JPWO2006101205A1 - T cell activation method and kit for producing activated T cells - Google Patents

Abstract

A method capable of activating T cells efficiently in a short time and effective for immunotherapy, and capable of reducing the risk due to contamination such as bacteria by a simple operation And a method for activating T cells comprising contacting a carrier on which an anti-CD3 antibody or a fragment thereof is immobilized, while moving a solution containing T cells, and anti-CD3 while moving a solution containing T cells. Activation comprising contacting a carrier immobilized with a CD3 antibody or fragment thereof to activate T cells, and culturing the obtained activated T cells in a medium containing lymphokine to proliferate activated T cells. The present invention relates to a method for producing T cells.

Description

The present invention relates to a method for activating T cells, a method for producing activated T cells, a method for producing an immunotherapeutic agent, and a kit for producing activated T cells.

There are three main types of treatment for cancer: “surgical treatment”, “chemotherapy”, and “radiotherapy”, each of which is performed alone or in combination, but in recent years it has been called “immunotherapy”. The method has attracted attention as a fourth therapy for cancer. Humans originally have the power (immunity and immunity) to cure themselves against illness and injury, and T cells in the blood play a role as the center of their immunity. It is said that the higher the attacking power against the disease, the less likely it is to get sick or the stronger the ability to fight the disease. “Immunotherapy” is a therapy that focuses on the human immune system and treats cancer by enhancing the ability of T cells to kill cancer cells (enhancing immunity).

With recent advances in immunology, immunological monitoring mechanisms by various immunocompetent cells have been clarified. Immunotherapy that recognizes and destroys antigens such as tumor cells has been developed by enhancing the function of the mechanism. For example, lymphocytes collected and isolated from a patient are cultured in the presence of interleukin (IL) -2 or IL-4 to induce lymphokine-activated killer (LAK) cells, and these LAK cells are reintroduced by the patient. LAK therapy (adoptive immunization) that returns to the body has been developed.

However, since LAK cells exhibit non-specific cytotoxic activity against antigens such as tumor cells, it is currently difficult to expect a therapeutic effect. In view of this, attention has been paid to a technique for performing treatment similar to LAK therapy using antigen-specific activated T cells instead of antigen-nonspecific killer cells.

Conventionally, for the induction of activated T cells, a method of culturing using a T cell culture carrier in which an anti-CD3 antibody is immobilized on the surface of a culture substrate such as a flask or a petri dish or an insoluble carrier has been proposed ( (See Patent Document 1). As this T cell culture carrier, each experimenter prepares and uses a T cell culture carrier on which an anti-CD3 antibody is immobilized by adding an anti-CD3 antibody to the carrier before culturing. The present condition is that the prepared carrier for cultivating T cells is stored in a solution in which an anti-CD3 antibody is added to an insoluble carrier and allowed to stand at a low temperature. The T cells activated by the above operation are added to a culture solution containing lymphokines such as IL-2 in a cell culture bag and amplified.

However, for the experimenter, the preparation of the T cell incubator requires skill and many complicated operations. In addition, when a culture substrate such as a flask or petri dish is used, there is a risk of contamination such as various bacteria when added to a cell culture bag for amplification. In addition, if the carrier for T cell culture prepared with the anti-CD3 antibody solution added is used as it is for culturing, the proliferation of T cells is suppressed due to the presence of free non-immobilized anti-CD3 antibody. The Therefore, it is necessary to remove the non-immobilized free anti-CD3 antibody solution at the time of use and culturing, and further wash with an appropriate washing solution such as a phosphate buffered saline solution (PBS). . However, even in the removal and washing treatment of the anti-CD3 antibody solution, there is a risk due to contamination of germs via droplets attached to the opening or lid of a culture flask as a carrier for T cell culture.

Furthermore, when conventional culture substrates and carriers are used, it takes about 1 to 3 days to activate T cells in a state of standing on the culture substrate. In addition to these, in the conventional T cell activation, when the phenotype of the amplified T cells was examined, there was often a tendency that the ratio of cells having no effect on treatment was high. Therefore, for clinical application, a method that enables efficient activation in a shorter period of time, and a carrier for T cell culture and T cell production that are less dangerous due to contamination such as bacteria with a simple operation. A kit is desired.
Japanese Patent Laid-Open No. 3-80076

In view of the above-mentioned present situation, the present invention can activate T cells efficiently in a short time so as to be effective for immunotherapy. Moreover, the present invention is free from the risk of contamination such as bacteria by a simple operation. An object of the present invention is to provide a method for activating T cells, a method for producing activated T cells, and a kit for producing activated T cells, which can be reduced.
It is another object of the present invention to provide a method for producing an immunotherapeutic agent containing activated T cells that is effective for treating cancer, infectious diseases, and the like.

As a result of intensive studies, the present inventors have found that activated T cells can be obtained by bringing a solution containing T cells into contact with a carrier on which an anti-CD3 antibody or a fragment thereof is immobilized while moving the solution. The headline and the present invention were completed.
That is, the present invention relates to the following (1) to (9).
(1) (a) A method for activating T cells, which comprises bringing a solution containing T cells into contact with a carrier on which an anti-CD3 antibody or a fragment thereof is immobilized while moving the solution.

(2) (a) While moving the solution containing T cells, the T cells are activated by contacting with a carrier on which an anti-CD3 antibody or fragment thereof is immobilized, and (b) the obtained activated T cells are treated with lymphokine A method for producing activated T cells, comprising culturing activated T cells by culturing in a medium containing
(3) The production method according to (2), wherein the lymphokine is at least one selected from the group consisting of IL-2, IL-12, and IFN-γ.
(4) The production method according to (2) or (3), wherein the carrier is packed in a column.
(5) The production method according to any one of the above (2) to (4), wherein a costimulatory signal donor is further immobilized on the carrier.
(6) The above-mentioned costimulatory signal donor is at least one selected from the group consisting of an anti-CD48 antibody, an anti-CTLA-4 antibody, an anti-CD2 antibody, an anti-CD26 antibody, an anti-CD28 antibody, and a fragment thereof. 5) The manufacturing method as described.

(7) A method for producing an immunotherapeutic agent comprising activated T cells as an active ingredient, comprising producing activated T cells by the production method according to any one of (2) to (6) above.

(8) An activated T cell production kit for carrying out the production method according to any one of (2) to (6) above,
A kit comprising a carrier for immobilizing an anti-CD3 antibody or a fragment thereof and a column for packing the carrier.
(9) The kit for producing activated T cells according to (8), further comprising an anti-CD3 antibody.
The present invention will be described below.

(1) Activation of T cells In the method for activating T cells and the method for producing activated T cells according to the present invention, in order to activate T cells by moving and contacting a solution containing T cells, A carrier on which a CD3 antibody or fragment thereof is immobilized is used.

The carrier for immobilizing the anti-CD3 antibody or a fragment thereof is not particularly limited as long as it can immobilize the anti-CD3 antibody and is suitable for contact with a moving liquid (solution containing T cells). Not. For example, inorganic substances (for example, glass, silica, metal oxide, etc.), synthetic polymers (for example, polyolefins such as polyethylene and polypropylene, synthetic polymer plastics such as polystyrene and polyvinyl chloride, polyvinyl, polyacrylamide, etc.), agarose And those made from crosslinked dextran, cellulose, hydrophilic vinyl polymer and the like. The surface of these carriers may be porous, rough or smooth.

The form of the carrier is not particularly limited, but a form that can be filled in a column or bag as described below is preferable. For example, particulate form, aggregate of particles, fibrous form (for example, non-woven fabric), film form, or hollow fiber And the like. There is no particular limitation on the size and shape.

The anti-CD3 antibody used in the present invention may be either a polyclonal antibody or a monoclonal antibody as long as it is an antibody against CD3, and is a commercially available OKT-3 antibody (manufacturer: Au (Sofa Matthematic) can also be used.

The fragment of the anti-CD3 antibody used in the present invention is not particularly limited as long as it is a fragment capable of binding to CD3. Examples thereof include Fab fragments and F (ab ′) ₂ fragments.
Only one kind of the above-mentioned anti-CD3 antibody fragment may be fixed to a carrier, or a plurality of kinds of fragments may be fixed to a carrier, or they can be used in combination with an anti-CD3 antibody.
In the present invention, only one type of anti-CD3 antibody or a fragment thereof may be immobilized on a carrier, or multiple types may be immobilized on a carrier.

In the present invention, in addition to the anti-CD3 antibody, a costimulatory signal donor is preferably immobilized on a carrier. In establishing an immune response by activation of antigen-specific T cells, there are not only antigen-specific signals via TCR (T cell receptor) but also costimulatory signals provided from antigen-presenting cells. It is because it is possible to do.

The costimulatory signal donor is not particularly limited as long as it transmits a costimulatory signal via a T cell membrane surface protein, membrane surface carbohydrate, membrane lipid, or the like. Examples thereof include B7 molecule family, anti-CD48 antibodies, anti-CTLA (cytolytic T lymphocyte associated antigen) -4 antibodies, anti-CD2 antibodies, anti-CD26 antibodies, anti-CD28 antibodies and other anti-CD antibodies, and fragments of these antibodies.
Although it does not specifically limit as said B7 molecule family, B7.1, B7.2, MB7.2 etc. are illustrated. These may be derived from cells or may be prepared by genetic engineering techniques.
The fragment of the antibody is not particularly limited as long as it is a fragment capable of binding to the antigen, and examples thereof include a Fab fragment and an F (ab ′) ₂ fragment.
Only one type of the above-mentioned costimulatory signal donor may be fixed to the carrier, or a plurality of types may be fixed to the carrier.

In the present invention, in addition to the anti-CD3 antibody, an antibody against a sugar chain-binding lectin can also be immobilized on a carrier. The anti-sugar chain-binding lectin antibody is not particularly limited, and examples thereof include L-selectin antibody that is a sialyl Lewis sugar chain-binding lectin. Only one kind of the above-mentioned anti-sugar chain-binding lectin antibody may be immobilized on a carrier, or multiple kinds may be immobilized on a carrier. The anti-sugar chain-binding lectin antibody can be immobilized on a carrier together with the anti-CD3 antibody and the costimulatory signal donor.

Binding between the carrier and an anti-CD3 antibody or the like (indicating an anti-CD3 antibody, a fragment thereof, a costimulatory signal donor, and an anti-sugar chain-binding lectin antibody) is covalent bond, ionic bond, physical bond, or live It can be performed by chemical specific binding or the like. Examples of the covalent bond include methods such as cyanogen bromide activation method, epoxidation method, acid azide derivative method, condensation reagent method, diazo method, alkylation method, carrier crosslinking method and the like. Among these, it is preferable to bind an anti-CD3 antibody or the like by covalent bond to a cyanogen bromide activated carrier or an epoxidized carrier. In the case of biochemical specific binding, a ligand for specifically adsorbing an anti-CD3 antibody or the like to a carrier (for example, protein A, protein G, antibody adhesion molecules such as antigen, derivatives thereof, partial peptide fragments, etc.) Prior to immobilization of the anti-CD3 antibody or the like, those coated in advance can also be used. Examples of the partial peptide fragment include a partial peptide fragment of protein G (Japanese Patent Laid-Open No. 2003-88381).

The method for immobilizing the anti-CD3 antibody or the like on the carrier is not particularly limited. Examples of known methods include the following methods. The solution is adjusted so that the anti-CD3 antibody or the like has a protein concentration of 0.001 to 0.05 as absorbance at a wavelength of 280 nm. The pH of the solution is about 5 to 10, and the salt concentration is about 0.01 to 1.0M. It does not specifically limit as said salt, A sodium salt, potassium salt, etc. are mentioned. Thereafter, an appropriate amount of the solution and a water-insoluble carrier are mixed or brought into contact with each other and allowed to stand at 0 to 37 ° C. for about 1 to 24 hours. After the treatment, the solution is removed and washed with an appropriate amount of washing solution.

Examples of the cleaning liquid include distilled water, physiological saline, and about 0.01 to 0.1 w / v% of albumin and nonionic surfactants [for example, polyoxyethylene sorbitan fatty acid ester, trade name Tween. )] And the like can be used. In addition, a cell culture medium can be used for washing.

Specifically, examples of the carrier on which the anti-CD3 antibody or the like is immobilized include those in which the anti-CD3 antibody or the like is immobilized on the surface of the carrier. In that case, as described above, an anti-CD3 antibody or the like may be immobilized on the surface of the carrier via a ligand for specifically adsorbing the antibody. Further, these carriers may be used as they are, or those obtained by cross-linking between carriers or those in which they are included may be used.

The amount of immobilization of anti-CD3 antibody and the like is as follows in terms of density. In the case of anti-CD3 antibodies, fragments thereof, anti-CD antibodies such as anti-CD28 antibodies, and anti-CTLA-4 antibodies, the amount is about 0.001 to 1000 μg / cm ² , preferably 0.001 to 10 μg / cm ² . In the case of the B7 molecule family, the number is about 10 to 10 ¹⁵ pieces / cm ² , preferably 10 to 10 ⁹ pieces / cm ² . Each of the above numerical values means the amount per unit area of the carrier.

Activation in the present invention is carried out by bringing a solution containing T cells into contact with a carrier on which an anti-CD3 antibody or the like is immobilized. In the present invention, by moving the solution containing T cells, the chance of contact with the anti-CD3 antibody and the above-mentioned costimulatory signal donor is increased, so that it can be efficiently performed in a short time (for example, within 24 hours). It is presumed that T cells can be activated to be effective.

The carrier on which the anti-CD3 antibody or the like is immobilized may be held in a state where the solution containing T cells can be brought into contact with the solution, for example, and is preferably packed in a column or bag. In particular, the column packed is preferable because the activation process is easy. In this case, the activation of the present invention can be carried out very easily by passing a solution containing T cells through the column. When using a bag, it can be carried out by moving a solution containing T cells (shaking, stirring, tilting, etc.) in the bag. It is also possible to carry out the method of the present invention by immobilizing an anti-CD3 antibody or the like on a filter or non-woven fabric, and passing and moving a solution containing T cells therethrough.

Although it does not specifically limit as T cell used by this invention, For example, a peripheral blood lymphocyte cell (PBL), a tumor wet lymphocyte, a lymph node cell, a cancer bearing (or tumor) local lymph node cell etc. are mentioned. In addition, when administering the activated T cell obtained by this invention to a patient, it is desirable to use the T cell extract | collected from this patient.

In the present invention, the amount of blood collected to obtain T cells and the number of T cells obtained therefrom are not particularly limited. The amount of blood collected is preferably 1 to 50 ml from the viewpoints of burden on the patient and separation of T cells. When the obtained activated T cells are used as a cancer metastasis inhibitor or a cancer therapy adjuvant, a tumor-specific antitumor effect is expected by using local lymph node cells of the target tumor. it can.

Although it does not specifically limit as a solution containing T cell used by this invention, For example, the culture solution containing T cell, what suspended T cell in the solution (for example, buffer solution), etc. are mentioned. The basic medium used in the culture medium containing T cells is not particularly limited as long as T cells can be cultured. An Eagle medium (IMDM) etc. are mentioned. Of these, KBM400 and RPMI 1640 are preferable.

The basal medium preferably contains lymphokine. Lymphokines include macrophage migration inhibitory factor (MIF), macrophage chemotactic factor (MCF), skin reaction factor (SRF), macrophage activation factor (MAF), leukocyte migration inhibitory factor (LIF), neutrophil chemotaxis. Factor (NCF), eosinophil chemotactic factor (ECF), basophil chemotactic factor (BCF), eosinophil stimulation promoting factor (ESP), lymphotoxin (LT), vascular permeability enhancing factor (VPF) Effector lymphokines such as blastogenic factor (BF or MF), osteoclast activator (OAF or IL-1β), γ-interferon (IFN-γ), and colony stimulating factor (CSF), IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-12, IL-2 receptor modulator (ATF), etc. And the like. Of these, IL-2, IL-12 and IFN-γ are desirable. IL-2 and IL-12 may be isolated from peripheral blood or prepared by genetic recombination. As the IFN-γ, those prepared by means such as cell culture and gene recombination can be used. What is mentioned later about addition amount is applicable. As a lymphokine, only 1 type may be used and 2 or more types can also be used together.

The basal medium may contain a drug for preventing contamination of germs. Although it does not specifically limit as said chemical | medical agent, For example, penicillin, streptomycin, gentamicin, ampicillin, kanamycin, chloramphenicol, streptomycin etc. are mentioned.

Components for further improving the T cell proliferation ability can be added to the basal medium. Although it does not specifically limit as said component, For example, insulin, transferrin, fetuin, catalase, O-phosphoryl ethanolamine, ethanolamine, sodium selenite, 2-mercaptoethanol, corticosterone etc. are mentioned.

As the insulin, any of those derived from humans, pigs, sheep, cows, horses, etc., or recombinants can be used, and the addition amount is preferably about 1 to 100 μg / ml, particularly about 10 μg / ml.
As transferrin, those derived from humans, cows, horses, dogs, mice, guinea pigs, rabbits and the like can be used, and the addition amount is preferably about 1 to 100 μg / ml, particularly about 10 μg / ml.
As fetuin, for example, a bovine-derived one can be used, and the amount added is preferably about 1 to 100 μg / ml, particularly about 10 μg / ml.
As the catalase, for example, those derived from cattle, dogs, mice, filamentous fungi and the like can be used, and the addition amount is preferably about 2 to 50 μg / ml, particularly about 30 μg / ml.
The amount of O-phosphorylethanolamine or ethanolamine added is preferably about 2 to 200 μg / ml, particularly about 20 μg / ml.
The amount of sodium selenite added is preferably 10 ⁻¹⁰ to 10 ⁻⁷ M, particularly about 10 ⁻⁸ M.
The addition amount of 2-mercaptoethanol is preferably 10 ⁻⁹ to 10 ⁻⁴ M, particularly about 5 × 10 ⁻⁵ M.
The amount of corticosterone added is preferably 10 ⁻⁹ to 10 ⁻⁶ M, particularly about 10 ⁻⁶ M.

In activating T cells in the present invention, the number of T cells contained in the solution is not particularly limited, but is preferably about 10 ³ to 10 ⁷ cells / ml, for example.

In the present invention, the time required for the contact between the T cell-containing solution and the carrier is not particularly limited as long as it is longer than 0 hours. In the present invention, since activation can be achieved efficiently even with a very short contact, the contact time may be less than 24 hours, and further, it should be sufficiently activated even with less than 7 hours. Can do. However, the method of the present invention is not limited to short-time contact, and for example, contact can be extended for about 10 days. In the case where a carrier on which an anti-CD3 antibody or the like is immobilized is packed in a column and a solution containing T cells is passed through the column, the total amount of the solution passes through the column within, for example, 1 hour, preferably within 30 minutes. The contact may be made as described above. In this case, the liquid may be passed only once (one pass), or the liquid may be passed repeatedly a plurality of times.

It does not specifically limit as temperature at the time of contact, What is necessary is just a temperature suitable for the activation of T cell, Usually, it is about 30-40 degreeC, and about 36-38 degreeC is preferable. Activation is preferably performed in an atmosphere containing 1 to 10% by volume of carbon dioxide gas.
The amount of the carrier used for T cell activation of the present invention can be appropriately selected depending on the scale and the number of cells. For example, the carrier may be used at a rate of about 1 to 10 ⁷ cm ³ per 1 ml of the solution containing T cells.

(2) Proliferation culture of activated T cells In the present invention, activated T cells obtained by the above method can be proliferated by culturing them in a medium containing lymphokine.
The culture time is not particularly limited, and may be appropriately determined according to the necessary amount of activated T cells, and is, for example, about 1 to 50 days.
The temperature at the time of growth culture is not particularly limited as long as it is suitable for T cell culture, and is usually about 30 to 40 ° C, preferably about 36 to 38 ° C. The culture is preferably performed in an atmosphere containing 1 to 10% by volume of carbon dioxide gas.

As the medium used for the growth culture, the basic medium described above can be used.
The medium used for growth culture contains lymphokine in order to induce LAK cells. As lymphokines, those isolated from peripheral blood or those prepared by genetic recombination can be used, and specific examples thereof include those described above. The amount of lymphokine is, for example, 10 to 1000 JRU / ml, preferably 10 to 100 JRU / ml in the case of IL-2, and 0.1 to 1000 U / ml, preferably 0.1 to 100 U in the case of IL-12. In the case of IFN-γ, it is 0.1 to 1000 IU / ml, preferably 10 to 1000 IU / ml.

As described above, the medium used for growth culture may contain a drug for preventing contamination of germs and a component for improving T cell proliferation ability.
In the present invention, the activation step and the growth culture step can be carried out as a series of closed system processes, thereby further reducing the risk of contamination such as germs.

(3) Method for producing immunotherapeutic agent The activated T cells obtained by the present invention are advantageous for immunotherapy, and infect mammals such as humans, cows, horses, dogs, mice and rats. Since it has effects such as defense, antiallergy, anti-AIDS, and cancer metastasis suppression, it can be used as an immunotherapeutic agent.

The above-mentioned immunotherapeutic agent is preferably formulated into a liquid preparation (for example, subcutaneous, intramuscular, intravenous injection) for parenteral administration by a conventional method, and has a desired therapeutic effect on the progress or disease state of the disease. It contains an effective amount of activated T cells to generate.

In addition to activated T cells, the above-mentioned immunotherapeutic agent may contain a drug useful for immunotherapy. Although it does not specifically limit as a chemical | medical agent which can be used together, For example, IL-2, IL-12, IFN-gamma etc. are illustrated. Furthermore, a suspending agent, a preservative, a pH buffering agent, an isotonic agent, a local anesthetic, an antibiotic and the like can be blended as necessary.
The above-mentioned immunotherapeutic agent is useful as an infection protective agent, antiallergic agent, anti-AIDS agent, cancer metastasis inhibitor or cancer therapy adjuvant.

(4) Kit for preparing activated T cells The kit for producing activated T cells of the present invention is a kit for carrying out the activation and amplification culture described above, and a carrier for immobilizing the anti-CD3 antibody and the like. And a column for filling it. In the kit of the present invention, the carrier may be packed in the column.

The kit for producing activated T cells of the present invention further comprises an anti-CD3 antibody, a lymphokine, a basic medium, a container for growing and cultivating activated T cells (for example, a cell culture bag having gas permeability), the above column And a device (for example, a tube) for connecting the container and the container. By connecting the column and the growth culture vessel, activation and growth culture can be carried out as a series of closed processes.
When an anti-CD3 antibody is attached to the kit for producing activated T cells of the present invention, the anti-CD3 antibody is preferably attached in a state where it can be stably stored.
The kit for producing activated T cells of the present invention produces activated T cells by immobilizing the anti-CD3 antibody attached to the kit or the separately obtained anti-CD3 antibody on the carrier. Can be used for

According to the above configuration, the present invention can activate T cells efficiently in a short time (for example, within 24 hours) so as to be effective for immunotherapy. According to the present invention, it is possible to reduce the risk of contamination such as bacteria by a simple operation.

According to the present invention, T cells can be differentiated and induced into activated T cells such as CD4 ⁺ , CD8 ⁺ (activated) T cells better than conventional methods. In addition, the proportion of CD4 ^- CD8 ^- T cells that are not activated (that is, not required for practical use in immunotherapy) can be reduced as compared with conventional methods.

The T cell activation method, activated T cell production method, and activated T cell production kit of the present invention examine and diagnose pathological conditions such as autoimmune diseases, bacterial infections, allergies, AIDS, and cancer progression. It is extremely useful. For example, after activating and amplifying T cells collected from patients with various medical conditions according to this method, the amplification rate or the expression ratio of cytokines such as CD4 and CD8 on the surface of the amplified T cells is compared. It is also possible to estimate the degree of progress.

Hereinafter, examples and experimental examples will be given to describe the present invention in more detail, but the present invention is not limited to these examples.

Example 1 Activation of human-derived T cells with anti-CD3 antibody-immobilized carrier and amplification (1) Preparation of anti-CD3 antibody-immobilized carrier As a carrier, cyanogen bromide-activated agarose carrier (CNBr activated Sepharose 6MB (commercial product) Name), Amersham Biosciences) was used. 1 g of the carrier was weighed, suspended in 3.5 ml of 1 mM hydrochloric acid, and washed on a glass filter with about 200 ml of 1 mM hydrochloric acid. The carrier was suspended in 5 ml of coupling buffer (0.1 M NaHCO ₃ , 0.5 M NaCl, pH 8.3), 1 mg of anti-CD3 antibody (OKT-3, manufactured by Ortho Pharmaceutical Corporation) was added, and room temperature was added. And mixed for 2 hours while rotating (10 rpm) in a 15 ml tube (manufactured by Asahi Techno Glass). Thereafter, only the carrier was recovered on a glass filter, which was suspended in 2 ml of a coupling buffer containing 0.2 M glycine, and allowed to stand at room temperature for 2 hours.

Next, the carrier is recovered with a glass filter, washed with about 100 ml of buffer A (0.1 M carbonate buffer containing 0.5 M NaCl, pH 4), and then about 100 ml of buffer B (containing 0.5 M NaCl). Washed with 0.1 M Tris buffer, pH 8). Thereafter, this AB washing operation was repeated two more times. Thereafter, it was washed with about 100 ml of PBS buffer (0.157 g of NaH ₂ PO ₄ .H ₂ O, 1.98 g of Na ₂ HPO ₄ , 8.1 g of NaCl, 1,000 ml), and about 5 ml of IL-2 was further added. After washing with the contained medium (KBM400, manufactured by Eugene Bio), the carrier was finally suspended in 5 ml of the same medium (note that the medium contained here was discarded when cells were added).

This suspension was added to a column prepared by cutting a cell culture bag (NIPRO culture bag 1000N, manufactured by Nipro) to complete a T cell activation column. In this column, 3 cm × 16 cm small bags are connected by upper and lower three-way stopcocks, and a carrier is added to the upper small bag (FIG. 1).

Moreover, what fixed the anti-CD3 antibody to the flask as a conventional method was prepared. Anti-CD3 antibody (OKT-3, manufactured by Ortho Pharmaceutical Corporation) was diluted with a phosphate buffered saline solution (PBS) to a concentration of 5 μg / ml, and 1 ml was injected into a flask having a surface area of 25 cm ² (manufactured by Asahi Techno Glass). This solution was spread evenly on the bottom of the flask and left at room temperature for 1 hour. Thereafter, the OKT-3 diluted solution was removed by suction, washed 3 times with PBS, and air-dried at room temperature in a clean bench to prepare a control flask for T cell activation.

(2) Preparation of human T cells, activation of human T cells with a carrier, and amplification of human peripheral blood 8 ml collected using a 10 ml syringe, and a vacuum blood collection tube (sterilized vacutina containing heparin) And collected in a blood collection tube (manufactured by Nippon Becton Dickinson) and centrifuged at 1,600 × g for 15 minutes. Thereafter, the T cell fraction was collected, T cells were collected by centrifugation (1,600 × g, 10 minutes), and the plasma fraction was collected. The T cells were washed once with PBS buffer, and a medium containing IL-2 (KBM400, Eugene Bio) was added thereto so that the number of cells was about 2 × 10 ⁵ cells / ml. In addition, the plasma fraction was added to this medium so that it might become 8% (w / w).

5 ml of this T cell suspension (about 1 × 10 ⁶ cells) was added to the T cell activation column and the T cell activation control flask prepared in (1) to initiate T cell activation. In addition, for the T cell activation column, the culture medium is passed from the upper small bag to which the carrier is added to the lower small bag every hour (so that the whole amount passes in about 10 minutes), and the upper part is again passed. The operation of returning to the small bag was repeated. The control flask was activated in a stationary state.

After 7 hours or 1 day of activation treatment (in the case of a T-cell activation control flask, 3 days of activation treatment was also performed), 1 ml of the culture solution was added to a 12-well plate (Nippon Becton Dickinson) After 3 days, 1 ml of the above medium was added (2 ml of culture solution). After 5 days, the entire volume was transferred to a 6-well plate (Nihon Becton Dickinson) and 2 ml of the above medium was added (cultured). 7 ml later, transferred to a petri dish (Asahi Techno Glass)) and added 10 ml of the above medium to make a total volume of 14 ml, followed by further culturing for 9 days (culture after completion of the activation treatment) The total period is 16 days). For each, the number of cells was counted immediately after the end of the activation treatment and on the third, fourth, fifth, seventh, tenth, twelfth, fourteenth, and sixteenth days after the activation treatment.

FIG. 2-1 shows the growth curve when cultured until the 16th day after the end of the activation treatment by the T cell activation column and the conventional T cell activation control flask, when the activation treatment time is 7 hours. The case where the activation treatment time is 1 day is shown in FIG.
Furthermore, FIG. 2-3 shows a growth curve when the activation treatment is performed for 7 hours using the T cell activation column and a growth curve when the activation treatment is performed for 3 days using the control flask for T cell activation. Indicated.

From FIG. 2-1 and FIG. 2-2, the number of T cell amplification cells, that is, the efficiency of proliferating activated T cells (activation efficiency) is greater when using a column than when using a conventional flask. It can be seen that the amplification is clearly good, and even when the activation time is only 7 hours, sufficient amplification is exhibited.

From Fig. 2-3, when activation for 3 days was performed using a flask as usual, not only did the number of days for activation increase, but also compared with the case of activation for 7 hours using a column. It can be seen that the number of cell amplification is small and the start-up of cell amplification is slow.

Example 2 Activation of 3 human-derived T cells by anti-CD3 antibody-immobilized carrier and amplification of 3 human-derived T cells (3 specimens were designated as sample A, sample B, and sample C, respectively) These were prepared in the same manner as 1 (2), and these were activated using a T cell activation column and a T cell activation control flask prepared in the same manner as in Example 1 (1).
In the same manner as in Example 1 (2), after 7 hours or 2 hours of activation treatment, 1 ml of the culture solution was collected, and T cells were amplified in the same manner.

Fig. 3-1 shows the growth curve when cultivated for 2 weeks after completion of the activation treatment using the T cell activation column and the conventional T cell activation control flask, when the activation treatment time is 2 hours. The case where the processing time is 7 hours is shown in FIG.
From these figures, it can be seen that when a column is used, all three samples show sufficient amplification with a short activation treatment as compared with the case of a conventional flask. Further, it can be seen that even an extremely short activation process of 2 hours has a sufficient effect.

Example 3 Effect of human anti-CD3 antibody fixation on activation of human-derived T cells Cyanogen bromide activated agarose carrier (CNBr activated Sepharose 6MB (trade name), manufactured by Amersham Biosciences) according to Example 1 (1) ) Was immobilized with an anti-CD3 antibody. In this case, the amount of antibody used was 1 mg, 500 μg, 100 μg, and 5 μg. These are CNBr1000, CNBr500, CNBr100, and CNBr5, respectively.

FIG. 4 shows a growth curve when these were used in the same manner as in Example 1 (2), and the activation treatment of human-derived T cells was performed for 2 hours and then cultured for 2 weeks.
From this figure, it can be seen that sufficient amplification can be obtained even with a small amount of antibody immobilized.

Example 4 Activation of human-derived T cells by one-pass stimulation using different carriers (1) Preparation of epoxidized carrier Sufficient water for cellulose carrier (prepared by the method described in Japanese Patent No. 1905101, average particle size 450 μm) Then, the sample was suspended in water and the sedimentation volume after 2 hours was measured. Cellulose carrier: Water: 2N NaOH: Epichlorohydrin (manufactured by Wako Pure Chemical Industries) = 1: 1: 0.53: 0.18 (volume ratio) is weighed, and the carrier and water are measured in a reaction vessel (made of polyethylene). Shake for about 15 minutes at 40 ° C. 2N NaOH was added to this and stirred for 30 minutes, and then epichlorohydrin was added and shaken. After the reaction for 2 hours, the filtrate was sufficiently washed until the filtrate became neutral to prepare an epoxidized carrier.

(2) Immobilization of anti-CD3 antibody on epoxidized carrier The carrier prepared in Example 4 (1) was collected on a glass filter, washed with about 300 ml of sterilized water, and further carbonated buffer (0.05 M NaHCO ₃ , Washed with about 300 ml of 0.1 M NaOH, pH 10). After washing, the carrier is suspended in about 7 ml of carbonate buffer, 5 ml of which is added to a 15 ml tube (Asahi Techno Glass), and 5 μg of anti-CD3 antibody (OKT-3, manufactured by Ortho Pharmaceutical Corporation). ) And mixed at 37 ° C. for 17 hours while rotating (10 rpm). Thereafter, the mixture was allowed to stand, the supernatant was discarded, 10 ml of glycine buffer (0.05 M NaHCO ₃ , 0.1 M NaOH, 0.2 M glycine, pH 10) was added, and the mixture was allowed to stand at 37 ° C. for 4 hours. Thereafter, the cells were washed with about 100 ml of sterilized water, washed with PBS buffer, and finally washed with a medium containing IL-2 (KBM400, manufactured by Eugene Bio). Using this carrier suspended in 5 ml of the same medium, a T cell activation column (epoxy) was prepared in the same manner as in Example 1 (2).

(3) Activation of T cells by one-pass stimulation Using the T cell activation column (epoxy) prepared in Example 4 (2) and the T cell activation column (CNBr5) prepared in Example 3 Then, in the same manner as in Example 1 (2), activation was carried out by passing the culture medium containing human-derived T cells once from the upper small bag to the lower column small bag over about 20 minutes. (One-pass stimulation). Thereafter, the result of amplification for 2 weeks in the same manner as in Example 1 (2) is shown in FIG.
FIG. 5 shows that even when the type of carrier is changed, it can be sufficiently activated, and efficient T cell amplification can be achieved by simple short-time treatment such as one-pass stimulation.

Example 5 Phenotype of amplified human T cells (CD4, CD8)
CD4 positive and CD8 positive ratios were measured for T cells amplified after activation by one-pass stimulation using a T cell activation column (CNBr5) or T cell activation column (epoxy) in Example 4 (3) did. Moreover, it measured also about the T cell amplified by the conventional flask method (activation time: 3 days).

^Five amplified 2 × 10 ⁵ T cells were recovered by centrifugation (430 × g, 5 minutes), and 200 μl of chilled PBS buffer was added. To this tube, 10 μl of antibody reagent (Tritest CD4 / CD8 / CD3, manufactured by Nippon Becton Dickinson) was added and allowed to react in ice for 15 minutes. Thereafter, 500 μl of PBS buffer was added, and the cells were collected by gentle stirring and centrifugation (430 × g, 5 minutes). The supernatant was removed, 1 ml of PBS buffer was added thereto, and the mixture was stirred and then centrifuged (430 × g, 5 minutes) to recover the cells. The cells were suspended in 500 μl of PBS buffer. Using this cell suspension, FACS analysis (BD FACSCanto, Nippon Becton Dickinson) was performed, and the ratio of CD3 positive, CD4 positive, and CD8 positive cells was calculated. The results are shown in Table 1.

In the case of lymphocyte therapy, a higher ratio of CD8 positive T cells is desirable. From Table 1, activation by the column ensures a sufficient ratio of CD8 positive T cells, and compared to the conventional flask method, the ratio of CD4 negative CD8 negative unnecessary for lymphocyte therapy is remarkably small and effective. It can be seen that the abundance ratio of T cells is high.

Example 6 Activation and amplification of human-derived T cells using an anti-CD3 antibody immobilized on a carrier to which a protein A ligand has been added (1) Immobilization of protein A on an epoxidized carrier (of protein A carrier Production)
The carrier prepared in Example 4 (1) was collected on a glass filter, washed with about 300 ml of sterilized water, and further washed with about 300 ml of carbonate buffer (0.05 M NaHCO ₃ , 0.1 M NaOH, pH 10). After washing, the carrier is suspended in about 7 ml of carbonate buffer, 5 ml of which is added to a 15 ml tube (Asahi Techno Glass), and 50 μg of Protein A (Zymed) is added to this at 37 ° C. The mixture was mixed while rotating (10 rpm) for 17 hours. Then, it was left still and the supernatant was discarded.
The recovered carrier was added with 5 ml of glycine buffer (0.05 M NaHCO ₃ , 0.1 M NaOH, 0.2 M glycine, pH 10), and allowed to stand at 37 ° C. for 4 hours. Then, it was washed with about 100 ml of sterilized water, then washed twice with about 100 ml of PBS buffer, and finally suspended in 5 ml of PBS buffer. This was used as a protein A carrier.

(2) Immobilization of anti-CD3 antibody on protein A carrier (production of protein A-anti-CD3 antibody carrier)
10 μg of anti-CD3 antibody (OKT-3, manufactured by Ortho Pharmaceutical Corporation) was added to the protein A carrier prepared in Example 6 (1), and the mixture was placed in a 15 ml tube (manufactured by Asahi Techno Glass) for 2 hours at room temperature. Mixing while rotating (10 rpm). Thereafter, only the carrier was recovered on a glass filter and washed twice with about 100 ml of PBS buffer. Thereafter, the carrier was washed twice with a medium (KBM400, manufactured by Eugene Bio) containing about 5 ml of IL-2, and finally the carrier was suspended in 5 ml of the same medium (the medium contained here) Were discarded when adding cells). This was designated as protein A-anti-CD3 antibody carrier (1).

Similarly, an agarose carrier (Protein A Sepharose 6MB (trade name), manufactured by Amersham Pharmacia) on which protein A is immobilized is prepared by immobilizing an anti-CD3 antibody, and this is prepared as a protein A-anti-CD3 antibody carrier (2). It was.

Each of these was added to a column prepared by cutting a cell culture bag (NIPRO culture bag 1000N, manufactured by Nipro) to complete a T cell activation column. In this column, a small bag of 3 cm × 16 cm is connected by upper and lower three-way stopcocks, and a carrier is added to the upper small bag.

As a conventional method, an anti-CD3 antibody immobilized on a flask was prepared. Anti-CD3 antibody (OKT-3, manufactured by Ortho Pharmaceutical Corporation) was diluted with a phosphate buffered saline solution (PBS) to a concentration of 5 μg / ml, and 1 ml was injected into a flask having a surface area of 25 cm ² (manufactured by Asahi Techno Glass). This solution was spread evenly on the bottom of the flask and left at room temperature for 1 hour. Thereafter, the OKT-3 diluted solution was removed by suction, washed 3 times with PBS, and air-dried at room temperature in a clean bench to prepare a control flask.

(3) Activation of T cells using protein A-anti-CD3 antibody carrier In the same manner as in Example 1 (2), a column containing protein A-anti-CD3 antibody carrier (1), protein A-anti-CD3 antibody carrier Using the column containing (2) or the control flask, two human-derived T cell specimens (sample A and sample B) were activated. For the column containing the protein A-anti-CD3 antibody carrier (1) or the column containing the protein A-anti-CD3 antibody carrier (2), all the culture solution passes from the upper small bag to the lower small bag. The activation was performed with a one-pass stimulation to allow the whole amount to pass through in about 20 minutes. The control flask was activated over 3 days.

FIG. 6 shows a growth curve when cultured until the 14th day after the activation treatment. From this figure, activation by one-pass stimulation using a protein A-anti-CD3 antibody carrier is based on the conventional flask method in terms of the number of T cell amplification cells, that is, the efficiency of activating T cells (activation efficiency). It can be seen that the result is comparable or better than the case of activation for 3 days.

Schematic diagram of T cell activation column used in Examples Cell number growth curve after performing T cell activation treatment in Example 1 in a column or control flask for 7 hours Cell number growth curve after performing T cell activation treatment in Example 1 in a column or control flask for 1 day Cell number growth curve after T cell activation treatment in Example 1 for 7 hours on column or 3 days in control flask Cell number growth curve after activation of 3 T cell specimens (Sample A, Sample B and Sample C) in Example 2 for 2 hours in a column or control flask Cell number proliferation curve after activation of 3 T cell specimens (Sample A, Sample B, and Sample C) in Example 2 using a column or control flask for 7 hours Cell number growth curve after 2 hours of activation treatment with columns (CNBr1000, CNBr500, CNBr100 and CNBr5) prepared in Example 3 with the amount of antibody used at the time of fixation being 1 mg, 500 μg, 100 μg, 5 μg Cell number growth curve after activation treatment by one-pass stimulation using column (epoxy) or column (CNBr5) in Example 4 In Example 6, the activation treatment of 2 specimens of T cells (sample A and sample B) was carried out using a column containing protein A-anti-CD3 antibody carrier (1) (one-pass stimulation), protein A-anti-CD3 antibody carrier (2). Cell number growth curve after using a column (one-pass stimulation) containing or control flask (3 days)

Explanation of symbols

1. Upper small bag 2, lower small bag 3, three-way stopcock 4, carrier

Claims (9)

(A) A method for activating T cells, which comprises bringing a solution containing T cells into contact with a carrier on which an anti-CD3 antibody or a fragment thereof has been immobilized. (A) While moving the solution containing T cells, the T cells are activated by contacting with a carrier on which an anti-CD3 antibody or fragment thereof is immobilized, and (b) the obtained activated T cells are treated with a medium containing lymphokine Proliferate activated T cells by culturing in,
A method for producing activated T cells. The production method according to claim 2, wherein the lymphokine is at least one selected from the group consisting of IL-2, IL-12 and IFN-γ. The production method according to claim 2 or 3, wherein the carrier is packed in a column. Furthermore, the costimulatory signal donor is fix | immobilized by this support | carrier, The manufacturing method of any one of Claims 2-4. 6. The costimulatory signal donor is at least one selected from the group consisting of an anti-CD48 antibody, an anti-CTLA-4 antibody, an anti-CD2 antibody, an anti-CD26 antibody, an anti-CD28 antibody, and fragments thereof. Production method. The manufacturing method of the immunotherapeutic agent which uses activated T cell as an active ingredient including manufacturing activated T cell by the manufacturing method of any one of Claims 2-6. An activated T cell production kit for carrying out the production method according to any one of claims 2 to 6,
A kit comprising a carrier for immobilizing an anti-CD3 antibody or a fragment thereof and a column for packing the carrier. The kit for producing activated T cells according to claim 8, further comprising an anti-CD3 antibody.

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