TW202000900A - A method for in-vitro expansion of nature killer cells (nk cells) and nature killer t cells (nkt cell) and the pharmaceutical composition thereof. - Google Patents

Abstract

This invention relates to a method for in vitro expansion of nature killer cells (NK cells) and nature killer T cells (NKT cells). The method includes the following steps: culturing cells at an initial cell density of 0.5x10<SP>6</SP> to 5x10<SP>6</SP> cells/mL, and adjusting the cell density again to 0.5x10<SP>6</SP> to 5x10<SP>6</SP> cells/mL at a specific time point after culturing the cells, so as to maintain a better cell growth environment and further to increase the cell expansion efficiency. Accordingly, compared with prior arts, the method of this invention has the advantages of obtaining more cells within a shorter period of time and achieving higher survival rate of the cells and higher ratio of the NK cells to the NKT cells. The ratio of the NK cells to the NKT cells which are in an activated state and have cytotoxicity is relatively high as well.

Description

Method and medical composition for in vitro expansion of natural killer cells and natural killer T cells

The invention relates to a method for in vitro expansion of natural killer cells (NK cells) and natural killer T cells (NKT cells), in particular to a method capable of expanding NK/NKT cells three to seven thousand times in vitro, and the method The expanded NK/NKT cells have a cell survival rate higher than 85%, the active NK/NKT cells are over 50%, and the cytotoxic effect on cancer cells is more than 76%.

Immunotherapy generally refers to the way to prevent and treat diseases by strengthening the organism's own immune system, or to impart immunity to the organism, and it has the characteristics of high specificity, high efficiency and durability; nearly ten Over the years, immune cell therapy derived from the concept of immunotherapy has gradually been widely explored.

In 2011, Immune Cell Therapy was first presented with a potential review in the international journal Nature , which is regarded as a possible cure for cancer; Immune Cell Therapy focuses on stimulating the immune system and can disrupt surgery (such as radiation therapy or chemotherapy) ) Cancer cells remaining afterwards, thereby improving the efficacy, and reducing the toxic effects of surgery or chemotherapy, radiotherapy, to improve the quality of life and prognosis of patients; for example, the use of immune cell therapy to treat cancer is isolated from the blood of patients Immune cells are cultured, amplified and activated in vitro, and then returned to the patient's body to produce a specific immune response to tumors in specific tissues in the body. The use of self-immune cells to reinject for treatment can also avoid immunity Rejection; among the immune cells mentioned, natural killer cells (NK cells) can directly kill the target cells without presenting antigen through antigen presenting cells, so it is predicted that it has Higher anti-tumor efficiency.

Natural killer cells serve as the first line of defense against foreign substances in the human immune system. Since the cell surface lacks a specific antigen receptor (TCR), the immune response it triggers is a non-specific defense. Natural killer cells It is located in the peripheral blood circulation and can attack virus-infected cells to protect the body from virus infection. It can also generate rejection reactions to tumor cells and bone marrow transplants to attack these foreign cells. When natural killer cells and these heterogeneous cells After contact, it activates its activating receptor, thus activating itself, and prompts natural killer cells to release factors such as perforin and granzyme; perforin will form holes in the target heterogeneous cell membrane, After the granzyme enters the target heterogeneous cell, it changes the permeability of the mitochondrial membrane and activates apoptosis-related proteins to initiate the apoptosis reaction, thereby fragmenting the DNA in the target heterogeneous cell It also causes cell breakdown; in addition, a group of natural killer T cells is a group of heterogeneous immune cells that simultaneously express T cell surface antigens (eg: CD3, TCRαβ) and natural killer cell surface antigens (eg: CD56), so they can be induced simultaneously as T Cell-specific immune responses, as well as non-specific immune responses like NK cells, are also regarded as a major focus of the development of immune cell therapy.

However, natural killer cells only account for 5-10% of lymphocytes in the blood around the human body, and natural killer T cells account for less than 1%. Therefore, they can efficiently expand natural killer cells and natural killer T cell lines in vitro A major issue, up to now, researchers are still striving to improve the in vitro expansion efficiency of natural killer cells and natural killer T cells, their cell survival rate, cell purity and cytotoxicity, and further enhance their medical and medical Applicability.

The present invention in one aspect, provides an in vitro amplification-based natural killer cells (NK cells) and natural killer T cells Method (NKT cells), the Department of the process carried out in one of the initial cell density 0.5x10 ⁶ ~ 5x10 ⁶ cells / mL Cultivate to provide a better cell growth environment, and when the total number of cells reaches 2.5x10 ⁶ ~5x10 ⁶ cells/mL, update the cell culture medium for the first time and readjust the cell density to 0.5x10 ⁶ ~5x10 ⁶ cells/mL, that is, when After the cells are in a stable state, the cell density is readjusted to increase the growth efficiency of the cells.

In another aspect of the present invention, there is provided a system and method for natural killer cells in vitro NK T cells of amplification, the method based 2-3 days every update the cell culture medium, and re-adjust the cell density of 0.5x10 ⁶ ~ 5x10 ⁶ cells/mL, so it can continue to provide a better cell growth environment.

In another aspect, the present invention provides a method for in vitro expansion of natural killer cells and natural killer T cells. The cell culture fluid used in the method includes human recombinant interleukin-2 (rhIL-2) for activation NK/NKT cells, in turn, promote NK cells to secrete perforin to bind the target cells and cause cytotoxic effects, and at the same time help the growth of immune cells to increase the total number of expanded NK/NKT cells.

In another aspect, the present invention provides a method for in vitro expansion of natural killer cells and natural killer T cells. The cell culture fluid used in the method further includes Anti-CD3 monoclonal antibody to promote the activation of NK/NKT cells , In order to enhance the cytotoxicity of NK/NKT cells to cancer cells without affecting the growth of NK/NKT cells.

Therefore, one aspect of the present invention relates to a method for in vitro expansion of natural killer cells and natural killer T cells, which comprises the steps of: suspending a peripheral blood mononuclear cells (PBMCs) in a cell culture solution To obtain a cell mixture; adjust the cell density of the cell mixture to 0.5x10 ⁶ ~5x10 ⁶ cells/mL as an initial cell density, and use the cell culture solution to culture the cell mixture on a cell In the culture plate; when the total number of cells in the cell culture plate reaches 2.5 x10 ⁶ ~5x10 ⁶ cells/mL, update the cell culture solution, and readjust the cell density to 0.5x10 ⁶ ~5x10 ⁶ cells/mL; every Renew the cell culture fluid in 2 to 3 days, and readjust the cell density to 0.5x10 ⁶ ~5x10 ⁶ cells/mL, culture for at least 10 to 16 days; and wash the cell mixture and obtain a cell in the cell mixture Community, the cell community is the mixture of the natural killer cells and natural killer T cells upon expansion.

In some embodiments of the present invention, before the step of suspending the peripheral blood mononuclear cells, the method further includes the step of collecting the peripheral blood mononuclear cells, which includes: centrifuging a peripheral blood specimen to make the peripheral The blood sample is layered into a plasma layer, a white blood cell layer (buffy coat) and a red blood cell layer; the plasma layer is removed and the white blood cell layer is obtained, the white blood cell layer is mixed with Hanks balanced salt solution (HBSS) and a mononuclear cell In the cell separation liquid, a white blood cell mixture is obtained; centrifugation is used to separate the white blood cell mixture; and the supernatant is removed, and the middle layer of white liquid is obtained to obtain the peripheral mononuclear cells.

In other specific embodiments of the present invention, the cell culture solution includes: a stem cell growth medium (SCGM); a human interleukin-2 recombinant protein (rhIL-2), the concentration range of which is from 250 to 1,000IU/mL; a serum with a volume percentage ranging from 1 to 10%; and a primary anti-CD3 monoclonal antibody (anti-CD3 monoclonal antibody) with a concentration ranging from 10 to 500ng/mL.

In other specific embodiments of the present invention, in the step of adjusting the initial cell density, the cell density of the cell mixture is adjusted to 0.5×10 ⁶ to 2.5×10 ⁶ cells/mL.

In other embodiments of the present invention, in the step of updating the cell culture fluid and readjusting the cell density, the cell density of the cell mixture is adjusted to 0.5 x 10 ⁶ ~2.5 x 10 ⁶ cells/mL.

In other specific embodiments of the present invention, in the step of culturing the cell mixture, the cell mixture is cultured in an environment of 37° C. and 5% carbon dioxide.

In other embodiments of the present invention, before the step of obtaining the cell community, the method further includes: analyzing the ratio of the natural killer cells and the natural killer T cells in the cell mixture, and the ratio is between 30 and 90% The cell surface antigen of the natural killer cell line is a cell colony of CD16 ⁺ CD56 ⁺ CD3 ^- , and the cell surface antigen of the natural killer T cell line is a cell colony of CD16 ⁺ CD56 ⁺ CD3 ⁺ .

In other specific embodiments of the present invention, after the step of washing the cell mixture and obtaining the cell colony, the method further includes the step of suspending the cell colony with a preservation solution, which is a phosphate buffer solution or an extracellular Solution (Plasma-Lyte A).

In other specific embodiments of the present invention, the cell survival rate of the amplified natural killer cell and natural killer T cell mixture is at least 80%, and the natural killer cell and natural killer T cell mixture have The cytotoxicity of the natural killer cells and natural killer T cell lines reaches at least 50%.

Another aspect of the invention relates to a pharmaceutical composition of natural killer cells and natural killer T cells, which comprises the amplified natural killer cells and natural killer T cell mixture prepared by the foregoing method; and medicine Acceptable solvent.

S10~S18‧‧‧Step

S101~S104‧‧‧Step

Figure 1 is a schematic flow diagram of the first embodiment of the present invention; Figure 2 is a schematic flow diagram of the second embodiment of the present invention; Figures 3A-3B are expanded natural killer cells (NK cells) / natural killer T cells ( NKT cells) ratio analysis; Figure 4A-4B is the ratio of amplified natural killer cells (NK cells) / natural killer T cells (NKT cells); Figure 5 is the amplified natural killer cells (NK cells) )/Analysis of cytotoxicity of natural killer T cells (NKT cells).

In view of the fact that the conventional technology still has many improvements to expand natural killer cells (NK cells) and natural killer T cells (NKT cells) in vitro, because NK cells only account for about 5-10% of human immune cells, NKT cells are more Less than 1%, in order to make it more suitable for medical applications, it is necessary to obtain a larger number of cells in a shorter culture period, and to be able to expand NK/NKT cells in the cell community to enhance NK/NKT cells Ratio, which in turn improves cell purity. In addition, it is necessary to maintain the survival rate of the expanded cells, and in the process of cell expansion, effectively stimulate and activate NK/NKT cells to enhance their immunotoxicity efficacy; accordingly The present invention proposes a method for in vitro expansion of natural killer cells (NK cell)/natural killer T cells (NKT cells) to improve the deficiencies of conventional technologies; the technical means and features of the present invention will be described below.

definition

As used in this specification, the term "nature killer cell" (Nature killer cell, NK cell) means a cytotoxic lymphocyte that is part of the innate immune system and can be induced to perform non-specificity Cytotoxic effect.

As used in this specification, the term "Nature killer T cell" (NKT cell) means an immune cell that simultaneously expresses αβT cell receptor (TCR) and molecular markers associated with natural killer cells, and it also has specificity Cellular immunotoxicity and non-specific cellular immunotoxicity.

As used in this specification, the term "peripheral mononuclear cells" (PBMCs) means a cell with a single nucleus and a round nucleus morphology, which includes lymphocytes such as T cells, B cells, and NK cells , Mononuclear cells (monocytes) and dendritic cells (dendritic cells), mononuclear ball cells located in peripheral blood can also be called peripheral blood mononuclear cells (PBMC).

Materials and Methods

Reagent preparation

Cell culture medium

One embodiment of the present invention uses Clonetics ^™ CellGro® SCGM BulletKit ^™ (CC-3205) as the basic medium, and its 500 ml (mL) package contains Stromal Cell Basal Medium, 0.5 mL Human fibroblast growth factor (h-FGF-B), 0.5 mL insulin (Insulin), 0.5 mL fetal bovine serum (FBS) and 0.5 mL Gentamicin sulfate/Amphotericin; GA-1000).

Recombinant human interleukin-2 (rhIL-2)

In a sterile working environment, take 1 mL (mL) of sterile water in a sterile syringe and pour it into a container containing 1.1 mg (mg) rhIL-2 powder (Proleukin ^® ). After uniformly dissolving, the concentration is 1.1 mg/ml (mg /mL) of rhIL-2 stock solution, then remove the rhIL-2 stock solution with a sterile syringe and place in a 50mL centrifuge tube, then add 43mL of cell culture medium to adjust the concentration of rhIL-2 solution to 500 units/microliter (U/μl) to obtain rhIL-2 diluted solution; divide rhIL-2 diluted solution into micro-sterile centrifuge tubes and store in -20℃ refrigerator for later use.

Anti-cluster of differentiation-3 monoclonal antibody (Anti-CD3 mAb)

In a sterile working environment, take the Anti-CD3 monoclonal antibody stock solution (anti-CD3 monoclonal antibody concentration of 1mg dissolved in 1mL solvent) from the original packaging (Takara), and then mix the Anti-CD3 monoclonal antibody stock solution To 4mL of cell culture medium, dilute the concentration of Anti-CD3 monoclonal antibody solution to 20μg/mL; divide the diluted solution of Anti-CD3 monoclonal antibody solution into micro-sterile centrifuge tubes and store in a -20℃ refrigerator for use .

Cell culture medium for expansion of natural killer cells (NK cell)/natural killer T cells (NKT cell)

Add the aforementioned rhIL-2 dilution solution, Anti-CD3 monoclonal antibody dilution solution and serum to the cell culture medium, mix evenly to configure a cell culture solution for expanding natural killer cells/natural killer T cells. The culture medium contains rhIL-2 with a final concentration of 250 to 1,000 IU/mL, Anti-CD3 monoclonal antibody with a final concentration of 10 to 500 ng/mL, and serum with a final concentration of 1 to 10%.

Peripheral blood sample

The peripheral blood samples of the subjects are collected by the hospital physicians' outpatient department and stored in the sterile blood collection tube, and the samples are transported to the core laboratory of Good Tissue Practice (GTP) for follow-up treatment according to the specifications.

Cell

Preparation of peripheral blood mononuclear cells (PBMC)

Transfer the peripheral blood from the blood collection tube to a 50mL centrifuge tube for gradient centrifugation. The centrifuge (AUBOCA, 4000/4200) is set to a centrifugal force of 600xg, and the brake function of the centrifuge is turned off. Peripheral blood samples are divided into three layers, from top to bottom are plasma (Plasma), white blood cell layer (Buffy coat) and red blood cells (Erythrocytes); then remove the plasma layer and transfer the white blood cell layer to a new sterile centrifuge tube, and then Use Hanks Balanced Salt Solution (HBSS) to slowly mix leukocytes to obtain a leukocyte dilution solution; on the other hand, take 1~3mL of mononuclear cell separation solution (Ficoll-Paque Plus) into a mononuclear ball separation centrifuge tube, Centrifuge at 600xg centrifugal force for 30 seconds at room temperature, and then stand by; then, take the aforementioned leukocyte dilution solution into a single-nuclear ball separation centrifuge tube containing mononuclear cell separation solution, and centrifuge at 600xg centrifugal force for 10 minutes at room temperature Layer, then remove the supernatant, transfer the white intermediate layer (ie peripheral blood mononuclear cells) to a new sterile centrifuge tube, add HBSS to mix evenly to wash the cells, centrifuge at room temperature for 10 minutes to remove the supernatant Then, resuspend the cells with HBSS again, centrifuge at room temperature for 10 minutes, and collect the supernatant and peripheral blood mononuclear cells separately for use.

Cell test

Cell surface antigen analysis

Take 1x10 ⁶ cells in a 15mL centrifuge tube, centrifuge at 500g for 5 minutes, remove the supernatant, resuspend the cells in 4mL Dulbeccos Phosphate Buffered Saline (DPBS), and repeat this step twice. Then add 5μl of cell fluorescent labeling reagent and react for 15 minutes in the dark at 4°C. Then, add 4mL DPBS to wash the cells. After centrifuging at 500g for 5 minutes, remove the supernatant. Finally, add 0.5mL DPBS to resuspend the cells. Analyze and quantify the fluorescent label on the cell surface by flow cytometry (SONY; SH800Z); the aforementioned fluorescent labeling reagent for cells contains the fluorescent label Anti-CD3 mAb (Invitrogen; Cat# 11-0038-42 related to T cells) ), and fluorescent-labeled Anti-CD16 mAb (Invitrogen; Cat# 17-0168-42), Anti-CD56 mAb (Invitrogen; Cat#12-0567-41) and Anti-CD314 (NKG2D) mAb related to NK cells (BD; Cat# 562365).

cell counts

Obtain the cell sample to be analyzed for cell viability from the cell culture dish, suspend the cell sample uniformly with a micropipette to obtain a cell suspension, then take 20 μl of the cell suspension and 20 μl of 0.4% trypan blue dye (Trypan blue; Gibco; Cat# 15250-061) Mix evenly to obtain a cell mixture, and then use a micropipette to take 10~20μl of the cell mixture into the groove between the cover glass and the cell counting disc (Marienfeld; Cat# AP-0650030) Then, place the cell counting disc under the microscope and observe it. After the cells are at rest, they start to count the cells.

The four corners of the cell counting disk include a 4*4 area, and the counting range includes the four areas mentioned above; count the bright cells in the four areas to obtain the number of viable cells, and then count the four areas For dark cells and dark blue cells, obtain the number of dead cells, and then use these values to calculate cell density, total cell count, and cell survival rate; among them, cell density (cells/ml) = (number of viable cells in four areas/ 4) x (dilution factor 2) x 10 ⁴ /mL; total cells (cells) = cell density (cells/ml) x cell culture fluid volume (mL); cell survival rate% = viable cells (cells) / (live Cells + dead cells) (cells) x%.

Cytotoxicity test

In the experimental department, the expanded NK/NKT cells were used as effector cells and the K562 cell line was used as the target cells. The effector cells and the target cells were mixed in a 1:1 ratio and incubated for reaction. After 4 hours, stain the cells with dye 7-AAD. Since 7-AAD cannot permeate the cell membrane of normal cells, it can only permeate cells that are in the process of apoptosis or have died, so it can be detected by detecting the cells The 7-AAD signal is used as the basis for apoptosis.

Next, please refer to FIG. 1, which is a schematic flowchart of the first embodiment of the present invention; as shown in the figure, the in vitro expansion of natural killer cells (NK cells) and natural killer T cells (NKT cells) described in this embodiment The method includes the following steps: Step S10: suspend a peripheral blood mononuclear cells (PBMCs) in a cell culture solution to obtain a cell mixture; Step S12: cells of the cell mixture The density is adjusted to 0.5x10 ⁶ ~5x10 ⁶ cells/mL as an initial cell density, and the cell mixture is used to culture the cell mixture in a cell culture dish; Step S14: When the total in the cell culture dish Update the cell culture medium when the number of cells reaches 2.5 x10 ⁶ ~5x10 ⁶ cells/mL, and readjust the cell density to 0.5x10 ⁶ ~5x10 ⁶ cells/mL; Step S16: Update the cell culture medium every 2 to 3 days , And re-adjust the cell density to 0.5x10 ⁶ ~5x10 ⁶ cells/mL, culture for at least 10 to 16 days; and Step S18: wash the cell mixture and obtain a cell community in the cell mixture, the cell community is Once expanded, the natural killer cell and natural killer T cell mixture.

Before step S10, it further includes a schematic flow chart of the second embodiment shown in FIG. 2; as shown, the collection of the peripheral blood mononuclear cell line of this embodiment includes the following steps: Step S101: centrifugation 1 Peripheral blood specimens, layering the peripheral blood specimens into a plasma layer, a white blood cell layer (buffy coat) and a red blood cell layer; Step S102: removing the plasma layer and obtaining the white blood cell layer, mixing the white blood cell layer with Hanks Balanced salt solution (HBSS) and a mononuclear cell separation solution to obtain a leukocyte mixture; step S103: centrifugation to layer the leukocyte mixture; and step S104: removing the supernatant and obtaining the intermediate layer of white liquid, That is, the peripheral mononuclear cells are obtained.

This embodiment is a process for preparing peripheral blood mononuclear cells as described in the materials and methods; wherein, a layered peripheral blood specimen is obtained in step S11, and then in step S12, it is removed with a 10mL sterile pipette and an auxiliary device Plasma, in the process of aspiration, stop when the liquid level is close to 0.4~0.5 cm away from the white blood cell layer to avoid the consumption of white blood cells and the destruction of the peripheral blood stratification interface. Then mix and dilute the white blood cells with HBSS to It is suitable for the subsequent separation of mononuclear balls; in step S14, during the process of sucking up the supernatant, it stops when the liquid level is close to 0.5~1 cm away from the white liquid in the middle layer. And to avoid destroying the layered interface; through the method of this embodiment, peripheral blood mononuclear cells can be obtained, which is the initial cell source for expanding NK/NKT cells in the first embodiment.

Next, in step S10, the peripheral blood mononuclear cells obtained in steps S11 to S14 are suspended in the cell culture medium. The cell culture medium needs to provide sufficient nutrients during cell growth and expand the cells. At the same time, provide appropriate factors to stimulate and activate NK/NKT cells. Based on the above purpose, the cell culture solution of this embodiment uses a stem cell growth medium (SCGM) as the basic medium, and the concentration range is 150~1,500IU /mL of human interleukin-2 recombinant protein (rhIL-2), and a concentration range of 10 to 800ng/mL of anti-CD3 monoclonal antibody (anti-CD3 monoclonal antibody, anti-CD3 mAb) as activated NK /NKT cell stimulating factor. In this embodiment, rhIL-2 can also promote the growth of NK/NKT cells to increase the total number of NK/NKT cells. In addition, this embodiment also adds a volume percentage between 1~ 10% of serum; the above-mentioned additives, in a preferred embodiment, the final concentration of added rhIL-2 is 250~1,000IU/mL, 50-500ng/mL anti-CD3 mAb and 1~ 5% serum. The serum may be the autologous serum of the provider of peripheral blood samples, or may be serum of other animal origin.

Continuing in step S12, the cell density of the cell mixture obtained in step S11 is adjusted to 0.5x10 ⁶ ~5x10 ⁶ cells/mL, and the initial cell density is placed on the cell culture tray carrying the cell culture solution Then, place the cell culture tray in the cell culture incubator, start the cultivation of mononuclear cells at 37°C and 5% carbon dioxide, and define this step as day 0 of the culture. This initial cell density can provide cells Appropriate contact with cells and good growth space to promote the cells to stabilize and facilitate subsequent growth and division; in a preferred embodiment, 0.5 x 10 ⁶ ~2.5 x 10 ⁶ cells/mL The cell number was used as the initial cell density, and the cells were cultured in a thin-necked flask.

Continuing in step S14, when the total number of cells in the cell culture plate grows to about 2.5x10 ⁶ ~5x10 ⁶ cells/mL, the cell culture solution in the cell culture plate is updated, and the cell density is adjusted to 0.5x10 again ⁶ ~ 5x10 ⁶ cells/mL to maintain a good growth environment and sufficient growth nutrients in the cell culture dish; in a preferred embodiment, it grows to 2.5x10 ⁶ ~3.5x10 ⁶ cells/mL in total cells Or culture until the 5th day, at this time, the cells are in a stable state and have better cell growth and division efficiency, that is, adjust the cell density to 0.5 x 10 ⁶ ~ 5 x 10 ⁶ cells/mL again, and continue to expand NK/NKT cells.

Then, after step S14, update the cell culture fluid every 2 to 3 days, and readjust the cell density to 0.5x10 ⁶ ~5x10 ⁶ cells/mL to continue to maintain a good growth environment and sufficient Growing nutrients and continuously cultivating from the 10th day to the 16th day; in a preferred embodiment, in the process of expanding NK/NKT cells, it is on the 5th day, 7th day, 9th day, The cell density was readjusted on day 12, day 14 and day 16, and the cell density was adjusted to 0.5x10 ⁶ ~5x10 ⁶ cells/mL.

Next, before step S16, a part of the cell mixture in the cell culture plate is first obtained as a test sample for cell counting and cell characteristic analysis; the cell characteristic analysis is based on the analysis of cells as described in the materials and methods surface antigen CD16, the CD56 cell population and CD3 amplified to the definition contained in the NK / NKT cells, to analyze the proportion of NK cells and percentage of NKT cells, wherein the cell line NK-cell surface antigen CD16 ⁺ CD56 ⁺ CD3 ^- of The cell community, and the NKT cell is a cell community of cell surface antigens CD16 ⁺ CD56 ⁺ CD3 ⁺ , and the proportion of NK/NKT cells in the cell community expanded through the process of this embodiment can reach up to 90%.

Finally, in step S18, after washing the cell colony, the amplified mixture of natural killer cells and natural killer T cells is obtained, which is suspended with physiological grade sterile phosphate buffer solution, and centrifuged for 10 minutes Remove the supernatant and repeat this step at least three times to ensure that the cell culture fluid and the added substances it contains are removed.

The amplified natural killer cell and natural killer T cell mixture obtained through the foregoing steps can be mixed and stored in an appropriate solvent, or further prepared as a pharmaceutical composition, and the solvent can be a phosphate buffer solution or a cell External fluid (Plasma-Lyte A)

Other features and advantages of the present invention will be further exemplified and described in the following embodiment examples, and the embodiment examples are only used as an auxiliary description and are not intended to limit the scope of the present invention.

Examples

Example 1. Optimization of initial cell number

The purpose of this embodiment is to establish a better initial cell density used in the method of in vitro expansion of natural killer cells (NK cell)/natural killer T cells (NKT cells) of the present invention, which is to obtain peripheral blood mononuclear cells as described above Cells were cultured with autologous serum from the peripheral blood samples (including rhIL-2 of 250 IU/ml and Anti-CD3 monoclonal antibody of 50 ng/ml), and then the cell density was evaluated by the aforementioned cell counting method; as shown in table I, which lines were cultured at an initial cell density of ^{1.00 x10 6, 2.00x10 6, 3.00} x10 6 and 5.00 x10 ⁶ cells / ml, and after cultivation 5,7,9,12,14,16 , 18, and 20 days, remove the cells from the culture plate to count, and readjust the cell density to 1.00 x 10 ⁶ cells/ml. From the results, we can know that when these initial cell densities are used to expand NK/NKT cells in vitro, cell viability can be maintained at 90% or more, wherein, when cultured to 1.00 x10 ⁶ cells / ml of the initial cell density, the cells of the best amplification efficiency, the number of cells that can be obtained after the amplification also up; data here, one preferred embodiment of the present invention, the choice of ^{1.00 x10 6, 2.00x10 6, 3.00} x10 6 and 5.00 x10 ⁶ cells / ml initial cell density of the culture.

Example 2. Optimization of cell culture days

According to the above, in order to achieve better cell growth efficiency, this embodiment continues to discuss the required cell culture days and the time interval for readjusting the cell density; in this embodiment, the aforementioned peripheral blood mononuclear cells are obtained in order to A preferred embodiment 1.00 x 10 ⁶ cells/mL is the initial cell density, using autologous serum from the peripheral blood sample source (including 250IU/ml of rhIL-2 and 50ng/ml of Anti-CD3 monoclonal antibody) For culture, count the cells at the culture time indicated in each group in Table 2. After the cell count, readjust the cell density in the culture plate to 1.00 x 10 ⁶ cells/mL and continue the culture.

As shown in Table 2, it is divided into three test groups, in which the first group performs the first cell density adjustment on the 5th day after the cells are placed in the culture plate, and then readjusts the cell density to 1.00 every 2 days x10 ⁶ cells/mL, followed by continuous culture for 3 days on the 9th day, and then readjust the cell density to 1.00 x10 ⁶ cells/mL every 2 days from the 12th day of culture, as can be seen from the results, with the second group Compared with the third group, the total number of cells in the first group continued to rise steadily after the 12th day of culture. The total number of cells reached on the 20th day was 2 to 20 times more than other groups. And the cell survival rate is also maintained above 95%; accordingly, in a preferred embodiment of the present invention, the cell density adjustment interval described in the first group of Table 2 is used as the culture condition for expanding NK/NKT cells, To achieve better cell growth efficiency.

Example 3. Optimization of human recombinant interleukin-2 (rhIL-2) concentration in cell culture fluid

Since the amount of additives used in the cell culture solution also affects the cell growth efficiency during cell expansion culture, this example continues to discuss how to adjust the cell density and adjust the cell density time interval at the beginning of the establishment of examples 1 and 2. under culture conditions may further enhance the concentration of rhIL 2-cell growth efficiency; that the lines made of peripheral blood mononuclear cells ball to a preferred embodiment of 1.00 x10 ⁶ cells / mL for the initial cell density, the use of peripheral blood tests Autologous sera from individuals (including rhIL-2 and 50 ng/ml Anti-CD3 monoclonal antibody) were cultured, and the test groups were rhIL-2 containing 250, 500, 750, or 1,000 IU/ml, respectively.

As shown in Table 3, the group cultured with 500 IU/ml rhIL-2 had the most stable cell expansion trend, and the total number of cells obtained on the 20th day of culture was 1-3 times more than the other groups; According to this, a preferred embodiment of the present invention can be cultured with rhIL-2 from 250 to 1,000 IU/ml. In addition, from the results of this embodiment, it can also be seen that the cell culture solution used to expand cells of the present invention, It can further help the growth of immune cells, that is, the concentration of rhIL-2 selected in one embodiment of the present invention can increase the total number of NK/NKT cells in the expanded cell population.

Example 4. Optimization of anti-differentiation cluster-3 (Anti CD-3) monoclonal antibody concentration and number of additions in cell culture fluid

In vitro expanded immune cell lines are used in the human body to enhance immunity, and then toxic to tumor cells. In order to improve the immune toxin killing effect of the expanded in vitro expanded immune cells into the human body, in addition to the need to proliferate a large number of cells The obtained cells also need to possess cytotoxicity. Here, this example further discusses the anti-CD-3 monoclonal antibody used to activate NK/NKT cells in vitro, which is the better dosage in the process of expanding cultured cells ; Department of which made the cell of the peripheral monocytes, in a preferred embodiment 1.00 x10 ⁶ cells / mL for the initial cell density, use of autologous serum derived by peripheral blood examination of the body (containing 500IU / ml of rhIL-2 And Anti-CD3 monoclonal antibodies) were cultured, and the test groups were Anti-CD3 monoclonal antibodies containing 50, 100, 250, or 500 ng/ml, respectively.

As shown in Table 4, the group with 50 ng/ml and 500 ng/ml Anti-CD3 monoclonal antibody cultured can reach a higher total cell number on the 20th day of culture compared to other groups. In order to reduce the cost of preparation and reduce the residual of foreign substances in the prepared cell population, and thereby reduce the immune response induced by the foreign substances, so as to reduce the risk of side effects when applied to the human body, a better implementation of the present invention In the example, 50-500ng/ml Anti-CD3 monoclonal antibody was used for cultivation. In addition, as shown in Table 5, the conditions for adding 50ng/ml Anti-CD3 monoclonal antibody to the cell culture medium were After the cells were cultured, they were added once on the 0th, 5th and 7th days respectively. Compared with other groups, the cells could be expanded more steadily. In addition to maintaining the total number of cells after expansion that can be achieved in Example 3, while increasing the number of cells after expansion The proportion of NK/NKT cells with cytotoxic activity in the cell population.

Example 5. Proportion of natural killer cells (NK cells) and natural killer T cells (NKT cells) obtained by expansion

According to the data of the above examples, the method of in vitro expansion of natural killer cells and natural killer T cells of the present invention can obtain 3,000 to 7,000 times the expansion of cells after obtaining peripheral mononuclear cells and culturing for 18 days. And the cell survival rate is higher than 85%; Then, this embodiment further establishes that the method of the present invention can indeed expand NK/NKT cells in the initial cells (ie, peripheral mononuclear cells).

In this embodiment, the expanded cell community is obtained through the foregoing embodiment, and then the flow cytometry (Flow cytometry) is used to analyze the effective cells (NK/NKT cells) in the expanded cell community The ratio is based on the cell specific markers of NK cells and NKT cells, and the ratio of NK/NKT cells in the expanded cell community is quantified by flow cytometry. The specific markers of the NK cells are CD56. And NK cell activation receptors CD16 and NKG2D, in addition, CD3 is also used as a T cell specific marker to analyze NKT cells; the test is based on the cell surface antigen analysis process described in the materials and methods for cell population analysis.

Please refer to FIG. 3A and FIG. 3B. FIG. 3A is the analysis result of cell surface granularity and cell particle size. As shown in the result, most of the cell samples analyzed in this example are concentrated in the same community, showing that their cell types are consistent. And the cell samples are not clearly divided into multiple communities, showing the consistency of their cell survival rate; Figure 3B is the analysis result of the proportion of NK/NKT cells in the cell community, as shown in the figure, the cell group in the upper left corner In the cell population collected in Figure 3A, the cell population that does not express CD3 but express CD56 (CD3 ^- CD56 ⁺ ) is NK cells, and the proportion of total cells is 5.64%. In addition, the upper right corner shows CD3. The cell population expressing CD56 (CD3 ⁺ CD56 ⁺ ) is NKT cells, and its cell ratio is 55.82%. It can be seen that the ratio of NK/NKT cells in the cell community expanded by the method of the present invention reaches 62.5 % (NK cell 5.64 + NKT cell 55.82).

Please continue to refer to 4A and 4B. The upper right corner of Figure 4A shows the cells that express CD16 and CD56 in the cell community at a rate of 23.03% of the total cells. After selecting the cell population of CD16 ⁺ CD56 ⁺ from Figure 4A, analyze CD16 ⁺ In the cell population of CD56 ⁺ , the distribution of NKG2D and CD3 cells is shown. The results are shown in FIG. 4B. The cell population of CD16 ⁺ CD56 ⁺ NKG2D ⁺ CD3 ^-in the upper left corner is NK cells, and the CD16 ^{+ is in the} upper right corner ^. The cell population of CD56 ⁺ NKG2D ⁺ CD3 ⁺ is NKT cells. According to the quantitative results of flow cytometry, it can be known that the ratio of NK/NKT cells expressing NK cell specific markers CD16, CD56 and NKG2D is 16.9% ( 23.03%*(15.37+58.3)%=16.9%).

Example 6. The cytotoxicity of the amplified natural killer cells (NK cells) and natural killer T cells

Finally, in order to establish the method for in vitro expansion of NK/NKT cells of the present invention, the prepared NK/NKT cells can meet the needs of preparation as a pharmaceutical composition. In this embodiment, the cytotoxicity described in the materials and methods is used Test to further analyze the cytotoxic effect of the amplified NK/NKT cells on cancer cells; please refer to Figures 5A and 5B. Figure 5A shows that the cell samples present a consistent cell type and cell survival rate, and Figure 5B can be clearly known The ratio of K562 cells co-cultured with the expanded NK/NKT cells in the early stage of apoptosis or apoptosis process is about 76.30%, which is the cytotoxic effect of the expanded NK/NKT cells on cancer cells Up to 76.30%.

Based on the description of the above embodiments, the method for expanding natural killer cells and natural killer T cells in vitro disclosed by the present invention has been verified by testing, the initial cell density used in the steps, and the cell culture renewal at specific time points The cell density in the dish can indeed give the cell a better growth environment, thereby improving the expansion efficiency of the cell. In addition, the cell culture medium of this method contains additives rhIL-2 and anti-CD3 monoclonal antibodies, in addition to In addition to improving cell growth efficiency, it can also activate the expanded NK/NKT cells. Thus, compared with the conventional technology, the cells obtained by the method of the present invention can not only obtain a higher total number of cells, but also maintain a higher Excellent cell survival rate, higher NK/NKT cell ratio, and even more, the expanded NK/NKT cells also have better cytotoxicity, that is, the method of the present invention can indeed be targeted at the cell community NK/NKT cells are effectively expanded and activated, which can be further applied to the preparation of pharmaceutical compositions.

Although the foregoing description has cited and detailed various implementation examples, those with ordinary knowledge in the field to which the present invention belongs can easily understand the essence and features of the present invention. However, it should be understood that, without departing from the spirit and scope of the invention, the present invention can also undergo certain changes and modifications from the foregoing description to make it more suitable for various uses and conditions. Therefore, the scope of this specification and the patent applications described therein should be for exemplary purposes, and not to limit the scope of the present invention in any way.

S10~S18‧‧‧Step

Claims (10)

A method for in vitro expansion of natural killer cells (NK cells) and natural killer T cells (NKT cells), which comprises the steps of: suspending a peripheral blood mononuclear cells (PBMCs) in a cell culture solution To obtain a cell mixture; adjust the cell density of the cell mixture to 0.5x10 ⁶ ~5x10 ⁶ cells/mL as an initial cell density, and use the cell culture solution to culture the cell mixture in a cell culture In the plate; when the total number of cells in the cell culture plate reaches 2.5x10 ⁶ ~5x10 ⁶ cells/mL, update the cell culture solution, and readjust the cell density to 0.5x10 ⁶ ~5x10 ⁶ cells/mL; every 2 Renew the cell culture fluid within 3 days, and readjust the cell density to 0.5x10 ⁶ ~5x10 ⁶ cells/mL, cultivate for at least 10 to 16 days; and wash the cell mixture and obtain a cell colony in the cell mixture The cell community is the mixture of the natural killer cells and natural killer T cells once expanded. The method as described in item 1 of the patent application scope, wherein before the step of suspending the peripheral blood mononuclear cells, the method further includes the step of collecting the peripheral blood mononuclear cells, which includes: centrifuging a peripheral blood specimen, The peripheral blood sample is layered into a plasma layer, a white blood cell layer (buffy coat) and a red blood cell layer; the plasma layer is removed and the white blood cell layer is obtained, the white blood cell layer is mixed with Hanks balanced salt solution (HBSS) and a Obtain a white blood cell mixture in the mononuclear cell separation solution; centrifuge to layer the white blood cell mixture; and remove the supernatant and obtain the intermediate layer of white liquid to obtain the peripheral mononuclear cells. The method as described in item 1 of the patent application scope, wherein the cell culture fluid system comprises: a stem cell growth medium (SCGM); a human interleukin-2 recombinant protein (rhIL-2) whose concentration range is between 250 Up to 1000IU/mL; a serum with a volume percentage ranging from 1 to 10%; and a primary anti-CD3 monoclonal antibody (anti-CD3 monoclonal antibody) with a concentration ranging from 10 to 500ng/mL. The application method of claim 1 patentable scope clause, wherein in the step of adjusting the initial cell density, the cell line The cell density of the mixture was adjusted to ^{0.5 x10 6 ~ 2.5 x10 6 cells} / mL. The application method of claim 1 patentable scope clause, wherein the updating in the cell culture medium and cell density of the re-adjustment step, the cell line The cell density of the mixture was adjusted to ^{0.5x10 6 ~ 2.5x10 6 cells / mL} . The method as described in item 1 of the patent application scope, wherein in the step of culturing the cell mixture, the cell mixture is cultured in an environment of 37°C and 5% carbon dioxide. The method as described in item 1 of the patent application scope, wherein before the step of obtaining the cell community, the method further comprises: analyzing the ratio of the number of cells of the natural killer cells and the natural killer T cells in the cell mixture, and the ratio is between At 30 to 90%, the cell surface antigen of the natural killer cell line is a cell community of CD16 ⁺ CD56 ⁺ CD3 ^- , and the cell surface antigen of the natural killer T cell line is a cell community of CD16 ⁺ CD56 ⁺ CD3 ⁺ . The method as described in item 1 of the patent application scope, wherein after the step of washing the cell mixture and obtaining the cell colony, the method further includes the step of suspending the cell colony with a preservation solution, which is a phosphate buffer solution or An extracellular fluid (Plasma-Lyte A). The method as described in item 1 of the patent application scope, wherein the cell survival rate of the amplified natural killer cell and natural killer T cell mixture is at least 60%, and the amplified natural killer cell and natural The natural killer cell and natural killer T cell line with cytotoxicity in the killer T cell mixture reach at least 50%. A pharmaceutical composition of natural killer cells and natural killer T cells, which comprises the amplified natural killer cells and natural killer T cell mixture prepared by the method described in item 1 of the patent scope; and a pharmaceutical Acceptable solvent.

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