A method for in vitro expansion and activation of gamma delta-t cells

Abstract

The present application relates to a method for in vitro expansion and activation of γδ-T cells, which stimulates γδ-T cells with audible sound waves, or co-cultures with autologous dendritic cell exosomes, effectively increases the cell expansion multiple, and produces high purity and high toxicity to cancer cells. The present application also relates to a pharmaceutical composition for inhibiting tumor cell proliferation, which comprises γδ-T cells.

Description

[0001] This application is a divisional application of the invention patent application No. 202010475465.8, filed on May 29, 2020, entitled "A method for in vitro expansion and activation of γδ-T cells". The entire contents of the original application are incorporated herein by reference. Technical Field

[0002] This invention relates to a method for in vitro expansion and activation of γδ-T cells. This invention also relates to a pharmaceutical composition comprising γδ-T cells. Background Technology

[0003] Immunotherapy is a treatment method that involves proliferating and activating the patient's own immune cells in vitro before reinfusing them into the body. Currently, immunotherapy is increasingly being used in 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, such as NK cells or T cells, based on the cancer patient's condition and genetic characteristics.

[0004] T cells in the human immune system are broadly classified into two categories: Alpha-Beta T cells and Gamma-Delta T (γδ-T) cells. γδ-T cells are present in peripheral blood, accounting for only 1% to 5% of all T cells in the blood. They distinguish between normal and abnormal cells by recognizing isopentenyl pyrophosphate (IPP) molecules on the cell surface. IPP is an intermediate product of cell metabolism, and its production increases in cancer cells, especially when the p53 gene of cancer cells mutates. γδ-T cells that recognize IPP proliferate and activate, enhancing their ability to attack tumor cells. This is a unique function of γδ-T cells. Even if other immune cells do not detect cancer cell markers, γδ-T cells can still find and attack cancer cells. Generally, abnormal cells possess molecules that differ from those of normal cells; γδ-T cells recognize and attack these as cancer cell markers. In addition to IPP molecules, γδ-T cells can also recognize 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.

[0005] A key characteristic of γδ-T cells is that they do not require HLA to identify abnormal cells, thus being independent of an individual's HLA type. This characteristic allows γδ-T cells to be used in anyone without causing graft-versus-host disease (GVHD). Currently, numerous clinical applications of cell therapy using γδ-T cells have been conducted in the United States, Europe, and Japan, and these clinical applications have all demonstrated that the use of γδ-T cells is safe and reliable.

[0006] Because 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 lies in whether they can be rapidly and massively expanded and activated in vitro. In recent years, it has been discovered that zoledronic acid can induce massive proliferation of γδ-T cells, and the technology has been established. Besides its use in culturing γδ-T cells, injecting zoledronic acid into cancer patients has revealed that cancer cells exhibit a greater degree of IPP (Intracytoplasmic Proliferation), thereby increasing the sensitivity of γδ-T cells to cancer cells. However, zoledronic acid is a drug used to treat osteoporosis, and its use in culturing cells or injecting it into patients still raises certain concerns. Therefore, there is a strong need to develop a convenient, effective, and safe method for in vitro expansion and activation of γδ-T cells. Summary of the Invention

[0007] In one aspect, the present invention provides a method for in vitro expansion and activation of γδ-T cells, comprising the following steps: isolating peripheral blood mononuclear cells from a blood sample; isolating γδ-T cells from the peripheral blood mononuclear cells; suspending the γδ-T cells in a culture medium and culturing them in a cell culture dish; and continuously applying visible light or audible sound stimulation to the γδ-T cells in the cell culture dish for 12 to 16 days.

[0008] In one specific embodiment, the wavelength of the aforementioned visible light wave is between 400nm and 700nm; in a preferred embodiment, the wavelength of the aforementioned visible light wave is between 550nm and 700nm.

[0009] In one 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.

[0010] In some specific embodiments of the present invention, the aforementioned culture medium comprises 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 culture medium further comprises zoledronic acid; in another preferred embodiment, the aforementioned culture medium further comprises basal culture medium and cytokines.

[0011] In another aspect, the present invention provides a cell prepared according to the aforementioned method.

[0012] In another aspect, the present invention provides a pharmaceutical composition for inhibiting the proliferation of tumor cells, comprising the aforementioned cells and pharmaceutically acceptable excipients.

[0013] The foregoing overview, and the following detailed description of the invention, will be better understood when read in conjunction with the accompanying drawings. To illustrate the invention, the accompanying drawings show some, but not all, alternative specific embodiments. However, it should be understood that the invention is not limited to the precise arrangements and means shown. These drawings, which are incorporated into and form part of this specification, help to explain the principles of the invention. Attached Figure Description

[0014] Figure 1 This is a flowchart of the in vitro expansion and activation method for γδ-T cells of the present invention.

[0015] Figure 2A This invention analyzes the fold expansion of γδ-T cells in cell culture trays after continuous stimulation with visible light of different wavelengths.

[0016] Figure 2B This invention analyzes the cytotoxicity efficiency of γδ-T cells against Daudi cells after continuously applying visible light of different wavelengths to γδ-T cells in cell culture trays.

[0017] Figure 2C This invention analyzes the cytotoxicity efficiency of γδ-T cells against A549 cells after continuously applying visible light of different wavelengths to γδ-T cells in cell culture trays.

[0018] Figure 2D This invention provides the results of cell analysis using flow cytometry after continuously applying visible light of different wavelengths to γδ-T cells in a cell culture dish.

[0019] Figure 3A This invention analyzes the fold increase of γδ-T cells in cell culture trays after continuously applying audible sound waves of different waveforms.

[0020] Figure 3B This invention analyzes the cytotoxicity efficiency of γδ-T cells against Daudi cells after continuously applying audible sound waves of different waveforms to γδ-T cells in cell culture trays.

[0021] Figure 3C This invention analyzes the cytotoxicity efficiency of γδ-T cells against A549 cells after continuously applying audible sound waves of different waveforms to γδ-T cells in cell culture trays.

[0022] Figure 4A This invention relates to the analysis of the fold expansion of γδ-T cells after adding autologous dendritic cell exosomes to the culture medium.

[0023] Figure 4B This invention analyzes the cytotoxicity efficiency of γδ-T cells against Daudi cells after adding autologous dendritic cell exosomes to the culture medium.

[0024] Figure 4C This invention analyzes the cytotoxicity of γδ-T cells against A549 cells after adding autologous dendritic cell exosomes to the culture medium. Detailed Implementation

[0025] 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 involves applying visible light stimulation, audible sound stimulation, and autologous dendritic cell exosomes to expand γδ-T cells in vitro, effectively increasing the expansion rate of γδ-T cells and producing γδ-T cells with high purity and high cancer cell killing activity.

[0026] definition

[0027] 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 pertains. In case of conflict, this document and its definitions shall prevail.

[0028] As used herein, “about,” “approximately,” or “roughly” generally refers to within 20%, preferably within 10%, and even more preferably within 5% of a specific value or range. The values ​​used herein are approximate and mean that unless explicitly stated otherwise, the words “about,” “approximately,” or “roughly” can be inferred to apply.

[0029] In this invention, "gamma-delta T cell" refers to a cell whose surface antigen presents CD3+. + It also showed TCR Vγ9 and TCR Vδ2 in cells.

[0030] In this invention, the "cell expansion factor" 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.

[0031] In this invention, "cancer cell killing efficiency" is defined as follows: γδ-T cells are used as the effector cells, and Daudi cell line or A549 cell line is used as the target cells. The killing test is conducted when the ratio of effector cells to target cells (E:T ratio) is 0.5, 1 or 5, and the proportion of target cells that die is taken as the killing efficiency.

[0032] Materials and Methods

[0033] The peripheral blood sample described in this invention is obtained by collecting whole blood from the subject's arm according to a plan approved by the ethics committee, placing it in a sterile blood collection tube, and storing it at room temperature for subsequent processing.

[0034] The basal culture medium used in this invention can be selected from commercially available basal culture media such as CellGro SCGM (CellGenix), KBM 501 (Kohjin Bio), AIM-V (Thermo Fisher), X-VIV015 (Lonza), DMEM, or RPM1-1640.

[0035] The culture medium described in this invention sometimes contains appropriate proteins, cytokines, antibodies, serum, compounds, and other components. 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).

[0036] Methods for isolating peripheral blood mononuclear cells from blood samples

[0037] 7.5-8 ml of blood was drawn into a blood collection tube containing the anticoagulant heparin and Ficoll-Hypaque reagent, with a polyester gel septum to separate the two liquids. The blood collection tube was centrifuged at 1800g for 20 minutes at room temperature. After centrifugation, the plasma was collected and separated for subsequent cell culture, leaving a 5-10 mm plasma layer on the interface, without disturbing the cell layer during the procedure. Next, the peripheral blood mononuclear cell (PBMC) layer from the interface was collected using a pipette into a 15 ml conical tube. The PBMCs were washed with 10 ml of phosphate-buffered saline (PBS), and the conical tube was inverted 5 times. The tube was then centrifuged at 400g for 5 minutes. This washing step was repeated twice, and the cells were resuspended in 5 ml of PBS. The cell count was calculated; typically, 1.3 x 10⁻⁶ cells could be separated from 1 ml of whole blood. 6 PBMCs were then analyzed. Finally, flow cytometry was used to confirm the proportion and phenotype of γδ-T cells in the PBMCs.

[0038] Methods for in vitro expansion and activation of γδ-T cells

[0039] Peripheral blood mononuclear cell suspension in a 15 ml conical tube was centrifuged at 400 g for 5 minutes at room temperature, and the supernatant was discarded. Cell culture medium was prepared by adding interleukin-2 (IL-2) and zoledronic acid (Zometa) to a final concentration of 1000 IU / ml and 5 μM, respectively. Zoledronic acid was added in liquid form, with 50 μl of zoledronic acid (concentration of 4 mg / 5 ml) added to every 30 ml of culture medium. The cell pellet was then resuspended in the culture medium and adjusted to 1 x 10⁻⁶ cells / ml. 6 Cells. Use a 24-well cell culture dish, add 1 x 10⁶ cells to each well. 6 Cells are cultured. For large-scale culture, a density of 0.5 x 10⁻⁶ cells per square centimeter is recommended. 6 Cell density is adjusted based on the surface area of ​​the culture dish or flask used, using a principle of control. Next, add autologous plasma, human AB serum, fetal bovine serum, or autologous dendritic cell exosomes, making it approximately 10% of the total culture medium volume (equivalent to 100 μl of cells per well in a 24-well cell culture dish). Incubate the cell culture dish at 37°C in a 5% CO2 incubator for 24–48 hours. Maintain the cell density at 0.5 x 10⁻⁶ cells / mL. 6 ~2x 10 6Cells were cultured in fresh medium containing 1000 IU / ml interleukin-2 every 2 to 3 days. If necessary, cells were transferred to new culture dishes or flasks for further culture depending on the degree of cell expansion. The serum concentration in the culture medium was maintained at at least 1% during the culture process. Cells were harvested on day 12 and the number, phenotype, and function of γδ-T cells were confirmed by flow cytometry.

[0040] Analyzing cell surface antigens using flow cytometry

[0041] With 2x10 5 Cells / 200 μl: Amplified and activated cells were placed in a 96-well plate, and 3 μl of fluorescently labeled antibody was added. The mixture was incubated at 4°C for 15 minutes. After washing three times with PBS, 400 μl of PBS was added to suspend the cells, and the fluorescent labeling on the cell surface was analyzed by flow cytometry. The fluorescently labeled antibodies included anti-CD3 antibody, anti-TCR Vγ9 antibody, and anti-TCR Vδ2 antibody.

[0042] Test methods for assessing the ability of γδ-T cells to kill cancer cells

[0043] 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) were used as target cells. The effector and target cells were mixed and cultured at a ratio of 0.5:1, 1:1, or 5:1, and reacted for 4 hours. Cells were then stained with 7-AAD to determine the number of apoptotic cells.

[0044] Methods of stimulating γδ-T cells with visible light waves

[0045] To create a stable light source within the same culture environment, a standard 8-watt fluorescent tube was placed 15 cm above the culture trays in the same incubator to ensure that all cultured cells received the same light stimulation. The light intensity of the culture trays was set to 1,000 lumens using a photometer, with wavelengths of 400 nm, 550 nm, or 700 nm. The control group's culture trays were also placed in the same environment, 15 cm away from the light source, but covered with white cardboard to completely block light.

[0046] Method of stimulating γδ-T cells with audible sound waves

[0047] The sound waves used in this invention are 110Hz sine waves, triangle waves, and square waves, all generated by NCH audio generator software. The waveforms and spectra are analyzed using SP4Win software. A Fostex 6301NB full-range speaker is used to output sound waves 15 cm above the culture dish. A RION NL-31 sound level meter is used to detect the sound pressure level next to the cell culture dish, and the sound pressure level is set to 70 dB.

[0048] Preparation and purification of autologous dendritic cell exosomes

[0049] The preparation of autologous dendritic cell exosomes involved replacing the dendritic cell culture medium from day 5 with fresh cell culture medium, adding granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4, and continuing to culture the dendritic cells for another 24 hours. The culture medium was collected and centrifuged at 300g and 1000g for 10 minutes each, then filtered through a 0.45μm pore size membrane to remove cells and debris. The filtered medium was then concentrated by centrifugation at 1000g for 45 minutes using a Centricon Plus-70 Millipore membrane, followed by ultracentrifugation at 100,000g for one hour to obtain the dendritic cell exosomes. These exosomes were then washed twice with PBS, centrifuged at 1000g for 25 minutes using an Amicon Ultra-15 membrane, and finally resuspended in 200μL of PBS. Quantification of dendritic cell exosomes was performed using the BCA protein analysis kit (Thermo Scientific).

[0050] The expanded and activated γδ-T cells obtained by the aforementioned method can be stored in a suitable excipient, such as a phosphate buffer, and finally prepared into a pharmaceutical composition.

[0051] Example

[0052] Example 1: Results of γδ-T cell culture stimulated by visible light waves

[0053] Figure 2A This study analyzed the fold expansion of γδ-T cells in cell culture dishes after continuous stimulation with visible light of different wavelengths. Figure 2AOn day 12 of culture, the cells in the 700nm visible light group expanded 4,989 times; the cells in the 550nm visible light group expanded 3,433 times; the cells in the 400nm visible light group expanded 2,335 times; and the cells in the control group (without visible light stimulation) expanded 2,750 times. Figure 2B To analyze the cytotoxicity of γδ-T cells against Daudi cells after continuous stimulation with visible light of different wavelengths in cell culture dishes. Figure 2B The study found that, with an E:T ratio of 5, the cytotoxicity of the group irradiated with 700 nm visible light was 62.1%; the cytotoxicity of the group irradiated with 550 nm visible light was 66.8%; the cytotoxicity of the group irradiated with 400 nm visible light was 60.5%; and the cytotoxicity of the control group (without visible light stimulation) was 63.4%. Figure 2C To analyze the cytotoxicity of γδ-T cells against A549 cells after continuous stimulation with visible light of different wavelengths in cell culture dishes. Figure 2C The study found that, with an E:T ratio of 5 and the addition of zoledronic acid, the cytotoxicity of the group irradiated with 700 nm visible light was 48.9%; the cytotoxicity of the group irradiated with 550 nm visible light was 52.3%; the cytotoxicity of the group irradiated with 400 nm visible light was 45.1%; and the cytotoxicity of the control group (without visible light stimulation) was 42.7%. Figure 2D This is the result of flow cytometry analysis of γδ-T cells in cell culture dishes after continuous stimulation with visible light of different wavelengths. Figure 2D The study found that on day 12 of cell culture, the purity of γδ-T cells irradiated with 700 nm visible light was 92.1%; the purity of γδ-T cells irradiated with 550 nm visible light was 91.7%; the purity of γδ-T cells irradiated with 400 nm visible light was 90.7%; and the purity of γδ-T cells in the control group was 73.3%. In conclusion, continuous stimulation with visible light of a specific wavelength during cell culture can effectively enhance the proliferation rate, purity, and cancer cell cytotoxicity of γδ-T cells.

[0054] Example 2: Results of γδ-T cell culture stimulated with audible sound waves

[0055] Figure 3A This study analyzed the fold increase in γδ-T cells in cell culture dishes after continuous application of audible sound waves of different waveforms. Figure 3AThe study found that continuous stimulation with audible sound waves of 110 Hz and 70 dB sine wave resulted in a 3,006-fold increase in cell size on day 12 of culture; continuous stimulation with audible sound waves of 110 Hz and 70 dB triangular wave resulted in a 3,226-fold increase in cell size on day 12 of culture; continuous stimulation with audible sound waves of 110 Hz and 70 dB square wave resulted in a 2,864-fold increase in cell size on day 12 of culture; and the control group (without audible sound wave stimulation) showed a 3,108-fold increase in cell size on day 12 of culture. Figure 3B To analyze the cytotoxicity efficiency of γδ-T cells against Daudi cells after continuous application of audible sound waves of different waveforms to cell culture dishes. Figure 3B The study found that, with an E:T ratio of 5, the cytotoxicity of γδ-T cells after continuous stimulation with a 110Hz, 70dB sine wave audible sound was 75.6%; the cytotoxicity of γδ-T cells after continuous stimulation with a 110Hz, 70dB triangular wave audible sound was 78.1%; the cytotoxicity of γδ-T cells after continuous stimulation with a 110Hz, 70dB square wave audible sound was 72.6%; and the cytotoxicity of the control group (without audible sound stimulation) was 61.9%. Figure 3C To analyze the cytotoxicity efficiency of γδ-T cells against A549 cells after continuous application of audible sound waves of different waveforms to cell culture dishes. Figure 3C The study found that, with an E:T ratio of 5 and the addition of zoledronic acid, the cytotoxicity of γδ-T cells after continuous stimulation with a 110Hz, 70dB sine wave audible sound was 55.1%; the cytotoxicity of γδ-T cells after continuous stimulation with a 110Hz, 70dB triangular wave audible sound was 58.0%; and the cytotoxicity of γδ-T cells after continuous stimulation with a 110Hz, 70dB square wave audible sound was 60.4%. The cytotoxicity of the control group (without audible sound stimulation) was 43.3%. In conclusion, continuous application of audible sound stimulation with specific waveforms during cell culture can effectively improve the cell purity of γδ-T cells and their cytotoxic activity against cancer cells.

[0056] Example 3: Culture results of adding autologous dendritic cell exosomes to γδ-T cell culture medium

[0057] Figure 4A Analysis of the fold expansion of γδ-T cells after the addition of autologous dendritic cell exosomes to the culture medium. Figure 4A The study found that using a culture medium containing 25 μl / ml of autologous dendritic cell exosomes resulted in a 6,236-fold increase in cell size on day 12, while the control group (without autologous dendritic cell exosomes) showed a 3,754-fold increase in cell size. Figure 4BThis study analyzed the cytotoxic efficiency of γδ-T cells against Daudi cells after the addition of autologous dendritic cell exosomes to the culture medium. Figure 4B It was found that, with an E:T ratio of 5, the cytotoxicity of γδ-T cells cultured in a medium containing 25 μl / ml autologous dendritic cell exosomes was 78.3%, while the cytotoxicity of γδ-T cells in the control group (without autologous dendritic cell exosomes) was 64.4%. Figure 4C This study analyzed the cytotoxicity of γδ-T cells against A549 cells after the addition of autologous dendritic cell exosomes to the culture medium. Figure 4C The study found that, with an E:T ratio of 5 and the addition of zoledronic acid, the cytotoxicity of γδ-T cells cultured in a medium containing 25 μl / ml autologous dendritic cell exosomes was 58.9%, while the cytotoxicity of the control group (without autologous dendritic cell exosomes) was 43.7%. In conclusion, adding autologous dendritic cell exosomes to the γδ-T cell culture medium can effectively enhance the expansion rate of γδ-T cells and their cytotoxic activity against cancer cells.

[0058] The table below summarizes the data on the expansion fold, cell purity, and cytotoxic activity of γδ-T cells under the aforementioned different culture conditions.

[0059] Table 1. Summary of γδ-T cell expansion fold, cell purity, and cytotoxic activity under various culture conditions.

[0060]

Claims

1. A method for in vitro expansion and activation of γδ-T cells, comprising the following steps: (a) Isolation of peripheral blood mononuclear cells from a blood sample; (b) γδ-T cells were isolated from the peripheral blood mononuclear cells; (c) The γδ-T cells were suspended in a culture medium and cultured in a cell culture dish; and (d) Continuously apply an audible sound wave to the γδ-T cells in the cell culture dish, wherein the waveform of the audible sound wave is a sine wave, a triangular wave or a square wave, and the frequency is 110 Hz and the intensity is 70 dB, and culture for 12 to 16 days.

2. The method as described in claim 1, characterized in that, The culture medium contains an autologous dendritic cell exosome.

3. The method as described in claim 2, characterized in that, The culture medium also contains zoledronic acid.

4. The method as described in claim 2, characterized in that, The culture medium also includes basal culture medium and cytokines.

5. A method for in vitro expansion and activation of γδ-T cells, comprising the following steps: (a) Isolation of peripheral blood mononuclear cells from a blood sample; (b) Isolation of γδ-T cells from peripheral blood mononuclear cells; and (c) The γδ-T cells were suspended in a culture medium and cultured in a cell culture dish for 12 to 16 days; wherein the culture medium contained an autologous dendritic cell exosome at a concentration of 25 μg / ml. Step (c) of the method further includes continuously applying an audible sound wave stimulus to the γδ-T cells in the cell culture dish, wherein the waveform of the audible sound wave is a sine wave, a triangular wave or a square wave, and the frequency is 110 Hz and the intensity is 70 dB.

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