JPH09275976A - Preparation of activated t cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a large amount of an activated T cell useful for treating and diagnosing infection, allergy, AIDS, cancer, etc., by culturing a lymphocyte in the presence of a lymphokine using an immobilized water-insoluble carrier such as a parastimulation signal donor or an anti-CD3 antibody. SOLUTION: A solution containing a parastimulation signal donor and/or an anti-CD3 antibody is added to each well of a plastic plate, allowed to stand for one night, the solution is removed and the plate is washed to give a carrier for preparing an activated T cell in which the parastimulation signal donor and/or the anti-CD3 antibody is immobilized to the water-insoluble carrier. Then a lymphocyte is cultured in the presence of a lymphokine such as interleukin-2 (IL-2), interleukin-12 (IL-12), interferon-γ (IFN-γ) to efficiently give a large amount of an activated cell useful for phylaxis, antiallergy, anti- AIDS, metastasis control, cancer adjuvant thereby and effective for treating and diagnosing the condition of an illness such as autoimmune disease, bacterial infection, allergy, AIDS, cancer, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a carrier for preparing activated T cells, a method for preparing activated T cells using the carrier, and a preparation kit.

[0002]

2. Description of the Related Art With the recent advances in immunology, the immunological surveillance mechanism by various immunocompetent cells has been clarified. Immunotherapy for recognizing and destroying antigens such as tumor cells has been developed by enhancing the function of the mechanism. For example, lymphocytes collected and separated 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 again collected from the patient. LAK therapy (adoptive immunization method) for returning to the body has been developed.

However, since LAK cells show nonspecific cytotoxic activity against antigens such as tumor cells,
At present, the therapeutic effect cannot be expected so much. Therefore, attention is being focused on a method of performing the same treatment as LAK therapy by using antigen-specific activated T cells instead of antigen-nonspecific killer cells.

[0004]

In this therapy using activated T cells, it is an important requirement to activate, proliferate, and obtain a large amount of T cells. For example, as in the case of LAK therapy, Anti-CD (cluster differentiation) 3 antibody or high concentration (50 to 2000 JRU / ml) of IL-2
There is known a method of differentiating lymphocytes into activated T cells by using.

[0005]

Means for Solving the Problems As a result of intensive studies based on the above findings, the present inventors have found that a costimulatory signal donor and / or an anti-CD3 antibody-immobilized water-insoluble carrier is used. By culturing lymphocytes in the presence of lymphokines, they have been found to be able to differentiate into activated T cells and proliferate more efficiently than in the conventional method, and have completed the present invention.

The carrier for preparing activated T cells of the present invention is characterized in that a costimulatory signal donor and / or an anti-CD3 antibody is immobilized on a water-insoluble carrier. The method for preparing activated T cells of the present invention is characterized by culturing lymphocytes in the presence of lymphokines using the carrier of the present invention. Furthermore, the activated T cell preparation kit of the present invention is characterized by comprising the carrier of the present invention, a lymphokine, and a basal medium. The above inventions will be described below.

(1) Carrier for Preparing Activated T Cells The carrier for preparing activated T cells of the present invention is a carrier for lymphocyte culture used for preparing activated T cells, and includes a costimulatory signal donor and And / or an anti-CD3 antibody immobilized on a water-insoluble carrier.

[0008] An antigen-specific signal via TCR (T cell receptor) is not sufficient for establishment of an immune reaction by activation of antigen-specific T cells, and a costimulatory signal provided from an antigen-presenting cell. (Costimulatory signa
l) is mandatory. In the present invention, a substance that provides such a costimulatory signal, that is, a costimulatory signal donor, transmits a costimulatory signal via a T cell membrane surface protein, a membrane surface carbohydrate, or a membrane lipid. If it is a thing, it will not be specifically limited. For example, B
7 molecule family, CD48, anti-CTLA (cytolytic
T lymphocyte associated antigen) -4 antibody, anti-CD
Anti-CD such as 2 antibody, anti-CD26 antibody, anti-CD28 antibody
Examples include antibodies. Examples of the B7 molecule family include B7.1, B7.2, MB7.2 and the like. These may be derived from cells or may be prepared by genetic engineering techniques.

The anti-CD3 antibody may be either a polyclonal antibody or a monoclonal antibody as long as it is an antibody against CD3, and an antibody against TCR α or β chain can be substituted.

The water-insoluble carrier is applicable to cell culture, and is a costimulatory signal donor and / or anti-C.
There is no particular limitation as long as it can immobilize the D3 antibody. Materials include inorganic substances (eg glass, silica), polyalkylene resins (eg polyethylene, polypropylene, polystyrene, polyvinyl)
Plastic, agarose, cross-linked dextran, etc.
Examples include cellulose and hydrophilic vinyl polymers.
In addition, the form is flask, beads, (micro)
Examples include plates and fibers. Specific examples include those in which the costimulatory signal donor and / or the anti-CD3 antibody are immobilized on the inner surface of the plastic flask, particularly on the bottom, or on the surface of the beads.

Co-stimulatory signal donor and / or anti-C
As a method for immobilizing the D3 antibody on the water-insoluble carrier, a known method can be adopted. For example, a costimulatory signal donor and / or an anti-CD3 antibody may be used at a wavelength of 280 nm.
The solution is adjusted so that the protein concentration is 0.001 to 0.05 as the absorbance at. The pH of the solution is about 5-10,
The salt concentration is, for example, about 0.01 to 0.5M. After that, an appropriate amount of the solution and a water-insoluble carrier are mixed or brought into contact with each other, and left still at 0 to 37 ° C. for about 1 to 24 hours. After the treatment, remove the solution and wash with an appropriate amount of washing solution. As the cleaning liquid, distilled water, physiological saline, or 0.0
It is possible to use those to which about 1 to 0.1 w / v% of albumin, a nonionic surfactant [eg, polyoxyethylene sorbitan fatty acid ester, trade name: Tween], or the like is added.

The immobilized amounts of the costimulatory signal donor and the anti-CD3 antibody are shown below in terms of density. Anti-CD3
Antibodies, anti-CD antibodies such as anti-CD28 antibody, anti-CTLA
-4 antibody, 0.001 to 1000 μg / cm ²
Degree, preferably 0.01 to 100 / cm ^2, more preferably 0.01~1μg / cm ^2. In the case of the B7 molecule family, about 10 to 10 ¹⁵ / cm ² , preferably 10 to 10 ⁹ / cm ² , and more preferably 10 ³ to.
It is 10 ⁴ / cm ² .

(2) Activated T Cell Preparation Kit The activated T cell preparation kit of the present invention comprises the carrier (1), lymphokine and a basal medium.

Lymphokines include macrophage migration inhibitory factor (MIF) and macrophage chemotactic factor (M
CF), skin response factor (SRF), macrophage activating factor (MAF), leukocyte migration inhibitory factor (LIF), neutrophil chemotactic factor (NCF), eosinophil chemotactic factor (EC)
F), basophil chemotactic factor (BCF), eosinophil stimulatory factor (ESP), lymphotoxin (LT), vascular permeability factor (VPF), juvenile factor (BF or MF),
Osteoclast activating factor (OAF or IL-1β), γ-
Effector lymphokines such as 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 regulatory factor (AT
F-2) and other immunoregulatory lymphokines, with IL-2, IL-12 and IFN-γ being particularly preferred.
These lymphokines can be used alone or in combination of two or more kinds. IL-2 and IL-12 may be isolated from peripheral blood or may be prepared by gene recombination. As IFN-γ, those prepared by means such as cell culture and gene recombination can be used.

As the basic medium, the minimum basic medium (ME
M), RPMI1640, Ham's F12, Dulbecco's modified Eagle medium (DMEM), Iscove's modified Eagle medium (IMDM) and the like can be used. Preferably R
PMI 1640 is illustrated.

Various known components may be added to the basal medium in order to further improve its cell growth ability. Specific examples thereof include insulin, transferrin, fetuin, catalase, O-phosphorylethanolamine, ethanolamine, sodium selenite, 2-mercaptoethanol, corticosterone and the like.

Insulin includes human, pig, and sheep.
Derived from di, cattle, horses, etc., or recombinant
Any of them can be used, and the addition amount is 1 to 100 μg / ml,
Particularly, about 10 μg / ml is preferable. With transferrin
Humans, cows, horses, dogs, mice, guinea pigs
Tomato, rabbit, etc. can be used, and the amount added is
1-100 μg / ml, especially about 10 μg / ml is preferred
Yes. As the fetuin, for example, one derived from bovine is used
It can be added in an amount of 1-100 μg / ml, especially 10 μg.
About g / ml is preferable. Examples of catalase include
It is possible to use those derived from shishi, dog, mouse, filamentous fungus, etc.,
The addition amount is 2 to 50 μg / ml, especially about 30 μg / ml
Degree is preferred. O-phosphorylethanolamine or
The amount of ethanolamine added is 2-200 μg / ml,
It is preferably about 20 μg / ml. Sodium selenite
Is 10^-Ten-10^-7M, especially 10^-8About M
preferable. The amount of 2-mercaptoethanol added is 10
^-9-10^-FourM, especially 5 × 10^-FiveAbout M is preferable. Col
The amount of thicosterone added is 10^-9-10^-6M, especially 10
^-6About M is preferable.

(3) Method for preparing activated T cells The method for preparing activated T cells of the present invention is characterized by culturing lymphocytes in the presence of lymphokines using the carrier of (1) above. For example, the following operation is performed. First, lymphocytes (10 ⁴ to 10 ⁶ cells / ml) are cultured with the carrier of (1) above in a basic medium for about 1 to 3 days. The carrier of the above (1) is 10 ³ to 10 ⁷ per 1 ml of lymphocytes.
It can be applied at a rate of about cm ³ .

Examples of lymphocytes which are the starting cells include peripheral blood lymphocyte cells (PBL), lymph node cells, tumor infiltrating lymphocytes, tumor-bearing (or tumor) local lymph node cells and the like. When the activated T cells obtained by the preparation method of the present invention are administered to a patient, it is desirable to use lymphocytes collected from the patient. In addition, when used as a cancer metastasis suppressor and a cancer therapy adjunct, by culturing the local lymph node cells of the target tumor,
A tumor-specific antitumor effect can be expected.

Next, 1 to 5 in a medium containing lymphokine
Incubate for about 0 days. As the lymphokine, one isolated from peripheral blood or one prepared by gene recombination can be used. For example, in the case of IL-2, the blending amount of lymphokine is 10 to 1000 JRU / ml, preferably 10 to 100 JRU / ml, and more preferably 20 to
50 JRU / ml, and in the case of IL-12, 0.1-1
000 U / ml, preferably 0.1 to 100 U / ml, more preferably 1 to 50 U / ml. In the case of IFN-γ,
0.1-1000 IU / ml, preferably 10-1000
IU / ml, more preferably 20 to 500 IU / ml. The above-mentioned culture of the first stage and the second stage is preferably carried out at 36 to 3 in an atmosphere containing 3 to 7% by volume of carbon dioxide.
Perform at 8 ° C.

According to the method for preparing activated T cells of the present invention, lymphocytes are satisfactorily better than CD4 ⁺ and CD in comparison with the conventional method.
Differentiated and induced into activated T cells such as 8 ⁺ (activated) T cells. Activated T cells can be used alone or in combination with known agents to protect mammals such as humans, cows, horses, dogs, mice and rats from infection prevention, anti-allergy, anti-AIDS, cancer metastasis suppression, etc. Have an effect. Known drugs that can be used in combination include IL-2, IL-12, IFN-
γ and the like are exemplified.

The pharmaceutical composition containing activated T cells is administered parenterally in the form of a liquid formulation and is activated in an amount sufficient to produce the desired therapeutic effect on the progress or condition of the disease. It should contain T cells. The pharmaceutical composition is useful as an infection preventive agent, an antiallergic agent, an anti-AIDS agent, a cancer metastasis inhibitor, or a cancer therapy adjunct.
This pharmaceutical composition contains a suspending agent, preservative, p
An H buffer, an isotonic agent, a local anesthetic, an antibiotic, etc. are added, and subcutaneous, intramuscular, and intravenous injections can be produced by a conventional method.

The carrier for preparation of activated T cells, the preparation method and the preparation kit of the present invention are used for examining and diagnosing pathological conditions such as autoimmune diseases, bacterial infections, allergies, AIDS, and cancer progression. Is also extremely useful.

[0024]

EXAMPLES Examples and experimental examples will be given below in order to explain the present invention in more detail, but the present invention is not limited thereto.

Example 1 An anti-CD3 antibody solution (10 μg / ml) was added to a 24-well plastic plate in an amount of 1 ml per well, and the mixture was left standing overnight and the solution was removed and washed. Further, an anti-CD28 antibody solution (10 μg / ml) was added in an amount of 1 ml per well, the solution was removed after standing overnight, and the plate was washed to prepare an immobilization plate. Lymphocytes from allergic patients (10 ⁶ / well) were added to this immobilization plate.
And RPMI 1640 (1 ml / well) were added and 3
Activated T cells were prepared by culturing at 7 ° C. for 24 hours.

Experimental Example 1 IL-2 produced by the activated T cells obtained in Example 1 using the usual 24-well plastic plate (with no antibody immobilized) as a control.
The amount of 5 was measured, and the results are shown in Table 1.

[0027]

[Table 1]

According to the present invention, the production amount of IL-5 was increased about 100 times or more as compared with the case of no stimulation. Also, I
The amount of L-5 produced correlated with the severity of allergy. That is, since the production of cytokines is increased about 100 times or more as compared with the conventional method, it is about 100 times or more when allergic symptoms of patients are examined and diagnosed using the amount of IL-5 produced by T cells as an index. It can be detected with high sensitivity.

Example 2 The same as Example 1 except that the lymphocytes derived from allergic patients were changed to local lymphocytes (10 ⁶ cells / well) of a mouse experimental autoimmune encephalomyelitis model in Example 1. And activated T cells were prepared.

Experimental Example 2 As in Experimental Example 1, the activation T obtained in Example 2 was compared with the case where a normal 24-well (well) plastic plate (with no antibody immobilized) was used as a control. The amount of IFN-γ produced by the cells was measured, and the results are shown in Table 2.

[0031]

[Table 2]

According to the present invention, as in Experimental Example 1, the IF
The amount of N-γ produced increased about 100-fold. Therefore, when the symptoms of an autoimmune disease in a patient are examined and diagnosed using the amount of IL-γ produced by T cells as an index, it can be detected with about 100 times sensitivity.

Example 3 Lymph node cells from naive C57BL / 6 mice were treated with anti-C.
CD4 ⁺ T cells were prepared by treatment with D8 monoclonal antibody and anti-HSA (human serum albumin) antibody.
These CD4 ⁺ T cells (2 × 10 ⁵ cells / well) were treated with anti-CD3
Stimulation was performed by culturing at 37 ° C. for 2 days using a plastic plate on which a monoclonal antibody (10 μg / well) and an anti-CD28 antibody (10 μg / well) were immobilized. Further, IL-2 (20 JRU / well) was added, and the culture was continued at 37 ° C. for 5 days. The basic medium is
RPMI 164 with 10% FCS (fetal calf serum)
0 medium was used (also in the following examples). As a result, T
The cells grew to a total number of 19 × 10 ⁵ cells / well, of which CD4 ⁺ T cells were 98% and CD8 ⁺ T cells were 2%.
Met.

Example 4 Untreated C57BL / 6 mice were subcutaneously inoculated with B16 melanoma, and 10 to 15 days later, tumor local lymph node cells were collected. This lymph node T cell (50% CD4, CD8
45%) 2 × 10 ⁵ cells / well were stimulated by culturing at 37 ° C. for 2 days using the immobilized plastic plate of Example 3. In addition, IL-2 (20 JRU /
well) and IL-12 (100 U / well) were added, and the culture was continued at 37 ° C. for 5 days. As a result, T cells proliferated to a total number of 160 × 10 ⁵ cells / well, the breakdown of which was CD.
91% of 4 ⁺ T cells and 9% of CD8 ⁺ T cells.

Example 5 Lymph node T cells obtained in the same manner as in Example 4 (CD4 was 5
0%, CD8 45%) 2 x 10^FiveExample / well
2 at 37 ° C using 3 immobilized plastic plates
It was stimulated by culturing for a day. Furthermore, IL-2
(20 JRU / well) was added and cultured at 37 ° C for 5 days
Continued. As a result, the total number of T cells was 560 × 10.^FivePieces/
Proliferate well and the breakdown is CD4⁺50% T cells
CD8 ⁺T cells were 50%.

Example 6 Lymph node T cells (CD4 5) obtained in the same manner as in Example 4
0%, CD8 45%) 2 x 10^FiveExample / well
2 at 37 ° C using 3 immobilized plastic plates
It was stimulated by culturing for a day. Furthermore, IL-2
Add (100 JRU / well) and incubate at 37 ℃ for 5 days.
I continued feeding. As a result, the total number of T cells is 900 x 10^FivePieces
/ Well, the breakdown is CD4⁺9% T cells
CD8 ⁺The T cells were 91%.

Experimental Example 3 A lung metastasis tumor model of 1 × 10 ⁷ activated T cells derived from a B16 melanoma tumor-bearing mouse prepared in Example 6 (a mouse to which B16 melanoma was intravenously administered 3 days before)
, IL-2 (30,000 JRU) was intraperitoneally administered twice a day for a total of 4 days.

According to the evaluation experiment based on the survival rate, the group to which the activated T cells derived from the B16 melanoma tumor-bearing mouse was administered showed an antitumor effect without one death. This effect was tumor-specific. On the other hand, all untreated mice died.

Experimental Example 4 Picibanil was administered around the cancerous part of B16 melanoma-bearing mice, local lymph node cells in the tumor were collected, and activated T cells were cultured and expanded according to the method of Example 6. .
Using the activated T cells thus prepared, IL
-2 is not used together, and IL-2 (10,000 JRU)
The effect of using the above-mentioned drug twice a day for a total of 4 days was evaluated by the survival rate.

As a result, in the group to which activated T cells derived from B16 melanoma tumor-bearing mice were administered without using IL-2 together, 5 out of 7 mice were completely cured. On the other hand, in the group in which activated T cells derived from B16 melanoma-bearing mice were administered in combination with IL-2, all 7 cases were completely cured, which is more remarkable than in the case where IL-2 is not used in combination. It showed an antitumor effect.

Example 7 Balb / c mice were challenged with DNP-KLH (dini
4 μg of trophenol-keyhole limpet hemocyanin) and 4 mg of aluminum hydroxide were intraperitoneally administered to give an allergic state (indicating IgE production). The mouse spleen cells (2 × 10 ⁵ cells / well) were stimulated by culturing at 37 ° C. for 2 days using the immobilized plastic plate of Example 3. Further, IL-2 (50 JRU / well) and IL-12 (100 U / well) were added, and the temperature was 37 ° C.
Culture was continued for 5 days. As a result, the total number of T cells is 150
It proliferated to × 10 ⁵ cells / well, and the content was 90% of CD4 ⁺ T cells and 10% of CD8 ⁺ T cells.

Experimental Example 5 The activated T cells prepared according to Example 7 were added to IL-12 (1000) to other mice in the same example allergic state.
When administered intravenously together with ~ 10,000 U), the allergic condition was relieved.

[0043]

INDUSTRIAL APPLICABILITY According to the present invention, activated T cells can be prepared more efficiently and in large quantities as compared with the conventional method. In addition, CD4 ⁺ or CD8 ⁺ (activated) T
It is also easy to grow only cells.

Therefore, the present invention provides means for facilitating clinical application of a therapeutic method utilizing activated T cells (so-called LAT therapy). That is, it can be greatly expected to be used for infection prevention, anti-allergy, anti-AIDS, suppression of cancer metastasis, and adjuvant cancer therapy.

The present invention is also a very useful means as a test or diagnostic reagent when testing and diagnosing conditions such as autoimmune diseases, bacterial infections, allergies, AIDS, and cancer progression. Is.

Front Page Continuation (72) Inventor Kamehisa Nomoto 3-25-52 Higashi, Kashii Station, Higashi-ku, Fukuoka City, Fukuoka Prefecture

Claims (3)

[Claims] 1. A carrier for preparing activated T cells, wherein a costimulatory signal donor and / or an anti-CD3 antibody is immobilized on a water-insoluble carrier. 2. A method for preparing activated T cells, which comprises culturing lymphocytes using the carrier according to claim 1 in the presence of lymphokines. 3. A kit for preparing activated T cells, which comprises the carrier according to claim 1, a lymphokine, and a basal medium.