells of B cells (CD45RA ^+). Similarly, the LAP positive rate decreased in the collection material of the present invention, but did not decrease in the column of the comparative collection material in many cases.

The life and death of rats may involve factors other than the measured items such as white blood cell count and LAP positive rate, and it is difficult to make an accurate analysis only by these analyzes, but the survival rate in the examples is higher than that in the comparative examples. Certainly expensive. In Comparative Example 1, it is presumed that the reason why the death occurred despite the decrease in the proportion of LAP-positive cells was that the decrease in the immunosuppressive cell marker TIGIT was insufficient as shown in Table 4.

[Test Example 2] (Septicemia Treatment Experiment-2)
After LPS administration in 4 rats, septic rats were anesthetized, and 4 hours later, extracorporeal circulation therapy was performed using the columns of the collectors of the present invention-2 to 5 for 30 minutes. At the beginning and end of extracorporeal circulation, 0.3 mL of blood was collected from the artery and surface antigen analysis of leukocytes was performed to examine the decrease in the LAP positive rate. The results are shown in Table 5. All five rats survived, but all showed a marked decrease in LAP positivity.

[Test Example 3] (Cancer-bearing rat treatment experiment-1)
On the 7th day after the inoculation of the cancer cells, the cancer-bearing rat was anesthetized, and extracorporeal circulation therapy was performed for 30 minutes on the column of the collector-1 of the present invention. At the beginning and end of extracorporeal circulation, 0.3 mL of blood was collected from the artery and surface antigen analysis of leukocytes was performed. The results are shown in Table 6. Since the ratio of LAP-positive cells decreased in both T cells and B cells, and TIGIT also decreased remarkably, it can be seen that the immunity was activated by the column of the collector-1 of the present invention.

[Test Example 4] (Cancer-bearing rat treatment experiment-2)
Cancer-bearing rats were anesthetized 7 days after inoculation with cancer cells, and extracorporeal circulation therapy was performed for 30 minutes on the column of the collector-2 of the present invention. At the beginning and end of extracorporeal circulation, 0.3 mL of blood was collected from the artery and surface antigen analysis of leukocytes was performed. The results are shown in Table 7. Since the ratio of LAP-positive cells decreased in both T cells and B cells, and TIGIT also decreased remarkably, it can be seen that the immunity was activated by the column of the collector-2 of the present invention.

Claims

From a molded product containing a linear aromatic polymer in which non-polar side chain amino acid residues are bonded as aromatic substituents at a ratio of 1 to 100 per 200 aromatic nuclei via a spacer having a length of 10 atoms or more. An immunosuppressive cell collector.
The collection material according to claim 1, wherein the non-polar side chain amino acid residue is at least one selected from the group consisting of leucine, isoleucine, norleucine, valine and a linear peptide containing these.
The collector according to claim 1 or 2, wherein the spacer is a linear polyamine.
The collector according to any one of claims 1 to 3, wherein the linear aromatic polymer is polysulfone, polyetherimide, polyimide or a derivative thereof.
The collector according to any one of claims 1 to 4, wherein the linear aromatic polymer is applied to fibers.
The collector according to any one of claims 1 to 5, wherein the immunosuppressive cell is a cell having a latency-associated protein on the cell surface.
The collection material according to any one of claims 1 to 5, wherein the immunosuppressive cell is a cell having TIGIT on the cell surface.
An immunosuppressive cell collection column filled with the collection material according to any one of claims 1 to 7.
The column according to claim 8, which is for extracorporeal circulation.
The column according to claim 8, which is for cell therapy.
The column according to any one of claims 8 to 10, which is used for treatment of infectious diseases, treatment of burns, treatment of cancer, prevention of recurrence of cancer after surgery to remove cancer, or prevention of sepsis.
A method for reducing the concentration of immunosuppressive cells in a patient's blood, which comprises a step of circulating the patient's blood extracorporeally in the column according to any one of claims 8 to 10.
A method for treating infectious diseases, treating burns, treating cancer, preventing recurrence of cancer after surgery to remove cancer, or preventing septicemia, and these treatments are listed in the column according to any one of claims 8 to 10. Alternatively, a method comprising the step of extracorporeally circulating the blood of a patient in need of prophylaxis.