KR101415039B1 - Medium Composition and Method for Massive Culture of Autologous Activated Lymphocyte - Google Patents

Abstract

The present invention relates to a medium composition for mass propagation of autologous activated lymphocytes and to a method for culturing autologous activated lymphocytes using the same. In particular, the present invention relates to a method for massively culturing autologous activated lymphocytes by adding an anti-CD3 antibody, an autoplasma, interleukin-2 (IL-2), interleukin-18 (IL-18), and poly I:C in a medium for culturing cells and to a medium for culturing cells to which the anti-CD3 antibody, the autoplasma, the interleukin-2 (IL-2), the interleukin-18 (IL-18), and the poly I:C are added. When lymphocytes are cultured by means of the method of the present invention, the ratio of NK cells in the overall cells is increased, and cells with an increased production amount of interferon-gamma and increased cytotoxicity of lymphocytes can be massively cultured.

Description

Technical Field [0001] The present invention relates to a medium composition and a culture method for mass proliferation of a self-derived activated lymphocyte,

The present invention relates to a culture composition for mass-proliferation of self-derived activated lymphocytes and a method for culturing self-derived activated lymphocytes using the same. Specifically, a method of mass-culturing a self-derived activated lymphocyte by adding anti-CD3 antibody, autologous plasma, interleukin-2 (IL-2), interleukin-18 (IL-18) and poly I: C to a cell culture medium (IL-2), interleukin-18 (IL-18) and poly I: C are added to the culture medium for cell culture, wherein the anti-CD3 antibody, autologous plasma, interleukin-2

Surgical surgery, radiation therapy, and chemotherapy are the three major treatments for cancer that are widely used clinically. Such cancer treatment methods have been used for a long time and have achieved many effects, but they have limitations that cause side effects in patients by inhibiting the immune system of cancer patients (immune suppression).

A new cancer treatment modality that minimizes these side effects has been continuously studied, and tumor cell vaccines, tumor-specific antibodies, gene therapy, dendritic cells, New therapies such as dendritic cell vaccination, natural killer cell, and activated killer cell have been developed. In particular, in the mid-1980s, the use of immunotherapy was rapidly increasing in the world as a result of reports of good results in animal experiments. Currently, more than 400 clinical trials are underway. have.

Immunocytochemistry has the advantage of using the immune system of the human patient to remove the cancer cells present in the body and to activate the immune function to prevent recurrence. Natural Killer cells, T cells (T cells, T lymphocytes), and dendritic cells are typical examples.

Among these, NK cells are morphologically cytoplasmic granulocyte-like lymphocytes that express CD3-CD56 + phenotype and have the function of killing tumor cells and protecting the body from infection. Unlike T cells, NK cells do not have cell surface receptors such as T cell receptors (TCRs) and have the ability to directly kill virus-infected cells or cancer cells without being sensitized to specific antigens. However, NK cells do not attack nonmagnetic cells indiscriminately. NK cells have an activation receptor and an inhibitory receptor, and the HLA-class I antigen of the target cell acts as a ligand to control the attack on the target cell. However, when the expression of MHC-class I is reduced like virus-infected cells or cancer cells, the action of the inhibitory receptor of NK cells is weakened and the function of the activating receptor becomes relatively strong, so that these cells directly attack. In addition, NK cells recognize the surface of tumor cells and recognize cytotoxicity by re-recognizing the antibodies associated with the recognition markers. This reaction is called antibody dependent cell-mediated cytotoxicity (ADCC) . In addition to the cell-mediated cytotoxicity described above, the ability of NK cells to kill tumor cells is demonstrated by the secretion of various cytokines such as Perforin and Granzyme.

As a conventional method for producing lymphocytes containing most commonly used NK cells, there is a method of culturing mononuclear cells isolated from peripheral blood using an interleukin-2 or an interleukin-15 and an OKT-3 antibody, This method has a limited number of finally obtained cells.

In order to overcome the problem of insufficient cell number, a method of cell proliferation using an inactivated feeder cell such as Korean Patent No. 1133185 may be used. However, a separate process such as irradiation is performed , And especially when tumor cells are used as auxiliary cells, safety issues must be taken into account when using them in actual patients.

Thus, the lymphocytes prepared using the conventional culture method are effective in increasing the proportion of NK cells, but culturing the activated lymphocytes in large quantities has not yet reached a satisfactory level.

Korean Registered Patent No. 1133185 (Green Cross Labcell, Seoul National University Hospital)

(IL-2), interleukin 18 (IL-18), and poly I: C in a culture medium for cell culture. And a method for culturing a self-derived activated lymphocyte in which a self-derived activated lymphocyte is mass-cultured.

It is still another object of the present invention to provide a culture medium for culturing a self-derived activated lymphocyte which is used in the culture method.

In order to achieve the above-mentioned object, the culture composition for culturing a self-induced activated lymphocyte according to the present invention comprises a culture medium for cell culture and an additive to be added to the culture medium for cell culture, In the composition, the additive is characterized in that it comprises an anti-CD3 antibody, autologous plasma, interleukin-2 (IL-2), interleukin 18 (IL-18) and poly I:

In addition, the self-derived activated lymphocytes cultured in the culture medium for self-induced activated lymphocyte culture may have a ratio of self-derived activated lymphocytes having CD3-CD56 + immunophenotype of 60 to 95%.

On the other hand, the self-derived activated lymphocyte culture method of the present invention

(A) isolating and obtaining mononuclear and autologous plasma from human peripheral blood, respectively;

(B) coating the cell culture vessel with an anti-CD3 antibody;

(C) a first culture step of suspending the mononuclear cells in the cell culture medium to which the autologous plasma and the interleukin-2 are added, and inoculating the coated mononuclear cells in the coated cell culture vessel and culturing them; And

(D) a second culturing step of culturing the culture obtained through the first cultivation step into a cell culture container not coated with an anti-CD3 antibody, and culturing by adding interleukin-18 and poly I: C do.

In the method for culturing a self-induced activated lymphocyte according to the present invention, the second culturing step of the step (D) may include the step of adding a cell culture medium supplemented with autologous plasma, interleukin-2, interleukin-18 and poly I: C May be further included.

In addition, in the method for culturing a self-induced activated lymphocyte of the present invention, the second culturing step of the step (D) comprises adding interleukin-18 and poly I: C and culturing the cell culture medium supplemented with the autologous plasma and interleukin- May be further included.

Further, in the method for cultivating a self-induced activated lymphocyte of the present invention, the second culturing step of the step (D) may further comprise the step of adding the cell culture medium to which the autologous plasma and the interleukin-2 are added.

In the step (A), the mononuclear cell layer is formed by centrifuging 2 to 20 blood vessels containing human peripheral blood, the mononuclear cell layer is transferred to 2 to 20 test tubes, Followed by washing again by centrifugation after filling the buffer with the buffer solution.

In addition, the composition ratio of the anti-CD3 antibody to 100 parts by weight of the culture medium for activating spontaneously activated lymphocytes may be 0.01 to 0.2 parts by weight.

In addition, the composition ratio of the autologous plasma to the 100 parts by weight of the culture medium for activating a spontaneously-derived activated lymphocyte may be 2 to 15 parts by weight.

In addition, the composition ratio of interleukin-2 to 1 ml of the culture medium for autologous-derived activated lymphocyte culture may be 200 to 2000 IU.

In addition, the composition ratio of interleukin-18 to 100 parts by weight of the culture medium for activating spontaneously-derived activated lymphocytes may be 0.007 to 0.15 parts by weight.

In addition, the composition ratio of poly I: C to 100 parts by weight of the culture medium for activating spontaneously activated lymphocytes may be 0.5 to 50 parts by weight.

Also, the first incubation step of step (C) may be performed for 30 minutes to 24 hours.

In addition, the second cultivation step of step (D) may be performed for 10 to 20 days.

Further, the self-derived activated lymphocyte may be a natural killer cell (NK cell).

The self-derived activated lymphocyte prepared by the present invention is a self-derived activated lymphocyte having a high proportion of NK cells in all cells, and is a cell having high interferon-gamma production and high cytotoxicity to tumor cells By mass culture, it can be useful as an immunotherapy for cancer patients.

1 is a graph showing the cell proliferation rate by the culturing method of the present invention.
2 is a graph showing the cell survival rate by the culture method of the present invention.
FIGS. 3 to 5 are graphs showing distribution patterns and phenotypes of lymphocytes cultured by the culture method of the present invention.
6 is a graph showing interferon-gamma production of activated lymphocytes cultured by the culture method of the present invention.
FIG. 7 is a graph showing the cytotoxicity amount of activated lymphocytes cultured by the culture method of the present invention. FIG.

Hereinafter, preferred embodiments of the present invention will be described in detail. In the following description, numerous specific details, such as specific elements, are set forth in order to provide a thorough understanding of the present invention, and it is to be understood that the present invention may be practiced without these specific details, It will be obvious to those who have knowledge of. In the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear.

In order to achieve the above object, the present invention provides a culture medium for culturing a self-induced activated lymphocyte, comprising a culture medium for cell culture and an additive added to the culture medium for cell culture, wherein the self-generated activated lymphocyte, (IL-2), interleukin-18 (IL-18), and poly (I): C, wherein the additive is selected from the group consisting of an anti-CD3 antibody, an autologous plasma,

Herein, the term "mononuclear cells separated from the initial blood" refers to peripheral blood-derived mononuclear cells (PBMCs), and the PBMCs may be immune cells such as B cells, T cells and natural killer cells And granulocytes such as basophils, eosinophils, and neutrophils, and the like.

NK cells in PBMC isolated from normal blood are about 5 to 15%, and NK cells present in PBMC have an interferon-gamma production ability in the range of about 0.5 to 6%. On the other hand, the self-derived activated lymphocytes cultured by the present invention correspond to 60 to 95% of NK cells, and these activated NK cells have a high cytotoxicity to cancer cells by secretion of interferon-gamma.

NK cells function at the forefront of defense mechanisms in the immune system, such as eliminating tumor cells and virus-infected host cells without antigen sensitization and controlling the immune response of dendritic cells and T cells. NK cells react with cytokines such as interleukin-2, interleukin-12 and interferon to promote proliferation and cytotoxicity against cancer cells. These phenotypes of NK cells are human CD16 (FcγRIII) and CD56, and CD16 and CD56 have no T-cell receptor complex (TRC) on the cell surface and are used as markers for NK cells.

The CD3 antibody is a protein specifically reactive with a CD3 antigen, which is a molecule group that associates with a T cell receptor (TCR) to form an antigen recognition complex. The CD3 molecule has a longer intracellular domain than the TCR, And is responsible for delivering.

Interleukin-2 (IL-2) is a glycoprotein with a molecular weight of 14 to 17 kDa, which is produced when T cells are recognized and activated by an antigen, and is produced by T cells, It reacts with itself and promotes the growth of the T cell. Interleukin-2 also acts on NK cells to promote growth, enhances NK cell kill ability, and acts on B cells to promote its growth. In the present invention, the composition ratio of interleukin-2 to 1 mL of the culture medium for self-induced activated lymphocyte culture may be 200 to 2000 IU.

Interleukin-18 (IL-18) is a proinflammatory cytokine produced by macrophages that causes an immune response to bacteria or virus-infected cells and increases the production of interferon-gamma in NK cells and T cells. In the present invention, NK cells are activated by inducing receptor expression of interleukin-18, an essential factor for the production of interferon-gamma. In the present invention, the composition ratio of interleukin-18 to 100 parts by weight of the culture medium for self-induced activated lymphocyte culture may be 0.007 to 0.15 parts by weight.

The culture medium composition of the present invention is characterized by containing poly-I: C (poly-amino-polycytidylic acid) as an active ingredient. Poly I: C is an agonist of the cytosolic melanoma differentiation-antigen-5 (MDA-5) and congenital endosomal toll-like receptor 3 (TLR3) Have. Poly I: C induces the production of proinflammatory cytokines including type I interferon (type I IFN) and stably and matured dendritic cells, the most potent antigen presenting cells in mammals. In addition, NK cells are activated not only in vivo but also in vigorous adjuvant activity against peptide antigens, and researches are actively carried out by vaccine agents. In the present invention, the composition ratio of poly I: C to 100 parts by weight of the culture medium for activating a self-induced activated lymphocyte may be 0.5 to 50 parts by weight.

The autologous-derived activated lymphocytes cultured in the culture medium for autologous-derived activated lymphocyte culture have a ratio of self-derived activated lymphocytes having CD3-CD56 + immunophenotypes, that is, NK cells of 60 to 95% .

On the other hand, the self-derived activated lymphocyte culturing method of the present invention starts from the step of firstly isolating and obtaining mononuclear cells and autologous plasma from human peripheral blood, respectively. In the step of obtaining the mononuclear cells, 2 to 20 blood vessels containing human peripheral blood are centrifuged to form a mononuclear cell layer, the mononuclear cell layer is transferred to 2 to 20 test tubes, each test tube is filled with a buffer solution By centrifuging and washing, the cell yield can be further increased.

The cell culture vessel is then coated with anti-CD3 antibody. The anti-CD3 antibody can be diluted with a phosphate buffer solution. In this case, it is also possible to increase the efficiency of the coating using gelatin, collagen or fibronectin.

Then, the first culturing step of suspending the mononuclear cells in the cell culture medium to which the autologous plasma and the interleukin-2 are added, and inoculating the monocyte into the coated cell culture container and culturing is carried out. Such a first incubation step is preferably carried out for 30 minutes to 24 hours.

The culture obtained through the first culturing step is transferred to a cell culture container not coated with the anti-CD3 antibody and subjected to a second culturing step in which interleukin-18 and poly I: C are added and cultured, Activated lymphocytes are obtained.

The second culturing step may include adding a medium for cell culture supplemented with autologous plasma, interleukin-2, interleukin-18, and poly I: C in addition to the step of propagating the lymphocytes to be cultured, or adding interleukin-18 and poly Adding a step of adding I: C and adding the medium for cell culture supplemented with the autologous plasma and interleukin-2, or adding the medium for cell culture supplemented with the autologous plasma and interleukin-2 . Such a period of the second culturing step is preferably 10 to 20 days.

The composition ratio of the anti-CD3 antibody to 100 parts by weight of the culture medium for activating spontaneously activated lymphocytes may be 0.01 to 0.2 parts by weight, and the composition of the autologous plasma may be 2 to 15 parts by weight.

Further, the self-derived activated lymphocyte may be an NK cell.

Hereinafter, embodiments of the present invention will be described.

Example

Example  : Self-Derived Activated Lymphocyte Culture

Isolation of mononuclear cells

Peripheral blood from healthy subjects was collected in a blood vessel (BD vacutainer CPT) containing sodium heparin and ficoll and the separation process was performed within 2 hours. The blood vessels were centrifuged at 2000 RCF and 20 ° C for 20 minutes, and the upper plasma layer and the middle layer mononuclear layer were collected in a new test tube, respectively. Plasma was stored at 56 ° C for 30 minutes and inactivated. After centrifugation at 2000 RPM at 20 ° C for 5 minutes, only the supernatant was collected and used for subsequent culture.

Mononuclear cells collected from blood vessels were filled up to 15 mL with phosphate buffered saline (PBS) and centrifuged at 300 rpm for 10 min at 20 ° C to remove supernatant. The cells were washed once more, suspended in the culture medium, and used as initial cells for culturing.

Mass culture of activated lymphocytes

1 mL of the anti-CD3 antibody solution prepared by adding 10 μL of anti-CD3 antibody (BD Pharmingen, USA) to 10 mL of phosphate buffer was added to the 12-well cell culture plate one day before the cell inoculation and left at room temperature Coating.

The monocyte cells obtained above were stained with trypan blue, counted, suspended at a concentration of 1 x 10 ⁶ cells / mL based on living cells, and inoculated on a 12-well cell culture plate coated with the anti-CD3 antibody. At this time, Alys505N (CSTI, Japan) supplemented with 10% autologous plasma and 1000 IU / mL of interleukin-2 (Norvatis, Swiss) was used as a basic culture medium for culturing. The plate inoculated with the cells was allowed to react for 1 hour in an incubator maintained at 5% carbon dioxide and 37 ° C, and then transferred to a new 12-well cell culture plate not coated with the anti-CD3 antibody. And 100 μg / mL of interleukin-18 (MBL, Japan) and 20 μg / mL of poly I: C (InvivoGen, USA), followed by incubation in an incubator maintained at 5% carbon dioxide and 37 ° C. .

After 2 to 3 days from the start of the culture, 10 μg / ml of interleukin-18 and 10 μg / ml of poly I: 1 mL of IU / mL added Alys505N (CSTI, Japan) was added and cultured.

After that, the same amount of culture medium or 2 to 3 times culture medium was added every 1 to 2 days depending on the cell growth rate. The culture medium used was a basic culture medium, that is, 10% of autologous plasma and 1000 IU / mL of interleukin- The added Alys505N was used. Depending on the growth rate of the cells and the amount of the culture medium added, the cells were transferred to a larger size container and maintained a proper cell concentration for cell growth.

Test Example  1: Confirm cell proliferation rate and survival rate

Three healthy persons (expressed as A, B, and C in FIGS. 1 and 2) were used to culture for 14 days by the above example. After completion of the culture, the cells were collected, suspended in phosphate buffer, and centrifuged at 2000 RPM and 20 ° C for 5 minutes to remove the supernatant. After this cell washing process was performed once more, the number of living cells and total cells was counted by staining with trypan blue and the cell proliferation rate was compared with the number of cells at the initial stage of culture.

As a result, it increased about 392 times on average after 14 days incubation compared with the initial number of inoculated cells, and maintained a high survival rate of about 96.8%.

Test Example  2: Distribution and phenotypic analysis of mass-cultured lymphocytes

In the Examples, cell distribution and phenotype of cells obtained before culturing (PBMC) and cells after mass culture were confirmed. Cells after mass culture were collected, centrifuged at 2000 RPM for 5 minutes at 20 ° C, and then the supernatant was removed. Cells were suspended in phosphate buffer and centrifuged at 2000 RPM for 5 minutes. After this process was performed twice, the supernatant was removed, stained with trypan blue, and diluted to a cell concentration of 1 × 10 ⁶ cells / mL based on living cells. The diluted cells were dispensed in 100 μL each to a test tube dedicated to flow cytometry, and then fluorescent-labeled antibodies for surface antigen analysis were added under the following conditions. Each antibody used a fluorescent antibody conjugated with a primary antibody that binds to the surface antigen and a secondary antibody that fluoresces at a specific wavelength band, and the isotype antibody conjugated with only the fluorescent antibody was used as a control.

FITC PC5 PC7 APC Examiner 1 IgG1-FITC IgG1-PC5 IgG1-PC7 IgG1-APC Test tube 2 anti-human CD3 anti-human CD16 anti-human CD56 anti-human CD69

Antibodies for surface antigen analysis were added to a test tube containing cells, incubated at 4 ° C for 30 minutes, washed with phosphate buffer, and fixed with 4% paraformaldehyde. Fixed cells were analyzed for cell distribution using Gallios (Beckman Coulter, USA) and data were analyzed using KALUZA software.

As a result of the distribution and phenotypic analysis of cells after culturing for 14 days using blood of three healthy persons (represented by A, B and C in FIGS. 3 to 5), CD3-CD56 + cells (NK Cells (T cells, left graph in Figs. 3 to 5) and CD3 + CD56- cells (indicated by AX- + or AW- + in the left graph of Figs. 3 to 5) represent an average of 72.38% The ratio of CD3 + CD56 + cells (represented by AX ++ or AW ++ in the left graph of FIG. 3 to FIG. 5) showed an average of 4.37%. In addition, the ratio of cells expressing CD16 and CD69, which are active markers of cells, is 73.07% (indicated by AW- +, AX- + or AY- + in the middle graphs of Figs. 3 to 5) and 71.53% 5), and most of the cells expressing this active marker were found to be CD3-CD56 +.

Test Example  3: Interferon of activated lymphocytes - gamma  Production amount measurement

The amount of interferon gamma (interferon-gamma) secreted into the supernatant was assayed using an ELISA IFN-gamma Duoset kit (R & D system, DY285, USA) during the proliferation of large-volume activated lymphocytes. Cells containing the culture solution obtained by the examples were centrifuged at 2000 RPM for 5 minutes, and the supernatant was collected in a 15 mL tube.

Before proceeding with this experiment, 56 μL of capture antibody was added to 10 mL of phosphate buffer, and 100 μL of each was dispensed into a 96-well plate, followed by standing at room temperature for 8 hours or more. The solution contained in each well was removed and washed three times with 400 μL of washing solution (phosphate buffer containing 0.05% tween 20, the same applies hereinafter). After removing the washing solution completely, 300 μL of block solution (phosphate buffer containing 1% BSA) was dispensed. After standing for more than 1 hour at room temperature, the block solution was removed and washed. Then, 100 μL each of the supernatant to be analyzed and 1.8 μL of the recombinant interferon-gamma solution was added to 1 mL of the prepared standard solution (manufactured by Cat. 1311704), and the mixture was dispensed at room temperature for 2 hours It was politics. After removing all the solutions, 100 μL of detection solution (adding 56 μL of detection antibody to 10 mL of Reagent diluent) was dispensed into each well and allowed to stand at room temperature for 2 hours. After removing the detection solution and washing, 100 μL of Streptavidin-HRP enzyme solution (containing 10 μL of Reagent diluent and 50 μL of Streptavidin-HRP) was dispensed into each well and allowed to stand at room temperature for 20 minutes. After removing the enzyme solution and washing, 100 μL of the substrate solution (prepared by mixing Color Reagent A (H ₂ O ₂ ) and Color Reagent B (TMB) at a ratio of 1: 1) was dispensed into each well, . After observing the color reaction, 50 μL of the reaction suspension (H ₂ SO ₄ ) was added, and the absorbance at 450 nm was measured using a microplate reader (Molecular device, USA).

As shown in FIG. 6, the amount of interferon-gamma produced was 355 pg / mL, as shown in FIG. 6, after incubation for 14 days using blood of three healthy persons (represented by A, B and C in FIG. 6) Interferon-gamma. In general, it is known that interferon-gamma increases the activity of T cells and increases cytotoxicity of NK cells. As described above, it is expected that cytotoxicity of activated lymphocytes producing a large amount of interferon-gamma to cancer cells is also high In order to confirm this, Test Example 4 was carried out.

Test Example  4: Cytotoxicity of Activated Lymphocytes cytotoxicity ) Measure

Cytotoxicity of activated lymphocyte cells cultured by the example was measured using K562, a lymphocyte extracted from the bone marrow of a patient with chronic myelogenous leukemia, as a target cell. Prior to reacting the two cells, K562 cells were stained with Carboxyfluorescein succinimidyl ester (Invitrogen, USA) for 20 minutes in a humidified incubator maintained at 37 ° C and 5% carbon dioxide. After that, the cells were washed twice with 10 mL of phosphate buffer and the cells were suspended in RPMI culture without phenol red to prepare target cells. When both cells were prepared, the target cells and activated lymphocytes were placed in a U-shaped round-bottomed 96-well plate at a ratio of 1:10, and then reacted for 4 hours in a humidified incubator maintained at 5% carbon dioxide and 37 ° C. At this time, untreated target cells were also divided into one well and used as a control.

After the reaction was completed for 4 hours, 1 μL of PI (SIGMA, USA) reagent was added to each well, stained for 5 minutes, and fixed with 4% paraformaldehyde. Fixed cells were analyzed using Gallios (Beckman Coulter, USA), and the cytotoxicity results were calculated by subtracting the PI (+) ratio of the group containing only the target cells from the CFSE (+) PI .

As shown in FIG. 7, the cytotoxicity of the activated lymphocytes after 14 days of culture using the blood of three healthy persons (represented by A, B and C in FIG. 7) resulted in an average cytotoxicity of 73.34% .

While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, Of course it is possible. Accordingly, the scope of the present invention should not be construed as being limited to the above-described embodiments, but should be determined by equivalents to the appended claims, as well as the following claims.

Claims (7)

delete delete delete (A) isolating and obtaining mononuclear and autologous plasma from human peripheral blood, respectively;
(B) coating the cell culture vessel with an anti-CD3 antibody;
(C) a first culture step of suspending the mononuclear cells in a mixture of the autologous plasma, the interleukin-2 and a cell culture medium, and inoculating the monocyte into a cell culture container coated with the anti-CD3 antibody; And
(D) transferring the culture obtained through the first culturing step to a cell culture container not coated with the anti-CD3 antibody, culturing the monocyte with the mixture of the culture obtained through the first culturing step, interleukin-18 and poly I: And a second culturing step of cultivating the activated lymphocyte. The method of claim 4,
After step (D) above,
(E) culturing the mononuclear cells with a mixture of a culture obtained through the second culturing step, interleukin-18, poly I: C, the autologous plasma, interleukin-2, and a medium for cell culture To a culture medium. The method of claim 4,
After step (D) above,
Culturing the mononuclear cells with a mixture of the culture obtained through the second culturing step, the autologous plasma and the interleukin-2, and the culture medium for cell culture. The method of claim 4,
Wherein the self-derived activated lymphocyte is a natural killer cell (NK cell).

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