JPH07120452A - Selective separation method for cell - Google Patents

Abstract

PURPOSE:To provide a method for peculiarly and efficiently separating only a specific cell. CONSTITUTION:This selective cell separation method consists of the following processes; a monoclonal antibody or a polyclonal antibody having binding quality with an antigen manifesting on the surface of an isolated cell, or protein or peptide containing an antibody fragment having such binding quality is bound with a compound containing an amino or imino group and having molecular weight equal to or less than 10000 (i.e., preparation of modified antibody). Then, blood, blood plasma, blood serum, body fluid or cell suspension is made to react and bound with the modified antibody. Cell sap bound with the modified antibody so prepared is treated in water insoluble carrier containing a carboxyl group on the surface. A target cell is thereby bound with a carrier, and other cells or substances are removed, thereby dissociating only the target cell from the carrier.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for efficiently separating cells or cell populations having a specific marker antigen by improving conventional affinity chromatography methods. The cell separation method according to the present invention is applied not only for the purpose of research in the field of biochemistry, but also for the treatment of various diseases by separating specific cells by extracorporeal circulation of blood. For example C
By separating and removing either D4-positive cells or CD8-positive cells, the ratio of the two can be controlled, whereby autoimmune diseases or allergic diseases in which the immune reaction acts excessively, or conversely AIDS It is useful for the treatment of such immunodeficiency diseases.
In addition, by separating CD34-positive cells, it is possible to efficiently isolate the most undifferentiated stem cells that exist in extremely small amounts from peripheral blood, and to apply chemotherapy or radiation therapy to cancers, malignant tumors, etc. By injecting this in the case of doing, the hematopoietic function can be restored.

[0002]

2. Description of the Related Art As a cell separation method currently applied, a centrifugation method, a method of killing cells other than desired cells, a fluorescent antibody-labeled cell separation method (hereinafter referred to as FACS),
There are a method of immobilizing a ligand having an affinity for a target cell on a water-insoluble carrier and directly or indirectly binding the target cell thereto, a separation by an immunoadsorption column and a separation method by an immunomagnetic bead.

[0003]

The centrifugal separation method is a method of separating cells by the difference in cell size and specific gravity, and is useful when there is a large difference in physical properties such as white blood cells, red blood cells and platelets. However, this is not applicable when separating cells having a small physical difference, such as T cells and B cells.

Various methods for killing cells other than the desired cells have been studied, but this is applied to the use of drugs or monoclonal antibodies for killing cancer cells present in autologous bone marrow during transplantation surgery. It is a degree. Although 4-hydroperoxycyclophosphamide is generally used as a drug, even normal bone marrow cells are often damaged. Antibodies that bind to cancer cells and T cells have been used to kill cells other than the desired cells, but in this case also other drugs must be used in combination. However, it is often difficult to completely kill cancer cells because the binding properties of antibodies and cancer cells are likely to change. In addition, the drugs used in combination also tend to damage target normal cells.

In FACS, the cell mixture is first incubated with a fluorescent-labeled monoclonal antibody that recognizes the membrane antigen expressed in the target cells, and then the treated cells are irradiated with laser light to produce antibodies. This is a method for separating cells bound with a fluorescent antibody by applying that only cells bound with fluoresce. This method is effective for separating a small amount of cells for research, but is not suitable for separating a large amount of cells required for diagnosis or treatment because the cell processing rate of 1 × 10 ⁷ cells / hour is limited. Is. For example, it takes several hours to process about 20 ml of blood containing about 2 × 10 ⁷ nucleated cells using a FACS machine, which is generally 1-2 × that required for bone marrow transplantation.
It takes several weeks to separate 10 ⁸ stem cells.
In addition, the FACS device is very expensive, requires highly skilled and sophisticated technology to operate, and is expensive to maintain.

WO87-04628 describes two types of cell separation methods. One is a method in which a monoclonal antibody against an antigen present on the membrane surface of a target cell is directly immobilized on the surface of a separation device and used. Cell separation is carried out for monoclonal antibodies immobilized on a carrier or device,
It is carried out by directly binding the antigen-positive cells. Another method is to first incubate the cell mixture with a monoclonal antibody that binds to the membrane antigen on the target cell, and then to immobilize the ligand, such as an anti-immunoglobulin antibody, that binds to the antibody on the cell surface. It is processed by a separation device. In the panning method included in these methods, the antibodies are separated on a plastic dish on which they are immobilized. To describe the procedure of this method, the cell mixture is first poured onto a plastic dish on which the antibody has been immobilized and incubated to bind the antibody to the membrane antigen on the target cells. After incubation, wash the plastic dishes to remove unbound cells and separate. Thus, although the panning method is very simple, it is necessary to incubate the cells on a plastic dish for a long time in order to efficiently combine the cells with the antibody due to the weak binding between the antigen and the antibody. However, there is a drawback that non-specific adhesion of other cells occurs and the purity decreases. Furthermore, since the binding between cells and antibodies is weak, many antibodies remain without binding to cells, and in order to bind a large amount of cells, it is necessary to immobilize a large amount of antibodies on a plastic dish, which reduces the cost of the device. Very high. Further, in order to prevent non-specific adsorption of red blood cells, this method has many drawbacks such that red blood cells must be removed from the cell mixture before use.

In the immunomagnetic bead method, target cells are labeled with magnetic beads by first incubating a cell mixture with magnetic beads having an antibody bound thereto. After labeling, labeled cells are separated from unlabeled cells using a magnetic device.
This technique has been applied to clinically remove cancer cells from the bone marrow fluid of patients. Incubation for a long time is required to efficiently bind the beads to the target cells, which causes nonspecific adhesion of cells other than the target to reduce the purity and at the same time to recover the target cells adsorbed to the small magnetic beads. Is very difficult to apply for treatment.

In the immunoadsorption column method, a ligand such as an antibody against a membrane antigen on a target cell is immobilized on the bead surface, and the bead is packed in the column for cell separation. In this case as well, as in the panning method and magnetic bead method, the binding force between the cell membrane antigen and the antibody is weak, so it is necessary to incubate the cells to bind them to the antibody on the bead surface. Adhesion occurs and the purity decreases. Further, since the binding between the antigen and the antibody is weak, a large amount of the antibody needs to be immobilized, and the cost of the column becomes very high, which makes it difficult to apply it to the therapeutic field requiring a large amount of cell separation. .

In WO91-16116, a biotin-labeled antibody against a membrane antigen on a target cell is added to a cell mixture and incubated to form a cell-antibody-biotin bond, and then a porous acrylamide gel. Describes a method in which target cells are bound to the column and separated by passing through a column packed with beads on which avidin is immobilized and utilizing strong binding of biotin-avidin. This method is improved in processing speed and purity as compared with the above-mentioned various methods, and has clinical application. However, since the biotin-avidin bond is very strong, it is difficult to regenerate the column by breaking this bond once all of the avidin immobilized on the beads once used for cell separation binds to biotin. Therefore, it is not possible to adopt a method of arranging columns in parallel and switching to the other column when one column becomes saturated, and regenerating the saturated column in the meantime, resulting in an increase in column capacity and an increase in priming capacity. . Further, since avidin is directly linked to the acrylamide beads, it is difficult for avidin to act effectively, and there are problems that the adsorption rate of the cell-antibody-biotin conjugate and its effect are reduced.

The present invention improves the above-mentioned drawbacks and problems and enables the target cells to be separated efficiently and at high speed, and also has a high degree of purity. And a device. Further, it is also a great feature of the present invention to provide a reproducible cell separation method and device.

[0011]

The present invention provides (1) a monoclonal antibody or polyclonal antibody having a binding property to an antigen expressed on the cell surface to be isolated, or a fragment thereof having a binding property. The containing protein or peptide is covalently bound to a compound having an amino group or an imino group and having a molecular weight of 10,000 or less, and (2) any of blood, plasma, serum, body fluid or cell suspension containing cells of interest ( The target cells are bound by reacting with the modified antibody obtained in 1), and the modified antibody-bound cell liquid obtained in (3) and (2) is treated with a water-insoluble carrier containing a carboxyl group on the surface. To bind the target cells to the carrier, remove other cells or other substances, and (4) dissociate only the target cells from the carrier by an appropriate method. It relates separation method and apparatus of a cell which comprises the step.

The cells that can be separated according to the present invention are not particularly limited, but particularly, endothelial cells, cancer cells, Langerhans islet cells, macrophages, monocytes, B lymphocytes, T lymphocytes and various cells of hematopoietic system, and pregnant women. And human cells such as fetal cells in blood. Among them, B lymphocytes and T lymphocytes are classified into many subpopulations. Particularly, in T lymphocytes, CD4 positive cells, CD8 positive cells,
CD19-positive cells, interleukin 2 receptor (I
L2R) positive cells, transferrin receptor (Tr
Separation of subpopulations such as R) positive cells is significant because it can be applied to various autoimmune diseases, AIDS, prevention of acute rejection after transplantation, and the like. Further, the isolation of hematopoietic stem cells expressing CD34 for autologous bone marrow transplantation therapy performed in the treatment of hematopoietic malignant tumors and cancers is currently attracting attention and is a field in which demand is expected to increase in the future.

As an antibody that recognizes these cells, either a monoclonal antibody or a polyclonal antibody can be used, but the monoclonal antibody is more preferable from the viewpoint of its singleness and binding specificity. Although the antibody molecule can be used as it is, Fab, F (ab ′) ₂ or Facb which is a fragment containing an antigen-binding site obtained by treatment with various proteases can also be applied. Furthermore, a peptide that constitutes the variable region, or a peptide having an amino acid sequence that constitutes the complementarity determining region, or a modified peptide thereof can be used. In addition, when the present invention is applied to the extracorporeal circulation of blood in a human body, a portion other than the variable region may be changed to a human antibody in consideration of side effects and the antigenicity when the antibody is released into the blood. It is also useful to use chimeric antibodies. Of these, Fab and F are considered when considering the binding strength with cells (marker antigens) and side effects when released.
It is preferable to use a peptide that constitutes (ab ′) ₂ or the variable region. The method for producing these antibodies is not particularly limited, but those purified to high purity must be used. In the case of a monoclonal antibody, a commonly used method is to obtain a hybridoma from a culture supernatant in a serum-free medium. Fragment peptides can be obtained by enzymatic treatment of antibody molecules, but can also be produced by bacteria, yeast, etc. by genetic engineering techniques.

The method for binding the above-mentioned antibody to a compound containing an amino group or an imino group is not particularly limited. As a general method, a carbodiimide compound is used to condense the carboxyl group in the antibody with the amino group or imino group, and the glycol moiety of the sugar chain bonded to the antibody molecule is cleaved to form an aldehyde group. Then, a method of forming a Schiff base between this and the amino group or imino group and then reducing the Schiff base, and the like can be mentioned.

The compound having an amino group or imino group is preferably a compound having at least two, preferably four or more amino groups or imino groups in one molecule. These compounds have a molecular weight of 100
To 20,000, preferably 150 to 10,000
Polyethyleneimine, polyvinylamine or polyallylamine and their derivatives. Of these, polyethyleneimine is particularly preferable. Other than the above, diamines or various compounds containing two or more amino groups or imino groups can be used if there is no problem in stability and safety.

In the case of a suspension, the binding between the antibody bound with the above compound and the cell fluid is 1% bovine serum albumin (hereinafter referred to as B).
(Referred to as SA) in a PBS solution containing
After incubating at 4 ° C. for about 30 minutes to 1 hour, it is preferable to wash with the same PBS solution 2-3 times. The easiness of binding varies depending on the type of cell or antibody, but it is approximately 1 x 1
5 to 500μ when treating 0 ⁷ cell fluids
It is preferable to mix and react g of antibody, preferably 10 to 100 μg.

The water-insoluble carrier used in the present invention may be synthetic or natural as long as the material itself or the eluate is not toxic, and any water-insoluble or water-insoluble polymer compound is applicable. These shapes are beads,
Both fibers and hollow fibers are possible, and can be used properly according to the size, shape, and properties of the cells to be separated. Of these, polyacrylamide, polyacrylic acid ester derivatives, polyvinyl alcohol, polystyrene and its derivatives, cellulose, agarose, chitosan, chitin, and other commercially available bead-like polymers are suitable as the material used in the form of beads. Of these, polyacrylamide, polyvinyl alcohol and cellulose having strong hydrophilicity are preferable in order to prevent non-specific adsorption. The particle size of the beads varies depending on the size of the cells to be separated, but generally 5 to 4000 μm is suitable. When the size of the target cells is 1 to 5 μm, the particle size of the beads is 5 to 500 μm, preferably 10 to 300 μm, and the size of the target cells is 5 to 5 μm.
If the diameter is 0 μm, the particle size of the beads is 30 to 4000 μm.
m, preferably 50 to 2000 μm. The beads are preferably porous, and their exclusion limit molecular weight is preferably about 5 × 10 ⁶ . Furthermore, the pore volume of the whole beads is 10 to 90%, preferably 20 to 80%.

The material of the fibrous carrier is polyester,
Polyamide, polyethylene (PE), cellulose, polypropylene (PP), polyethylene terephthalate (P
ET), rayon, polystyrene, polyvinyl acetal and the like. Among them, polyester, cellulose, rayon, PE from the viewpoint of easy modification and strength retention.
T, polystyrene is preferred. The shape of the fibers may be woven, cotton-like, or non-woven fabric, but non-woven fabric is preferable from the viewpoint of easy handling and adsorption efficiency. The fiber diameter is 1 to 200 μm, preferably 2 to 100 μm, more preferably 3 to 50 μm. These fibers may be porous and the pore size is preferably in the same range as in the case of beads.

Various methods can be used to introduce the carboxyl group into the water-insoluble carrier. For example, in the case of PE and PP, acrylic acid (AA), methacrylic acid (MAA)
Alternatively, there is a method in which these derivatives are copolymerized at the time of polymerization. Further, there is a method of irradiating these materials with an electron beam, an ultraviolet ray or ozone to generate radicals or ions and grafting AA, MAA or a derivative thereof. In this case, it is possible to coexist with another vinyl monomer.

In the case of cotton or viscose, it may be oxidized with periodate to cleave its glycol moiety to form an aldehyde group, which may be oxidized to introduce a carboxyl group, or the aldehyde group may be introduced. After reacting a compound having at least one primary amino group such as ammonia, ethylenediamine or hexamethylenediamine to form a Schiff base and reducing it to introduce an amino group, the amino group and AA, MAA and Or a polyacrylic acid (PAA), polymethacrylic acid (PMAA) or a derivative polymer thereof may be condensed. At this time, the molecular weight of PAA, PMAA and their derivative polymers is preferably 200 to 20,000, more preferably 500 to 10,000. The term "derivative polymer" as used herein means a copolymer with another vinyl monomer containing at least one type of AA or MAA monomer unit in an amount of 10 to 80 mol% or more.

Further, as a method for introducing a carboxyl group by electron beam irradiation, AA, MAA or PAA, P
It is possible to coat a carrier with a mixed solution of MAA and a derivative polymer thereof and a crosslinkable monomer having at least two vinyl groups in one molecule, which is dried and then irradiated with an electron beam to introduce a carboxyl group. it can. In this case, the molecular weight of the polymer is preferably 200 to 20,000, more preferably 500 to 10,000.

Examples of the crosslinkable monomer to be used include monomers having a plurality of vinyl groups such as methylenebisacrylamide, trimethylolpropane diacrylate, trimethylolpropane triacrylate, tetramethylolmethane tetraacrylate and triallyl isocyanurate. 1 includes the compound represented by the general formula. Among these crosslinkable compounds, the case of using the chemical formula 1 has the best carboxyl group introduction efficiency, and the carboxyl group concentration can be controlled to a desired value. R ₁ , R ₂ , and R ₃ in Chemical formula ₁ each represent a hydrogen atom or a methyl group. n is an integer of 1 to 100, but 3
The integer is preferably from 70 to 70, more preferably from 5 to 60.

[0023]

[Chemical 1]

The concentration of the coating solution is 0.1 to 20%, preferably 0.5 to 10%, and the solvent used is a solvent that dissolves both the crosslinkable compound and PAA, PMAA and their derivative polymers. However, water, methanol, ethanol, methylene chloride, chloroform, acetone, dioxane, tetrahydrofuran, or a mixed solvent thereof can be used. The mixing ratio of PAA, PMAA and their derivative polymers to the crosslinkable compound is 50: 1 to 1:50, preferably 30: 1 to 1:30. The mixture to be coated is 0.5 to 30% by weight, preferably 1 to 20% by weight, based on the carrier.

In particular, in the case of a non-woven fabric, various methods of introducing a carboxyl group have been studied. As a result, AA, MAA or PAA, PMAA and their derivative polymers are obtained.
The most preferable method is to coat with a compound represented by the formula (1) and then coat the mixture with an electron beam. As the material used in this case, PET, PE or cotton is preferable, and PET is particularly preferable. Irradiation dose is 1 to 20 Mr
ad, preferably 2 to 10 Mrad. The non-woven fabric introduced with a carboxyl group in this manner has a synergistic effect between the carboxyl group and polyethylene glycol, and particularly when using this to extracorporeally circulate blood, there is no adhesiveness of platelets, leukocytes, etc. It is also advantageous in that the activation of coagulation factors is also suppressed.

By providing the above-mentioned cell-bound amino group-containing compound to a column packed with a carboxyl group-containing carrier as described above and bringing them into contact with each other, an ionic bond between the two occurs and the target cells are collected on the carrier. It The cells can be easily dissociated by stirring or shaking treatment. The pH of the carboxyl group-containing carrier after the above treatment is
2.0 to 4.0, preferably pH 3.0 to 3.5
It is possible to regenerate with the acidic buffer solution.

[0027]

EXAMPLES Examples regarding cell separation according to the present invention are shown below. <Example 1> Separation of CD34-positive cells (1) Separation of buffy coat cells Bone marrow fluid to which heparin was added was centrifuged at 100 xg for 5 minutes, and the buffy coat cell layer (nucleated cell layer) above the precipitation layer To collect. Further, washing with a PBS solution was performed twice by centrifugation under the same conditions. The washed buffy coat cells were suspended in a PBS solution containing 1% BSA and dispersed so that the cell number became about 5 × 10 ⁷ cells / ml.

(2) Binding of amino group-containing compound to anti-CD34 antibody 100 μg of anti-CD34 antibody HPCA-1 (manufactured by Becton Dickinson) and 200 μg of sodium periodate were added to 0.1 M acetate buffer of pH 4.5. 20 ml
Was dissolved in and reacted at room temperature for 3 hours with shaking to introduce an aldehyde group into the sugar chain site of the antibody. After dialyzing this with the same buffer, 100M of 0.1M carbonate buffer of pH 10.0
The pH was adjusted to 9.0 by adding sodium carbonate to the mixture. Furthermore, polyethyleneimine 10 having a molecular weight of 2000 and having amino groups at both ends
μg was added, and the mixture was reacted at room temperature for 20 hours with shaking. After the reaction, the solution was dialyzed with the same carbonate buffer solution as an external solution, 100 μg of sodium borohydride was added, and a reduction reaction was carried out for 3 hours while shaking at room temperature. After the reaction, the antibody was recovered by salting out with ammonium sulfate, redissolved, and dialyzed with a PBS solution (pH 7.4) as an external solution and stored at 4 ° C.

(3) Binding of Buffy coat cells with antibody To the Buffy coat cell suspension obtained in (1), the modified antibody obtained in (2) was added to a concentration of 10 μg / ml, and the mixture was added at 4 ° C. It was kept warm for 30 minutes. Then P containing 1% BSA
It was washed twice with a BS solution (pH 7.4). The cells to be treated were redispersed in the same buffer solution to a concentration of about 1 × 10 ⁷ cells / ml.

(4) Introduction of carboxyl group into water-insoluble carrier In the chemical formula 1, R ₁ , R ₂ and R ₃ are hydrogen atoms and the value of n is 1
4 polyethylene diacrylate (CH ₂ = CH-
_{COO- (CH 2 CH 2 O)} 14 -OC-CH 2 = C
H ₂ ) 2 g, AA 0.72 g and ethylenediamine 0.15 g are dissolved in 200 ml of methanol,
A PET non-woven fabric having a fiber diameter of 3.5 μm cut into m squares was dipped in this solution, and then 10 Mrad of electron beam was irradiated on both sides of the non-woven fabric in total of 5 Mrad. The nonwoven fabric after the electron beam irradiation was washed with water and methanol three times each and air dried. The carboxyl group content of the obtained nonwoven fabric was 0.549 meq / g as determined by titration with a sodium hydroxide solution.

(5) Separation of cells The above non-woven fabric was cut according to the size and shape of a diamond-shaped polycarbonate module case having a side length of 5 cm to prepare a column filled with 20 sheets. 1 ml of suspension of antibody-bound cells prepared in (3) on this column
Was circulated for 10 minutes with a tubing pump passing through the column at 1 ml / min. Then PBS solution (p
After washing with H7.4), the column was gently shaken to detach the cells from the column. The detached cells were collected by running the same PBS solution. The recovery rate of CD34-positive cells was calculated from the quantification result by fluorescence-labeled flow cytometry. At the same time, the purity of the CD34-positive cells was calculated from the ratio with the CD34-negative cells. In addition, the survival rate of the recovered cells was determined by dye exclusion method using trypan blue to determine whether the cells were live or dead.
It was measured and calculated using an eubauer hemocytometer. The results are shown in Table 1.

[0032]

[Table 1]

Example 2 Separation of CD4-Positive Cells Human peripheral blood supplemented with 10% heparin (100 U / ml) was added to 3 cells.
After centrifuging at 50 × g for 10 minutes, the buffy coat cell layer was collected, mixed with an equal amount of McCoy's 5A medium (manufactured by GIBCO), layered on Ficoll-Conray solution in equal amounts, and centrifuged at 750 × g for 20 minutes. separated. The obtained mononuclear cell layer was collected in another test tube containing the above-mentioned medium, and 32
Washing by centrifugation at 0 × g for 10 minutes was repeated 3 times.

In the same manner as in Example 1, 100 μg of Leu-3a (manufactured by Becton Dickinson), which is an anti-CD4 antibody, was bound to polyethyleneimine having an amino group at both ends with a molecular weight of 2000 to give the above mononuclear cell. Combined with. The cells to be treated were redispersed in a PBS solution (pH 7.4) containing 1% BSA so that the cells were treated at about 1 × 10 ⁷ cells / ml.

A carboxyl group-containing nonwoven fabric (0.544 meq / g) prepared in the same manner as in Example 1 was used to prepare a packed column, and the separation and recovery rate, purity and viability of CD4-positive cells were evaluated by the same method. . The results are shown in Table 1.

<Example 3> Separation of CD8-positive cells Using 100 µg of Leu-2a (manufactured by Becton Dickinson) which is an anti-CD8 antibody, the same procedure as in Example 2 was carried out except that the recovery and purity of CD8-positive cells were separated. , The survival rate was evaluated. The results are shown in Table 1.

[0037]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, the method for separating cells enables specific cells to be collected specifically and efficiently with high purity. INDUSTRIAL APPLICABILITY The present invention is usefully developed in fields such as cell separation in biochemical research and specific cell collection for medical purposes.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication location // A61M 1/36 545 9052-4C (72) Inventor Masakazu Tanaka 2-chome Katata, Otsu, Shiga Prefecture No. 1 Toyobo Co., Ltd. Research Institute

Claims (1)

[Claims] 1. A method for selectively separating cells, which comprises the following steps. (1) A protein or peptide containing a monoclonal antibody or a polyclonal antibody having a binding property to an antigen expressed on the surface of a cell to be isolated or a fragment thereof having a binding property is treated with an amino group or an imino group. It is covalently bound to the contained compound having a molecular weight of 10,000 or less. (2) The target cell is bound by reacting any of blood, plasma, serum, body fluid or cell suspension containing the target cell with the modified antibody obtained in (1). (3) The cell solution to which the modified antibody obtained in (2) is bound,
Target cells are bound to the carrier by treatment with a water-insoluble carrier containing a carboxyl group on the surface, and other cells or other substances are removed. (4) Only target cells are dissociated from the carrier.