KR100569609B1 - Long-term lymphocyte storage method for the preparation of therapeutic drug - Google Patents

Abstract

The present invention is to collect the lymphocytes from the peripheral blood when the normal person is healthy for the preparation of cell immunotherapy treatment showing the effects of anti-cancer, anti-viral, and stored for a long period of time when the disease that requires the administration of immune cells in the future, fusion It is about how to restore.

The present invention provides a combination of interleukin 2, interleukin 1 alpha, interferon gamma, and anti-CD3 antibodies in a first step of collecting and isolating lymphocytes from peripheral blood in healthy individuals from normal individuals and in vitro. Presence of a second step of culturing activated lymphocytes, a third step of cryopreserving the activated lymphocytes for a period of time, and a fourth step of fusion and restoration of cells obtained in the cryopreservation step.

Lymphocytes, Culture, Proliferation, Activation, Immune Therapy

Description

Long-term storage method for lymphocytes for the preparation of cell-immune therapeutics {PROCESS FOR ITS PREPERATION, THE LYMPOCYTE LONGTERM CRYOPRESERVATION FOR A CELLULAR IMMUNOTHERAPY}             

Figure 1 is a graph of the immunoexpression of lymphocytes using flow cytometry (flowcytometry).

 The present invention is to separate lymphocytes from peripheral blood of healthy normal people to proliferate and activate lymphocytes in vitro, and to freeze-preserve the lymphocytes in case of a disease that requires the administration of immune cells to the cells. It is intended to be used as an immunotherapy agent.

 Conventional cell immunotherapy using lymphocytes is taking a method for producing cell immune treatment by collecting lymphocytes from the patient's blood in the situation where the disease has already occurred. This method presents several problems in terms of manufacturing and effectiveness. Most cancer patients undergo surgery, chemotherapy and radiotherapy, which can damage the patient's immune system, especially lymphocytes. In addition, cancer patients appear symptomatically poor systemic state of the body called cachexia, accompanied by a decrease in the function of lymphocytes. Therefore, the production of cell therapy products using lymphocytes after the onset should overcome the problem that the culture of lymphocytes, which is essential for the manufacturing process, cannot be easily achieved, and even if the damaged lymphocytes are cultured, it is difficult to obtain enough lymphocytes to effect. Even if this is a problem that the effect as a treatment is inferior.

  There is also a separate problem from the side effects of cell immunotherapy, which is very painful for the patient. Patients undergoing conventional chemotherapy already experience hardening of the veins, which makes it difficult to draw blood, which is essential for the manufacture of immune cell therapies, and thus, multiple blood collection attempts, and cancer patients usually have anemia. Many repeated blood collections may worsen anemia in patients.

Many attempts have been made to collect and store lymphocytes prior to onset to solve the problems described above, but methods have been introduced. However, cryopreservation and thawing have not solved the proliferative and immunological deterioration of lymphocytes.

SUMMARY OF THE INVENTION An object of the present invention is to solve the problems of the prior art as described above, by separating lymphocytes from peripheral blood when healthy from normal persons and proliferating and activating lymphocytes in vitro in a specific manner. After freezing and preserving lymphocytes, the self-administration of the lymphocytes after a certain period of time decreases their immunity and is likely to cause cancer or viral diseases. It is possible to obtain a preventive effect of the disease, and when a cancer or viral disease occurs, it is used as a cell immune treatment using stored lymphocytes.

The present invention relates to a long-term storage method of lymphocytes for the production of a cell immunotherapy agent,

 A first step of collecting and isolating lymphocytes from peripheral blood when healthy from a normal person; A second step of culturing activated lymphocytes by presenting the lymphocytes in vitro in combination with interleukin 2, interleukin 1alpha, interferon gamma, and anti-CD3 antibodies; And a fourth step of cryopreserving the activated lymphocytes by cryopreservation for a predetermined period and a fourth step of thawing and restoring the cells obtained in the cryopreservation step. Hereinafter, the specific contents of these inventions will be described sequentially.

 The first step refers to a step of collecting and separating lymphocytes from peripheral blood. In this case, lymphocytes are collected from one's own peripheral blood when healthy. Any material containing lymphocytes can be used. As the amount of blood collected, peripheral blood of about 0.001 ml to 500 ml is preferable, and peripheral blood collection within a range of about 10 ml to 100 ml is preferable.

 Heparin and citric acid may be added to the peripheral blood collected in the first step so that coagulation does not occur. The activated lymphocytes according to the present invention can be obtained by separating lymphocytes from the collected peripheral blood and proliferating and activating them through in vitro culture. In addition, with respect to the lymphocytes collected in the present invention, 'culture' means to artificially propagate lymphocytes in a culture medium containing the nutrients necessary for the lymphocytes to survive in vitro, and 'proliferation' means the test by the culture. In vitro, this means artificially increasing the amount of lymphocytes. In addition, 'activation' refers to controlling proliferation by actively adding a growth factor and an activating factor to the culture medium, and more specifically, antitumor and anti-infectious diseases and anti-immune disease characteristics. It means to have.

 The second step refers to a culturing step of activating and proliferating the lymphocytes. The method of culturing the lymphocytes obtained in the first step is not particularly limited, but any one of interleukin 2, interferon gamma, interleukin 1 alpha, and anti-CD3 antibody may be used. It is cultured by being present alone or in combination. In this case, culturing by combining these interleukin 2, interferon gamma, interleukin 1 alpha, and anti-CD3 antibody alone is superior to the anticancer effect. This will be described in detail below with reference to FIG. 1.

 Figure 1 is a result of examining the immune phenotype of lymphocytes using flow cytometry (a) and (b) in Figure 1 shows the immunophenotype of lymphocytes at the start of the culture, (c) and (d ) Shows the immunophenotype of lymphocytes after 7 days of culture. On the other hand, (a) and (c) is a case of culturing a combination of interleukin-2 and anti-CD3 antibody, (b) and (d) is an interleukin 2 and interferon gamma, interleukin 1 alpha, It shows the case where the culture was combined with anti-CD3 antibody. In addition, the x-axis of the graph of FIG. 1 is labeled with CD3 (T-lymphocyte marker) and the y-axis is labeled with CD56 (killer cell marker), and each graph is divided into four sections and analyzed. The upper right compartment represents the lymphocytes that are positive for both CD3 and CD56, and the lymphocytes that are positive for both CD3 and CD56 represent the most effective anti-cancer lymphocytes. Therefore, according to the assay of the present invention, when interleukin 2, interferon gamma, interleukin 1 alpha, and anti-CD3 antibody were cultured in combination (b), 4.9% of lymphocytes positive for both CD3 and CD56 were shown. However, in (d), it increased to 50.7%. On the other hand, when the combination of interleukin-2 and anti-CD3 antibodies was cultured, in (a), lymphocytes positive for both CD3 and CD56 were 4.9%, but in (c) it was 18.1%. In the case of the anticancer drugs, the interleukin-2 and interferon gamma, interleukin 1 alpha, and anti-CD3 antibodies were used as the embodiments of the present invention rather than the combination of only interleukin-2 and anti-CD3 antibodies ((b) and (c)). When cultured in combination ((a), (c)) it can be judged that the anticancer effect is much better.

 As described above, in the proliferation and activation of lymphocytes, it is most preferable to cultivate a combination of interleukin 2, interferon gamma, interleukin 1 alpha, and anti-CD3 antibody from the viewpoint of proliferative effect and anti-cancer effect. Antigen-specific T lymphocytes, and the like, and then CD3 antibodies or C28 antibodies, or various mitogens, may be used to obtain antigen-specific activated lymphocytes. In addition, the antigen is not only purified by the antigen, but also can be used for extracts from cancer cells or viruses, cancer cells or viruses themselves, or analogues having cross-reactivity with them. Any substance can be used as long as it has a substance.

  Interleukin 2 and interferon gamma and interleukin 1 alpha used in the second step may be commercially available, and the culture medium is preferably used at a concentration of 1-2000 U / ml, and interleukin 2 and interferon gamma and interleukin 1 alpha. Is dissolved in water, physiological saline, Dulbecco (Dulbecco) phosphate buffer, RPMI-1640, DMEM, IMDM, AIM-V and other commonly used cell culture medium solution. Once dissolved, it is necessary to be refrigerated to prevent deterioration of activity, and the culture medium used in this case is not particularly limited as long as it is suitable for the culture of lymphocyte cells. Synthetic media including amino acids, vitamins, nucleic acid bases, etc. may be used in the salt solution, and RPMI-1640, AIM-V, DMEM, IMDM, etc. may be preferred, but RPMI-1640 is particularly excellent.

As the culture medium used in the second step, it is preferable to use bovine fetal serum because it is excellent in proliferative effect, but commercial products may be used as these mediums. It is also possible to use serum of normal persons in addition to the fetal bovine serum. It is also possible to use serum-free medium that does not contain serum. Cultivation can be done in conventional cell culture methods, such as in a CO ₂ incubator. The CO ₂ concentration is in the range of 1 to 10%, preferably in the range of 3 to 7%, and the temperature is in the range of 30 to 40 ° C., and particularly preferably 35 to 38 ° C.

 The number of days in the culture is not particularly limited. However, since the stimulation information of the anti-CD 3 antibody should be transmitted to the cells, it is preferable to carry out about 2 to 9 days, and it is particularly preferable to perform the cells for 3 to 7 days. It is able to transmit stable stimulus information and is also ideal in terms of culture efficiency. Within the incubation period, it is more preferable for the culture efficiency to add the culture medium while observing the cell state under a microscope and measuring the appropriate cell number. In addition, in the culture, there is no cell proliferation on the first or second day of incubation, but when the cell proliferation is observed from the third day and starts to proliferate smoothly, the culture medium is changed from orange to yellow, and the additional amount of the culture medium is the medium in the culture before the addition. The addition of about 0.1-5 times is preferable with respect to the quantity of liquid. On the other hand, the addition cycle is necessary 1 to 7 days, preferably every 3 to 5 days in order to prevent degradation of the culture medium and degradation of interleukin 2 and interferon gamma, interleukin 1 alpha activity.

 In the culturing in the second step, monocytes are suspended in a culture medium solution containing interleukin 2, interferon gamma, and interleukin 1 alpha, and the culture can be started by placing them in a culture vessel in which the anti-CD3 antibody is solidified. In addition, in this case, the use of various cytokines and mitogens in the culture medium, if necessary, further improves the efficiency of proliferation and activation of the lymphocytes. In addition, the anti-CD3 antibody used for stimulation of the lymphocyte cells can be used by purification of the antibody produced in the animal or cells, commercially available OKT-3 antibody. However, in addition to this, any antibody that can promote the proliferation and activation of the lymphocytes is not particularly limited, and anti-CD28 antibodies and the like can also be used.

 In addition, the anti-CD3 antibody can be used in the culture medium solution, but is preferably solidified from the viewpoint of lymphocyte proliferation efficiency and ease of operation. As a mechanism for solidifying an antibody, a culture vessel made of a material such as glass, polyurethane, polyolefin, or polystyrene may be mentioned. In this case, commercially available cell culture flasks or the like may be used, and the size thereof may be appropriately selected. Solidification can be used by adding a dilution of the anti-CD3 antibody to the solidified apparatus and left at 4 to 37 ° C. for 2 to 24 hours. In addition, the anti-CD3 antibody is used by diluting the anti-CD3 antibody to a concentration of 0.1 to 30 μg / ml in a physiological buffer solution such as sterile Dulbecco's phosphate buffer solution. After solidification, cooling and storage in the refrigerator (4 ° C) can be maintained until use. In this case, the solution may be removed during use and washed in a physiological buffer such as Dulbecco's phosphate buffer solution at room temperature, if necessary.

The third step, say the cryopreservation comprising: store a certain period freezing the activated lymphocytes, lymphocytes preservation may choose a density that is suspended in the cell preservative solution appropriately depending on the size of dog ^{1 × 10 3 / ml ~ 1} × Suspension and cryopreservation are required in the stock at a density of 10 ¹⁰ / ml. In addition, although the quantity of the cell preservation liquid used in this case is not determined, it can be used in the range of 0.1 ml to 1000 ml for convenience, but the inside of the range of 0.5 ml to 100 ml is more preferable.

 The cryopreservation liquid in the third step can be used not only commercially available cell preservation liquid, but also self-prepared. The components of the cell preservation fluid may be those containing high molecular weight substances such as serum, protein and polysaccharides, and dimethyl sulfate (hereinafter referred to as 'DMSO') in a suitable buffer or basal medium. It is not. Therefore, the composition is not limited as long as it can preserve cells. Lymphocytes are suspended in appropriate cell preservatives and cryopreserved at low temperatures. The prepared cryopreservation liquid may be stored in a refrigerator (4 ° C.) until use after manufacture.

The freezing tube in which the frozen cells are to be preserved in the third step is preferably a commercially available cell freezing tube, and a freezing bag may be used, and the size thereof may be appropriately selected. The number of cells to freeze is 1 ~ 5 × 10 ^{7 for} each freezing tube is appropriate, the number of freezing tubes depends on the volume of blood collected and can store up to 2 to 1000 units, thaw, thaw when used Then restore and administer. On the other hand, in the case of freezing can be frozen through a refrigerator, freezer, cryogenic freezer, nitrogen tank, etc., it is preferable to use a freezing treatment container in terms of stability and growth efficiency of lymphocytes. The freezing container may be a freezing container such as Bicell, which is commercially available, and can be developed and used directly. In addition, the storage period of the frozen cells in the freezing container is within the range of 2 to 30 days, preferably between 3 and 9 days, and when moving from the freezing container to the nitrogen tank is a cane or freezing tube box, etc. It is preferable, but other commercial items that can enter the nitrogen tank may be used.

 Hereinafter, the long-term storage method of lymphocytes for use in the preparation of a cell immunotherapy agent according to the present invention will be described sequentially.

1. Isolation of blood / lymphocytes

60ml ~ 80ml of peripheral blood was collected from human vein and heparin was added to disinfect the syringe port of the collected blood sample aseptically in Clean Bench (JISICO), and blood was drawn using 50ml syringe (Korean vaccine). Then, 60 ml of washing medium (RPMI1640) was injected into a 250 ml centrifuge tube, and the blood collected was slowly injected into the centrifuge tube.

 Completely cover the centrifuge tube, mix 2-3 times, mix 10 ml of Ficoll (Amershamphamacia) into 4 50 ml centrifuge tubes, using a 10 ml pipette, and place 30 ml of the diluted blood into each centrifuge tube. Put slowly so as not to scatter. In addition, centrifuge this at 20 rpm for 20 minutes at 2000 rpm and centrifugation temperature with a centrifuge, and then slowly suck the lymphocyte cells up to about 1 cm above the lymphocyte layer in the sterile centrifuge tube. It was. Also, take a lymphocyte separation layer with a 5 ml pipette to collect the lymphocyte separation layer, collect it into two 50 ml centrifuge tubes containing 25 ml of washing medium (RPMI1640) in advance, and close the lid of the centrifuge tube to mix for 2-3 times. After centrifugation for 10 minutes at 1800rpm rpm, 20 ° C temperature again.

After centrifugation, the supernatant was discarded and the lymphocyte sediment was agitated to release well. After counting the lymphocytes, lymphocytes were measured in 300 ml of interleukin 2, 100 µl of interleukin 1, 100 µl / ml in 90 ml of medium (RPMI1640). Lymphocyte suspension was prepared by adding interferon gamma, 10 ml of fetal bovine serum, and 100 ml of culture medium (hereinafter referred to as 'culture medium') to conduct well-mixing. 10 µl of the lymphocyte suspension was mixed with 40 µl of turk solution, and 10 µl was supplied to the hemocytometer and the number of lymphocytes was measured under a microscope (Olympus). The total lymphocyte count was 2.0 × 10 ⁷ to 1.0 × 10 ⁸ .

2. Presence of Solidified Flasks of Anti-CD3 Antibodies

10 ml of anti-CD3 antibody prepared at 5 μl / ml in PBS was placed in a culture flask with a floor area of 225 cm ² , and the solution was spread evenly on the bottom. The next day, the antibody solution of the flask was sucked into the aspirator, 50 ml of PBS was poured into the flask, and then the lid of the flask was closed and shaken. Then, the liquid in the flask and the lid was slowly sucked by the aspirator to prepare a solidified flask of anti-CD3 antibody.

3. Activating Lymphocyte Culture

100 ml of the lymphocyte suspension was poured into two solidification flasks of the anti-CD3 antibody prepared in 2. above, and cultured in the presence of carbon dioxide at 37 ° C and 5% concentration. After 3 days, 50 ml of the culture medium was added to each flask, and cultured in the presence of carbon dioxide at 37 ° C. and 5% concentration. After 4 days, 100 ml of the culture medium was added to each flask and incubated in the presence of carbon dioxide gas at 37 ° C and 5% concentration. In addition, after one day, by culturing in the presence of carbon dioxide gas at 37 ° C, 5% concentration, activated lymphocytes 3.0 × 10 ⁸ ~ 10 × 10 ⁸ were obtained.

4. Cryopreservation of activated lymphocytes

After centrifuging the activated lymphocyte mixture obtained in the above 3. The culture medium was removed by an aspirator to obtain an activated lymphocyte precipitate. 19.8ml of the cell preservation solution (CP-1 68ml, 20% albumin 32ml, medium (RPMI1640) mixed with 100ml) was added to the activated lymphocyte sediment, mixed well, and 1.8ml into a 1.8ml cell preservation tube (corning). Each tube was separately injected into 11 tubes. The tube was placed in the freezing container and stored at -80 ° C., and then transferred to a nitrogen tank after 3-7 days.

5. Thawing, Examination of Cryopreservation-activated Lymphocytes

After 30 days, one cryopreserved tube was taken out from 4., thawed for 4 minutes in a 37 ° C heat block, and the cryopreservation activated lymphocytes were fused and restored. About 1.8 ml of the cell stock solution containing the fused cryopreservation activated lymphocytes was transferred into a 15 ml centrifuge tube aseptically and suspended by injecting 7.2 ml of the medium. 2.5 ml of cell stock was transferred to a 15 ml tube, followed by centrifugation at 1600 rpm, 20 ° C for 10 min, and then the supernatant was removed and suspended by adding 40 ml of the culture medium. 10 μl of the cell suspension was mixed with 20 μl of trypan blue (Gibco) solution, and 10 μl was supplied to the hemocytometer and the survival rate was measured under a microscope (Olympus). The survival rate was 75% or more and the recovery rate was 70% or more. The activated lymphocyte suspension was dispensed into 2 wells per 24 well plate and tested for survival and recovery of lymphocytes after 2 days. The survival rate was 95% or more and the recovery rate was 100% or more.

As described above, the present invention overcomes the difficulties in the manufacture of cell-immune therapeutics and solves the problem that the anti-cancer effects of cell-immune therapeutics are inferior, thereby separating lymphocytes from peripheral blood when they are healthy and in vitro experiments (in vitro). By proliferating, activating and freezing and storing them for a long time, the disease can be used as a cell-immunotherapeutic agent in the event of a disease that requires immune cells to be administered later, and lymphocytes can be treated in the culture stage in vitro. The presence of a combination of interleukin 1 alpha, interferon gamma, and anti-CD3 antibodies can significantly enhance anticancer effects.

Claims (4)

A first step of collecting and isolating lymphocytes from peripheral blood in healthy individuals from normal persons; activating the lymphocytes by presenting a combination of interleukin 2, interleukin 1alpha, interferon gamma, and anti-CD 3 antibodies in vitro Culturing lymphocytes; And a fourth step of cryopreserving the activated lymphocytes by cryopreservation for a predetermined period of time and a fourth step of thawing and restoring the cells obtained in the cryopreservation step. The method of claim 1, The anti-CD3 antibody of the second step is a method for organ storage of lymphocytes for the preparation of a cell-immunotherapy, characterized in that the solidified use. Cryopreservation activated lymphocytes, which are fused and restored by the method of claim 1. The method of claim 3, wherein The cryopreservation activated lymphocytes have the effects of anticancer and antiviral.

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