TWI439275B - An ex vivo method for expanding human natural killer cells - Google Patents

Description

In vitro proliferation method of human natural killer cells

The invention provides a method for proliferating human natural killer cells, which can achieve the killing activity of natural killer cells by at least 500 times the number of cells before the proliferation of natural killer cells by the method of the invention, and is a natural killer for human beings. Cells provide a practical method when used to prepare a combination of medical drugs such as tumor vaccines.

According to the Department of Health, cancer has consistently ranked first among the top ten causes of death in Taiwan. The extent of cancer impact and the cost of social costs are evident. At present, there are at least five categories of anticancer therapies that are well known to humans, including surgical resection, chemotherapy, and radiation therapy, as well as combination therapy and folklore therapy.

Although the surgical treatment is very effective, some tumors cannot be surgically removed, and there are still residual cancer cells that are likely to cause recurrence or metastasis after surgery. This is the main reason why cancer is terrible and causes death. Cancer cells that have been transferred or cannot be surgically removed must be treated with radiotherapy or chemotherapy, or even in the current medical treatment.

However, radiotherapy or chemotherapy is not specific, killing both good cells and bad cells at the same time, leading to serious side effects, which are the most serious damage to the immune system that is most critical to the patient's own anti-cancer ability. At present, one of the problems that medicine has been unable to overcome, so there is still room for improvement in such treatment in terms of prolonging life or reducing side effects. Therefore, for cancer treatment, all walks of life are actively seeking treatments that can fight cancer without side effects and strengthen immune function.

Natural killer cells (NK cells) are one of the first native immunity members in the human body and the strongest and most effective cell against cancer cells or viruses. Compared with other anti-cancer immune cells, such as Cytotoxic T cells or Dendritic cells, NK cells are more toxic, more broad-spectrum, and not subject to histocompatibility. (MHC-Restriction) does not require the initiation of sensitization (Primimg) to directly launch an attack on cancer cells. In addition, NK cells have the ability to modulate immunity and interact with the nervous system. In theory, NK cells can be an effective treatment for cancer. However, the number of such cells in the body is usually not much, only about 2 to 6% of white blood cells in the blood, plus disease, age, stress, and malnutrition. Due to lifestyle and other environmental factors, the number and activity of NK cells often decrease with age, directly or indirectly contributing to the occurrence or deterioration of cancer. Clinically, it is often observed that the number of NK cells in cancer patients is insufficient or The phenomenon of decreased activity. The conventional technology cannot proliferate a large amount of NK cells in a short time in vitro, and at the same time retains the cytotoxic activity of NK cells. Furthermore, the NK cells used in the prior art are taken from patients who have already obtained cancer, The NK cells obtained in the state are not strong enough to kill cancer cells. Even if they are regenerated and then returned to the patient, the cancer cells can not be killed. The idea of using NK cells to treat cancer is very attractive. Force, but the actual implementation is not practically applied to cancer treatment due to the lack of conventional technology.

The object of the present invention is to provide a method for in vitro proliferation of human natural killer cells, which can proliferate CD3 ^- CD56 ⁺ subtype natural killer cells from cryopreserved peripheral blood mononuclear cells (PBMCs). In the method, after the natural killer cells are proliferated by the method, the number of cells at least 500 times higher than that of the proliferating cells can be achieved, and the killing activity of the natural killer cells is maintained, which is a natural killer cell for human beings when preparing a medical drug combination such as a tumor vaccine. Provide a practical method.

Still another object of the present invention is to provide a practical medical solution and technique for tumor NK cell therapy, for example, NK cells cultured using the proliferation technique of the present invention are used for preparing a medical composition such as a tumor vaccine.

For a better description and understanding of the present invention, we will explain the terminology involved in the present invention in detail.

Natural killer cells (NK cells): NK cells are a group of large granular lymphocytes different from T and B lymphocytes. They are distributed in various lymphoid organs and blood circulation systems. They can be used without pre-stimulation and activation of antigens. The cell kills the effect and secretes a variety of cytokines and chemokines.

Activation of NK cells: NK cells express a series of activating receptors and inhibitory receptors, and the balance between the two is an important mechanism for controlling whether NK cells are activated. At the same time, interferon and macrophage-derived interleukins are highly potent activators of NK cells. In the present invention, recombinant human interleukin (rhIL) 2 (rhIL-2), 12 (rhIL-12), 15 (rhIL-15), 18 (rhIL-18), and 21 (rhIL-) are utilized. 21) and combinations thereof to activate NK cells.

Proliferation of NK cells means that NK cells reach a certain number of proliferation through a series of cell divisions in vitro. The NK cells proliferated in the present invention are mainly NK cells of the CD3 ^- CD56 ⁺ subgroup.

Cytotoxic activity: refers to a simple cell killing event caused by cells. The cytotoxic activity of NK cells is not limited to tumor cells, but also includes killing effects on virus-infected cells, damaged and dysfunctional cells. The NK cell toxicity activity involved in the present invention mainly refers to its killing effect on tumor cells. The determination of NK cell cytotoxic activity is mainly based on the use of erythrocyte-derived erythroleukemia cell line K562 and NK cell-resistant lymphoma cell line Raji as target cells, and the killing rate of NK cells is determined. In the present invention, K562 cell line is used to determine NK cell virulent activity.

NK Cell Therapy: It is a kind of high-tech cell culture technology that can multiply the NK cells in the short-term (Ex vivo) patients themselves to fight cancer cells, and then inject them into the patient to directly improve the patient's own resistance. Cancer ability, a medical technology that achieves anti-cancer and cancer treatment goals. The premise of NK cell therapy is the initiation and proliferation of NK cells, and maintains high cytotoxic activity against tumor cells.

The immunocytotherapy of autoimmune cells, especially NK cells, is simply a high-tech cell culture technique that allows a large number of patients in vitro to excrete NK cells against cancer cells in a short period of time. Then, it is injected into the patient to directly improve the patient's own anti-cancer ability. The actual practice is that after the patient has carefully evaluated and determined that it is suitable for NK cell therapy, about 30 ml of peripheral blood is collected from the patient for 15 consecutive days in a GTP/GMP-compliant dust-free, sterile laboratory. Specially formulated to specifically culture and proliferate NK cells, increase the number to hundreds of times or even thousands of times before injecting into the body, directly increasing the number of NK cells to achieve the purpose of treating cancer.

The use of NK cell therapy to treat cancer has the following various academic advantages: First, NK cells are the most potent anti-cancer cells in the human body, which can directly kill cancer cells to inhibit tumor growth and spread; Second, NK cells will inhibit tumors nearby. The proliferation of new blood vessels can limit the tumors to obtain the nutrients needed to limit the growth of tumors. Third, NK cells can directly improve and regulate the patient's immunity and nervous system, and indirectly improve the quality of life of patients; Fourth, NK cells will Secreting a brain endorphin called b-endorphin can reduce the pain of the patient and make the patient feel happy, and indirectly improve the quality of life. 5. Low side effects or almost no side effects. At present, some studies have pointed out that the peripheral blood natural killer cells have a range of poisoning in addition to melanoma, head and neck cancer, as well as lung, ovary, cervix, bladder, prostate, liver cells, pancreas, esophagus, breast. And many combined cancers. Especially for cancers such as lung cancer, lymphoma, esophageal cancer, breast cancer, and liver cancer, which have strong blood flow and dense blood vessels.

Under normal circumstances, we will have 5 to 15 cancer cells in our body. At that time, as long as one NK cell can kill these cancer cells. However, once cancer is acquired, every cancer cell needs 250 NK cells in the body to kill. Therefore, storing NK cells with high fighting power before cancer can be copied into a large number in the future, and then injected into the body to help fight cancer. It is currently the most effective and no side effect.

The method for proliferating NK cells provided by the invention satisfies the conditions of NK cell therapy and provides a practical medical technique for tumor NK cell therapy.

The present inventors have attempted to solve the problem of tumor NK cell therapy, and have found a method for starting and proliferating NK cells from frozen peripheral blood mononuclear cells. The proliferating NK cells are a CD3 ^- CD56 ⁺ subpopulation with high tumor cell toxicity. Cell culture conditions are described in detail in the examples. According to the results of the embodiments of the present invention, after the peripheral blood mononuclear cells are cultured for 5 days in vitro, the NK cells start to proliferate, and the inventors call it a start-up phase. In the subsequent 1 to 10 days of process culture, the inventors call it For the proliferative phase, NK cells will continue to proliferate 100 to 500 times, or even 1000 times.

According to the results of the examples of the present invention, the NK cells activated and proliferated have a cytotoxic activity increased by at least 100%, preferably by 150% to 200%, and even more preferably by 400. %. The NK cells which are not activated and proliferated in vitro in the present invention refer to fresh NK cells which are positively sorted using CD56 antibody magnetic beads. The increased cytotoxic activity of NK cells that initiate and proliferate may be due to an effect mediated by up-regulation of binding receptor expression.

Specific methods of NK cell initiation and proliferation are described in the Examples of the present invention. It should be emphasized that in a culture system in which cryopreserved peripheral blood mononuclear cells initiate and proliferate NK cells, peripheral blood mononuclear cells are a mixture containing different cell populations, such as NK of CD3 ⁺ CD56 ^- subpopulation. Cells, NKT cells (CD3 ⁺ CD56 ⁺ ) and T cells (CD3 ⁺ ), so after the initiation of proliferation, in addition to the large proliferation of CD3 ^- CD56 ⁺ subgroup NK cells, there are other cell types in the culture system that initiates proliferation. presence.

According to the results of the examples of the present invention, the inventors have also elaborated a clinical administration scheme of NK cells in detail. The inventors found that the optimal state of NK cells was on the 9th to 14th day of culture during a series of observations of NK cell culture and its state. According to the state of the cells, the inventors will change the serum-free medium on days 9-10 and 13-14, change the solution after 48 hours, and then reach the optimal state after 24 hours. The number of NK cells reaches 10-8 billion. between. Therefore, this is the best time to return the cells to the human body.

According to the cytotoxic activity of NK cells against tumor cells in the present invention, the present invention encompasses the administration or application of NK cells in combination with other cancer treatments. For example, in combination with surgery, radiation therapy, and cytotoxic drugs. At the same time, the related research results of the present invention suggest that the proliferating NK cells in vitro have obvious anti-tumor ability compared with NK cells which are not activated in vivo. Therefore, the present invention also includes the killing effect of NK cells on tumors. The said tumor cell refers to an autologous tumor cell. Autologous tumor cells refer to cells that are sourced and reimplanted in the same individual, that is, the donor and recipient are the same person. The above autologous tumor cells include hematological tumors and solid tumors.

Compared to previous methods of NK cell proliferation invention, the present invention encompasses the following distinct differences or advantages:

1. The present invention extracts peripheral blood of a healthy human individual, and extracts peripheral blood mononuclear cells (PBMCs) therein for proliferation of NK cells, and ensures that the vitality, quality, and toxicity of NK cells are in comparison with conventional techniques. A healthy and effective state.

2. The invention stimulates the proliferation of NK cells by using rhIL-2, rhIL-12, rhIL-15, rhIL-18, rhIL-21 and a combination thereof in the process of NK cell proliferation, and more effectively ensures that NK cells have high cells. Toxic killing activity.

3. The proliferation method of the present invention can cause NK cells to reach a cell number at least five times higher than the number of pre-proliferation cells after proliferation.

4. The invention adopts autologous serum to culture in the process of NK cell proliferation, so that the NK cell tumor treatment scheme is safer.

In the present invention, the use of different stem cell culture medium to mix and culture NK cells is effective in reducing the cost of NK cell proliferation, and also provides a practical method for NK cells to be used in the preparation of a combination of medical drugs such as tumor vaccines.

In the following, the inventors will elaborate on how to initiate proliferation of NK cells from cryopreserved peripheral blood mononuclear cells. At the same time, the inventors will further explain the cytotoxic activity of the activated proliferating NK cells. These data provide a solid basis for tumor NK cell therapy.

Embodiment 1: Please refer to FIG. 1(A), FIG. 1(B), and FIG. 1(C). Research sample source:

The peripheral blood comes from 5 healthy blood donors, who are clinically diagnosed with no hematological malignancies and severe autoimmune diseases. The study was approved by the local research ethics committee and an informed letter was signed with all blood donors.

Extraction, cryopreservation and resuscitation of peripheral blood mononuclear cells (PBMCs):

All operations are strictly in accordance with the Standard Operating Procedure (SOP) program. 20-40 ml of peripheral blood of the subject (about 3-6 X10 ⁷ mononuclear cells) was collected. Mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation, and the isolated mononuclear cells were cryopreserved with a mixture of 90% autologous serum and 10% dimethylarsine. When cryopreserving cells, the preferred embodiment is to first place them at 4 degrees Celsius for 2 hours, then place them at minus 20 degrees Celsius overnight, and finally store them in a cryopreservation device containing liquid nitrogen for a long time, or first contain 90% autologous serum and 10% dimethyl sulfoxide and peripheral blood mononuclear cells (PBMCs) were placed at 4 degrees Celsius for 10 minutes, then placed at minus 20 degrees Celsius for 30 minutes, then placed at minus celsius After 80 ° overnight, it was finally stored in a cryopreservation device containing liquid nitrogen for a long time. Another suitable method of cooling is to place a mixture containing 90% autologous serum and 10% dimethyladenine and peripheral blood mononuclear cells (PBMCs) in a programmed-controlled cooling machine for cooling. When cryopreserving cells, the speed of cooling should preferably be maintained at 1 to 3 degrees Celsius per minute, so as to avoid cell rupture if the temperature is too fast, or the cell is inactive due to too slow cooling rate. When proliferating peripheral blood mononuclear cells (PBMCs), the frozen cell tubules were taken out and rewarmed in a 37-to 40-degree water bath. After the contents of the tubules were all melted, the cells were aspirated and diluted in 5 volumes of serum-free RPMI 1640 medium at 1200 rpm. Centrifuge at /min for 5 minutes, discard the supernatant, wash twice, and resuspend in serum-free RPMI 1640 medium.

A method for in vitro initiation of proliferation of cryopreserved peripheral blood PBMCs in NK cells:

The obtained PBMCs were centrifuged, resuspended in 5% human autologous serum stem cell culture medium, and placed in a culture flask at a cell density of 0.5×10 ⁶ /ml, and recombinant human interleukin 2 (rhIL-) was added. 2) The final concentration was 500-750 U/ml and the mouse anti-human CD3 mAb, 10-20 ng/ml, was cultured for 5 days. The cells were counted on the 6th day, and the Hanks solution was washed 1-2 times, and then the stem cell culture medium containing 3-5% human autologous serum was added, and the stem cell culture medium may contain rhIL-2 (final concentration of 500 U/ml), rhIL-12. (final concentration is 200 U/ml), rhIL-15 (final concentration 20-30 ng/ml), rhIL-18 (final concentration 100 U/ml), rhIL-21 (final concentration 100 U/ml), and combinations thereof Then, the cells were resuspended to maintain the cell density at about 0.5 x 10 ⁶ /ml. In a preferred embodiment, the cells are counted at 0, 10-11, and 14-15 days, the cell ratio is measured by flow cytometry, and the number of cell proliferation is recorded in detail (Fig. 2), and supplemented according to the proliferation of the cells. Stem cell culture medium containing 3-5% human autologous serum, which may be rhIL-2 (final concentration 500 U/ml), rhIL-12 (final concentration 200 U/ml), rhIL-15 (final concentration is 20-30ng / ml), rhIL-18 ( final concentration of 100U / ml), rhIL-21 ( final concentration of 100U / ml), and combinations thereof, the cell density is maintained at about 0.5x10 ⁶ / ml.

The natural killer cells obtained by the method of the present invention can be used together with an appropriate volume of the human autologous serum and an appropriate volume of physiological saline to prepare a medical drug combination such as a tumor vaccine, for example, by intravenous injection. Lost into the human body.

In order to verify the efficiency of NK cell initiation of proliferation, the inventors performed proliferation experiments of NK cells in peripheral blood PBMCs of five donors cryopreserved. In the PBMCs of 5 healthy donors, flow cytometry analysis was performed before the initiation of proliferation, and it was confirmed that the CD3 ^- CD56 ⁺ subgroup NK cells were 9.7% ± 2.1% of the total cells, and the CD3 ⁺ cells, mainly T cells were the total. 61% ± 8%. After 15 days of initiation of proliferation, the total number of cells proliferated by 135 ± 11 times (Fig. 3). The proportion of NK cells increased from 9.7%±2.1% before proliferation to 62%±3.6% (Fig. 4). Due to the increase in the proportion of NK cells, the inventors calculated that the proliferation ratio of NK cells was 888±165 (Fig. 5 times. The above results confirmed that the inventors can effectively proliferate NK cells from cryopreserved peripheral blood PBMCs, and the proliferated NK cells are mainly CD3 ^- CD56 ⁺ subpopulation cells.

According to the actual production of NK cells in vitro, the cells can be administered intravenously on the 9th-16th day of cell culture, and the preferred embodiment is cell culture days 10-11 and 14-15. It is expected that a large number of NK cells will continue to proliferate in the case.

Example 2. Detection method of NK cell toxicity activity:

K562 cells in logarithmic growth phase were collected, NK cells were activated on the 5th and 15th day of culture, and NK cells were positively sorted by magnetic beads in vitro. K562 cells were used as target cells, and the cell concentration was adjusted to 1 x 10 ⁵ /ml. NK cells as effector cells in order to adjust the different ratios of effector to target cells according to the concentration of ^{1x 10 5 /ml,1.5x 10 5 / ml} , 1x 10 6 / ml. The effector cells and the target cells were mixed according to different target ratios (1:1, 5:1, 10:1), and corresponding target cell wells, effector cell wells and medium blank control wells were set. Each set has 3 parallel holes. After incubating in a 37 ° C, 5% CO 2 , saturated humidity incubator for 12 hours, 10 ul of Cell Counting kit-8 (Japan Tongren Chemical) was added to each well, and mixed in a 37 ° C, 5% CO 2 , saturated humidity incubator. After continuing to culture for 4 hours, the OD value at a wavelength of 450 nm per well was measured with a microplate reader. Calculation method of cytotoxic activity: The average value of parallel wells was determined, and the NK cell toxicity activity of each group was calculated by the following method, and the killing rate was expressed by %.

Cytotoxic activity = [1 - (target cell pore OD value - effector cell pore OD value) / target cell pore OD value] x 100%

The present inventors examined and compared the cytotoxic activity of the activated proliferating NK cells, using K562 cells as target cells, comparing NK cells positively sorted by cell magnetic beads in vitro, and NK cells proliferating for 5 and 15 days using the present invention. Cytotoxic activity. At a target-to-effect ratio of 1:10, the NK cell cytotoxic activity that initiated proliferation in 15 days was 65% ± 10%, and the NK cell cytotoxic activity that initiated proliferation in 5 days was 51% ± 6.3%, while the cell magnetic beads The positively sorted NK cell cytotoxic activity was only 9% ± 3.2%. At a target-effect ratio of 1:5, the cytotoxic activity of the three was 51%±8.2%, 39%±5.1%, and 7%±2.4%, respectively. At a target-to-effect ratio of 1:1, the cytotoxic activity of the three was 31% ± 5.6%, 22% ± 3.7%, and 4% ± 1.4%, respectively (Fig. 6). The above results revealed that the proliferating NK cells of the present invention have high killing activity against tumor cells.

The above embodiments are intended to be illustrative of the present invention, and are not to be construed as limiting the scope of the invention. The principles are changed and modified to achieve an equivalent purpose, and such changes and modifications are to be included in the scope defined by the scope of the patent application as described later.

Figure 1 (A) is a flow chart of a preferred embodiment of the method of the present invention.

Figure 1 (B) is a flow diagram of a preferred embodiment of cryopreservation of peripheral blood mononuclear cells (PBMCs) in the method of the present invention.

Figure 1 (C) is a flow diagram of a preferred embodiment of natural killer cells in proliferating peripheral blood mononuclear cells (PBMCs) in vitro in the methods of the present invention.

Figure 2 is a flow cytometric analysis of NK cell ratios on days 11 and 15 of proliferation of NK cells in accordance with the methods of the present invention.

Figure 3 is a graph showing the results of culture growth using five healthy human peripheral blood mononuclear cells (PBMCs) according to the method of the present invention.

Figure 4 is a graph showing changes in the proportion of NK cells during proliferation of peripheral blood mononuclear cells (PBMCs) of five healthy individuals according to the method of the present invention.

Figure 5 is a graph showing the results of NK cell culture proliferation in peripheral blood mononuclear cells (PBMCs) of five healthy persons according to the method of the present invention.

Fig. 6 shows the results of detecting cytotoxic activity by proliferating NK cells according to the method of the present invention and using K562 cells as target cells.

Claims (9)

An in vitro proliferation method of natural killer cells (NK cells), which is a kind of natural killer cells that proliferate CD3 ^- CD56 ⁺ subtypes from frozen peripheral blood mononuclear cells (PBMCs). The method, wherein the method comprises the steps of: (a) obtaining a peripheral blood sample of a human subject; (b) obtaining a peripheral blood mononuclear cell (PBMCs) from the peripheral blood sample; (c) obtaining the peripheral blood Mononuclear cells (PBMCs) are suspended in a mixture and stored frozen; wherein the composition of the mixture is 90% of one of the human individual autologous serum and 10% dimethylammonium (DMSO); (d) The peripheral blood mononuclear cells (PBMCs) are thawed; and (e) the peripheral blood is cultured in vitro using a stem cell culture solution containing the human serum and a recombinant human interleukin. The mononuclear cells, in the peripheral blood mononuclear cells on the 15th day of in vitro culture, the number of natural killer cells is at least 888 times higher than the number of cells before proliferation in vitro; step (e) comprises the following steps: (i) culturing the peripheral blood mononuclear cells (PBMCs) after thawing with a stem cell culture solution containing 5% of the autologous serum of the human individual, and adding a recombinant human leukapherin 2 (final concentration of 500) -750 U/ml) and a mouse anti-human CD3 mAb (final concentration of 10-20 ng/ml); and (ii) after 6 days, cultured with a stem cell culture medium containing 3-5% of the human individual's autologous serum And adding the recombinant human leukocyte interstitial to continue culture; the recombinant human leukocyte interstitial component comprises recombinant human leukocyte interleukin 2 (final concentration of 500 U/ml), recombinant human leukocyte interleukin 12 (final concentration of 200 U /ml), recombinant human leukocyte interleukin 15 (final concentration 20-30 ng/ml), recombinant human leukapherin 18 (final concentration 100 U/ml), and recombinant human leukapherin 21 (final concentration is 100U/ml). The proliferation method according to claim 1, wherein the human individual can be a healthy human individual or a human tumor patient. The proliferation method according to claim 1, wherein the step (c) cryopreserving the peripheral blood mononuclear cells (PBMCs) comprises the steps of: (i) preparing one containing 90% human individual autologous serum and 10%; a mixture of dimethyl hydrazine (DMSO); (ii) mixing the peripheral blood mononuclear cells (PBMCs) obtained from the peripheral blood sample with the mixture; (iii) containing the peripheral blood mononuclear cells (PBMCs) the mixture is placed between 1 and 10 degrees Celsius for a period of one minute to three hours; (iv) the mixture containing the peripheral blood mononuclear cells (PBMCs) is placed in Celsius Between zero and minus 40 degrees, the length of time is between ten minutes and forty-eight hours; (v) placing the mixture containing the peripheral blood mononuclear cells (PBMCs) between minus 50 degrees Celsius and minus 100 degrees Celsius The length of time is from 20 minutes to 48 hours; and (vi) the mixture containing the peripheral blood mononuclear cells (PBMCs) is placed in a cryopreservation device containing liquid nitrogen for long-term cryopreservation. The proliferation method according to claim 3, wherein the steps (iii) to (v) are replaced by the step of: placing the mixture containing the peripheral blood mononuclear cells (PBMCs) in a program-controlled manner In a cooling machine, and cooling down at a rate of 0.1 to 5 degrees Celsius per minute. The proliferation method of claim 3, wherein the step (v) can be omitted. The method for in vitro proliferation of human natural killer cells according to claim 1, wherein the step (e) further comprises a step (f): culturing in a serum-free cell culture medium starting from the 9th to 10th day of the in vitro culture. The in vitro proliferation method of human natural killer cells according to claim 6, wherein in the peripheral blood mononuclear cells proliferated by the step (e) and the step (f), the ratio of the number of the natural killer cells after the proliferation is greater than Or equal to 62%. The in vitro proliferation method of human natural killer cells according to claim 1, wherein the cytotoxic activity of the natural killer cells proliferated by the step (e) reaches at least 65% at a target ratio of 1:10. 2. The proliferation method according to 2, 4, 5, 6, 7, or 8, wherein the natural killer cell obtained by the proliferation method can be used for preparing a medicine within a period of 9-16 days of cell culture. Composition.