JPH10290689A - Production of lymphocytic cell and immunotherapeutic agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a lymphocytic cell having cytotoxicity to tumorous cell and useful for an immunotherapeutic agent, etc., with low-toxic interleukin 15 (IL-15) by culturing the lymphocytic cell in the presence of an anti-CD3 antibody and the IL-15. SOLUTION: A physiological saline solution is added to dilute a human peripheral total blood collected by using a syringe treated with a heparin and the diluted blood is then added to a medium for density-gradient centrifugation, superposed and centrifuged to separate a lymphotic cell. The first-stage culture of the resultant lymphocytic cell is then carried out in the presence of an anti-CD3 antibody converted into the solid phase and interleukin 15 and the second-stage culture is subsequently performed in the absence of the anti-CD3 antibody converted into the solid phase and in the presence of the IL-15 to thereby provide the objective lymphocytic cell, having cytotoxicity to tumorous cells and useful as an immunotherapeutic agent, etc., suitable for the adoptive immunotherapy used for treating cancers, infectious diseases, etc., in a large amount by using the low-toxic IL-15.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the selective expansion of lymphocyte subpopulations having cytotoxicity on tumor cells, in particular CD4 + T cells, CD8 + T cells, CD16 + NK cells and / or gamma delta T cells. And an immunotherapeutic agent useful for adoptive immunotherapy.

[0002]

2. Description of the Related Art In the past, various immunotherapies have been tried for the purpose of treating cancer. As a typical method, one is
Specific or non-specific immune activators were administered in hopes of enhancing the host immune response of the cancer bearing patient to the cancer cells. Certain species, such as interferon, interleukin 2 (IL-2), interleukin 12 (I
L-12) and other cytokines, PSK, OK-43
2. Experiments with immunostimulatory substances such as lentinan have suggested the possibility of application to cancer treatment. However, the inability to strongly induce an immune response to the target tumor antigen, and the immunity of cancer patients who have basically decreased There have been no successful examples yet because of the inability to achieve the expected increase and the side effects of the administered substance itself.

[0003] Another method is to separate and stimulate immune cells outside the body to enhance immunity and re-introduce them into a living body, directly or indirectly irrespective of the immune response of a cancer host with a reduced immune response. It was expected to damage cancer cells, clinical trials were attempted, and some effects were confirmed. Representative examples of immunologically active cells used for such treatment include lymphokine-activated killer (LAK) cells, tumor-infiltrating lymphocytes (TIL) cells, and the like.

Japanese Patent Application Laid-Open No. 62-116518 discloses that a patient's blood can be collected within 10 hours using a cell separator.
After processing 20 liters, each time about 5 × 105-5 ×
Described is to collect 1010 mononuclear cells, separate lymphocytes with Ficoll Hypaque, stimulate with IL-2 to obtain LAK cells, and finally administer LAK cells with a large amount of IL-2 to patients Have been. As a result, effects such as partial regression of metastasis have been confirmed in some patients or some cancer types. On the other hand, however, the majority of treated patients also reported relatively serious side effects such as fever, chills, nausea, vomiting, dyspnea, malaise, erythema or skin rash.
One of the major drawbacks of these methods is that they require a large amount of cells to be used for treatment, and the serious side effects of administering a large amount of the stimulant simultaneously with the lymphocytes after stimulation appear. .

[0005] In Japanese Patent Application Laid-Open No. Hei 3-80076, a small number of lymphocytes are stimulated with an anti-CD3 antibody immobilized on a culture vessel, and further stimulated with an anti-CD3 antibody and IL-2 to thereby increase the cell count. About 100-1 when departure
It is reported to grow 0000-fold. In addition, we are conducting clinical trials in which these lymphocytes and drugs to suppress host immunity that increases tumors are administered simultaneously.
All patients who received lymphocytes reported that their tumors shrank by more than 50%. However, this method also uses an immune-suppressing drug and / or IL-2 simultaneously with lymphocytes.
Is administered, it is presumed that side effects occur similarly to the above-mentioned method.

[0006] Interleukin 15 (IL-15)
A new cytokine discovered as a T cell growth factor (WO95 / 27722), T cells, NK
It is known to activate cells. However, IL-
Unlike the case of No. 2, they are not produced from lymphoid cells, but rather are produced from non-lymphoid cells such as monocyte endothelial cells and smooth muscle cells. In addition, it has a binding ability to, for example, vascular endothelial cells other than T cells. IL-1
5 is also known to signal through the IL-2 receptor β and γ chains together with the IL-15 receptor α chain. However, the effect of IL-15 on lymphocytes I
The difference from L-2 is not clear.

[0007] In this connection, when mice were previously administered with IL-2 or IL-15 intraperitoneally and subsequently with 125I-labeled bovine serum albumin (BSA) intravenously, I
It was reported that in the L-2 administration group, radioactivity in lung tissue was significantly increased by a factor of 6 compared to IL-15, and that lung weight was also significantly increased, indicating that IL-2 administration caused vascular leak syndrome. (Vascular Leak Syndrome: VLS) and the occurrence of pulmonary edema have been confirmed (William Mu
nger et al., Cellular Immunology, 165, 289-293
P. 1995). Thus, IL-2 is six times more toxic than IL-15, which may be related to the problem of side effects identified in human clinical trials.

[0008]

According to the first and second aspects of the present invention, there is provided a method for producing lymphocytes which proliferate a large amount of lymphocytes having cytotoxicity to tumor cells using IL-15 having low toxicity. It provides the law. Claim 3
The described invention provides an immunotherapeutic agent suitable for adoptive immunotherapy used for treatment of cancer, infectious disease and the like.

[0009]

SUMMARY OF THE INVENTION The present invention relates to a method for producing lymphocyte cells, which comprises culturing lymphocyte cells in the presence of an anti-CD3 antibody and interleukin-15. Further, the present invention provides an anti-CD
A first-stage culture step of culturing in the presence of Antibody 3 and Interleukin 15, and a second-stage culture step of culturing in the absence of immobilized anti-CD3 antibody and in the presence of Interleukin 15 And a method for producing lymphocyte cells, characterized in that: Furthermore, the present invention relates to an immunotherapeutic agent containing the lymphocyte cells obtained by the above-mentioned production method.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The lymphocyte cells used in the present invention include all lymphocytes existing in a living body, such as peripheral blood lymphocytes, epithelial lymphocytes, infiltrating lymphocytes in tumors, and infiltrating lymphocytes infiltrating ascites and pleural effusion. Is not particularly limited,
Peripheral blood lymphocytes, especially fresh peripheral blood lymphocytes immediately after blood collection, provide a physical burden to blood collection patients, ease of lymphocyte separation,
It is preferable in terms of the good survival rate of lymphocytes after separation. The number of cells is preferably about 1 × 10 ⁶ to 50 × 10 ⁶ , and the blood volume is 10 to 20 ml, so that the cells can be cultured without any problem and the burden on the patient during blood collection. Is also preferred because it is light.

[0011] The above lymphocyte cells are cultured in the presence of an anti-CD3 antibody. Here, CD3 is a molecule that exists on the cell surface of T lymphocytes and is important when recognizing a target antigen together with a T cell receptor (TCR). T cells begin to proliferate by transmitting stimulation information into the cells via the CD3 molecule. A similar phenomenon occurs with the stimulation of a specific antibody against the CD3 molecule (anti-CD3 antibody). An anti-CD3 antibody used for stimulating lymphocyte cells can be produced in an animal or a cell using a purified CD3 molecule, but a commercially available product excellent in stability, cost and the like can also be used.

It is preferable to use the anti-CD3 antibody in the form of a solid phase because the effect of proliferation is high. For immobilization, it is preferable to dilute the anti-CD3 antibody with a sterilized phosphate buffer to a concentration of 1 to 10 μg / ml before use. As a device for immobilizing the antibody, a sterilized cell culture flask made of plastic such as polystyrene is preferably used, and its size can be appropriately selected. As the solid phase immobilization method, a method is preferable in which the diluent of the anti-CD3 antibody is immobilized on a solid solution and left at 4 to 37 ° C. for 2 to 24 hours. After solidification, it is preferable to store in a refrigerator (4 ° C.) until use. At the time of use, the solution is removed and washed with a phosphate buffer or the like, preferably at room temperature.

In the present invention, interleukin 15 (IL-15) is used for culture. IL-15 is present in culture during 1
It is preferable to use a concentration of １０００1000 ng / ml. A commercially available IL-15 can be used. IL-15 is composed of water, physiological saline, phosphate buffer,
RPMI-1640, DMEM, IMDM, AIM-V
Can be used by dissolving it in a commonly used cell culture solution or the like. Once dissolved, it is preferable to store it in a refrigerator in order to prevent a decrease in activity.

The culture of lymphocytes is preferably started by suspending the lymphocytes in a culture solution containing IL-15, placing the cells in a culture vessel having an anti-CD3 antibody immobilized thereon. The culture medium is not particularly limited as long as it is suitable for culturing lymphocytes, and amino acids, vitamins, nucleic acid bases, etc. are added to a culture medium derived from a living organism such as serum, a balanced salt solution, and, if necessary, cattle. Synthetic medium containing fetal serum or bovine serum albumin can be used, and RPMI-1640, AI
MV, DMEM, IMDM and the like are mentioned as preferable ones, and RPMI-1640 is mentioned as a particularly preferable one. The culture solution to which normal human serum is added is preferable because of its excellent growth effect.

Commercially available media can be used for these media. Culture can be performed according to a general cell culture method. For example, it can be performed in a CO2 incubator. The CO ₂ concentration is preferably 1 to 10%, particularly 5%, and the temperature is preferably 30 to 40 ° C, particularly preferably 37 ° C. The number of days for this culture is not particularly limited, but it is premised that the stimulation information of the anti-CD3 antibody is transmitted to the cells. During this culture period, observe the state of the cells under a microscope,
It is preferable to appropriately add a culture solution while appropriately counting the number of cells. In this culture, there is usually no noticeable cell growth on the first or second day of the culture, but cell growth is observed from about the third day. I will be. The amount of the culture solution to be added is preferably about 0.1 to 5 times the amount of the solution during the culture before the addition. The ratio of the addition is preferably once every 1 to 7 days in order to prevent the deterioration of the culture solution and the decrease of the IL-15 activity.

In the present invention, the above-mentioned culturing is defined as the first culturing step, and the culturing is carried out in the absence of immobilized anti-CD3 antibody
It is preferable to provide a second culture step of culturing in the presence of L-15 because lymphocytes can be proliferated and maintained in large quantities. Long-term culture in a culture vessel having an anti-CD3 antibody immobilized thereon may suppress the proliferation of lymphocytes. Second
As a culture vessel used for the stage culture, a plastic flask for cell culture, a gas permeable bag permeable to CO ₂ gas, or the like can be used. In this step, lymphocytes rapidly grow in large quantities. For this reason, it is preferable to increase the number of incubators every 2 to 4 days. As a standard for the number of cells, it is preferable to add a culture solution or increase the number of incubators so as to be 5 × 10 ⁵ to 10 × 10 ⁵ cells / ml.

According to the production method of the present invention, mainly CD4 +
T cells, CD8 + T cells, gamma delta T cells and / or CD16 + NK cells proliferate. Furthermore, at least one of these cells exhibits cytotoxicity to tumor cells. In other words, the stimulation of IL-15, a new cytokine, enables efficient and large-scale induction of a lymphocyte population having an activity of damaging target cells from a small amount of lymphocytes.

Here, CD4 + T cells are conventionally used as helpers /
Inducer T cells are cells that recognize histocompatibility complex (MHC) class II molecules and antigen molecules of the target cells. Further, CD8 + T cells, which are conventionally called killer / suppressor T cells, are cells that recognize and attack histocompatible antigen (MHC) class I molecules and antigen molecules of target cells. CD16 + NK (natural killer) cells are cells that express the CD16 molecule as a surface antigen and have the ability to attack target cells without being bound by the histocompatibility antigens of the target cells. In addition, gamma delta T cells have at least CD3 + C
These cells have the D16-surface antigen and express the T cell receptor gamma-delta chain.

Furthermore, the lymphocyte cells obtained by the production method of the present invention highly express the IL-2 receptor and the IL-12 receptor. In order for a cell such as a lymphocyte to receive stimulus information from a cell stimulating substance such as a cytokine and to transmit information into the cell, it is necessary to receive information using a receptor specific to each substance. That is, if a receptor is present on the cell surface, it can receive stimulation from a substance that specifically binds to the receptor. The fact that the lymphocytes cultured according to the present invention highly express the IL-2 receptor and the IL-12 receptor means that the lymphocytes express at least IL-2 and IL-12 in addition to the stimulation of IL-15. This indicates that the stimulus is received and activated, and an additive or synergistic effect between IL-15 and these cytokines is also expected. In addition, IL-4, an IL that shares part of the IL-2 receptor
It is shown that similar results can be obtained with -7, IL-9 and the like. Therefore, the lymphocyte cells produced according to the present invention can be used as an active ingredient of an immunotherapeutic agent used for adoptive immunotherapy useful for treating cancer, various infectious diseases, and the like. Further, it can be used for various research purposes as a research reagent.

When used as an immunotherapeutic agent, the dosage is 1
Preferably, 10 ⁶ to 10 ¹² cells are used per cell. As a dosage form, a liquid such as an injection or a drip is preferable.
Injections or drops dispersed in a physiological saline solution added so as to be more preferable. As an administration method, intravenous drip or injection into a vein, an artery, a local area or the like is preferable. The amount of liquid to be administered varies depending on the administration method, administration place, and the like.
It is preferable that the amount of cells is included in the liquid volume. Dosing frequency is 1
It is preferably from once / day to once / month, and the number of administrations is at least once, preferably 5 or more.

[0021]

【Example】

(1) Preparation of culture flask having immobilized anti-CD3 antibody An anti-CD3 antibody (OKT3, manufactured by Ortho Pharmaceutical Corporation) was diluted to a concentration of 5 μg / ml with a phosphate buffered saline solution (PBS) to give a surface area of 25 cm ^2.
5 ml was poured into the flask. The solution was spread evenly on the bottom of the flask, and allowed to stand in a refrigerator (4 ° C.) overnight or longer until use. The flask into which the solution was injected was washed three times with normal-temperature PBS before use.

(2) Preparation of culture solution (medium) containing IL-15 10% (vol / vol) human normal serum (fresh frozen plasma was thawed at 37 ° C.) in RPMI-1640 medium (manufactured by Sigma), Inactivated by heating at 56 ° C for 30 minutes, 18000 rp
The precipitate was removed by centrifugation at 4 ° C. for 60 minutes at 60 ° C., and further filtered through a 0.22 μm filter.
ml penicillin (Meiji Seika Co., Ltd.), 100γ / ml streptomycin (Meiji Seika Co., Ltd.) and IL-1
5 (manufactured by Immunotech) was adjusted to contain 50 ng / ml.

(3) Culture of lymphocytes 20 ml of physiological saline was added to 20 ml of human peripheral blood whole blood collected using a 20 ml syringe treated with heparin, and diluted twice to obtain Ficoll, a medium for density gradient centrifugation. -Paque (Pharmacia) 4ml, 10ml of diluted blood is layered on 16
The lymphocyte layer was collected by centrifugation at 00 rpm for 25 minutes at 20 ° C., and washed three times with RPMI-1640 medium.
About 2 × 10 ⁷ lymphocytes were obtained. The above IL-1
5 was suspended at a cell density of about 2 × 10 ⁶ cells / ml, and about 5 ml of the cell suspension was added to the anti-CD3 antibody-immobilized culture flask to start culture. The next day (Culture 1
From day (1), 1 ml of a culture solution containing IL-15 was added, and this operation was repeated until the fourth day of culture. Anti-CD on day 5 of culture
(3) The cell suspension was transferred to a 75 cm ² flask on which no antibody was immobilized, and about 10 ml of a culture solution containing IL-15 was added. Thereafter, the amount of the cell suspension per flask is about 30 ml as a guide, the color of the culture changes from orange to yellow, and the number of cells increases (1 to 10 × 10 ⁵ cells / ml).
According to the above, a new flask was basically divided into two to increase the number thereof, and about 10 ml of a medium containing IL-15 was added. This operation was repeated, and the lymphocyte population was cultured for 8 days.

(4) Measurement of Phenotype of Cultured Lymphocytes 5 × 10 ⁵ cultured lymphocytes obtained above were collected in a test tube, added with RPMI-1640 medium, centrifuged once, and the supernatant was removed. For direct staining, 10 μl of FITC (fluorescein isothiocyanate) or PE (phycoerythrin) -labeled monoclonal antibody was added, and for indirect staining, 1 μg of unlabeled monoclonal antibody was added, and the mixture was stirred well. After standing in ice for 30 minutes, RPMI-164
The cells were centrifugally washed twice with medium 0. In the case of indirect staining, 10 μl of an appropriately diluted FITC-labeled sheep anti-mouse antibody or anti-rat antibody (manufactured by Kappel) was added, the mixture was allowed to stand on ice for 30 minutes, and then centrifuged twice with RPMI-1640 medium. Finally, 0.5 ml of RPMI-1640 medium was added,
After stirring well, the surface antigen reacted with each antibody was measured by FACScan (Becton Dickinson).

The monoclonal antibodies used for the measurement were: FITC-labeled anti-CD3 antibody, FITC-labeled anti-CD16 antibody, FITC-labeled anti-TCR-α / β antibody, F
ITC-labeled anti-TCR-γ / δ antibody, FITC-labeled anti-CD
4 + PE-labeled anti-CD8 antibody, PE-labeled anti-CD56 antibody (Becton Dickinson), rat anti-IL-2 receptor α-chain antibody (anti-CD25, Serotech), mouse anti-IL-2 receptor for indirect staining Body β-chain antibody (M
ik-β1, Nichirei Co., Ltd.), mouse anti-IL-2 receptor γ chain antibody (AG184, Pharmingen) and mouse anti-IL-12 receptor β1 antibody (Hoffman La Roche) and FITC-labeled anti-rat IgG or FI
TC-labeled anti-mouse IgG (all manufactured by Kappel) was used. Table 1 shows the results.

[0026]

[Table 1]

From the data in Table 1, it can be seen that the phenotype of the obtained lymphocyte cells was mostly CD3 + CD4 +
And CD3 + CD8 + T cells and have T cell receptor alpha-beta chains. Also, other T cells with a small number of T cell receptor gamma-delta chains
Cells and CD16 + NK cells were present. These lymphocytes also contain the IL-2 receptor α chain, β chain, γ chain and I
It strongly expresses the L-12 receptor, and in addition to IL-15, for example, I-2 such as IL-2, IL-4, IL-7, and IL-9
It may be strongly activated by stimuli such as cytokines that transmit information by sharing a part of L-2 receptor and cytokines that transmit information via IL-12 receptor such as IL-12. Can imagine. That is, an additive or synergistic effect on lymphocyte activation is expected by the combination of IL-15 and these cytokines.

(5) Measurement of Damage Activity on Cultured Tumor Cells As effect cells, lymphocytes obtained by culturing were
(Volume / volume) RPMI-1 supplemented with fetal bovine serum (FBS)
After washing with 640 medium three times, suspending the cells in the same medium, counting the number of cells, seeding 200 μl each on the top of a 96-well U-bottom plate (manufactured by Falcon), and diluting by 2 times from the second step onward. A dilution step was made. Subsequently, 51Cr (ICN
Cells (cultivated cancer cell line K56)
2, Daudi and Raji cells) were seeded at 100 μl (1 × 10 ⁴ cells) at 1 × 10 ⁵ cells / ml, mixed with lymphocytes, and cultured at 37 ° C. in 5% CO ₂ for 4 hours. After completion of the culture, 100 μl of the supernatant was collected, and the radioactivity (cp) of 51Cr released by a gamma counter (manufactured by Packard) was measured.
m, test free group). The cytotoxicity rate (%) was calculated by the following equation.

(Equation 1) Incidentally, ⁵¹ Cr-labeled tumor cells in 3% Triton X-100 was added as a maximum free group of lysing the cells, ⁵¹ Cr-labeled tumor cells alone was spontaneous release group.

FIG. 1 shows the results. These lymphocytes effectively impaired the established tumor cells as shown in FIG. 1, indicating that they were an effective cell group for use in adoptive immunotherapy.

(6) Preparation of immunotherapeutic agent and method of using the same Immunotherapeutic agent Lymphocyte cells derived from a cancer patient were produced by the above-described method, and a commercially available separating agent (Lymphoprep, Nycomed) was used. And purify it according to the protocol. The obtained 2 × 10 ⁹ cells are suspended in 200 ml of a physiological saline solution containing 0.1% of human serum albumin to prepare an immunotherapeutic agent in the form of drops. How to use the immunotherapeutic agent The whole amount of the above injection is intravenously infused into a cancer patient. By repeating this operation once a month, it is possible to cure the cancer of the cancer patient or to stop or slow down the progress of the symptoms.

[0031]

According to the method for producing lymphocytes according to claims 1 and 2, a sufficient number of activated lymphocytes having an activity of damaging tumor cells and a small number of activated lymphocytes for use in adoptive immunotherapy are used. It is obtained efficiently starting from blood. The immunotherapeutic agent according to claim 3 contains a large amount of activated lymphocytes having an activity of damaging tumor cells, and is useful for adoptive immunotherapy. Furthermore, it is suggested that the combination with other cytokines and other immunostimulants will provide more activated lymphocytes.

[Brief description of the drawings]

FIG. 1 is a graph showing the cytotoxic activity (Cytotoxicity) of lymphocytes (effect cells) obtained by the production method of the present invention on cancer cell lines (target cells).

Claims (3)

[Claims] 1. A method for producing lymphocyte cells, comprising culturing lymphocyte cells in the presence of an anti-CD3 antibody and interleukin 15. 2. A first-stage culturing step of culturing lymphocyte cells in the presence of an immobilized anti-CD3 antibody and interleukin 15, wherein no immobilized anti-CD3 antibody is present and interleukin 15 is present. A method for producing lymphocyte cells, which comprises the steps of a second culture step of culturing below. 3. An immunotherapeutic comprising a lymphocyte cell obtained by the production method according to claim 1.