TW202104578A - A method for in vitro expansion and activation of gamma delta T cells - Google Patents

Abstract

The present invention relates to a method for in vitro expansion and activation of γδ-T cells, which comprises stimulating γδ-T cells by visible light wave or audible sound wave, or using culture medium which contains autologous dendritic cell exosome. Such method provides an optimized environment for γδ-T cells and could efficiently increase expansion fold with high purity and cytotoxicity. The present invention also relates to a pharmaceutical composition for inhibiting tumor growth, which comprises γδ-T cells.

Description

A method for in vitro expansion and activation of γδ-T cells

The present invention relates to a method for in vitro expansion and activation of γδ-T cells. The present invention also relates to a pharmaceutical composition containing γδ-T cells.

Immune cell therapy is a treatment method in which the patient's own immune cells are proliferated and activated in vitro, and then infused back into the body. At present, immune cell therapy has been gradually applied to cancer treatment. The key to success lies in understanding the characteristics and functions of various immune cells, and selecting the most suitable immune cell type according to the condition and genetic characteristics of cancer patients, such as NK cells and T cells. Cells and so on.

T cells in the human immune system are roughly divided into two categories: Alpha-Beta T cells and Gamma-Delta T (γδ-T) cells. γδ-T cells exist in the peripheral blood, and their number only accounts for 1% to 5% of all T cells in the peripheral blood. It distinguishes normal and abnormal cells by identifying isopentenyl pyrophosphate (IPP) molecules on the cell surface. IPP is an intermediate product of cell metabolism, and its production in cancer cells will increase, especially when the p53 gene of cancer cells is mutated. Γδ-T cells that recognize IPP will proliferate and activate and increase the power to attack tumor cells. This is a special function of γδ-T cells. If other immune cells do not detect cancer cells If it is written, γδ-T cells can still find cancer cells and attack them. Generally speaking, abnormal cells have different molecules from other normal cells, and γδ-T cells use these as cancer cell markers to identify and attack. In addition to IPP molecules, γδ-T cells can also be identified by markers such as MIC A/b, HMB-PP, intercellular adhesion molecule-1, and CD166. Therefore, γδ-T cells can recognize and kill a variety of cancer cells.

γδ-T cells have a very important feature, that is, they do not need HLA when identifying abnormal cells, so they have nothing to do with the individual's HLA type. This feature allows γδ-T cells to be used on anyone without graft-versus-host disease (GVHD). At present, there have been many human clinical applications of cell therapy in the United States, Europe and Japan using γδ-T cells. These clinical applications have confirmed that the use of γδ-T cells is safe.

Since γδ-T cells are scarce in the blood and their proliferation ability varies from person to person, the challenge of using γδ-T cells for cell therapy is whether they can expand and activate γδ-T cells in large numbers and quickly in vitro. In recent years, it has been discovered that the use of zoledronic acid (Zoledronic acid) can make γδ-T cells proliferate in large quantities, and its technology has also been established. In addition to the use of zoledronic acid in culturing γδ-T cells, injection of zoledronic acid into cancer patients can reveal that cancer cells express IPP in greater numbers, thereby increasing the sensitivity of γδ-T cells to cancer cells. However, zoledronic acid is a drug for the treatment of osteoporosis. There are still some doubts about the use of culturing cells or injection into patients. Therefore, there is still a great need to develop a convenient, effective and safe in vitro amplification and activation γδ- T cell method.

In one aspect, the present invention provides a method for in vitro expansion and activation of γδ-T cells, which comprises the following steps: isolating peripheral blood mononuclear cells from a blood sample; Γδ-T cells are separated from blood mononuclear cells; the γδ-T cells are suspended in a medium and placed in a cell culture dish for culture; and the γδ-T cells in the cell culture dish are continuously stimulated by a visible light wave Or an audible sound wave stimulation, cultivate for 12 to 16 days.

In a specific embodiment, the wavelength of the visible light wave is between 400 nm and 700 nm; in a preferred embodiment, the wavelength of the visible light wave is between 550 nm and 700 nm.

In a specific embodiment, the waveform of the aforementioned audible sound wave is a sine wave, a triangle wave, or a square wave; in a preferred embodiment, the frequency of the aforementioned audible sound wave is 110 Hz, and the intensity is 70 decibels.

In some embodiments of the present invention, the aforementioned medium contains an autologous dendritic cell exosome; in a preferred embodiment, the concentration of the aforementioned autologous dendritic cell exosome is 25 μg /ml. In a preferred embodiment, the aforementioned medium further contains zoledronic acid; in another preferred embodiment, the aforementioned medium further contains a basic medium and cytokines.

In another aspect, the present invention provides a cell prepared according to the aforementioned method.

In yet another aspect, the present invention provides a pharmaceutical composition for inhibiting the proliferation of tumor cells, which comprises the aforementioned cells and pharmaceutically acceptable excipients.

The foregoing overview, as well as the following detailed description of the present invention, will be better understood when read in conjunction with the drawings. To illustrate the present invention, the accompanying drawings show some, but not all, alternative specific embodiments. However, it should be understood that the present invention is not limited to the precise arrangements and instrumentalities shown. These drawings, which are incorporated and constitute a part of the specification, help explain the principle of the present invention.

Figure 1 is a flow chart of the method for in vitro expansion and activation of γδ-T cells of the present invention.

Figure 2A shows the cell expansion analysis after continuous application of different wavelengths of visible light to the γδ-T cells in the cell culture plate according to the present invention.

Figure 2B shows the cytotoxicity analysis of the γδ-T cells on the Daudi cells after the present invention continues to stimulate the γδ-T cells in the cell culture plate with different wavelengths of visible light waves.

Figure 2C shows the cytotoxicity analysis of the γδ-T cells on the A549 cells after the present invention continues to stimulate the γδ-T cells in the cell culture plate with different wavelengths of visible light waves.

Figure 2D shows the results of cell analysis by flow cytometry after the present invention continues to stimulate the γδ-T cells in the cell culture plate with different wavelengths of visible light waves.

Figure 3A shows the cell expansion analysis after continuous application of different waveforms of audible sound waves to the γδ-T cells in the cell culture dish according to the present invention.

Figure 3B shows the cytotoxicity analysis of γδ-T cells on Daudi cells after the present invention continues to irritate the γδ-T cells in the cell culture plate with different waveforms of audible sound waves.

Figure 3C shows the cytotoxicity analysis of γδ-T cells on A549 cells after continuous application of audible sound waves with different waveforms to γδ-T cells in the cell culture plate according to the present invention.

Figure 4A shows the expansion factor analysis of γδ-T cells after adding autologous dendritic cell exosomes to the culture medium of the present invention.

Figure 4B shows the cytotoxicity analysis of γδ-T cells on Daudi cells after autologous dendritic cell exosomes are added to the culture medium of the present invention.

Figure 4C shows the effect of γδ-T cells on A549 after adding autologous dendritic cell exosomes to the culture medium of the present invention. Analysis of cytotoxicity efficiency.

In view of the above-mentioned problems to be solved, the present invention proposes a method for in vitro expansion and activation of γδ-T cells. The method applies visible light wave stimulation, audible sound wave stimulation, and autologous dendritic cell exosomes to γδ-T cells. Expansion of cells in vitro can effectively increase the expansion times of γδ-T cells, and produce γδ-T cells with high purity and high cancer cell cytotoxicity.

definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. In case of conflict, this document and its definition shall prevail.

As used herein, "about", "approximately", or "approximately" should generally mean within 20% of a specific value or range, preferably within 10%, and more preferably within 5%. The numerical values used here are approximate, meaning that words such as "approximately", "approximately", or "approximately" can be inferred to apply unless stated explicitly.

In the present invention, "gamma-delta T cell" (gamma-delta T cell) refers to a cell ^{whose cell surface antigen presents CD3 +} and expresses TCR Vγ9 and TCR Vδ2.

In the present invention, "cell expansion factor" is judged by the following method: "the number of cells after 12 days of in vitro culture" divided by "the initial number of γδ-T cells isolated from peripheral blood mononuclear cells".

In the present invention, the "cancer cell killing efficiency" is based on the use of γδ-T cells as the effector cell, and the Daudi cell line or A549 cell line as the target cell. Use the ratio of cells to target cells (E:T ratio) to be 0.5, 1 or 5 to perform the poisoning test, and use the ratio of target cell death as the poisoning efficiency.

Materials and Methods

The peripheral blood specimen of the present invention is based on the plan approved by the ethics committee, collect whole blood from the arm of the subject, place it in a sterile blood collection tube, and store it at room temperature for subsequent processing.

The basic medium used in the present invention can be selected from: CellGro SCGM (CellGenix Company), KBM 501 (Kohjin Bio Company), AIM-V (Thermo Fisher Company), X-VIV015 (Lonza Company), DMEM or RPM1-1640, etc. Basic medium for sale.

The culture medium of the present invention may sometimes contain appropriate proteins, cytokines, antibodies, serum, compounds and other components. The cytokine is sometimes interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-7 (IL-7), interleukin-12 (IL-12) , Interleukin-15 (IL-15), Interleukin-18 (IL-18) or Interleukin-21 (IL-21).

Method for separating peripheral blood mononuclear cells from blood sample

Draw 7.5-8ml of blood into a blood collection tube, which contains an anticoagulant-heparin, and Ficoll-Hypaque reagent, plus a polyester gel barrier to separate the two liquids. Centrifuge the blood collection tube at 1800 g at room temperature for 20 minutes. After the centrifugation is completed, the plasma layer is collected for subsequent cell culture, and a plasma layer of 5 to 10 mm is left on the interface without disturbing the cell layer during operation. Then use a pipette to collect the peripheral blood mononuclear cell (PBMC) layer of the interface into a 15ml conical tube, wash the PBMC with 10ml Phosphate buffer saline (PBS for short) and turn it over Conical tube 5 times, and then centrifuge at 400g for 5 minutes. After repeating the aforementioned washing step twice, the cells were resuspended in 5 ml of PBS. Calculating the number of cells, typically 1ml whole blood can be isolated in PBMC 1.3x10 ^6. Finally, flow cytometry was used to confirm the proportion and phenotype of γδ-T cells in PBMC.

Method for expanding and activating γδ-T cells in vitro

After centrifuging the peripheral blood mononuclear cell suspension in a 15 ml conical tube at room temperature at 400 g for 5 minutes, the supernatant was discarded. Prepare the cell culture medium, add interleukin-2 (IL-2) and zoledronic acid (Zometa) to the culture medium to a final concentration of 1000IU/ml and 5μM, respectively. Among them, zoledronic acid is added in liquid form, and 50μl of zoledronic acid (concentration of 4mg/5ml) is added to every 30ml of medium. Then the cell pellet was resuspended in the culture medium and adjusted to contain 1×10 ⁶ cells per milliliter of culture medium. 24-well cell culture plate, each well of 1x10 ⁶ cells were added to culture. If a large amount of culture is required, ^{the density of 0.5x10 6} cells per square centimeter can be used as the principle, and it can be adjusted according to the surface area of the culture plate and flask used. Then add autologous plasma, source AB serum, fetal bovine serum or autologous dendritic cell exosomes to account for about 10% of the total culture medium volume (equivalent to a 24-well cell culture plate, 100μl cells per well) . Place the cell culture plate in a 37°C, 5% CO ₂ cell incubator for 24 to 48 hours. Maintain the cell density at 0.5 x 10 ⁶ ~ 2 x 10 ⁶ cells per milliliter. Add fresh medium containing 1000IU/ml interleukin-2 every 2 to 3 days. If necessary, transfer cells to Continue the culture in a new culture plate or flask. Maintain the serum concentration in the culture medium at least 1% during the cultivation process. The cells were harvested on the 12th day and the number, phenotype and function of γδ-T cells were confirmed by flow cytometry.

Analysis of cell surface antigens by flow cytometry

Place the expanded and activated cells in a 96-well plate at 2x10 ⁵ cells/200μl, and add 3μl of fluorescently labeled antibody to react at 4°C for 15 minutes, then wash 3 times with PBS, and then add 400μl of PBS to suspend the cells. Then analyze the fluorescent markers on the cell surface by flow cytometry. The aforementioned fluorescently labeled antibodies include anti-CD3 antibodies, anti-TCR Vγ9 antibodies, and anti-TCR Vδ2 antibodies.

γδ-T cytotoxicity test method for killing cancer cells

The amplified and activated γδ-T cells are used as effector cells, and Daudi cell lines (lymphoma cell lines) or A549 cell lines (lung cancer cell lines) are used as target cells. After mixing the affected cells and target cells at 0.5:1, 1:1 or 5:1, react for 4 hours, and then stain the cells with 7-AAD to determine the number of apoptotic cells.

Method of stimulating γδ-T cells with visible light waves

In order to create a stable light source in the same culture environment, a standard 8-watt fluorescent tube is set up in the same incubator 15 cm above the culture plate to ensure that all cells in culture can receive the same Light stimulation. The light intensity of the culture plate is set to 1,000 lumens by the photometer, and the wavelength is set to 400nm, 550nm or 700nm. The culture plate of the control group was also placed in the same environment, 15 cm away from the light source, but covered with a white paper card to completely block light.

Method of stimulating γδ-T cells with audible sound waves

The sine wave, triangle wave and square wave with the sound wave frequency of 110Hz used in the present invention are all generated by NCH audio sound generator software. The wave shape and frequency spectrum are analyzed by SP4Win software, and the Fostex 6301NB full-range speaker is used on the top of the culture plate. The sound wave is output at 15 cm, and the sound pressure level is detected by the RION NL-31 sound level meter next to the cell culture plate, and the sound pressure level is set to 70 decibels.

Preparation and purification of autologous dendritic cell exosomes

Autologous dendritic cell exosome The preparation system replaced the dendritic cell culture medium cultured to the fifth day with fresh cell culture medium, and added Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 and continued Culture the dendritic cells for 24 hours. The cultured medium was collected and centrifuged at 300 g and 1000 g for 10 minutes, respectively, and then filtered through a filter membrane with a pore size of 0.45 μm to remove cells and their debris. The filtered medium is concentrated by centrifugation at 1000g for 45 minutes with Centricon Plus-70 Millipore membrane, and then ultracentrifuged at 100,000g for one hour. The exosomes of dendritic cells can be separated from the medium and washed twice with PBS. , Centrifuge through Amicon Ultra-15 membrane at 1000g for 25 minutes, and finally resuspend the dendritic cell exosomes in 200μL PBS. The BCA protein analysis kit (Thermo Scientific) was used for the quantification of dendritic cell exosomes.

The expanded and activated γδ-T cells obtained by the foregoing method can be mixed with a suitable excipient for storage, and the excipient can be a phosphate buffer solution, and finally prepared into a pharmaceutical composition.

Example

Example 1. The results of culturing γδ-T cells stimulated by visible light waves

Figure 2A shows the results of cell expansion analysis after continuous stimulation of γδ-T cells with different wavelengths of visible light waves in the cell culture plate. From Figure 2A, it is found that on the 12th day of culture, the cell amplification factor of the 700nm visible light wave group was 4,989 times; the cell amplification factor of the 550nm visible light wave group was 3,433 times; the cell amplification factor of the 400nm visible light wave group was irradiated The multiple is 2,335 times; the cell expansion multiple of the control group (without visible light wave stimulation) is 2,750 times. Figure 2B shows the effects of γδ-T cells on Daudi after continuous stimulation of γδ-T cells with different wavelengths of visible light waves Analysis of cytotoxicity efficiency. It is found from Figure 2B that when the ratio of E:T is 5, the cytotoxicity efficiency of the 700nm visible light wave group is 62.1%; the cytotoxicity efficiency of the 550nm visible light wave group is 66.8%; the cytotoxicity efficiency of the 550nm visible light wave group is 66.8%; that of the 400nm visible light wave group The cytotoxicity efficiency was 60.5%; the cytotoxicity efficiency of the control group (no visible light wave stimulation) was 63.4%. Figure 2C shows the cytotoxicity analysis of γδ-T cells on A549 cells after continuous stimulation of γδ-T cells with different wavelengths of visible light waves. It is found from Figure 2C that when the E:T ratio is 5 and zoledronic acid is added, the cytotoxicity efficiency of the 700nm visible light wave group is 48.9%; the cytotoxicity efficiency of the 550nm visible light wave group is 52.3%; The cytotoxicity efficiency of the 400nm visible light wave group was 45.1%; the cytotoxicity efficiency of the control group (no visible light wave stimulation) was 42.7%. Figure 2D shows the results of cell analysis with flow cytometry after continuous stimulation of γδ-T cells with different wavelengths of visible light waves in the cell culture plate. From Figure 2D, it is found that on the 12th day of culture, the cell purity of γδ-T cells in the 700nm visible light wave group was 92.1%; the cell purity of γδ-T cells in the 550nm visible light wave group was 91.7%; and 400nm visible light was irradiated The cell purity of γδ-T cells in the wave group was 90.7%; the cell purity of γδ-T cells in the control group was 73.3%. In summary, continuous application of visible light waves of specific wavelengths during cell culture can effectively increase the expansion rate, cell purity and cancer cell cytotoxicity of γδ-T cells.

Example 2. Results of stimulating γδ-T cell culture with audible sound waves

Figure 3A shows the results of cell expansion analysis after continuous application of different waveforms of audible sound stimulation to the γδ-T cells in the cell culture plate. From Figure 3A, it is found that continuous stimulation with 110 Hz, 70 decibel sine wave audible sound waves, the cell expansion factor on the 12th day of culture is 3,006 times; continuous stimulation with 110 Hz, 70 decibel triangle wave audible sound waves, On the 12th day of culture, the cell expansion factor was 3,226 times; continuous stimulation was performed with 110 Hz, 70 decibel square wave audible sound waves, The cell expansion factor on the 12th day of culture was 2,864 times; the cell expansion factor of the control group (without audible sound wave stimulation) was 3,108 times. Figure 3B shows the analysis of the cytotoxicity of γδ-T cells on Daudi cells after continuous stimulus with different waveforms of audible sound waves on the γδ-T cells in the cell culture plate. From Figure 3B, it is found that when the E:T ratio is 5, the γδ-T cytotoxicity efficiency after continuous stimulation with 110 Hz, 70 decibel sine wave audible sound waves is 75.6%; at 110 Hz, 70 decibel triangle waves The cytotoxicity efficiency of γδ-T cells after continuous stimulation of audible sound waves was 78.1%; the cytotoxicity efficiency of γδ-T cells after continuous stimulation with 110 Hz, 70 decibel square waves of audible sound waves was 72.6%; the control group (unavailable The cytotoxicity efficiency of listening to sound wave stimulation is 61.9%. Figure 3C shows the analysis of the cytotoxicity efficiency of γδ-T cells on A549 cells after continuously stimulating γδ-T cells with different waveforms of audible sound waves. From Figure 3C, it is found that when the E:T ratio is 5 and zoledronic acid is added, the γδ-T cytotoxicity efficiency after continuous stimulation with 110 Hz, 70 decibel sine wave audible sound waves is 55.1%; The γδ-T cytotoxicity efficiency after continuous stimulation with 110 Hz, 70 decibel triangle audible sound waves is 58.0%; the γδ-T cytotoxicity efficiency after continuous stimulation with 110 Hz, 70 decibel square audible sound waves is 60.4% ; The cytotoxicity efficiency of the control group (no audible sound wave stimulation) was 43.3%. In summary, continuous application of audible sound waves with a specific waveform during cell culture can effectively enhance the cell purity of γδ-T cells and the cancer cell toxic activity.

Example 3. Culture results of autologous dendritic cell exosomes added to γδ-T cell culture medium

Figure 4A shows the expansion factor analysis of γδ-T cells after adding autologous dendritic cell exosomes to the culture medium. From Figure 4A, it is found that using a medium containing 25μl/ml autologous dendritic cell exosomes, the cell expansion factor on the 12th day of culture is 6,236 times; the control group (without autologous dendritic cells) The cell expansion factor of exosomes is 3,754 times. Figure 4B shows the cytotoxicity analysis of γδ-T cells against Daudi cells after autologous dendritic cell exosomes are added to the culture medium. From Figure 4B, it is found that when the E:T ratio is 5, the γδ-T cytotoxicity efficiency of culture medium containing 25μl/ml autologous dendritic cell exosomes is 78.3%; the control group (without The cytotoxicity efficiency of γδ-T cells from autologous dendritic cells (exosomes) was 64.4%. Figure 4C shows the cytotoxicity analysis of γδ-T cells against A549 cells after autologous dendritic cell exosomes are added to the culture medium. From Figure 4C, it is found that when the E:T ratio is 5 and zoledronic acid is added, the γδ-T cell cytotoxicity efficiency of cultured medium containing 25μl/ml autologous dendritic cell exosomes is 58.9% ; The cytotoxicity efficiency of γδ-T in the control group (without autologous dendritic cell exosomes) was 43.7%. In summary, adding autologous dendritic cell exosomes to the γδ-T cell culture medium can effectively increase the expansion rate of γδ-T cells and the cancer cell cytotoxicity.

The following table summarizes the data on the expansion ratio, cell purity, and toxic activity of γδ-T cells under the aforementioned various culture conditions.

Table 1. Consolidation of γδ-T cell expansion times, cell purity, and cytotoxic activity under various culture conditions.

Claims (15)

A method for in vitro expansion and activation of γδ-T cells, which comprises the following steps: (a) Separating peripheral blood mononuclear cells from a blood sample; (b) Isolating γδ-T cells from the peripheral blood mononuclear cells; (c) Suspend the γδ-T cells in a culture medium and place them in a cell culture dish for culture; and (d) Continuously administer a visible light wave stimulation or an audible sound wave stimulation to the γδ-T cells in the cell culture plate for 12 to 16 days. Such as the method of the first item in the scope of patent application, wherein the wavelength of the visible light wave is between 400nm~700nm. Such as the method of the second item of the scope of patent application, wherein the wavelength of the visible light wave is between 550nm~700nm. Such as the method of item 1 in the scope of patent application, wherein the waveform of the audible sound wave is a sine wave, a triangle wave or a square wave. Such as the method of item 4 in the scope of patent application, wherein the frequency of the audible sound wave is 110Hz and the intensity is 70dB. Such as the method of any one of items 1 to 5 in the scope of the patent application, wherein the medium contains an autologous dendritic cell exosomes. Such as the method of item 6 of the scope of patent application, wherein the medium further contains zoledronic acid. Such as the method described in item 6 of the scope of patent application, wherein the medium further includes a basic medium and a cytokine. A method for in vitro expansion and activation of γδ-T cells, which comprises the following steps: (a) Separating peripheral blood mononuclear cells from a blood sample; (b) Isolating γδ-T cells from the peripheral blood mononuclear cells; and (c) Suspend the γδ-T cell in a culture medium and place it in a cell culture dish for 12 to 16 days; wherein the culture medium contains an autologous dendritic cell exosomes. Such as the method of item 9 in the scope of patent application, wherein the concentration of autologous dendritic cell exosomes is 25 μg/ml. For example, the method according to any one of items 9 to 10 in the scope of the patent application, wherein the step (c) of the method further comprises continuously applying a visible light wave stimulation or an audible sound wave to the γδ-T cells in the cell culture plate stimulate. Such as the method of item 11 in the scope of patent application, wherein the wavelength of the visible light wave is between 550nm~700nm. Such as the method of item 11 in the scope of patent application, wherein the waveform of the audible sound wave is a sine wave, a triangle wave or a square wave, the frequency is 110 Hz, and the intensity is 70 dB. A γδ-T cell prepared according to the method of any one of items 1 to 13 in the scope of the patent application. A pharmaceutical composition for inhibiting the proliferation of tumor cells, which comprises the γδ-T cell of item 14 of the scope of patent application and pharmaceutically acceptable excipients.

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