JPH0632800A - Protein-immobilized carrier and separation of cell using the same - Google Patents

Abstract

PURPOSE:To provide a lymphotoxin-immobilized carrier for therapies, etc., capable of selectively adsorbing lymphotoxin receptor-bearing cells thereto, thus capable of separating and eliminating leukocytes from the blood for transfusion or the blood of patients with rheumatism or autoimmune diseases. CONSTITUTION:A lymphotoxin solution produced from Chinese hamster ovary cells by stimulating lymphocytes with a phorbol ester or mitogen, etc., or stimulating established lymphocyte-derived cells with e.g. mitogen, or by using a recombinant gene prepared with a cloned lymphotoxin CDNA, is incorporated with a 0.1M glycine buffer solution (pH 8.2) containing 0.15M NaCl, and the resultant solution is added to the wells of a carrier such as a plastic 96-well plate at 100mul per well and then allowed to stand overnight at 4 deg.C followed by addition of a 1% bovine serum albumin solution to effect blocking, thus obtaining the objective lymphotoxin-immobilized carrier capable of specifically binding lymphotoxin receptor-bearing cells thereto.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a carrier on which lymphotoxin (hereinafter referred to as LT) is immobilized, and a method for separating or removing cells using the carrier.

[0002]

2. Description of the Related Art LT is a type of cytokine also called TNF-β. Although the protein chemistry of LT has been studied by several groups, it is said that the smallest unit is a component having a molecular weight of about 20,000, and that the unit component is associated with or complex with other components. (Aggarwal, BB) et al., Journal of Biological Chemistry (J. Biol.che
m.), 259, 686 (1984)). Currently, L
Two types of T, namely soluble LT secreted as a trimer and a complex with other membrane proteins, are known to exist in the membrane (Androlewicz, MJ). Et al., Journal of Biological Chemistry, 267, 2542 (1992)).

It is known that LT is produced by lymphocytes stimulated by phorbol ester, mitogen and the like. It is also known that LT is induced inducibly when stimulated cells derived from established lymphocytes with mitogen or the like. However, the LT production method using lymphocytes or cell lines has not been a system suitable for mass production. In recent years, finally LT's cDN
A can be cloned, and LT can be produced in E. coli (see Gray, PW et al., Nature, 312, p. 721 (1984)),
It has also become possible to produce LT in recombinant animal culture cells by the method reported by Nakagawa et al. (Nakagawa, K. et al., Agricultural Biological Chemistry (Agric.
Biol. Chem.), 55, 501 (1991)).

Of the LTs produced in large quantities in this way, the soluble LT trimer is as follows:
Its activity as a cytokine has been sought, and research is being conducted to develop applications such as anticancer agents and immunomodulators. However, nothing has been known about a carrier on which LT is immobilized and a method for separating cells using the carrier.

The present inventors have found that when cultured cells are brought into contact with a carrier on which LT is immobilized, the cells adhere to the carrier, and the present invention has been completed by conducting more research.

[0006]

An object of the present invention is to provide a carrier on which LT is immobilized and a method for separating or removing cells using the carrier.

[0007]

The present invention relates to a carrier on which lymphotoxin is immobilized. The present invention also relates to a method for separating or removing cells using the carrier.

[0008]

Examples LT which can be used for immobilization of the present invention may be any LT as long as it has an affinity for cells when immobilized. Specific examples of such LT include cultured lymphocytes stimulated with, for example, mitogen, LT produced by human T cells, LT expressed in Escherichia coli using cDNA of human LT, recombinant animal cultured cells. , For example, LT produced by CHO cells. Further, not only ordinary LT, but also one in which a mutation such as substitution, deletion or insertion of one or more amino acids is introduced into its amino acid sequence may be used. The LT having such a mutation can be designed and produced by a gene recombination technique. LT produced by gene recombination technology is preferable for mass production.

As the carrier used for immobilization, any carrier can be used as long as it can immobilize proteins. Specific examples of such a carrier include plastic beads, plastic plates, plastic petri dishes, fibers, and non-woven fabrics.

Immobilization of LT on a carrier is carried out by hydrophobic bond,
It can be immobilized by a bond such as a covalent bond or an ionic bond.

Here, the immobilization by a hydrophobic bond means immobilization of a protein on a carrier having strong hydrophobicity, for example, a carrier made of polystyrene by utilizing a hydrophobic interaction. The immobilization by covalent bond means that a protein is covalently immobilized by a dehydration condensation reaction between a carboxyl group and an amino group or between a carboxyl group and a hydroxyl group using carbodiimide, for example. Immobilization by ionic bond means immobilization of a protein by utilizing electrostatic interaction with an ion exchanger.

Here, the carrier on which LT is immobilized means LT
Not only does this define a carrier immobilized on a carrier by a bond such as a hydrophobic bond, a covalent bond or an ionic bond, but it also includes a carrier in which LT and a substance other than LT are immobilized on the carrier. As substances other than LT,
Examples thereof include proteins such as bovine and human, such as proteins present in human serum such as albumin and globulin. These can influence the cell adhesion activity of the carrier on cells that have an affinity for LT.

The carrier on which LT is immobilized can be put into a suitable housing according to its application and used.

The cells to which the method for separating or removing cells using the carrier of the present invention can be applied may be any cells as long as they have an affinity for LT. Specific examples of such cells include, for example, Jurkat which is a T cell of a leukemia patient, MOLT-4 (ATCC CRL1582) which is a peripheral blood cell of a leukemia patient, and ARH77 (ATCC CRL1 which is a B cell of a leukemia patient.
621) or U937 (ATCC CRL1593) which is a tissue macrophage of leukemia patients.

Next, a method for separating or removing cells using the carrier of the present invention will be described.

A cell solution containing cells having an affinity for LT is prepared, and the solution is loaded into the housing containing the carrier of the present invention. After the cells having an affinity for LT are adsorbed on the carrier, the cells can be separated or removed.

As a cell solution containing cells having an affinity for LT, the cells may be a buffer or a medium such as isotonic phosphate buffer containing 5% bovine serum albumin and 1% glucose or 10%. A solution prepared by suspending in RPMI1640 medium containing fetal bovine serum or the like can be mentioned. The cell concentration in such cell solution is 1 x 10
^{5 to} 5 × 10 ⁷ cells / ml, preferably 5 × 10 ⁵ to 1 × 10 ⁷
cells / ml, more preferably 1 × 10 ^{6 to} 5 × 10 ⁶ cells / ml
ml. In addition, human blood, blood for transfusion, blood of patients with autoimmune diseases, blood of leukemia patients, and the like are also included.

The housing containing the carrier of the present invention may be, for example, the plate itself having the LT immobilized thereon, or a column packed with beads having the LT immobilized thereon.

Adsorption of cells having an affinity for LT to the carrier can be carried out, for example, by incubating in the case of a plate and flowing a cell solution in the case of a column. The flow rate in the case of the column is 0.01 to 0.5 ml / min, preferably 0.05 to 0.25 ml / m.
in, more preferably 0.05 to 0.1 ml / min. The temperature condition for performing the adsorption may be any temperature that does not affect the cell solution, 4-37 ° C, preferably 10-30 ° C.
More preferably, it is 20 to 25 ° C.

Next, the LT-immobilized carrier of the present invention, the method for separating cells and the method for removing cells using the carrier will be described based on Examples. However, the present invention is not limited to such Examples. is not.

Example 1 Adsorption of cells onto 96-well plate immobilized with LT produced in animal culture cells The method reported by Nakagawa et al. (Nakagawa, Kei et al., Agricultural Biological Chemistry, 55, 501 ( 1991)), a solution of human LT produced in CHO cells (Chinese hamster ovary cells) (3 mg / ml, isotonic phosphate buffer (hereinafter,
Dissolved in PBS)) containing 0.15M NaCl 0.1M
Glycine buffer (pH 8.2) (hereinafter referred to as GBS)
Was added to prepare an LT solution. Add 100 μl / well of the solution to a 96-well plate (Coaster, Catalog No. 3590), leave it at 4 ° C overnight,
It was immobilized by coating T. After removing the LT solution from each well, 200 μl of 1% bovine serum albumin dissolved in GBS was added to each well, and the mixture was allowed to stand at 37 ° C. for 1 hour. Then remove the bovine serum albumin solution,
2 × 10 ⁵ cells (100 μl) suspended in PBS containing 5% bovine serum albumin and 1% glucose in each well.
1) was added and incubated at 4 ° C. for 2 hours. After washing each well with PBS three times, cells adhering to the bottom of the well were observed. The cells used in the test were Jurkat (Schneider, U., et al., International Journal of Cancer, 19).
Vol. 621 (1977)), MOLT-4 (ATCC
CRL1582), ARH77 (ATCC CRL1
621) and U937 (ATCC CRL1593)
Is.

As a result, all the cells were attached to the bottom of the well coated with LT, but were not attached to the bottom of the well only coated with bovine serum albumin.

Example 2 Adsorption of cells to 96-well plate immobilized with LT produced in Escherichia coli Method reported by Gray et al. (See Gray, Pea Brew et al., Nature, 312, p. 721 (1984)) Solution of human LT produced in E. coli by 1 ml (1.
5 mg of GBS was added to 2 mg / ml, dissolved in PBS) to prepare an LT solution. 100 μl of the solution was added to a 96-well plate (manufactured by Coaster, Catalog No. 3590) per well, and LT was coated at 4 ° C. overnight for immobilization. GBS after removing the LT solution
200 μl of 1% bovine serum albumin dissolved in each well was added to each well and allowed to stand at 37 ° C. for 1 hour. Next, the solution of bovine serum albumin was removed, and each well was suspended in PBS containing 5% bovine serum albumin and 1% glucose.
2 × 10 ⁵ cells (100 μl) of at cells were added and incubated at 4 ° C. for 1 hour and 30 minutes. After washing the wells three times with PBS, cells adhering to the bottom of the wells were observed.

As a result, Jurkat cells were attached on one side to the bottom of the well coated with LT, but were not attached to the bottom of the well just treated with bovine serum albumin.

Example 3 Adsorption of cells to plastic beads on which LT was immobilized 1 ml of a solution of LT produced by CHO cells (1.79 mg / ml, dissolved in PBS) was suspended in 5 ml of GBS in the same manner as in Example 1. It was added to 500 mg of turbid polystyrene beads (diameter 0.2 mm) (produced by Kanegafuchi Chemical Industry Co., Ltd.) and incubated at 37 ° C. for 1 hour with shaking to immobilize LT on the beads. Next, bovine serum albumin was added to a final concentration of 1%, and the mixture was allowed to stand at 37 ° C. for 1 hour. The beads having LT immobilized thereon were packed in a glass column having a diameter of 10 mm and washed with RPMI1640 medium containing 10% fetal bovine serum. Next, Jurkat cells (1.5 × 10 ⁶ cells / 2 ml) suspended in the same medium were passed at room temperature at a flow rate of 0.1 ml / min, and the column was washed with 5 ml of the same medium.

As a result, Jurkat cells were adsorbed on the LT-immobilized bead column and were not detected in the column passage solution or the washing solution. However, in a bead column that was only treated with bovine serum albumin, Jur
The kat cells passed through.

[0027]

INDUSTRIAL APPLICABILITY The carrier of the present invention can easily separate or remove cells, for example, separate or remove leukocytes from blood. More specifically, leukocytes can be removed from blood for transfusion, leukocytes can be removed from blood of patients with rheumatism or autoimmune disease, or leukemia cells can be removed from blood of patients with leukemia.

The LT-immobilized carrier of the present invention can selectively adsorb cells having an LT receptor. Since LT is involved in various immune reactions, cells having LT receptors are cells responsible for LT immune reactions, and removal of these cells suppresses LT immune reactions.

Further, the carrier on which LT is immobilized may give a signal as a cytokine of LT to cells contacting the carrier via the immobilized LT. Therefore, a carrier having LT immobilized thereon can substitute for LT,
May be used for immune regulation or cancer control.

The presence of soluble TNF receptor is known in the living body. The physiological significance of this soluble TNF receptor and its causal relationship with disease are not known. If there is a relationship between the presence of soluble TNF receptor in serum and the disease of the patient, the carrier of the present invention provides a therapeutic method for removing soluble TNF receptor from the patient of such disease. become.

Claims (6)

[Claims] 1. A carrier on which lymphotoxin is immobilized. 2. The carrier according to claim 1, wherein the lymphotoxin is human lymphotoxin. 3. The carrier according to claim 1, wherein the lymphotoxin is a recombinant lymphotoxin. 4. The carrier according to claim 1, 2 or 3 for the purpose of separating or removing cells. 5. The carrier according to claim 1, 2 or 3 for the purpose of separating or removing cells from human blood. 6. A method for separating or removing cells using the carrier according to claim 1, 2, 3, 4 or 5.