TWI768357B - 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 of expanding and activating γδ-T cells in vitro

The present invention relates to a method for expanding and activating γδ-T cells in vitro. The present invention further relates to a pharmaceutical composition comprising 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 returned to 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 types of immune cells, and selecting the most suitable immune cell types according to the condition and genetic characteristics of cancer patients, such as NK cells, T cells cells etc.

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 recognizing isopentenyl pyrophosphate (IPP) molecules on the cell surface. IPP is an intermediate product of cellular metabolism, and its production is increased in cancer cells, especially when cancer cells have mutations in the p53 gene. γδ-T cells that recognize IPP will proliferate and activate and will increase their power to attack tumor cells. This is a special function of γδ-T cells. If other immune cells do not detect cancer cells γδ-T cells can still find cancer cells and attack them. In general, abnormal cells have molecules that are different from other normal cells, and γδ-T cells recognize these as markers of cancer cells and attack them. 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.

An important feature of γδ-T cells is that they do not require HLA to identify abnormal cells and are therefore independent of an individual's HLA type. This feature allows γδ-T cells to be used in anyone without developing graft-versus-host disease (GVHD). At present, there are many human clinical applications of cell therapy in the United States, Europe and Japan using γδ-T cells, and 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 proliferative capacity varies from person to person, the challenge of using γδ-T cells for cell therapy is whether to rapidly expand and activate γδ-T cells in vitro. In recent years, it has been found that the use of Zoledronic acid can make γδ-T cells proliferate in large quantities, and the technology has also been established. In addition to the use of zoledronic acid in culturing γδ-T cells, injecting zoledronic acid into cancer patients can find that cancer cells express IPP in greater quantities, thereby improving 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 zoledronic acid for culturing cells or injecting it into patients. Therefore, there is still a great need to develop a convenient, effective and safe in vitro expansion and activation of γδ- Methods of T cells.

In one aspect of the present invention, it provides a method for expanding and activating γδ-T cells in vitro, comprising the following steps: isolating peripheral blood mononuclear cells from a blood sample; Separating γδ-T cells from blood mononuclear cells; suspending the γδ-T cells in a medium and culturing them in a cell culture plate; and continuously applying a visible light wave stimulation to the γδ-T cells in the cell culture plate Or an audible sonic stimulation, cultured for 12 to 16 days.

In a specific embodiment, the wavelength of the visible light wave ranges from 400 nm to 700 nm; in a preferred embodiment, the wavelength of the visible light wave ranges from 550 nm to 700 nm.

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

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

In another aspect of the present invention, there is provided a cell prepared according to the aforementioned method.

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

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the appended drawings show some, but not all, alternative embodiments. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. These drawings, which are incorporated in and constitute a part of the specification, help explain the principles of the invention.

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

Fig. 2A shows the cell expansion fold analysis of the γδ-T cells in the cell culture plate after the present invention continuously stimulates the γδ-T cells with different wavelengths of visible light waves.

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

Figure 2C shows the analysis of the killing efficiency of γδ-T cells on A549 cells after the present invention continuously stimulates γδ-T cells in cell culture dishes with visible light waves of different wavelengths.

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

Figure 3A is the analysis of the cell expansion fold after the present invention continuously applies different waveforms of audible acoustic wave stimulation to the γδ-T cells in the cell culture dish.

Fig. 3B shows the analysis of the poisoning efficiency of γδ-T cells to Daudi cells after continuously applying different waveforms of audible acoustic wave stimulation to γδ-T cells in the cell culture dish according to the present invention.

Figure 3C shows the analysis of the killing efficiency of γδ-T cells to A549 cells after the present invention continues to stimulate the γδ-T cells in the cell culture plate with different waveforms of audible acoustic waves.

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

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

Figure 4 C 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 the killing efficiency of cells.

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, which is to apply visible light wave stimulation, audible sound wave stimulation and autologous dendritic cell exosomes to γδ-T cells. In vitro expansion of cells can effectively increase the expansion multiple 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 one of ordinary skill in the art to which this invention belongs. In case of conflict, this document and its definitions will control.

As used herein, "about", "approximately", or "approximately" shall generally mean within 20%, preferably within 10%, and more preferably within 5% of a particular value or range. Numerical values used herein are approximations, meaning that words such as "about," "approximately," or "approximately" can be inferred to apply if not expressly stated.

In the present invention, "gamma-delta T cells" refer to cells that display CD3 ⁺ on the cell surface antigen and express TCR Vγ9 and TCR Vδ2.

In the present invention, "cell expansion fold" is determined by dividing "the number of cells after 12 days of in vitro culture" by "the number of initial γδ-T cells isolated from peripheral blood mononuclear cells".

In the present invention, "cancer cell killing efficiency" refers to using γδ-T cells as effector cells, and using Daudi cell line or A549 cell line as target cells. The poisoning test was performed when the ratio of cells to target cells (E:T ratio) was 0.5, 1 or 5, and the ratio of target cell death was taken as the poisoning efficiency.

Materials and Methods

The peripheral blood sample of the present invention is based on the plan passed by the ethics committee. Whole blood is collected from the subject's arm, placed in a sterile blood collection tube, and stored at room temperature for subsequent processing.

The basal 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 contain appropriate components such as proteins, cytokines, antibodies, serum, and compounds. The cytokines are 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 isolating peripheral blood mononuclear cells from blood sample

7.5-8ml of blood was drawn into a blood collection tube containing anticoagulant-heparin and Ficoll-Hypaque reagent, plus a polyester gel separator to separate the two liquids. The blood collection tubes were centrifuged at 1800 g for 20 minutes at room temperature. 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, and the cell layer is not disturbed during operation. Then, the peripheral blood mononuclear cell (PBMC) layer of the interface was collected by a pipette into a 15ml conical tube, and the PBMC was washed with 10ml phosphate buffered saline (PBS) and turned over. Conical tube 5 times and centrifuged at 400g for 5 minutes. After repeating the preceding washing steps twice, the cells were resuspended in 5 ml of PBS. Counting the number of cells, usually 1.3x10 ⁶ PBMCs can be isolated from 1 ml of whole blood. Finally, the proportion and phenotype of γδ-T cells in PBMC were confirmed by flow cytometry.

Methods of expanding and activating γδ-T cells in vitro

After centrifugation of the peripheral blood mononuclear cell suspension in a 15 ml conical tube at 400 g for 5 minutes at room temperature, the supernatant was discarded. Cell culture medium was prepared, and interleukin-2 (IL-2) and zoledronic acid (Zometa) were added to the medium to final concentrations of 1000 IU/ml and 5 μM, respectively. The zoledronic acid was added in liquid form, and 50 μl of zoledronic acid (concentration of 4 mg/5 ml) was added to each 30 ml of culture medium. The cell pellet was then resuspended in medium and adjusted to 1 x ¹⁰⁶ cells per milliliter of medium. Using a 24-well cell culture dish, add 1x10 ⁶ cells to each well for 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 culture bottle used. Then add autologous plasma, incoming AB serum, fetal bovine serum or autologous dendritic cell exosomes to make up about 10% of the total culture volume (equivalent to a 24-well cell culture plate, add 100 μl of cells to each well) . Place the cell culture plate in a cell culture incubator at 37°C, 5% CO ₂ for 24-48 hours. Maintain the cell density at 0.5 x 10 ⁶ ~2 x 10 ⁶ cells per ml, add fresh medium containing 1000IU/ml interleukin-2 every 2 to 3 days, and transfer the cells to Continue culturing in a new culture dish or flask. Maintain a serum concentration of at least 1% in the medium during incubation. Cells were harvested on day 12 and the number, phenotype and function of γδ-T cells were confirmed by flow cytometry.

Analysis of cell surface antigens by flow cytometry

The expanded and activated cells were placed in a 96-well plate at 2×10 ⁵ cells/200 μl, and 3 μl of fluorescently labeled antibody was added to react at 4°C for 15 minutes, then washed three times with PBS, and then added 400 μl of PBS to suspend the cells. Fluorescent markers on the cell surface were then analyzed by flow cytometry. The above fluorescently labeled antibodies include anti-CD3 antibody, anti-TCR Vγ9 antibody and anti-TCR Vδ2 antibody.

Test method for the ability of γδ-T cells to kill cancer cells

The expanded and activated γδ-T cells were used as effector cells, and Daudi cell line (lymphoma cell line) or A549 cell line (lung cancer cell line) was used as target cell. The effected cells and target cells were mixed and cultured at 0.5:1, 1:1 or 5:1, reacted for 4 hours, and then stained with 7-AAD to determine the number of apoptotic cells.

Method for stimulating γδ-T cells with visible light waves

In order to create a stable light source in the same culture environment, set a standard 8-watt fluorescent tube 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 was set to 1,000 lumens with a photometer, and the wavelength was set to 400 nm, 550 nm or 700 nm. 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 the light.

Method for 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 the NCH audio sound generator software, the wave pattern and spectrum are analyzed by the SP4Win software, and a Fostex 6301NB full-frequency 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 line replaced the dendritic cell 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 Dendritic cells were cultured for 24 hours. The cultured medium was collected and centrifuged at 300 g and 1000 g for 10 minutes, respectively, followed by filtration through a 0.45 μm pore size filter to remove cells and debris. The filtered medium was concentrated by centrifugation at 1000g for 45 minutes with a Centricon Plus-70 Millipore filter, followed by ultracentrifugation at 100,000g for one hour, and dendritic cell exosomes could be isolated from the medium and washed twice with PBS. , centrifuged at 1000 g for 25 min through an Amicon Ultra-15 filter, and finally dendritic cell exosomes were resuspended in 200 μL of PBS. Quantification of dendritic cell exosomes was performed using the BCA Protein Assay Kit (Thermo Scientific).

The expanded and activated γδ-T cells obtained by the aforementioned method can be stored in a suitable excipient, which can be a monophosphate buffer, and finally prepared into a pharmaceutical composition.

Example

Example 1. Culture results of γδ-T cells stimulated with visible light waves

Figure 2A shows the results of cell expansion fold analysis after continuous stimulation of γδ-T cells with different wavelengths of visible light waves in the cell culture dish. It can be seen from Figure 2A that on the 12th day of culture, the cells in the group irradiated with 700 nm visible light wave expanded by 4,989 times; those in the group irradiated with visible light at 550 nm were multiplied by 3,433 times; those in the group irradiated with 400 nm visible light wave expanded 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 effect of γδ-T cells on Daudi after continuous stimulation of γδ-T cells with different wavelengths of visible light waves in the cell culture dish. Analysis of the killing efficiency of cells. It can be found from Figure 2B that when the ratio of E:T is 5, the cytotoxicity efficiency of the 700nm visible light group is 62.1%; the 550nm visible light group is 66.8%; the 400nm visible light group The cytotoxicity efficiency was 60.5%; the cytotoxicity efficiency of the control group (without visible light wave stimulation) was 63.4%. Figure 2C shows the analysis of the killing efficiency of γδ-T cells on A549 cells after continuous stimulation of γδ-T cells with different wavelengths of visible light waves in the cell culture dish. It can be found from Figure 2C that when the ratio of E:T 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 (without visible light wave stimulation) was 42.7%. Fig. 2D shows the results of cell analysis by flow cytometry after continuous stimulation of γδ-T cells with different wavelengths of visible light waves in the cell culture dish. From Figure 2D, it was found that on the 12th day of culture, the γδ-T cells in the 700nm visible light wave group had a cell purity of 92.1%; the γδ-T cells in the 550nm visible light wave group had a cell purity of 91.7%; 400nm visible light irradiation The γδ-T cells in the wave group had a cell purity of 90.7%; the γδ-T cells in the control group had a cell purity of 73.3%. In conclusion, continuous application of visible light wave stimulation of a specific wavelength during cell culture can effectively improve the expansion rate, cell purity and cancer cell killing activity of γδ-T cells.

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

Figure 3A shows the results of cell expansion fold analysis after continuous stimulation of γδ-T cells with different waveforms of audible acoustic waves in the cell culture dish. From Figure 3A, it can be found that continuous stimulation with audible sound waves of 110Hz, 70dB sine wave, the cell expansion factor on the 12th day of culture is 3,006 times; continuous stimulation with audible sound waves of 110Hz, 70dB triangle wave, On the 12th day of culture, the cell expansion factor was 3,226 times; continuous stimulation with audible sound waves of 110Hz, 70dB square wave, On the 12th day of culture, the cell expansion multiple was 2,864 times; the cell expansion multiple of the control group (without audible acoustic stimulation) was 3,108 times. Figure 3B shows the analysis of the killing efficiency of γδ-T cells on Daudi cells after continuously applying different waveforms of audible acoustic wave stimulation to the γδ-T cells in the cell culture dish. It is found from Figure 3B that when the E:T ratio is 5, the γδ-T cell killing efficiency after continuous stimulation with audible sound waves of 110Hz, 70dB sine wave is 75.6%; with 110Hz, 70dB triangle wave After continuous stimulation with audible sound waves, the killing efficiency of γδ-T cells was 78.1%; after continuous stimulation with audible sound waves of 110 Hz and 70 dB square wave, the killing efficiency of γδ-T cells was 72.6%; The cytotoxicity efficiency of listening to sonic stimulation) was 61.9%. Figure 3C shows the analysis of the killing efficiency of γδ-T cells on A549 cells after continuously applying different waveforms of audible acoustic wave stimulation to the γδ-T cells in the cell culture dish. From Figure 3C, it was found that when the E:T ratio was 5 and zoledronic acid was added, the γδ-T cell killing efficiency was 55.1% after continuous stimulation with audible sound waves of 110 Hz and 70 dB sine wave; The killing efficiency of γδ-T cells was 58.0% after continuous stimulation with audible sound waves of 110 Hz, 70 dB triangular wave; after continuous stimulation with audible sound waves of 110 Hz, 70 dB square wave, the killing efficiency of γδ-T cells was 60.4% ; The cytotoxicity efficiency of the control group (without audible sonic stimulation) was 43.3%. In conclusion, continuous application of audible acoustic wave stimulation with a specific waveform during cell culture can effectively improve the cell purity and cancer cell killing activity of γδ-T cells.

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

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

The following table summarizes the data of γδ-T cell expansion fold, cell purity, and poisoning activity under various culture conditions mentioned above.

Table 1. Summary of expansion times, cell purity, and cytotoxicity of γδ-T cells under various culture conditions.

Claims (13)

A method for expanding and activating γδ-T cells in vitro, comprising the following steps: (a) isolating peripheral blood mononuclear cells from a blood sample; (b) isolating gamma cells from the peripheral blood mononuclear cells δ-T cells; (c) suspending the γδ-T cells in a culture medium and culturing them in a cell culture dish; and (d) continuously administering a wavelength of medium to the γδ-T cells in the cell culture dish; Visible light wave stimulation at 400nm ~ 700nm or an audible sound wave stimulation with a waveform of sine wave, triangle wave or square wave, cultured for 12 to 16 days. According to the method of claim 1 of the claimed scope, the wavelength of the visible light wave is between 550nm and 700nm. According to the method of claim 1, the frequency of the audible sound wave is 110Hz and the intensity is 70dB. The method of any one of claims 1 to 3, wherein the culture medium comprises an autologous dendritic cell exosome. The method of claim 4, wherein the medium further comprises zoledronic acid. The method of claim 4, wherein the medium further comprises a basal medium and interleukin-2 (IL-2). A method for expanding and activating γδ-T cells in vitro, comprising the following steps: (a) isolating peripheral blood mononuclear cells from a blood sample; (b) isolating γδ-T cells from the peripheral blood mononuclear cells; and (c) suspending the γδ-T cells in a culture medium and placing them in a cell culture dish for 12 to 16 days; wherein the culture medium Contains an autologous dendritic cell exosome. The method of claim 7, wherein the concentration of the autologous dendritic cell exosomes is 25 μg/ml. The method according to any one of claims 7 to 8 of the claimed scope, wherein step (c) of the method further comprises continuously administering to the γδ-T cells in the cell culture dish a wavelength ranging from 400 nm to 700 nm. Visible light wave stimulation or an audible sound wave stimulation whose waveform is a sine wave, triangle wave or square wave. According to the method of claim 9, the wavelength of the visible light wave is between 550nm and 700nm. According to the method of claim 9, the frequency of the audible sound wave is 110Hz and the intensity is 70dB. A γδ-T cell prepared according to the method of any one of items 1 to 11 of the claimed scope. A pharmaceutical composition for inhibiting the proliferation of tumor cells, comprising the γδ-T cells of item 12 of the patent application scope and a pharmaceutically acceptable excipient.

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