KR20140145585A - Method for producing lymphocyte cell group consisting mainly of memory t cells - Google Patents

Abstract

In the peripheral blood-derived lymphocyte cells, about 50% of the inexperienced T cells are occupied before the activation culture, but the rate is decreased by the activation culture. On the other hand, the share of memory T cells, which was less than half of that before the start of culture, increased to 95% due to the activation culture, but gradually decreased when the culture was continued again. It is an object of the present invention to provide a method for massively preparing a CD45RO positive T cell group in a simple and rapid manner without using an antibody or the like, a pharmaceutical composition containing a memory T cell group prepared by the above method as a main component, and the like do. The present invention detects activated lymphocyte cells activated and cultured and shows that the percentage of the memory T cells in the lymphocyte cell group has reached a desirable value with the index of the cell density of the lymphocyte cell group in culture exceeding a predetermined threshold value, The cell group is harvested, cryopreserved, and the cryopreserved cells are thawed, cultured, and proliferated to thereby produce a large number of lymphocyte cell groups containing memory T cells as a main component.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a lymphocyte cell group containing a memory T cell as a main component,

The present invention relates to a method for mass production of a lymphocyte cell group mainly comprising memory T cells for obtaining a large amount of CD45RO positive CD62L positive T cell group containing T cells, or CD45RO positive CD62L negative T cell group, , A pharmaceutical composition containing such a lymphocyte cell group, and a system (a combination of means such as an apparatus) for preparing the above lymphocyte cell group.

Lymphocyte cells have received much attention in recent years as an important means of dealing with the immune system for biological defense. On the surface of lymphocyte cells, antigens, called differentiation antigens, are expressed, and lymphocytes are classified into various subsets according to these antigens. For example, lymphocyte cells can be detected by detecting the presence or absence of CD3 (CD is an abbreviation for cluster of differentiation) according to the reactivity of monoclonal antibody, thereby detecting T lymphocytes involved in cellular immunity (CD3-positive, CD3 + ), And B lymphocytes involved in humoral immunity (also referred to as CD3 negative and CD3-). These lymphocyte cells also have different functions. Moreover, T lymphocytes (also referred to as T cells) involved in cellular immunity are classified into CD4-positive cells expressing CD4 and CD8-positive cells expressing CD8. In the past, CD4-positive cells have been considered to accomplish helper function, and CD8-positive cells have been believed to accomplish cell-damaging functions. Thus far, many studies have been conducted to separate CD4-positive cells and CD8-positive cells.

Although it is difficult to distinguish lymphocyte cells in form, many of the functions are divided into subset based on the anti-circularity as described above. However, most of them were mainly monoclonal antibodies against the anti-circular forms of the respective cells, for example, anti-CD4 monoclonal antibodies or anti-CD8 monoclonal antibodies. Then, the antibody bound to the magnetic microbeads is reacted with the lymphocyte, and the magnetic microbeads are collected from the outside of the container by the magnetic force of the Dan magnet, and the bound beads are separated through the antibody or separated using a separation column . Further, in order to obtain a large number of CD4-positive cells or CD8-positive cells, each of them is separated according to the microbeads method and then activated again to carry out amplification culture (see Patent Document 2). In recent years, lymphocyte cells are stained with a fluorescently labeled antibody against each surface antigen by the progress of measuring instruments, and a method of discriminating them by using a cell sorter has also been carried out. In this method, the antibody against the cell surface antigen is labeled with a fluorescent dye in advance, the labeled antibody is bound to the lymphocyte, the lymphocyte is subjected to the measurement by the cell sorter to detect the expression state of the surface antigen, . However, this method is problematic in that it is applied to a small scale (see Non-Patent Document 2). No matter what conventional method is employed, complicated operations such as labeling of lymphocytes with expensive antibodies can not be avoided, and cost and effort are required to obtain a large number of cells.

Activated T lymphocyte cells were first reported by Sekine et al. This is the first report on a method for obtaining a proliferated activated T lymphocyte cell group by selectively stimulating proliferation of an activated T lymphocyte cell group. Specifically, human peripheral blood mononuclear cells (PBMCs) The first culture is performed in a culture flask in which an OKT3 antibody as an antibody is solidified and the thus cultured cells are cultured in the second phase in the absence of the OKT3 antibody. The activated lymphocyte cell group obtained in this manner can be administered to a patient who has undergone culturing in the presence of interleukin 2 (IL-2) and has taken the peripheral blood by quantum immunotherapy of cancer (see Patent Document 1). In the same manner, the donor-derived peripheral blood lymphocyte cells were cultured in the presence of IL-2 in a culture container in which the OKT3 antibody was solidified, CD4-positive cells were separated from the obtained activated lymphocyte group, A pharmaceutical composition for preventing and treating infectious diseases, tumor recurrence, autoimmune diseases and the like can be administered to a patient (refer to Patent Document 2). As these lymphocyte cell sources, TIL (Tumor Infiltrating Lymphocyte), remnant blood, bone marrow, etc. can be used (see Patent Document 2).

From another functional point of view, there are cases where lymphocyte cells are classified as naive lymphocytes and memory lymphocytes. Inexperienced lymphocyte cells are lymphocyte cells expressing the CD45RA antigen on the cell surface, which have not yet been subjected to antigen stimulation, and are believed to be activated during stimulation with the antigen in the local lymph nodes. Membrane lymphocytes are lymphocyte cells already expressing CD45RO antigen by antigen stimulation, regardless of specific stimulation or nonspecific stimulation. T lymphocytes and B lymphocytes can be divided into inexperienced T cells and memory T cells, inexperienced B cells, and memory B cells, depending on their respective states. Memory T cells can be further divided into effector memory (EM) T cells and central memory (CM) T cells. Central memory T cells have a function of fighting against foreign bodies or antigens that invade the body by homing with lymph nodes. Effective memory T cells have a function of capturing foreign bodies or antigens belonging to the original site. Therefore, it is expected that a preparation containing a memory T cell group as a main component can achieve a high therapeutic effect in a quantum immunotherapy. In order to elucidate the mechanism of action of activated lymphocytes used in quantum immunotherapy, Osumi et al. Focused on a memory T cell subset and analyzed the function of the activated lymphocyte cell group obtained by activation amplification culture. As a result, the effect memory T cells contained in the activated lymphocyte cell group expressed cytotoxic activity against cancer cells 24 hours after the co-culture with the tumor cells. On the other hand, the central memory T cells started to co- , It was revealed that the cells were differentiated into effective memory T cells at that time (see Non-Patent Document 2).

A report on the collection of memory T cells dates back to the literature of 2000 by Sekine et al. (Refer to Patent Document 3), in which lymphocytes derived from hematopoietic cells were activated and cultured in the presence of anti-CD3 antibody and IL-2, As a result of activation proliferation, a population of cells was obtained in which the proportion of CD3-positive cells was 98% and the proportion of CD45RO-positive cells was 73%. However, there was a problem that the cells at this stage did not have sufficient proliferation and thus the number of cells was small.

The activated lymphocytes need to be rapidly administered to patients after proliferating culture, and it is difficult to perform rapid administration and stable quality control according to the disease state. Considering the convenience of the patient, there is a report that when lymphocyte cells are prepared in patient's blood, the lymphocyte cells are cryopreserved after activation culture (see Non-Patent Document 1). Here, we conclude that the share of CD4 lymphocytes and CD8 lymphocytes in activated lymphocytes obtained by re-activating freezing-thawed lymphocyte cells was calculated, and almost all of the populations proliferated in almost parallel. Non-Patent Document 3 discloses a result of tracking a subset change in culturing period when lymphocyte cells derived from hematopoietic and peripheral blood are separated, frozen once, frozen and then activated. All lymphocyte cells derived from the remnant blood and peripheral blood showed that the central memory T cell, a subset of memory T cells, proliferated rapidly after the start of culture, proliferation peaked at 6 to 7 days and then decreased. T cells have been reported to proliferate in the later stages of culture. In addition, Sekine et al. Have shown that lymphocyte cells of a patient are in vitro proliferated / activated and then frozen / preserved in a cryocooler. After thawing from the frozen state at the time of use, the suspension is suspended with physiological saline solution, (See Patent Document 4) is disclosed.

As described above, the use of memory T cells is suggested in the quantum immunotherapy, and it is a social demand to collect a large amount of memory T cells richly. However, the current situation is that there is no easy way to collect a large amount of memory T cells necessary for clinical provision without imposing a large burden on lymphocyte donors.

Patent Document 1: JP-A-3-80076 Patent Document 2: Japanese Patent No. 4389039 Patent Document 3: Japanese Patent Application Laid-Open No. 2002-171966 Patent Document 4: Japanese Patent No. 4395644

Non-Patent Document 1: Sekine T., Human Cell, 5 (3) 243-245 (1992) Non-Patent Document 2: The 32nd Cancer Immunology Research Society Program / Abstract Page 103 April 2011 Non-Patent Document 3: Shikamura M., et al., Biotherapy 23 (3) 257-262 (2009)

In the peripheral blood-derived lymphocyte cell group, about 50% of the inexperienced T cells are occupied before the activation culture, but the ratio is lowered by the activation culture. On the other hand, the share of memory T cells, which were less than half of those before the start of culture, increased by activating culture to 95%. The present invention relates to a method of massively preparing a CD45RO positive T cell group in accordance with a simple and rapid high proliferation culture method without using an antibody or the like, And to provide a pharmaceutical composition and the like.

DISCLOSURE OF THE INVENTION The present inventors have conducted various studies under various conditions for the purpose of raising the ratio of memory T cells contained in activated lymphocytes. As a result, peripheral blood lymphocyte cells were cultured in a culture medium containing IL-2 (CD45RO +, CD62L +) or CD45RO-positive (CD45RO +, CD62L-) T cell group, that is, the memory T cell group, with CD45RO positive And the percentage of the lymphocyte cell group was found to increase. However, at this point it is still necessary to continue culturing because the total number of cells is still small, but it has been found that the rate of the memory T cell group is reduced by continuing the culture. Thus, the present inventors have extensively studied a method for mass-producing a group containing a memory T cell group as a main component by proliferating lymphocyte cells without reducing the ratio of the memory T cell group. As a result, in order to obtain large amounts of the memory T cells, the culture was continued until the proportion of the surface antigen in the lymphocyte cell group under the activation incubation ratios of CD45RO +, CD62L + and / or CD45RO +, CD62L- When harvesting the lymphocyte cell group that has reached the preferable ratio, the above-mentioned harvested cells were cryopreserved with the cell density exceeding a predetermined threshold value, and then the cells were activated and amplified again to solve the above problems.

That is, by finding the relationship between the increase in the share of the memory T cell group and the cell density of the activated cultured lymphocyte cell group, it is understood that the cell density above a certain threshold value, which is an index of cell harvesting, . This makes it possible to detect the optimal timing for harvesting a cell population having a sufficient number of cells and a high memory T cell group occupation rate by a simple and low-cost means of measuring cell density. In addition, by controlling the cell density at the time of sowing, it was also found that the culture period until harvest could be controlled optimally, and cell groups having sufficient cell numbers and high memory T cell group occupation rates could be harvested in a short time. Thus, the present inventors have developed a method for mass-producing lymphocyte cell lines which are composed of memory T cells as a main component at a simple and low cost without using an antibody or the like.

That is, the present invention relates to a method for producing a lymphocyte, comprising the steps of [1] (a) activating cultured collected lymphocyte cells, (b) detecting the increase of the occupancy of the memory T cell group based on the lymphocyte cell density, And (c) thawing the frozen lymphocyte to activate and culturing the lymphocyte cell population, wherein the step (a) to (c) of The method according to the above [1], wherein the detection of the increase in the occupancy rate of the memory T cell group based on the lymphocyte cell density in the step (b) is performed using the number of lymphocyte cells floating in the culture medium as an index , [3] the lymphocyte cell density of the step (b) is determined by the production method described in the above [1] or [2], wherein the number of lymphocyte cells floating in the culture medium is 4 × 10 ⁵ cells / The culture was incubated with IL-2 Immobilized anti made by the method according to any one of the above [1] to [3], wherein the incubation in the presence of a CD3 antibody.

[6] The method according to any one of [1] to [4], wherein the step (c) is completed within two weeks after thawing, [6] The method according to any one of the above [1] to [5], wherein the lymphocyte cell group is a lymphocyte cell group having a 75% or more occupancy of the memory T cells of the lymphocyte cell group, [7] The method according to any one of [1] to [6] above, wherein the cell comprises at least 70% of the lymphocyte cell group. [8] The method according to [1], wherein the isolated lymphocyte is derived from human peripheral blood [9] the production method according to any one of [1] to [7] above, wherein the lymphocyte cell group is for administration to a third person.

 The present invention also relates to [10] a lymphocyte cell group characterized by containing at least 75% of the lymphocyte cell line memory T cells, [11] a cell line comprising 70% or more of the central memory T cells of the lymphocyte cell group, (A) a lymphocyte cell group according to any one of (1) to (10), (12) a pharmaceutical composition comprising the lymphocyte cell group described in (10) or (11) as an active ingredient, (C) means for cryopreserving lymphocyte cells, and (D) means for thawing frozen lymphocyte cells; (A) to (D) means of And a lymphocyte cell group based on the T cell.

According to the present invention, it is possible to provide a large number of lymphocyte cell lines mainly consisting of CD45RO +, CD62L +, CD45RO +, and CD62L-, which are contained in activated lymphocyte cells derived from peripheral blood. In order to track the proliferation of the memory T cell population contained in the activated lymphocyte group, the number of cells in the activated culture medium, particularly floating lymphocytes, can be measured without using a complicated method of analyzing cell surface antigens conducted in the conventional method, It is possible to grasp the state of the proliferation and the occupancy of the memory T cells by an extremely simple and low-cost method of measuring the number, calculating the cell density, and comparing the cell density with a preset threshold value. Thus, not only is it possible to harvest the cells to find the optimal time for cell harvesting, but also to cryopreserve the harvested cells, the lymphocyte cell population in which the memory T cell share is increased can be appropriately used. In addition, by thawing the frozen memory T cell group and re-activating it, the cell population is amplified while maintaining a high memory T cell group occupation ratio, and as a result, a large number of lymphocyte cell groups mainly containing the memory T cell group can be obtained. In addition, the production method of the present invention has examined in detail the number of lymphocyte cells, the incubation time, and the amount of the lymphocyte cell group mainly composed of the memory T cells finally obtained. As a result, Can be produced in large quantities.

In the present invention, an increase in occupancy due to activation cultivation of a memory T cell group including a central memory T cell and an effect memory T cell is detected and a process of performing a freezing treatment is performed to produce a lymphocyte cell group having a high memory T cell share do. Central memory T cells have the function of fighting against foreign bodies or antigens invading the body by taking the lymph nodes. Effective memory T cells have a function of capturing foreign substances or antigens belonging to the original site. Therefore, the lymphocyte cell group containing the memory T cell group as a main component can be used as a quantum immunotherapy for returning to the same living body, or as a pharmaceutical composition for administration to a third person, and the occupancy rate of the memory T cells used conventionally in the quantum immunotherapy, A much higher therapeutic effect than the low lymphocyte group can be expected.

FIG. 1 is a graph showing the results of classification of lymphocyte cells on days 1, 5, 6, and 13 of activation culture according to presence or absence of cell surface antigens (CD45RO, CD62L, CD3). The share of memory T cells (memory), the sum of T lymphocyte (CM) and CD45RO + CD62L-T lymphocyte (EM) cells in peripheral blood lymphocyte cells, exceeded 90% at day 5 but continued to grow .
FIG. 2 is a graph showing that the memory T cell group in which the occupancy rate of the cell group increased before the cryopreservation and after the activation culture was kept at a high abundance ratio in the activation and amplification cultures after freeze thawing (n = 3).
Fig. 3 is a diagram showing the outline of a process for producing a medical composition containing a memory cell of the present invention as a main component.
4, the presence ratio of each cell in the activated lymphocyte cell group (A) prepared by the conventional method without cryopreservation and the activated lymphocyte cell group (B) prepared according to the method of the present invention was compared with the cell surface antigen (CD45RO, CD62L, CD3 ) On the basis of the presence or absence of the test. PBMC: Mononuclear cells (lymphocyte-rich fraction) collected from peripheral blood vessels at the start of culture, +50: Cells immediately before addition of 50 ml of medium at day 4 after the start of culture, +150 and Freeze: Cells immediately before carrying out the second medium addition or freezing treatment BP: Cells immediately before enlargement of the culture scale at +150 without being sent to the freezing step. Life H: Cells immediately before culturing of the raw BP, Cells, TW (Thaw): Cells immediately after thawing of frozen cells, OKT: Activation culture by OKT3 antibody after completion of the expansion culture. ② Cells immediately before thawing. BP: Cells immediately before entering the amplification culture Harvest: Harvest indicates cells after amplification culture.

In the present invention, the term "memory T cell" refers to a T cell expressing CD45RO as a lymphocyte cell surface antigen. Examples of such a memory T cell include a central memory T cell or an effect memory T cell. Lymphocyte cells express various cell surface antigens on their surface and can be classified into multiple subsets based on surface antigens. For example, lymphocyte cells can be divided into T cells and B cells, which are differentiated from B cells by having CD3 surface antigens on their surface. As surface antigens, there are CD45RA antigens such as isoforms of CD45RO and CD45RO, and classified according to the presence or absence of these antigens. CD45RO is an anti-circular form that is one of the indicators of the memory cell that expresses CD45RA when the uninfected lymphocyte expressing CD45RA is stimulated with antigen, whether specific or non-specific. CD62L is a 74 kDa glycoprotein belonging to the selectin family. It is expressed in most lymphocyte cells and is a cell adhesion molecule involved in homing to the lymph nodes of lymphocytes. It is expressed on the cell surface antigen as well as in the less-experienced cells. However, when CD45RO expression is accompanied, it becomes possible to distinguish the subset of memory cells called central memory or effect memory depending on the expression intensity of CD62L. T cells with CD45RO + and CD62L + surface antigens are central memory T cells, while T cells with CD45RO + and CD62L- surface antigens are considered to be effector memory T cells.

Among CD3-positive (CD3 +) T cells expressing antigen CD3 on the surface of lymphocytes, the cell surface antigen CD45RO positive and CD62L positive T cell group (CD45RO +, CD62L +) were the central memory T cell group, cell surface antigen CD45RO positive, Negative T cell groups (CD45RO +, CD62L-) are effect memory T cell groups. These central memory T cells and effect memory T cells together constitute a memory T cell group. These cell surface antigens can be detected by staining lymphocyte cells using an antibody labeled with a fluorescent dye (anti-CD3 antibody, anti-CD45RO antibody, anti-CD62L antibody) and using a flow cytometer such as FACS Calibur (Becton Dickinson) cytometer to measure the presence and number of the fluorescent dye-labeled antibodies. In addition, lymphocyte cells can be stained with a fluorescent dye-labeled antibody or a primary antibody and a fluorescent-labeled secondary antibody, and observed with a confocal laser fluorescence microscope.

Normally, the share of memory T cells in the peripheral blood lymphocyte cell group of a normal person is around 40%, but the occupancy rate can be increased according to the manufacturing method of the present invention. In the present invention, a 'lymphocyte cell group consisting mainly of memory T cells' (hereinafter also referred to as 'memory T cell hypertrophic lymphocyte cell group') is characterized in that the occupancy rate of the memory T cells in the memory lymphocyte- , More preferably 60% or more, more preferably 70% or more, 75% or more, 80% or more, 85% or more, more preferably 90% or more, still more preferably 95% or more. Among them, it is preferable that the occupancy rate of the memory T cells in the lymphocyte cell group is 75% or more. As the lymphocyte cell group of the present invention, 70% or more, more preferably 75% or more, 80% or more, 85% or more, more preferably 90% or more preferably 95% T cells. In addition, the lymphocyte cell group mainly comprising the memory T cells of the present invention does not exclude the inclusion of T cells other than memory T cells.

In addition, in the method for producing a lymphocyte cell group containing the memory T cells of the present invention as a main component, the proliferation-regulating culture method comprising the steps (a) to (c) It is possible to provide a sufficient amount of a memory T cell-rich lymphocyte cell group for preparation of a pharmaceutical composition to be administered to three persons. Here, the high proliferation culture method is a culture method in which the number of lymphocytes in a blood sample can be increased to 500 times or more, preferably 600 times or more, 700 times or more, 800 times or more, more preferably 900 times or more , And through such a culture method, the number of total lymphocytes in a lymphocyte cell group based on, for example, a memory T cell that is obtained from 50 mL of blood is preferably 5 x 10 ⁸ or more, more preferably 8 x 10 < ⁸ , more preferably 1 x 10 ⁹ or more, and most preferably 3 x 10 ⁹ or more, of a lymphocyte cell group highly enriched in a memory T cell can be obtained. It is also preferable that the cell density of the lymphocyte cell group mainly containing the memory T cells is 5 x 10 ⁴ / mL or more, preferably 1 x 10 ⁵ / mL or more, 5 x 10 ⁵ / mL or more, more preferably 1 10 < ⁶ > cells / mL or more can be obtained.

The 'collected lymphocyte' in the step (a) can be exemplified not only from the peripheral blood or the hematopoietic stem cells obtained by a conventional method but also from the bone marrow-derived lymphocyte. However, from the viewpoint of harvesting opportunity and ease of harvesting Peripheral blood-derived lymphocyte cells are preferred. Such peripheral blood is exemplified by peripheral blood derived from a non-human or peripheral blood derived from a human, but self-derived peripheral blood is advantageously used when used in a quantum immunotherapy. From the viewpoint of prevention and treatment of tumor and recurrence thereof, and viral infection or autoimmune disease, in the case of a patient who has undergone organ or bone marrow transplantation, it is preferable that the peripheral blood is derived from a donor organs or bone marrow. In addition, it is preferable to use isolated lymphocyte cells as collected lymphocyte cells. Such isolated lymphocyte cells are collected from peripheral blood collected from blood vessels, pheresis, or the like, preferably from a vein By treating the peripheral blood in a usual manner. Separation of lymphocyte cells from peripheral blood can be accomplished by well-known lymphocyte separation methods such as discontinuous density gradient centrifugation using sucrose or a commercially available lymphocyte cell separator. Peripheral blood for lymphocyte cell separation may be supplemented with heparin or citric acid to prevent blood from clotting. The amount of the peripheral blood is not particularly limited and may be suitably set based on the burden of the donor, the blood collection time, the separation operation of the lymphocyte, etc. The amount of the blood collected at one time is about 0.01 mL to 100 mL, more preferably 5 mL to 50 mL And more preferably from 10 mL to 20 mL.

In addition, the origin of the lymphocyte is not particularly limited to a person, and examples include monkeys, horses, sheep, goats, pigs, dogs, cats, rats, mice and hamsters. However, It is preferable that it is human-derived. The lymphocyte cell group of the present invention may be derived from a subject to be administered or may be derived from an individual other than the subject to be administered. That is, the donor and recipient may or may not be the same.

Examples of the 'activation culture' in the step (a) or the step (c) include a method of increasing the occupancy of the memory T cell group through in vitro culture, for example, Japanese Patent Laid-Open No. 3-80076 (IL-2). It is more preferable to culture in the presence of IL-2 and an anti-CD3 antibody. For example, culturing in a culture vessel in which lymphocyte cells are suspended in a culture medium containing IL-2 and in which the anti-CD3 antibody is solidified can be preferably exemplified. In addition, activation of lymphocyte cells can be performed using various mitogens as needed. As the anti-CD3 antibody, any antibody that recognizes CD3 surface antigen on the lymphocyte cell surface and can promote proliferation / activation can be used. An anti-CD3 antibody used for activation of lymphocyte cells can be produced in an animal or a cell using purified CD3 molecule, but an OKT-3 antibody (eBIO), which is a commercially available product excellent in stability and ease of use, can be advantageously used . As means for activating lymphocyte cells in a system for producing lymphocyte cell groups containing a memory T cell as a main component, a means commonly used for culturing cells, for example, a cell culture container, a cell culture device, a cell culture tank, etc. .

The conditions for the culturing in the activated culture are not particularly limited as long as the lymphocyte cell group containing the memory T cells as a main component is mass-produced at a high growth rate after completion of the high proliferation culture method. Activation cultures are activated by stimulation of the anti-CD3 antibody to lymphocytes, because the memory T-cell group proliferates and increases the share of the lymphocyte population, and since the increased share is not significantly reduced, The incubation period of the culture is from 1 to 10 days, more preferably from 2 to 8 days, still more preferably from 3 to 7 days, particularly preferably from 4 to 6 days, until the sufficient number of lymphocyte cells is obtained can do. There is no particular limitation on the frequency of replacement of the medium during this incubation period. However, in order to prevent deterioration of the culture medium and deterioration of IL-2 activity, it is preferable to perform once once every 1 to 7 days, It is preferable to add an amount of about 5 times.

In step (a), activated lymphocyte cells are cultured. Typically, the activated lymphocyte cells adhere to the bottom surface of the flask and proliferate. Some cells that proliferate while floating in the culture after activation are also present. Adhered and proliferating cells are living cells as well as floating cells. A culture method in which the culture medium is the same amount as the culture medium at the start of the activation culture can be used. However, a culture method in which the culture medium is further added during the culture, and the activation culture is continued for several days, for example, for 1 to 2 days to further proliferate the lymphocyte It is possible. The amount of the culture liquid to be added is preferably 0.5 to 2 times the amount of the culture liquid at the start of the activation culture, and the culture liquid of the same amount as that of the culture liquid at the start of the activation culture can be preferably exemplified. When the above-mentioned culture medium is further added and cultured continuously, only the lymphocyte cells floating in the culture medium can be subcultured. Alternatively, all of the lymphocyte cells adhering to the culturecontainer together with the floating lymphocyte can be subcultured.

In the activation culture of step (a), there is no particular limitation on the number of lymphocyte cells at the start of culture. However, if the number of cells at the start of culture in cell culture is too small, the period until the cell growth curve occurs is delayed, If it is too much, it is not possible to adopt a method such that the total amount is increased by adding a culture solution to reach a plateau at an early stage, and therefore, a sufficient amount of cells can not be obtained. Therefore, it is necessary to adjust the number of seeding at the start so that the proliferation of the lymphocyte cell can be rapidly performed, and the proportion of the T cell group that is proliferated and occupied in the activated lymphocyte cell rapidly increases to obtain a sufficient amount of cells desirable. For example, 1.0 × 10 ⁵ to 1.0 × 10 ⁶ cells / mL, preferably 1.5 × 10 ⁵ to 6.0 × 10 ⁵ cells / mL, more preferably 4.0 × 10 ⁵ to 6.0 × 10 ⁵ cells / mL It is preferable to start culturing. The capacitor and the amount of the culture medium of the culture vessel, and can be appropriately selected in consideration of the operability, for example, there can be mentioned the use of a culture solution to the culture 50mL 5mL, or 25cm ² flask of the flask of 225cm ^2.

As described above, in the activation culture of the present invention, it is preferable that the anti-CD3 antibody is solidified and used from the viewpoints of the efficiency of propagation of lymphocyte cells and ease of manipulation. Examples of the support for solidifying the anti-CD3 antibody include various flasks, chalks, plates, and bags made of glass, polyurethane, polyolefin, or polystyrene. A commercially available plastic sterilizing cell culture flask or the like may be used, and the size thereof can be appropriately selected. The method for solidifying the antibody can be carried out by nonspecific adsorption or chemical binding, but any method may be used as long as it is a solid-phase method capable of stimulating lymphocyte cells with the solidified anti-CD3 antibody. The solid phase can be carried out by dispensing the diluted solution of the anti-CD3 antibody into a device for solidifying the solution, and leaving the solution at 4 to 37 DEG C for 2 to 24 hours. Preferably, the diluted solution of the anti-CD3 antibody is diluted to a concentration of 1 to 30 μg / mL in a physiological buffer such as sterilized dulbecco's phosphate buffer and used. The solidifying device can be stored in a refrigerator or a refrigerator (4 ° C) until use, and it is preferable to remove the diluent at the time of use and wash it with physiological buffer such as dulbecco's acid buffer at room temperature. As described above, in the activation culture of the present invention, it is preferable to use interleukin 2 (IL-2) in the culture medium for culture from the viewpoint of the propagation efficiency of lymphocyte cells. The IL-2 is preferably diluted so that the concentration of the culture medium for culture is 1 to 2000 U / mL. As the IL-2, a commercially available product can be used. IL-2 can be used by dissolving in a widely used culture medium for cell culture such as water, physiological saline solution, dulbecco phosphate buffer, RPMI-1640, DMEM, IMDA and AIM-V. In addition, it is preferable to store in a refrigerated state to prevent degradation of activity once it has been dissolved.

The culture medium in the activated culture is not particularly limited as long as it is suitable for culturing lymphocyte. It may be a synthetic medium in which amino acids, vitamins, nucleic acid bases and the like are added to a culture solution or a balanced salt solution containing biologically- Can be used. Specifically, RPMI-1640, DMEM, IMDA, and AIM-V are exemplified, among which AIM-V is particularly preferable. The medium for culture is preferably added with normal human serum because it has excellent proliferation effect. Such a medium and human serum can be commercially available. The culture can be carried out according to a general cell culture method. For example, in a CO ₂ incubator. The CO ₂ concentration is preferably 1 to 10%, particularly preferably 5%, and the temperature is preferably 30 to 40 ° C, particularly preferably 37 ° C, and the humidity is preferably 90 to 100%, particularly 95%.

In the high-proliferation culture method of the present invention, cultivation for proliferation of lymphocytes may be performed before and after the activation culture. Examples of the culture include culturing in the absence of the anti-CD3 antibody (expansion or amplification) in the presence of IL-2, and the medium components can be appropriately controlled. For example, in the amplification culture followed by activated culturing of thawed lymphocyte cells in the step (c), the suspension culturing of floating lymphocyte cells and adhesive lymphocyte cells is carried out for 4 to 13 days. The culture medium in the above-mentioned expansion or amplification culture is not particularly limited as long as it is suitable for culturing lymphocyte cells. Amino acids, vitamins, nucleic acid bases and the like are added to a culture solution or a balanced salt solution containing biologically- One synthetic medium can be used, and commercial products can be obtained. Specific examples thereof include RPMI-1640, DMEM, IMDA, and AIM-V, among which AIM-V is particularly preferable.

In addition, in the step (b), "detection based on the lymphocyte cell density" means that in the activation culture of the lymphocyte cell of the step (a) in vitro, the cell density is measured sequentially as needed, And judging whether or not to continue the activation cultivation until a certain threshold value or more is reached. When the cell density becomes a certain threshold value, for example, 4 x 10 < ⁵ > cells / mL or more, an increase in the share of memory T cells in the activated culture medium is detected. The index based on the lymphocyte cell density includes indirectly measuring the number of lymphocytes indirectly under a certain cell culture condition in addition to direct measurement of the number of lymphocyte cells using a microscope or direct measurement using a cell counting device. That is, detection is performed by indirectly deducing the number of lymphocytes based on measurement data of the number of cells when a lymphocyte of a certain cell number is cultured under a certain activation culture condition. In addition, when the lymphocytes are activated and cultured, some cells adhere to the bottom surface of the culture container to proliferate, and some cells float and proliferate in the culture medium. On the other hand, during the activation culture, the culture is continued while measuring the number of cells in the culture liquid sequentially. Therefore, the number of lymphocyte cells may be measured a plurality of times. In this case, if the lymphocyte cells attached to the bottom surface are peeled off using a pipette or the like and the peeled lymphocyte and floating lymphocyte cells are measured as a uniform suspension, the peeled lymphocyte cells may thereafter stop the proliferation. In this case, since there is a problem in terms of harvesting more lymphocyte cells, it is preferable to measure the number of suspended lymphocyte cells without peeling off the attached cells by measuring the number of cells in culture. Since the number of floating lymphocyte cells and the number of adherent lymphocyte cells or the total number of lymphocyte cells are considered to be somewhat soft, the trend of proliferation can be estimated by measuring the number of floating lymphocyte cells. Therefore, measurement of floating lymphocytes is useful in terms of simplicity of operation, prevention of contamination, and promotion of proliferation. In addition, when culturing is terminated, it is preferable to measure the total number of lymphocyte cells by peeling off the cells attached to the bottom surface of the culture container through tapping, from the viewpoint of yield and the like.

Therefore, the term "increase in the occupancy rate of the memory T cell group" in the step (b) means that the ratio of the memory T cells to the total number of activated lymphocytes in the step (a) Is 80% or more, preferably 85% or more, 90% or more, more preferably 95% or more. The share of such memory T cells can be calculated by directly irradiating cell surface antigens such as CD3, CD45RO, and CD62L with a flow cytometer using an antibody. As means for increasing the occupancy rate of the memory T cell group based on the lymphocyte cell density in the system for producing lymphocyte cell groups containing the memory T cells as a main component, a means commonly used for measuring the number of lymphocyte cells, for example, And cell counting apparatus of lymphocyte cells.

The 'cryopreservation' in step (b) can be carried out according to the general cell freezing method. For example, lymphocyte cells are suspended in cell-freezing storage products such as Bambanker (Rimportek) or CP-1 (Kyokuto Pharmaceutical Industrial Co., Ltd.), stored in freeze-preservation containers, Ltd.), temporarily stored at -80 ° C in a cryogenic cooler (SANYO Electric Co., Ltd), and transferred to a liquid nitrogen tank (MVE) two days later to be stored in liquid nitrogen as cryopreserved cells have. From the viewpoint of proliferation in the activated culture after freezing / thawing, it is preferable to adjust the number of lymphocyte cells stored in one cryopreservation vessel, and it is preferable to adjust the number of lymphocyte cells stored in one cryopreservation vessel to 1 x 10 ⁷ to 5.0 x 10 ⁷ It is desirable to keep the dog. Cells stored in a plurality of cryopreservation vessels may be used for culturing after freeze-thawing. However, from the viewpoints of ease of operation and quality control, lymphocyte cells from one cryopreservation vessel are transferred to one or more culture vessels and cultured . Examples of the means for cryopreserving lymphocyte cells in a system for producing lymphocyte cell groups containing a memory T cell as a main component include means commonly used for cryopreservation of cells, for example, a cryopreservation vessel, BICELL, a cryocooler, Tank, and the like.

The 'thawing' of the frozen lymphocytes in step (c) can be carried out according to the general thawing method of frozen cells. For example, it can be thawed by heat treatment for 4 minutes using a dry thermo unit (TAITEC CORPORATION) at 37 ° C. The thawed lymphocyte cells are transferred into an appropriate amount of the medium, and centrifuged at room temperature to remove the supernatant, thereby removing the cell freezing storage solution. Activation of lymphocyte cells after thawing may also be carried out after the activation culture in the presence of the anti-CD3 antibody, followed by continuing the amplification without activation stimulation of the anti-CD3 antibody. That is, the culture can be continued until a large number of cells are obtained in an apparatus in which the anti-CD3 antibody is not solidified, for example, a culture flask, a roller bottle, a gas permeable culture bag and the like. At this time, it is preferable to perform in the presence of interleukin 2 (IL-2). It is preferable that the culture of the lymphocyte cells under these conditions is the same as the condition of the culture with the activation stimulation such as the concentration of the human serum except that there is no stimulation of the anti-CD3 antibody. , Cost, safety, and the like. Examples of means for thawing frozen lymphocytes in a system for producing lymphocyte cell groups containing memory T cells as a main component include means commonly used for thawing frozen cells, for example, a dry thermo unit.

According to the method for producing a lymphocyte cell group containing a memory T cell of the present invention as a main component, it is possible to obtain a lymphocyte cell group containing at least 75%, preferably at least 80%, at least 85%, more preferably at least 90%, even more preferably at least 95% Cell lymphocyte population comprising 70% or more, more preferably 75% or more, 80% or more, 85% or more, more preferably 90% or more preferably 95% or more of the central memory T cells Can be obtained. In addition, the present invention can provide a quantum immunotherapeutic method comprising a lymphocyte cell group containing such a memory T cell as an active ingredient or a pharmaceutical composition for administration to a third person. The pharmaceutical composition may be used as an antitumor agent, an HIV therapeutic drug, a viral infection therapeutic drug, an autoimmune disease therapeutic drug, a transplantation cell or organ transplantation promoting agent for cell or organ transplantation, and a tumor recurrence prevention drug. In addition, since the ratio of the memory T cell group to the memory T cell group as the main component of the memory T cell of the present invention is as high as 80% or more, the memory T cell group can be obtained by using an antibody against the surface antigen present on the cell surface of the lymphocyte cell group Or the like, without suspending or separating the drug from the solution, or suspending it in a suitable solution as a medicine. In the form of the pharmaceutical composition of the present invention, parenteral administration is preferable, and a liquid form such as an injectable preparation or a drip agent suspending the lymphocyte cell group of the present invention is preferable. Particularly, injection or drip agent in which the lymphocyte cell group of the present invention is suspended in a physiological saline solution for a solution added with 0.01-5% of human serum albumin is more preferable, but is not limited thereto. As the administration method, injection into the vein or injection into the vein, artery, or the like is preferable. The amount of the administered liquid varies depending on the administration method and site to be administered, but is usually preferably 1 to 500 mL, and it is preferable that the lymphocyte cell count of the lymphocyte dose to be described later is included in this liquid amount. In addition, the pharmaceutical composition of the present invention may contain a stabilizer, a buffer component, another therapeutic agent or an adjuvant in addition to a lymphocyte group containing a memory T cell. The dosage of the pharmaceutical composition of the present invention, the number of times of administration, the period of administration, and the concentration of the lymphocyte cell group in the pharmaceutical composition of the present invention are not particularly limited and may vary depending on the condition to be administered, disease condition, It is preferable to set the dose of the lymphocyte per 1 Kg of the subject to be administered on the basis of 1 × 10 ² to 1 × 10 ⁹ . In order to make it more effective, it is preferable that the dose of lymphocytes per 1 kg of body weight is 1 x 10 ³ or more. However, since it is not expected to increase the efficacy even when the dose exceeds 5 x 10 ⁸ , Most preferably from ³ to 5 x 10 ⁸ . The frequency of administration is preferably from 1 time / day to 1 time / month, and the administration frequency is at least one time or more. The use of the pharmaceutical composition of the present invention may be combined with other surgery or medication.

Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited to the following description.

Example 1

[Activated proliferation of lymphocyte cells]

(1) preparation of peripheral blood lymphocyte cells

Heparin was added from the vein to 50 mL of peripheral blood of three normal healthy controls to collect blood. Each blood sample was transferred to a 250 mL centrifuge tube (BD FALCON; 352075) and washed with a washing solution (0.1% human albumin added physiological saline solution: 500 mL of physiological saline solution; Otsuka Pharmaceutical, 25 mL of human blood donation albumin 2.0 mL; Mitsubishi Tanabe Pharma Corporation) was added and carefully mixed and diluted 3-fold. The following operations including this operation were carried out under aseptic conditions. Blood diluted 3-fold in four 50 mL centrifuge tubes (BD FALCON; 352070) dispensed in 15 mL of Ficoll (GE healthcare bio-sciences) per sample was layered and the centrifuge tube was braked at 1800 rpm for 20 minutes at room temperature The suspension was centrifuged (hanging: KOKUSAN Co., Ltd., H-700). The mononuclear layer of the middle layer was recovered in a 50 mL centrifuge tube preliminarily dispensed with the washing solution. The lid of the centrifugal tube was closed and mixed by inverting 2 to 3 times and centrifuged at 1800 rpm for 15 minutes at room temperature. After centrifugation, the supernatant was removed, the pellet was dispersed, and the washing solution was added to make a total of 50 mL of the cell suspension. The suspension was centrifuged at 1800 rpm for 10 minutes at room temperature and 50 mL of medium 1 (AIM-V medium containing 2% human serum (Limpotech) and 350 U / mL rIL-2 (Proleukin: Novartis) (Invitrogen)) was added and suspended to prepare a cell suspension.

10 μl of the cell suspension was added to 10 ml of the cell suspension, and 10 μl of the mixed solution was added to a Neu- bauer cell counting medium (ERMA Co., Ltd., Japan) 9731), and the number of cells was calculated with a microscope (Olympus Optical Co., Ltd .; 211320).

In addition, 10 μl of the cell suspension was mixed with 5 ml of a round tube (BD FALCON; 352008) of 20 μl of trypan blue staining solution (SIGMA; 93595), and 10 μl of the mixed solution was added to Neubau blood cells The viable cells were counted by counting live cells that had not been stained with a microscope.

(2) Preparation of OKT3 solid phase flask

OKT3 (import balmaewon: Janssen-Kyowa Co., Ltd., Manufacturer: Ortho pharmaceutical: OKT3 week) was added to a physiological saline solution to prepare a 5㎍ / mL, the prepared solution to 10mL flask culture of the floor area 225cm ² (Sumitomo Bakelite Co., Ltd., MS-2180R), and the entire bottom surface thereof was covered with the OKT3 solution. After standing for more than 3 hours, the OKT3 solution in the flask was sucked into the aspirator, about 100 mL of physiological saline solution (manufacturer: Otsuka Pharmaceutical) was poured, the lid of the flask was closed and the inside liquid was discarded. Approximately 100 mL of physiological saline solution (Otsuka Pharmaceutical) was poured into the flask, and the flask was left standing at room temperature for 15 minutes. Then, the lid was closed, and the inside liquid was discarded by shaking hard for one minute. The remaining liquid in the flask and the lid was sucked carefully using an aspirator to prepare an OKT3 solidified flask. When not used on the day of preparation, a small amount of physiological saline solution was left at 4 ° C until use.

Solidification treatment was also carried out in a culture flask (Sumitomo Bakelite Co., Ltd., MS2105R) having a floor area of 25 cm ² as described above.

(3) Examination of sowing lymphocyte cell number -1

The cell suspension prepared in (1) was centrifuged at 800 rpm for 15 minutes at room temperature. The cell density was adjusted to 0.2 × 10 ⁵ / mL, 1.0 × 10 ⁵ / mL, 2.0 × 10 ⁵ / mL, 2.0 × 10 ⁶ / mL and 6.0 × 10 ⁶ / mL, respectively, It was prepared. 5 mL of the cell suspension of each cell density was inoculated in a 25 cm ² OKT3 solid-phase flask prepared in the above (2), and the cells were inoculated in a TE-HER CO ₂ incubator (HIRASAWA) at 37.0 ± 0.5 ° C., At a humidity of 95.0 ± 5.0% and a CO ₂ concentration of 5.0 ± 0.2%. During the culture period, the number of floating cells and the survival rate were appropriately measured / calculated in the same manner as in (1), and the medium 1 was added appropriately while observing the change in color of the culture solution according to the growth state, and the culture was continued for 10 days. At the end of the incubation, the culture flask was tapped to recover the lymphocyte cells attached to the bottom surface, and the total lymphocyte cell count and survival rate were measured / calculated in the same manner as in (1).

result:

In Table 1, since seeding cell density of 0.2 × 10 ⁵ pieces / were mL work when 10 days has even not sufficient cell culture is not obtained, and if ⁶ × 10 6 pieces / mL day proliferation is decreased survival rates also had poor, both The seeding cell density was determined to be inadequate. In addition, the seeding cell density was decided to be reviewed between 1.0 × 10 ⁵ / mL and 2.0 × 10 ⁶ / mL of sowing cell density with a survival rate of 90% or less. In this study, medium 1 was added appropriately with the change in hue of the culture medium and the number of floating cells as indicators. In order to simplify the operation and obtain more rapid cell proliferation, it is possible to determine the timing of addition of the medium from the cell density and the number of incubation days So we will review the timing of addition of the culture medium.

Dissociation cell density and proliferation and survival rate Seeding cell density 0.2 x 10 < ⁵ > cells / mL 1 x 10 < ⁵ > cells / mL 2 x 10 ⁵ cells / mL 2 x 10 < ⁶ > cells / mL 6 x 10 < ⁶ > cells / mL Days of culture Specimen Total cell number Survival rate Total cell number Survival rate Total cell number Survival rate Total cell number Survival rate Total cell number Survival rate One One 1.00 x 10 ⁵ 100 5.00 x 10 ⁵ 100 1.00 x 10 ⁶ 100 1.00 x 10 ⁷ 100 3.00 x 10 ⁷ 100 2 1.00 x 10 ⁵ 100 5.00 x 10 ⁵ 100 1.00 x 10 ⁶ 100 1.00 x 10 ⁷ 100 3.00 x 10 ⁷ 100 3 1.00 x 10 ⁵ 100 5.00 x 10 ⁵ 100 1.00 x 10 ⁶ 100 1.00 x 10 ⁷ 100 3.00 x 10 ⁷ 100 5 One 1.17 × 10 ⁵ 77.8 5.83 × 10 ⁵ 94.6 1.97 × 10 ⁶ 90.8 6.47 × 10 ⁶ 73.3 7.95 × 10 ⁶ 76.6 2 5.00 x 10 ⁴ 60 5.33 × 10 ⁵ 100 1.75 x 10 ⁶ 93.8 6.18 × 10 ⁶ 81.3 1.17 x 10 ⁷ 78.3 3 8.33 x 10 ⁴ 100 1.52 × 10 ⁶ 93.8 2.18 × 10 ⁶ 94.2 6.80 × 10 ⁶ 90.9 1.47 x 10 ⁷ 90.2 6 One 3.33 × 10 ⁵ 100 2.03 × 10 ⁶ 94.6 2.60 × 10 ⁶ 95.7 5.80 × 10 ⁶ 88.8 4.57 × 10 ⁶ 80.1 2 1.00 x 10 ⁵ 75 7.83 × 10 ⁵ 88.7 3.85 × 10 ⁶ 95.9 3.53 × 10 ⁶ 90.6 2.43 × 10 ⁶ 47.4 3 5.17 × 10 ⁵ 100 4.50 × 10 ⁶ 93.8 6.06 × 10 ⁶ 94.3 4.37 × 10 ⁶ 84.5 4.97 × 10 ⁶ 82.3 7 One 5.83 × 10 ⁵ 97.2 5.83 × 10 ⁶ 93.1 1.01 x 10 ⁷ 93.5 7.33 × 10 ⁶ 89.8 5.04 × 10 ⁶ 72.5 2 2.33 × 10 ⁵ 77.8 6.67 × 10 ⁶ 94.8 1.00 x 10 ⁷ 94.3 2.25 × 10 ⁶ 73.8 2.15 × 10 ⁶ 50.6 3 1.27 × 10 ⁶ 93.8 4.83 × 10 ⁶ 93.5 6.35 × 10 ⁶ 76 8 One 7.33 × 10 ⁵ 86.3 5.57 × 10 ⁶ 94.4 1.11 x 10 ⁷ 91.1 6.83 × 10 ⁶ 86.3 6.85 × 10 ⁶ 64.6 2 4.67 × 10 ⁵ 93.3 6.50 × 10 ⁶ 93.5 5.83 × 10 ⁶ 92.1 8.45 × 10 ⁶ 77.5 6.05 × 10 ⁶ 64 3 2.20 x 10 ⁶ 94.3 1.31 × 10 ⁷ 94.9 2.94 × 10 ⁷ 94.4 2.98 × 10 ⁷ 83.4 8.93 × 10 ⁶ 76.6 9 One 4.75 × 10 ⁶ 89.7 1.27 x 10 ⁷ 94.4 3.55 x 10 ⁷ 91.8 4.58 × 10 ⁷ 86.1 1.44 × 10 ⁷ 70.3 2 9.33 × 10 ⁵ 90.3 1.60 x 10 ⁷ 81.2 3.15 x 10 ⁷ 91.3 1.77 × 10 ⁷ 84.9 1.23 x 10 ⁷ 74.8 3 3.03 × 10 ⁶ 96.8 1.57 x 10 ⁷ 90.4 2.09 × 10 ⁷ 77.2 10 One 2.80 × 10 ⁶ 62.5 2.08 × 10 ⁷ 87.9 2.47 x 10 ⁷ 66.7 2 1.23 × 10 ⁶ 80.4 2.15 × 10 ⁷ 79 2.38 × 10 ⁷ 90.5 2.03 × 10 ⁷ 63.1 3 7.97 × 10 ⁶ 96.8 4.07 x 10 ⁷ 92.6

(4) Review of culture medium addition timing

Peripheral blood lymphocyte cells were prepared in the same manner as in (1), and the number of cells was measured.

The prepared lymphocyte cells were centrifuged in the same manner as in (3), and cell densities of 0.5 × 10 ⁵ / mL, 1 × 10 ⁵ / mL, 2.0 × 10 ⁵ / mL and 4 × 10 ⁵ / ⁵ / mL, and 8 × 10 ⁵ / mL, respectively. 50 mL of the cell suspension of each cell density was added to each well of 225 cm ² prepared in (2) The culture medium was dispensed into an OKT3 solid phase flask (Sumitomo Bakelite Co., Ltd., MS-2180R), and the medium 1 was used to culture the cells in a TE-HER CO ₂ incubator (HIRASAWA) at a temperature of 37.0 賊 0.5 캜, The culture was started at a CO ₂ concentration of 5.0 ± 0.2%. From the viewpoint of culture efficiency, 50 mL of the culture solution was added to each flask on the third day of the culture, and the number of floating cells before the addition of the culture solution and the total number of cells at the fourth day after the addition were measured by the same method as in (1) .

result:

In Table 2, the number of the sowing 2.0 × 10 ⁷ cells (more 4.0 × 10 ⁵ / mL) or ^{4.0 × 10 7 (8. 0 ×} 10 5 pieces / mL) by the number of seeded cells, the total number of cells after incubation in the case of And the proliferation rate was 2.5 times or more. That is, the fourth day (1.1 × 10 ⁸ and 1.3 × 10 ⁸ ) of the specimen 1 and the fourth day (1.0 × 10 ⁸ ) of the specimen 3 are. The sample to the total number of cells obtained after the culture solution was added in excess of 10 ^8, the number of floating cells prior to adding the culture of the samples 1.9 × 10 ⁷ dogs and 6.3 × 10 ⁷ (3 days after the subject 1) and 2.0 × 10 ⁷ pieces (specimen 3). Here, after 3 days from the start of culturing, when the number of floating cells reached 3.8 × 10 ⁵ cells / mL or 4.0 × 10 ⁵ cells / mL, the culture medium was added.

Seeding cell number
(Seeding cell density) Specimen 1 Specimen 2 Specimen 3 Day 3 Day 4 Day 3 Day 4 Day 3 Day 4 2.5x10 ⁶
(0.5 x 10 < ⁵ > cells / mL) 2.3x10 ⁶ 8.3x10 ⁶ 8.3x10 ⁵ 3.3 x 10 ⁶ 3.3x10 ⁵ 2.7x10 ⁶ 5.0x10 ⁶
(1.0 x 10 < ⁵ > cells / mL) 5.8 x 10 ⁶ 1.4x10 ⁷ 3.3 x 10 ⁶ 1.1x10 ⁷ 1.7 x 10 ⁶ 1.1x10 ⁷ 1.0x10 ⁷
(2.0 x 10 < ⁵ > cells / mL) 1.1x10 ⁷ 4.6 x 10 ⁷ 2.7x10 ⁶ 1.5x10 ⁷ 3.3 x 10 ⁶ 3.0x10 ⁷ 2.0x10 ⁷
(4.0 x 10 < ⁵ > cells / mL) 1.9x10 ⁷ 1.1 x ^{10 8}
(5.5 times) 6.2 x 10 ⁶ 5.0 x 10 ⁷ 7.7 x 10 ⁶ 5.7x10 ⁷ 4.0x10 ⁷
(8.0 x 10 < ⁵ > cells / mL) 6.3x10 ⁷ 1.3x10 ⁸
(3.3 times) 2.8 x 10 ⁶ 2.0x10 ⁷ 2.0x10 ⁷ 1.0 x ^{10 8}
(2.5 times)

(5) Examination of number of seeding lymphocyte cells -2

Peripheral blood lymphocyte cells were prepared in the same manner as in (1), and the number of cells was measured.

The prepared lymphocyte cells were centrifuged in the same manner as in Examination 1 of sowing lymphocyte cell number (3), and the cell density was 1.0 × 10 ⁵ / mL, 1.5 × 10 ⁵ / mL, 2.0 × 10 ⁵ cells / mL, 4.0 × 10 ⁵ cells / mL, 6 × 10 ⁵ cells / mL, and 8 × 10 ⁵ cells / mL. 25cm ² of each cell suspension was adjusted to 5mL in the above (2) OKT3 solid phase flask, and cultured in Medium 1 (TEOS-HER CO ₂ incubator, HIRASAWA) at 37.0 ± 0.5 ° C, 95.0 ± 5.0% humidity and 5.0 ± 0.2% CO ₂ . After the start of culture, the number of floating cells and the cell viability were gradually measured in the same manner as in (1). When the cell density reached 4 × 10 ⁵ cells / mL or more, ⁵ mL of the culture was added to each flask and the cells were cultured for a maximum of 6 days . At the end of the culture, lymphocytes attached to the bottom surface were treated in the same manner as in (3) to collect all the cells, and the total cell number and survival rate were measured / calculated in the same manner as in (1).

result:

In this study, it was aimed to obtain the most cells by performing the activation culture in the shortest number of days.

At the seeding cell density of 1.0 × 10 ⁵ cells / mL, the number of floating cells did not reach 4 × 10 ⁵ cells / mL in two of the three specimens even on the fifth day of culture. Plant cell density of 1.0 × 10 ⁵ gae / mL of the sample 3, 1.5 × 10 ⁵ gae / mL in sample 1-3, 2.0 × 10 ⁵ gae / mL in sample 1-3, and 4.0 × 10 ⁵ gae / mL In specimen 1, the density of suspended cells exceeded 4 × 10 ⁵ cells / mL was in the fifth day, and when 5 mL of the culture medium was added, the total cell count exceeded 1.2 × 10 ⁷ in the majority of cells on the next day. The specimens 2 and 3 of 4.0 × 10 ⁵ cells / mL, the specimens 1 to 3 of 6.0 × 10 ⁵ cells / mL, and the specimens 1 to 3 of 8.0 × 10 ⁵ cells / mL all had a floating cell density of 4 × 10 ⁵ cells / mL, and on the fifth day after the addition of the culture medium, the total cell count exceeded 1.2 × 10 ⁷ cells in all.

In summary, at the seeding cell density of 1.5 × 10 ⁵ cells / mL at the start of culture, the culture was terminated at 4 × 10 ⁵ cells / mL on the fifth day, and the culture was terminated on the next day. At 4.0 × 10 ⁵ cells / mL, in excess of 4 × 10 ⁵ gae / mL next day and the following day were terminate the incubation, 6.0 × 10 ⁵ gae / mL and 8.0 × 10 ⁵ gae / mL in excess of any example 4 × 10 ⁵ gae / mL on day 4 And cultivation was terminated. Thus, in order to shorten the culturing period, it was judged to be appropriate to set the seeding cell density to 4.0 × 10 ⁵ / mL or more and 8.0 × 10 ⁵ / mL or less.

 Relationship between cell number and proliferation at the start of culture Seeding cell number
(/ mL) Specimen Day 4 Day 5 Day 6 Cell number Cell density
(Number of cells / mL) Cell number Cell density
(Number of cells / mL) Cell number Cell density
(Number of cells / mL) 1.0x10 ⁵ One n / a n / a 6.5x10 ⁵ 1.3x10 ⁵ 2.6 x 10 ⁶ 5.1x10 ⁵ 2 n / a n / a 1.8 x 10 ⁶ 3.6x10 ⁵ 4.8x10 ⁶ 9.6x10 ⁵ 3 n / a n / a 2.5x10 ⁶ 5.0x10 ⁵ 9.1x10 ⁶ 9.1x10 ⁵ 1.5x10 ⁵ One n / a n / a 2.1 x 10 ⁶ 4.1 x 10 ⁵ 9.8x10 ⁶ 9.8x10 ⁵ 2 n / a n / a 3.5x10 ⁶ 7.0x10 ⁵ 1.2x10 ⁷ 1.2x10 ⁶ 3 n / a n / a 3.1x10 ⁶ 6.1x10 ⁵ 1.2x10 ⁷ 1.2x10 ⁶ 2.0x10 ⁵ One 1.2x10 ⁶ 2.4x10 ⁵ 3.2 x 10 ⁶ 6.4x10 ⁵ 1.2x10 ⁷ 1.2x10 ⁶ 2 1.7 x 10 ⁶ 3.3x10 ⁵ 5.5x10 ⁶ 1.1x10 ⁶ 1.4x10 ⁷ 1.4x10 ⁶ 3 1.6x10 ⁶ 3.2x10 ⁵ 5.7x10 ⁶ 1.1x10 ⁶ 1.3x10 ⁷ 1.3x10 ⁶ 4.0x10 ⁵ One 1.7 x 10 ⁶ 3.3x10 ⁵ 4.2 x 10 ⁶ 8.3x10 ⁵ 1.3x10 ⁷ 1.3x10 ⁶ 2 3.0x10 ⁶ 6.0x10 ⁵ 1.2x10 ⁷ 1.2x10 ⁶ n / a n / a 3 2.4 x 10 ⁶ 4.8x10 ⁵ 1.1x10 ⁷ 1.1x10 ⁶ n / a n / a 6.0x10 ⁵ One 2.1 x 10 ⁶ 4.2 x 10 ⁵ 1.4x10 ⁷ 1.4x10 ⁶ n / a n / a 2 3.6 x 10 ⁶ 7.1 x 10 ⁵ 1.4x10 ⁷ 1.4x10 ⁶ n / a n / a 3 2.7x10 ⁶ 5.3x10 ⁵ 1.2x10 ⁷ 1.2x10 ⁶ n / a n / a 8.0x10 ⁵

One 3.1x10 ⁶ 6.2x10 ⁵ 1.2x10 ⁷ 1.2x10 ⁶ n / a n / a 2 5.0x10 ⁶ 9.9x10 ⁵ 1.5x10 ⁷ 1.5x10 ⁶ n / a n / a 3 4.9x10 ⁶ 9.8x10 ⁵ 1.2x10 ⁷ 1.2x10 ⁶ n / a n / a n / a: not available

Example 2

[Transition of lymphocyte surface antigen upon activation culture]

(1) Preparation and Activation of Lymphocyte Cells

Peripheral blood lymphocyte cells were prepared in the same manner as in (1) of Example 1, and the number of cells was measured.

The prepared lymphocyte cells were centrifuged in the same manner as in (3) of Example 1, and the medium 1 was used to prepare the cells so that the cell density of the lymphocyte cells was 6 × 10 ⁵ cells / mL. 50 mL of this cell suspension was dispensed into a 225 cm ² OKT3 solid-phase flask (Sumitomo Bakelite Co., Ltd.: MS-2180R) prepared in (2) of Example 1, and in the same manner as in (3) 1, the cells were cultured in a TE-HER CO ₂ incubator (HIRASAWA) at a temperature of 37.0 ± 0.5 ° C., a humidity of 95.0 ± 5.0%, and a CO ₂ concentration of 5.0 ± 0.2%. At the time of culture, floating cells were collected from time to time, and the number of floating cells was measured in the same manner as in (1) of Example 1. On the 4th day of cultivation, since it became 4 × 10 ⁵ cells / mL or more, 50 mL of medium 1 was added and culturing was continued. On the fifth day of culture, 50 ml of Medium 1 was again added and the culture was continued. On the 6th day of culture, the lymphocyte cells attached to the bottom surface of the solid-phase flask were peeled off by tapping to obtain a cell suspension. This 25 mL was dispensed into a 225 cm ² flask in which OKT3 was not solidified, and 75 mL of Medium 1 was further added to continue culturing. On the 9th day of culture, 100 mL of Medium 1 was added and the culture was continued until the 13th day. On the 13th day of culture, the lymphocyte cells attached to the bottom surface of the flask were peeled off by tapping, collected as a cell suspension, and cultured.

At this time, part of the cell suspension was collected at the start of culture, on the 5th day of the culture (immediately before addition of the medium), on the 6th day (before / after the adherent cell detachment), and on the 13th day and immediately analyzed the surface antigen of the collected lymphocyte . In addition, it can be seen that almost 100% of the lymphocyte cells are CD3-positive cells (Non-Patent Document 3)

(2) Method of analysis of lymphocyte surface antigen

Each 5 mL of the cell suspension collected in the above (1) was taken in a 15 mL centrifuge tube (BD FALCON; 352097) and centrifuged at 1800 rpm for 5 minutes at room temperature. To each of the obtained pellets, albumin solution (Beckman Coulter: IsoFlow) was added to prepare each cell suspension at 1.0 x 10 ⁷ cells / mL. 10 [mu] l of each antibody labeled with a fluorescent dye (anti-CD3 antibody Becton Dickinson 349201, anti-CD45RO antibody conjugate 340438, anti-CD62L antibody Becton Dickinson Pharmingen Co. 555544) was dispensed into a 5 ml round tube (BD FALCON) 50 占 퐇 of the suspension was dispensed into each tube, followed by shading and standing reaction at 4 占 폚 for 30 minutes. To each tube after the reaction, 2 mL of albumin was added, and the mixture was centrifuged at 1800 rpm for 5 minutes at room temperature. The supernatant was aspirated and 500 μl of CellFix (Becton Dickinson) was added to each pellet to make a suspension. 10,000 cells were measured for a target antibody-stained lymphocyte cell population using a flow cytometer (Becton Dickinson; FACS Calibur) .

(3) Analysis of antibody-stained lymphocyte populations

Analysis of the antibody-stained lymphocyte cell population prepared by the method of (2) above was carried out.

result:

Typically, the ratio of CD45RO + CD62L + T lymphocytes to CD45RO + CD62L-T lymphocytes in peripheral blood lymphocytes is about 20-40% (Table 4). However, by carrying out the activation culture, its occupation rate already exceeded 90% on the fifth day and exceeded 95% on the sixth day. However, when the cultivation was continued, the increased percentage of the cells was found to decrease to the original level as 40% (Table 4, Fig. 1). CD45RO +, CD62L +, CD3 + mediated T cells, CD45RO +, CD62L-, and CD3 + effected T cells, and CD45RO +, CD62L + and CD3 + Effect memory T cells, CM + EM or memory represent CD45RO +, CD62L +/-, CD3 + memory T cells.

Therefore, the increase of the occupancy rate of CD45RO +, CD62L + T lymphocytes, CD45RO +, and CD62L-T lymphocytes could be confirmed the day after the addition of the medium with the density of floating cells in the activated culture being 4 × 10 ⁵ / mL or more. Here, in order to increase the number of harvesting memory cells, the activation culture was stopped before the decrease of the memory T cell share after the day after the addition of the medium with the density of floating cells being 4 x 10 ⁵ / mL or more, The lymphocyte cells attached to the surface were peeled off by tapping to make floating cells, and all the cells were recovered.

The percentage of lymphocyte surface antigen occupancy during activation culture (%) culture
term CD45RO-
CD62L +
CD3 +





(Naive) CD45RO-
CD62L-
CD3 +





(Effector) CD45RO +
CD62L +
CD3 +




(Central
Memory: CM) CD45RO +
CD62L-
CD3 +




(Effector Memory: EM) CD45RO +
CD62L +
CD3 +

CD45RO +
CD62L-
CD3 +

(CM + EM) 1 day

Specimen 1 52.03 6.95 30.36 10.66 41.02 Specimen 2 46.68 5.68 27.94 19.7 47.64 Specimen 3 28.31 12.43 23.7 35.56 59.26 5 days

Specimen 1 4.02 6.75 80.53 12.08 92.61 Specimen 2 7.12 6.17 81.61 8.93 87.08 Specimen 3 6.31 4.56 69.75 23.76 89.13 6 days

Specimen 1 1.26 2.97 82.85 12.91 95.76 Specimen 2 1.63 1.61 86.13 10.63 96.76 Specimen 3 4.01 6.22 79.53 10.24 89.77 13th

Specimen 1 49.26 5.89 42.65 2.2 44.85 Specimen 2 46.69 9.35 42 1.96 43.96 Specimen 3 39.79 14.08 42.53 3.59 46.12 Blood drawing
Immediately
Specimen 1 27.88 7.4 24.66 10.86 35.5 Specimen 2 49.4 8.22 10.14 12.29 22.43 Specimen 3 38.99 6.88 19.3 14.91 34.21

Example  3

[Examination of density of frozen cells in freezing process]

The preparation containing the T cell group obtained according to the method of the present invention is used for treatment, and it is more efficient that the number of lymphocytes finally obtained is large. Here, the possibility that the number of cells per tube at the time of cryopreservation affects the subsequent activation amplification culture was examined.

(1) Examination of density of frozen cells -1 Activation culture

Peripheral blood lymphocyte cells were prepared in the same manner as in (1) of Example 1, and the number of cells was measured.

The prepared lymphocyte cells were centrifuged in the same manner as in (3) of Example 1, and the medium 1 was used to prepare the cells so that the cell density of the lymphocyte cells was 6 × 10 ⁵ cells / mL. 50 mL of this cell suspension was dispensed in a 225 cm ² OKT3 solid-phase flask (Sumitomo Bakelite Co., Ltd., MS-2180R) prepared in (2) of Example 1, -HER CO ₂ incubator (HIRASAWA Co., Ltd.) at a temperature of 37.0 ± 0.5 ° C, a humidity of 95.0 ± 5.0% and a CO ₂ concentration of 5.0 ± 0.2%. On the third day after the start of culture, 50 ml of medium 1 was added at a rate of more than 4 x 10 < ⁵ > cells / ml, and the culture was continued until the next day. On the fourth day after the start of the culture, the activated culture was stopped, and the lymphocyte cells attached to the bottom surface of the flask were peeled off by tapping to obtain suspended cells, and all cells were collected to obtain a cell suspension by the medium 1. The number of cells in the suspension was measured in the same manner as in item (1) of Example 1.

(2) Examination of freezing cell density -2 Freezing preservation

Based on the cell density measured in (1) above, the number of lymphocyte cells per one frozen storage tube (CORNING) was 0.5 × 10 ⁷ , 1.0 × 10 ⁷ , 3.0 × 10 ⁷ , 5.0 x 10 < ⁷ >, and the mixture was divided into a 15-mL centrifuge tube and centrifuged at 1200 rpm for 5 minutes at room temperature. After centrifugation, the supernatant in each centrifugal tube was removed, and the obtained pellet was dispersed. The cell freezing storage solution (CP-1: Kyokuto Pharmaceutical Industrial Co., Ltd.) was added so as to have a liquid amount of 1.8 mL per one frozen storage tube The cell was suspended and dispensed into a cryopreservation tube. The cryopreservation tube was placed in BICELL (Nihon Freezer Co., Ltd.) and temporarily stored at -80 ° C in a cryogenic cooler (SANYO Electric Co., Ltd) And transferred into a nitrogen tank (MVE) to be stored as cryopreserved cells.

(3) Examination of density of frozen cells -3 Thawing treatment

Cryopreserved tubes frozen at each cell density were taken out of the liquid nitrogen tank and thawed frozen lymphocytes by heating in a dry thermo unit (TAITEC CORPORATION) set at 37 ° C for 4 minutes. Thawed lymphocyte cells were transferred into a 15-mL centrifuge tube while suspended in 10 mL of Medium 1, and centrifuged at 1200 rpm for 3 minutes at room temperature. After centrifugation, the supernatant was removed, the pellet was dispersed, and 10 mL of Medium 1 was added to make a cell suspension, and the number of cells was measured in the same manner as in Example 1 (1). Since thawed lymphocyte cells differ in the number of cells per tube, if suspension seeding is carried out in the same volume of the same medium, the difference in the number of seeding cells may affect the proliferation. To eliminate this possibility, in the process of sowing and culturing thawed cells, the number of lymphocyte cells is measured before and during sowing, and the cell density at the time of sowing as well as the amount of the culture liquid to be added are adjusted so that the cell density becomes almost constant (7.5 x 10 < ⁵ > cells / mL). This method of adjusting the liquid amount at the start of culturing with different cell numbers according to the frozen storage tube was also employed in the following expansion, activation, and amplification cultures. Table 5 shows the amounts of culture liquid and added liquid in each step.

With the operation at the time of thawing, the suspension having a liquid amount shown in Table 5 was inoculated into each well of a 24-well multiwell plate (Sumitomo Bakelite Co., Ltd.) at a rate of 2 mL per well. And cultured in a carbon dioxide gas incubator having a humidity of 95% and a CO ₂ concentration of 5.0% for 2 days.

The amount of the culture liquid in each step and the amount of the added medium 1 Cell number /
Cryopreservation tube Culture conditions Thawing Expansion culture Activation culture ② Amplification culture ① Amplification culture ② 0.5 × 10 ⁷ Culture vessel 24well 25 cm ² 75 cm ² 225 cm ² 225cm ² × 2 Amount of culture liquid 6 mL 14 mL 36 mL 149 mL 295 mL Amount of added liquid 8 mL 22 mL 113 mL 146 mL 1.0 x 10 ⁷ Culture vessel 24well 75 cm ² 225 cm ² 225cm ² × 2 1L bag Amount of culture liquid 13 mL 30 mL 78 mL 322 mL 639 mL Amount of added liquid 17 mL 48 mL 244 mL 317 mL 3.0 x 10 ⁷ Culture vessel 24well 225 cm ² 225 cm ² 1L bag 1L bag × 2 Amount of culture liquid 40 mL 90 mL 240 mL 1,000 mL 2,000 mL Amount of added liquid 50 mL 150 mL 760 mL 1,000 mL 5.0 × 10 ⁷ Culture vessel 24well 225 cm ² 225cm ² × 2 1L bag 1L bag × 2 Amount of culture liquid 66 mL 149 mL 396 mL 1,634 mL 3,238 mL Amount of added liquid 83 mL 247 mL 1,238 mL 1,604 mL

(4) Expansion and activation culture

On the third day, the lymphocyte cells cultured after the thawing treatment in (3) above were transferred to a plain flask in a 24-well multi-well plate and cultured in a well of a 24-well multiwell plate And the remaining cells were recovered, and the liquid amount in the flask was adjusted to the liquid amount described in Table 5. [ Each flask was again cultured in a TE-HER CO ₂ incubator (HIRASAWA Co., Ltd.) for 2 days in a carbon dioxide gas incubator at a temperature of 37.0 ± 0.5 ° C., a humidity of 95%, and a CO ₂ concentration of 5.0% (Table 5, expanded culture).

After the expansion, the cell suspension in the culture flask was transferred into an OKT3 solid-phase flask having the capacity shown in Table 5 prepared in the same manner as in item (2) of Example 1 so as to carry out the activation culture, The cells were washed and the remaining cells were finally recovered and the liquid amount in the OKT3 solidification flask was adjusted to the liquid amount described in Table 5. Each flask was maintained in a TE-HER CO ₂ incubator (HIRASAWA Co., Ltd.) %, And CO ₂ concentration of 5.0% (Table 5, activated cultivation 2).

After the activation culture, each OKT3 solid phase flask was tapped and the cells attached to the bottom surface were peeled off to prepare a cell suspension. The obtained cell suspension was transferred into a new plain culture flask (Sumitomo Bakelite Co., Ltd.) or a new gas permeable culture bag (1000 mL, Kohjin Bio Co., Ltd. 16087528) shown in Table 5, (AIM-V medium: Invitrogen containing 1% human serum (Limpotech), 175 U / ml rIL-2 (Proleukin; Novartis)) with the additive liquid amount shown in Table 5, The cells were incubated for 3 days in a TE-HER CO ₂ incubator (HIRASAWA Co., Ltd.) at a temperature of 37.0 ± 0.5 ° C., a humidity of 95% and a CO ₂ concentration of 5.0% .

After the above-mentioned amplification culture (1), Medium 2 of the additive amount described in Table 5 was added to the culture container, and the culture was continued for another 4 days in a carbon dioxide gas incubator at 37 캜, a humidity of 95%, and a CO ₂ concentration of 5.0%. Here, when the flask was newly added to the culture container, the medium 2 of the added liquid amount was added to the flask under culture, the flask was shaken vigorously to stir the cell suspension, and the culture was continued by transferring half of the capacity into the new flask. When the culture container was transferred from the flask to the bag, the medium 2 of the added liquid amount was dispensed into two flasks, and the flask was vigorously shaken to agitate the cell suspension, and all of the contents were transferred to a new bag and the culture was continued. When a new bag was used as a culture container, the medium 2 of the additive amount described in Table 5 was added to a culture bag to be newly used, and this was adhered to a culture bag cultured in the above amplification culture 1 under aseptic conditions, The contents were mixed well and cultured again (Table 5, Amplification culture 2).

(3) and (4), the number of cells and the survival rate of each specimen were measured / calculated in the same manner as in (1) of Example 1. The results are shown in Table 6.

RESULTS: The number of cells stored in the cryopreservation tube was frozen and adjusted by setting each value, but there was no significant difference among the three samples in each treatment step. However, the memory T lymphocyte cell group prepared by the method of the present invention is for therapeutic use, and from the viewpoint of efficiency, it is preferable that the number of cells obtained by a series of cultures is large. Therefore, since a suitable culture scale is preferable, it was judged that 1.0 x 10 ⁷ to 5.0 x 10 ⁷ cells per frozen storage tube were valid. In the steps (1) to (4) of the present Examples, the cell density was controlled to be constant according to the additive liquid as shown in Table 5, in order to exclude the influence of the sowing cell density on the culture growth. On the other hand, 1 × 10 ⁸ dogs can during culture starting cells of the amplification incubation ①, 2 × 10 ⁸ gae, 3 × 10 set ^{to eight,} and the constant during culture starting liquid volume subjected to the culture results, 1 × 10 ⁸ gae the Although the amplification was delayed by about 2 days, it was confirmed that a sufficient amount of the memory T lymphocyte cell group was obtained after the step of the amplification culture (2) without significant difference at 2 × 10 ⁸ and 3 × 10 ⁸ . When the number of cells per one frozen storage tube is 1.0 × 10 ⁷ to 5.0 × 10 ⁷ , this condition can be satisfied.

Cell number, survival rate and cell density (actual value) after the thawing treatment step Processing date Process Name
Specimen
The cell number, survival rate (%) and cell density (actual value) 0.5x10 ⁷ / pcs 1.0x10 ⁷ / pcs 3.0x10 ⁷ / pcs 5.0x10 ⁷ / pcs One Defrosting One 4.14x10 ⁶ (93.24)
(6.90 x ¹⁰ < ⁵ > / mL) 8.71x10 ⁶ (93.06)
(6.70 x ¹⁰ < ⁵ > / mL) 2.80x10 ⁷ (94.59)
(7.00 x ¹⁰ < ⁵ > / mL) 4.82x10 ⁷ (94.81)
(7.30 x ¹⁰ < ⁵ > / mL) 2 4.14x10 ⁶ (94.52)
(6.90 x ¹⁰ < ⁵ > / mL) 9.36x10 ⁶ (96.00)
(7.20 x ¹⁰ < ⁵ > / mL) 2.88x10 ⁷ (96.00)
(7.20 x ¹⁰ < ⁵ > / mL) 4.82x10 ⁷ (94.81)
(7.30 x ¹⁰ < ⁵ > / mL) 3 4.08x10 ⁶ (95.77)
(7.40 x ¹⁰ < ⁵ > / mL) 9.62x10 ⁶ (97.37)
(7.40 x ¹⁰ < ⁵ > / mL) 2.88x10 ⁷ (94.74)
(7.20 x ¹⁰ < ⁵ > / mL) 4.95x10 ⁷ (94.94)
(7.50 x ¹⁰ < ⁵ > / mL) 3

Expansion culture One 9.05x10 ⁶ (98.53)
(6.46 x ¹⁰ < ⁵ > / mL) 1.52x10 ⁷ (98.11)
(5.07 x ¹⁰ < ⁵ > / mL) 7.74x10 ⁷ (97.73)
(8.60 x ¹⁰ < ⁵ > / mL) 1.26x10 ⁸ (97.70)
(8.46 x ¹⁰ < ⁵ > / mL) 2 7.16 x 10 ⁶ (96.36)
(5.11x10 < ⁵ > / mL) 1.61x10 ⁷ (96.49)
(5.37 x ¹⁰ < ⁵ > / mL) 4.68x10 ⁷ (94.55)
(5.20 x ¹⁰ < ⁵ > / mL) 8.32x10 ⁷ (93.33)
(5.58 x ¹⁰ < ⁵ > / mL) 3 1.04x10 ⁷ (97.47)
(7.42 x ¹⁰ < ⁵ > / mL) 2.31x10 ⁷ (96.34)
(7.70 x ¹⁰ < ⁵ > / mL) 6.39 x 10 ⁷ (97.26)
(7.10x10 < ⁵ > / mL) 1.11x10 ⁸ (97.40)
(7.44 x ¹⁰ < ⁵ > / mL) 5

Activation culture ② One 2.97x10 ⁷ (97.35)
(8.25 x ¹⁰ < ⁵ > / mL) 6.18 x 10 ⁷ (97.69)
(7.92x10 < ⁵ > / mL) 2.68x10 ⁸ (98.03)
(1.12 x ¹⁰ < ⁶ > / mL) 3.76x10 ⁷ (98.06)
(9.55 x ¹⁰ < ⁵ > / mL) 2 2.88x10 ⁷ (96.38)
(8.00 x ¹⁰ < ⁵ > / mL) 7.65x10 ⁷ (97.75)
(9.81 x ¹⁰ < ⁵ > / mL) 2.91x10 ⁸ (97.29)
(1.21 x ¹⁰ < ⁶ > / mL) 4.57x10 ⁸ (97.78)
(1.15 x ¹⁰ < ⁶ > / mL) 3 3.02x10 ⁷ (96.97)
(8.39 x ¹⁰ < ⁵ > / mL) 8.29 × 10 ⁷ (97.25)
(1.01 x ¹⁰ < ⁶ > / mL) 3.04x10 ⁸ (97.69)
(1.26 x ¹⁰ < ⁶ > / mL) 4.43x10 ⁸ (97.70)
(1.12 x ¹⁰ < ⁶ > / mL) 7

Amplification culture ①

One 3.49x10 ⁷ (94.17)
(2.34 x ¹⁰ < ⁵ > / mL) 8.11x10 ⁷ (92.86)
(2.51 x ¹⁰ < ⁵ > / mL) 2.83x10 ⁸ (95.16)
(2.83x10 < ⁵ > / mL) 3.25 x ^{10 8} (96.47)
(1.99x10 < ⁵ > / mL) 2 3.53x10 ⁷ (93.30)
(2.37x10 < ⁵ > / mL) 1.08x10 ⁸ (95.83)
(3.35x10 < ⁵ > / mL) 3.41x10 ⁸ (95.95)
(3.41 x ¹⁰ < ⁵ > / mL) 3.60x10 ⁸ (92.80)
(2.20 x ¹⁰ < ⁵ > / mL) 3 3.28x10 ⁷ (93.81)
(2.20 x ¹⁰ < ⁵ > / mL) 9.44 x 10 ⁷ (93.80)
(2.93x10 < ⁵ > / mL) 3.19x10 ⁸ (95.00)
(3.19x10 < ⁵ > / mL) 3.76x10 ⁸ (92.23)
(2.30 x ¹⁰ < ⁵ > / mL) 10

Amplification culture ②

One 2.64x10 ⁸ (95.65)
(8.95x10 < ⁵ > / mL) 8.03x10 ⁸ (96.86)
(1.26 x ¹⁰ < ⁶ > / mL) 1.89x10 ⁹ (96.43)
(9.45 x ¹⁰ < ⁵ > / mL) 3.73x10 ⁹ (97.00)
(1.15 x ¹⁰ < ⁶ > / mL) 2 3.36x10 ⁸ (97.39)
(1.58 x ¹⁰ < ⁶ > / mL) 8.06x10 ⁸ (98.02)
(1.26 x ¹⁰ < ⁶ > / mL) 2.56 x ^{10 9} (98.08)
(1.28 x ¹⁰ < ⁶ > / mL) 4.65x10 ⁹ (97.58)
(1.43 x ¹⁰ < ⁶ > / mL) 3 4.25 x ^{10 8} (97.92)
(1.44 x ¹⁰ < ⁶ > / mL) 9.46 x ^{10 8} (98.31)
(1.48 x ¹⁰ < ⁶ > / mL) 3.21 x ^{10 9} (97.87)
(1.60 x ¹⁰ < ⁶ > / mL) 3.68x10 ⁹ (96.54)
(1.13 x ¹⁰ < ⁶ > / mL)

Example 4

[Transition of lymphocyte surface antigen in the step of activating and amplifying through cryopreservation]

(1) Preparation of cryopreserved cells

In Example 2, in the activation culture before cell freezing, it was found that the occupancy rate of the memory T cell group among the cells in the culture liquid increased at the stage when the cell density in the culture liquid reached 4 x 10 ⁵ cells / mL or more.

Here, in the same manner as in (3) of Example 3, peripheral blood lymphocyte cells were prepared in the same manner as in (1) of Example 1 in three healthy subjects, and the number of cells in the obtained cell suspension was measured, a density of 6 × 10 ⁵ pieces / formulated to have mL and dispensed to the 50mL in example 1 (2) and OKT3 immobilized flask of 225cm ² made of the same sequence, and TE-HER CO ₂ incubator (HIRASAWA Co. ) At a temperature of 37.0 ± 0.5 ° C, a humidity of 95.0 ± 5.0% and a CO ₂ concentration of 5.0 ± 0.2%. Since the number of floating cells exceeded 4 × 10 ⁵ cells / mL on the third day of culture, 50 mL of Medium 1 was added to the cells, and the culture was continued until the next day. On the next day, that is, on the fourth day after the start of the culture, the activation culture was stopped, and the lymphocyte cells attached to the bottom surface of the flask were peeled off by tapping to make suspended cells. All the cells were collected and used as a cell suspension using the medium 1. The cell count and the survival rate in the suspension were measured in the same manner as in item (1) of Example 1. The suspension of 7.0 x 10 ⁷ cells was transferred into a 50 ml centrifuge tube and centrifuged at 1200 rpm for 5 minutes at room temperature. The supernatant in the centrifuge tube was removed, and 3.6 mL of CP-1 cell freezing stock solution (Kyokuto Pharmaceutical Industrial Co., Ltd.) was added to the pellet. The suspension was dispensed into two cryopreservation tubes of 1.8 ml each, and frozen and preserved in the same manner as in (2) of Example 3.

(2) Thawing of cryopreserved cells

The cryopreserved cells were removed from the liquid nitrogen tank after one week, and thawed in the same manner as in (3) Review 3 of the density of frozen cells in Example 3. The frozen lymphocyte cells were thawed by heating in a dry thermo unit (TAITEC CORPORATION) set at 37 ° C for 4 minutes. Thawed lymphocyte cells were transferred into a 15 mL centrifuge tube while suspending using 10 mL of Medium 1, and centrifuged at 1200 rpm for 3 minutes at room temperature. After centrifugation, the supernatant was removed, and the pellet was dispersed. 40 mL of Medium 1 was added to the cell suspension, and the number of cells was measured in the same manner as in Example 1 (1). In addition, almost 100% of the lymphocyte cells after cryopreservation are CD3-positive cells (Non-Patent Document 1).

(3) Expansion activation of cryopreserved cells Amplification culture

The suspension was inoculated in a 24-well multiwell plate (Sumitomo Bakelite Co., Ltd.) at a rate of 2 mL per well in the same manner as in (4) of Example 3, %, And a CO ₂ concentration of 5.0% in a carbon dioxide gas incubator for 2 days. The cultured lymphocytes were transferred to a plain flask from a 24-well multiwell plate on day 3, washed in wells of a 24-well multiwell plate with medium 1, and the remaining cells were recovered to prepare a liquid suspension of the cell suspension in the flask did. The flask was again cultured in a TE-HER CO ₂ incubator (HIRASAWA Co., Ltd.) for 2 days (expansion culture) in a carbon dioxide gas incubator at a temperature of 37.0 ± 0.5 ° C., a humidity of 95%, and a CO ₂ concentration of 5.0%. After the culturing, the cell suspension in the culture flask was transferred into an 225 cm ² OKT3 solid-phase flask for activation culture, and the culture flask was washed with medium 1 to recover the remaining cells, and finally the liquid amount in the OKT3 solid-phase flask And the flask was again cultured in a TE-HER CO ₂ incubator (HIRASAWA Co., Ltd.) at 37.0 占 0.5 占 폚, 95% humidity and 5.0% CO _{2 for} 2 days. After the activation culture, the OKT3 solid-phase flask was tapped and the cells adhering to the bottom surface were peeled to prepare a cell suspension. The obtained cell suspension was transferred into a new gas permeable culture bag (1000 mL, Kohjin Bio Co., Ltd. 16087528), and further cultured in medium 2 containing 1% human serum (Limpotech), 175 U / mL rIL-2 (Novartis) (Invitrogen), and the washings were combined with the cell suspension in the gas-permeable culture bag to a volume of 1000 mL, and incubated in a TE-HER CO ₂ incubator (HIRASAWA, Inc.) The cells were cultured again at 37.0 ± 0.5 ° C, 95% humidity and 5.0% CO _{2 for} 3 days (Amplification culture 1). After the above cultivation, 1000 mL of Medium 2 was added to a new culture bag, which was then adhered to the gas-permeable culture bag cultured in the above-mentioned Amplification Culture (1) under aseptic conditions, the contents in the two bags were mixed well and the culture was continued . The culture container was incubated again for 4 days in a carbon dioxide gas incubator at 37 캜, a humidity of 95% and a CO ₂ concentration of 5.0% (Amplification culture 2).

(4) Analysis of antibody-stained lymphocyte populations

In the steps (2) to (3) of Example 4, the first day was the day when the frozen cells were thawed, the cultivation in the plain flask (225 cm ² ) was terminated and the activation culture in the OKT3 solid phase flask was started On the 5th day immediately before the start of the incubation, the activated culture was terminated and the 7th day immediately before starting the culture in the gas-permeable culture bag (1000 mL, Kohjin Bio Co., Ltd. 16087528) On the first day (immediately after thawing), on the fifth day (immediately before activation), on the seventh day (immediately after activation), on the day after the first culture (immediately after thawing) And on the 14th day (immediately before the termination), a part of the cell suspension in the culture was taken under aseptic conditions and analysis of a subset of the obtained cells was carried out in the same manner as in Example 2 (2) using an anti-CD3 antibody (Becton Dickinson 349201 ), Anti-human CD45RO antibody (Becton Dickinson 340438) and anti-human CD62L antibody (Becton Dickinson Pharmingen 555544).

result:

The results are shown in Table 7. It has been found that the memory T cell group in which the occupancy rate is increased in the activated cultured cells before the cryopreservation maintains a high high abundance ratio in the course of normal culture and activation culture after freeze thawing. On the seventh day, the rate of the presence of the central memory T cells showed a slight decrease, but showed recovery as the culture continued, showing a gradual decrease in the presence rate as the memory T cell group, but the occupancy rate exceeded 80% . As a result, it was found that compared with the lymphocyte subset image immediately after the collection of peripheral blood lymphocyte cells in the subject, activated lymphocyte cells in which the memory T cell group is present at a high rate can be obtained (FIG. 2). CD45RO +, CD62L +, CD3 + mediated T cells, CD45RO +, CD62L-, and CD3 + effected T cells, and CD45RO +, CD62L + and CD3 + Effect memory T cells, CM + EM or memory represent CD45RO +, CD62L +/-, CD3 + memory T cells.

 Changes in lymphocyte surface antigens (5%) in the process of activating proliferation after cryopreservation Processing date Specimen CD45RO-
CD62L +
CD3 +




(Naive) CD45RO-
CD62L-
CD3 +




(Effector) CD45RO +
CD62L +
CD3 +



(Central Memory: CM) CD45RO +
CD62L-
CD3 +



(Effector Memory: EM) CD45RO +
CD62L +
CD3 +
CD45RO +
CD62L-
CD3 +

(CM + EM) Immediately after thawing
1 day One 1.97 4.4 67.91 25.72 93.63 2 2.65 7.69 61.88 27.78 89.66 3 4.57 12.23 53.58 29.62 83.2 Just before activation
5 days One 4.43 0.45 93.6 1.52 95.12 2 6.87 0.27 91.82 1.04 92.86 3 4.68 0.48 91.49 3.34 94.83 Immediately after activation (immediately before amplification)
7 days One 6.6 6.6 58.28 31.06 89.34 2 3.6 3.6 66.84 25.96 96.76 3 5.46 5.46 64.36 28.08 92.44 Just before the end
13th One 12.25 6.82 71.55 9.38 80.93 2 16.29 3.85 76.34 3.58 79.92 3 15.12 5.71 73.67 4.71 78.38

(5) Stability of cryopreserved cells

The cells that were cryopreserved at the same time were stored under liquid nitrogen for 1 week, 1 month, 2 months, 4 months, and 6 months in the same manner as in Example 4 (1), and the stability of the cryopreserved cells was examined (n = 3).

The number of cells after thawing, the cell phenotype, the degree of cell amplification after thawing, and the cell phenotype of the amplified cells were analyzed, and the cell number, survival rate and cell phenotype after thawing, cell amplification rate in the culture process after thawing, There was no difference in cell phenotype. Thus, cryopreserved cells were stable for at least 6 months, and cryopreserved cells could be stably preserved for a long time.

Example 5

A medical composition containing the memory cell of the present invention as a main component was prepared. An outline of the manufacturing process is shown in Fig.

[Production of a medical composition containing a memory cell of the present invention as a main component]

1. Activation culture ① Process

50 mL of the cell suspension prepared in the same manner as in item (1) of Example 1 was dispensed into a 225 cm ² activated flask in which the anti-CD3 antibody (OKT3) was solidified. The cell suspension was incubated at 37.0 占 0.5 占 폚, ₂ concentration 5.0 ± 0.2% in a TE-HER CO ₂ incubator. When the prepared cell suspension amount exceeded 100 mL, 50 mL of the cell suspension was dispensed into two activated flasks. When the amount of the cell suspension was 50 mL or more and less than 100 mL, 50 mL was dispensed. Three days after the start of the culture, 0.01 mL of the culture was collected and the number of floating cells was measured (in-process test II). When the density of floating cells was 4 × 10 ⁵ cells / mL or more, 50 mL of the culture solution 1 was added (addition of the culture solution).

When the density of floating cells did not reach 4 × 10 ⁵ cells / mL in the in-process test II on the third day of culture, the culture was continued for one day, and the number of floating cells was measured the next day When the density was 4 × 10 ⁵ cells / mL or more, the culture medium was added to the process.

If the density of floating cells reaches 4 × 10 ⁵ cells / mL by the 6th day of culture, proceed to the culture medium addition step. If not, the culture is stopped and the blood is collected again.

2. Cryopreservation process

On the next day after the addition of the culture medium, the cells attached to the bottom of the flask were peeled off and uniformly suspended, and 1 mL was sampled as a sample for in-process test III. Cells were collected from the cell suspension by centrifugation at 1200 rpm for 5 minutes at room temperature, and the cells were frozen at 3x10 < ⁷ > cells / tube. Cells were suspended in cryopreservation tubes at 3 × 10 ⁷ cells / tube in cell-freeze-preserving solution CP-1 (Kyokuto Pharmaceutical Industrial Co., Ltd.), the tubes were placed in BICELL, And rapidly frozen. The next day, the frozen tube was taken out from the cooler at -80 DEG C and stored in liquid nitrogen.

3. Thawing process

The tube containing the cryopreserved cells was taken out of the liquid nitrogen tank and the freezing cells were thawed by heating in a dry thermo unit heated to 37 ° C for 4 minutes. The thawed cell lysate was added to a 15 ml centrifuge tube containing 10 ml of Medium 1, mixed by inverting and mixing well, and centrifuged at 1200 rpm for 3 minutes at room temperature. The supernatant was removed and the cells were dispersed and then suspended uniformly in 40 mL of Medium 1. Cells were well mixed and 0.5 mL was sampled into a sample of in-process test IV.

The remaining cell suspension was dispensed into a 24 well multiwell plate at 2 mL / well and cultured for 2 days in a CO ₂ incubator at a temperature of 37.0 ± 0.5 ° C., a humidity of 95.0 ± 5.0%, and a CO ₂ concentration of 5.0 ± 0.2%.

4. Enlarged culture process

On the third day of culture, the cells were transferred from a 24 well multiwell plate to a 225 cm ² plain flask. The 24 well multiwell plate was washed with medium 1 and the remaining cells were recovered. The cells were suspended in medium 1 to a total of 90 mL, and cultured for 2 days in a CO ₂ incubator at a temperature of 37.0 ± 0.5 ° C., a humidity of 95.0 ± 5.0%, and a CO ₂ concentration of 5.0 ± 0.2%.

5. Activation culture ② Process

After incubation for 2 days, cells in a 225 cm ² plain flask were transferred to an activated flask where the anti-CD3 antibody (OKT3) was immobilized. The plain-flask of 225 cm ² was washed with medium 1, and the remaining cells were collected, so that the final volume of the culture liquid was 250 mL. The cells were cultured for 2 days in a CO ₂ incubator at a temperature of 37.0 占 0.5 占 폚, a humidity of 95.0 占 .5%, and a CO ₂ concentration of 5.0 占 .2%.

6. Amplification culture ① Process

After culturing for 2 days, the cells attached to the bottom surface of the activated flask were peeled to obtain a cell suspension. 1 mL of the cell suspension was sampled and used as a sample of the in-process test V. [ 250 mL of the remaining cell suspension was transferred to a gas permeable culture bag filled with 750 mL of Medium 2 (1% human serum-containing medium containing 175 U / mL of IL-2), and the temperature was 37.0 ± 0.5 ° C., the humidity was 95.0 ± 5.0% And cultured in a CO ₂ incubator with a CO ₂ concentration of 5.0 ± 0.2% for 3 days (the total amount of the culture was about 1 L).

7. Amplification culture ② Process

Permeability culture bag filled with a culture bag for 3 days and a culture medium of 1 L was bonded under aseptic conditions and the two culture solutions were well mixed and the temperature was 37.0 ± 0.5 ° C, the humidity was 95.0 ± 5.0%, the CO ₂ concentration was 5.0 ± 0.2% Of CO ₂ incubator for 4 days (total amount of culture about 2 L).

8. Preparation and filling process

Amplification culture ② After the initiation, 250 mL of the culture medium was transferred from one side of the two connected culture bags to each of four 250 mL centrifuge tubes on the fourth day (14-17 days in total), and centrifuged at 1,500 rpm for 8 minutes at room temperature. The supernatant was removed and the culture of the remaining one bag was transferred to the 250 mL centrifuge tube. The supernatant was removed by centrifugation under the same conditions as the first bag. Four 250-mL centrifuge tubes were dispersed, suspended in 500 mL of 0.1% human albumin-added physiological saline, collected in two 250 mL centrifuge tubes, and centrifuged at 1,700 rpm for 8 minutes at 4 ° C to wash the cells. The same procedure was repeated to wash the cells twice. The washed cells were dispersed and suspended in 210 mL of 1% human albumin-added physiological saline cooled to 4 째 C. 10 mL of the suspended cell solution was collected and used as a sample for shipment inspection. The remaining 200 mL was charged into the product charging bag and made into a product.

[Analysis of a medical composition containing a memory cell of the present invention as a main component]

Analysis of the subset of activated lymphocyte cells obtained according to the method of the present invention and activated lymphocyte cells prepared by the conventional method was carried out in the same manner as in Example 2 (2) using anti-human CD45RO antibody (Becton Dickinson) and anti-human CD62L Antibody (Becton Dickinson Pharmingen) (Fig. 4).

The method of the present invention was carried out by the method described in Example 5. Mononuclear cells (lymphocyte-rich fraction) (PBMC) isolated from peripheral blood vessels at the start of culture were activated and cultured in an OKT solid-phase flask. The sample (+50) was sampled on the fourth day after the activation cultivation with the concentration of 4 × 10 ⁵ / mL or more, and then 50 mL of the medium 1 was added, and the sample was incubated for 1 day to collect a sample (Freeze) . Immediately after thawing of the frozen cells, a sample (TW (Thaw)) was taken and subjected to an expansion culture step, and then the activated culture (2) was carried out in an OKT solidification flask. A sample (OKT) was sampled immediately before the activation culture (2), and a sample (thawing BP) was sampled immediately thereafter. Then, lymphocyte cells were harvested by performing amplification culture (2) in a culture bag. Cell samples (Harvest) were collected immediately before harvesting.

In the conventional method, monocytic cells (lymphocyte-rich fraction) (PBMC) separated from peripheral blood vessels at the start of culture were used as a cell suspension using Medium 1, and 50 mL of the suspension was activated and cultured in an OKT solidification flask. The sample (+50) was collected on the fourth day after the activation cultivation with the concentration of 4 x 10 ⁵ / mL or more, and then 50 ml of medium 1 was added and cultured for 1 day. On day 5, a sample (+150) was collected and 150 mL of medium 1 was added and cultured. The next day (raw BP) was sampled again. 100 ml of Medium 1 was added and cultured, and a sample (raw H) was collected just before the completion of the culture. Also, the +150 and Freeze samples are samples taken immediately after being divided into frozen and non-frozen groups, so they are substantially the same.

As a result of examining the cell subset of the sample, the ratio of the memory T cells in the obtained lymphocyte cell group was about 40%, which was about the same as that of the PBMC (Fig. 4A) Using the inventive method, memory T cells accounted for about 75% of the obtained lymphocyte cell lines (Fig. 4B). Therefore, not only can convenience of the lymphocyte cells obtained by the method of the present invention be preserved by cryopreservation, but also lymphocyte cell lines based on memory T cells as an active ingredient can be obtained in quantum immunotherapy and the like.

In order to elucidate the mechanism of action of activated lymphocytes used in the quantum immunotherapy method by Osumi et al., The function of the activated lymphocyte cells obtained by activation amplification culture was focused on a memory T cell subset and analyzed. As a result, it was shown that effect memory T cells contained in activated lymphocytes showed cytotoxic activity to cancer cells at 24 hours, that central memory T cells were activated after recognition of tumor antigens and differentiated into effective T cells Lt; RTI ID = 0.0 > cytotoxic < / RTI > activity.

Hereinafter, the effects of central memory T cells and effect memory T cells on tumor cells are shown. Activated autoimmune lymphocytes (KC-362) were isolated from central memory T cells (Becton Dickinson) using a flow cytometer (Becton Dickinson; FACS Calibur) with or without anti-human CD45RO antibody (Becton Dickinson) and anti- human CD62L antibody (Becton Dickinson Pharmingen) (CM) and effective memory T cells (EM) (CM purity 99.6%, EM purity 98.5%). Then, cell division of each cell group and KT-362 (tumor-derived tumor cell line) were co-cultured to analyze cytotoxic activity (Table 8).

Effector Cell Relative Cell Number of
KT-362 24hr 48hr None (KT-362 alone) 2.51 5.25 Central Memory T cell 0.91 0.297 Effector Memory T cell 0.239 N.D.

In KT-362 alone (control), the number of cells increased 2.5 times in 24 hours and 5.3 times in 48 hours compared to the number of cells at the start of culture. On the other hand, in the co-culture with CM, the growth inhibition was 0.9 times at 24 hours and decreased 0.3 times at 48 hours. At 48 hours after culture, lymphocyte cells were recovered and analyzed by flow cytometry (Becton Dickinson; FACS Calibur). The anti-circularity of CM was changed to EM.

In the co-culture of KT-362 and EM, the number of cells decreased by 0.2-fold compared with the number of cells at the start of control culture, and no live cells appeared at 48 hours.

Therefore, both CM and EM have cytotoxic activity. Of these, CM has a time difference in the expression of the inhibitory activity, but its lifetime is much longer than that of EM. Therefore, CM is considered to be a stable source of EM over a long period of time. It is suggested that a composition containing a lymphocyte cell group that selectively amplifies CM in terms of an effective therapeutic agent.

The activated lymphocyte cell group obtained by the conventional method can not obtain an activated lymphocyte group having a high central cell T cell occupancy only for a limited period of about 3 to 7 days after the PBMC collection, And the percentage of the central memory T cells is reduced to 20% or less after the number of cells is sufficiently increased (Non-Patent Document 3). On the other hand, according to the method of the present invention, it is possible to obtain the activated lymphocyte cell group of the present invention in which the CM is overwhelmingly larger than that of EM while sufficiently increasing the number of cells. In cytotoxic activity, However, the activity itself is not inferior to that of EM, and it is thought that a long-term stable supply of EM having high cytotoxic activity can be obtained through the administration, thereby realizing an effective therapeutic effect. In addition, since the cell freezing step is included and the freezing period is not particularly limited, it is possible to rapidly prepare the memory T cells, particularly the activated lymphocyte cell population with a high central memory T cell share, even for long term storage and sudden requests The convenience is very high.

INDUSTRIAL APPLICABILITY The present invention can be suitably used in a therapeutic agent field including activated lymphocyte cells.

Claims (13)

A method for producing a lymphocyte cell group comprising a memory T cell as a main component according to a high proliferation culture method comprising the following steps (a) to (c) sequentially.
(a) activating the collected lymphocyte cells;
(b) detecting the increase in the occupancy rate of the memory T cell group based on the lymphocyte cell density, and cryopreserving the lymphocyte of the step (a); and
(c) thawing frozen lymphocyte cells and activating them. The method according to claim 1,
Wherein the detection of the increase in the occupancy rate of the memory T cell group based on the lymphocyte cell density in the step (b) is performed using the number of lymphocyte cells floating in the culture medium as an index. The method according to claim 1 or 2,
Wherein the lymphocyte cell density in the step (b) is 4 x 10 < ⁵ > cells / mL or more in terms of the number of lymphocyte cells floating in the culture liquid. 4. The compound according to any one of claims 1 to 3,
Wherein the activated culture is cultured in the presence of IL-2 and a solid phase anti-CD3 antibody. 5. The method according to any one of claims 1 to 4,
And the step (c) is completed within two weeks after thawing. 5. The compound according to any one of claims 1 to 5,
Wherein the lymphocyte cell group mainly comprising the memory T cells is a group of lymphocyte cells having a share of the memory T cells of the lymphocyte cell group of 75% or more. 7. The compound according to any one of claims 1 to 6,
Wherein the memory T cells comprise centrally-stored T cells at least 70% of the lymphocyte cell population. 7. The compound according to any one of claims 1 to 7,
Wherein the isolated lymphocyte cells are derived from human peripheral blood. 8. The method according to any one of claims 1 to 8,
Wherein the lymphocyte cell group is for administration to a third person. Wherein the lymphocyte cell population comprises more than 75% of the memory T cells of the lymphocyte cell population. 11. The method of claim 10,
Wherein the lymphocyte cell population comprises more than 70% of the central memory T cells of the lymphocyte cell line. A pharmaceutical composition comprising the lymphocyte cell group according to claim 10 or 11 as an active ingredient. A system for producing a lymphocyte cell group mainly comprising memory T cells according to a high proliferation culture method, comprising the following means (A) to (D):
(A) means for activating lymphocyte cells;
(B) means for increasing the occupancy rate of the memory T cell group based on the lymphocyte cell density;
(C) means for cryopreserving lymphocyte cells; and
(D) means for thawing frozen lymphocytes.

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