CN115678847A

CN115678847A - Method for in vitro amplification and activation of gamma delta-T cells

Info

Publication number: CN115678847A
Application number: CN202211403552.8A
Authority: CN
Inventors: 张顺浪; 林杰良; 杨智雅; 孙境新; 刘威廷; 唐晓艳
Original assignee: Qianjing Biotechnology Co ltd
Current assignee: Qianjing Biotechnology Co ltd
Priority date: 2019-07-24
Filing date: 2020-05-29
Publication date: 2023-02-03
Also published as: TW202104578A; CN112300990A; TW202118858A; TWI768357B; CN112300989A; TWI768356B

Abstract

The invention relates to a method for in vitro amplification and activation of gamma delta-T cells, which uses audible sound waves to stimulate the gamma delta-T cells or efficiently improves the cell amplification multiple under the condition of co-culture of autologous dendritic cell exosomes and generates the gamma delta-T cells with high purity and high toxic activity to cancer cells. The invention also relates to a pharmaceutical composition for inhibiting tumor cell proliferation, which comprises gamma delta-T cells.

Description

Method for in vitro amplification and activation of gamma delta-T cells

This application is a divisional application of the invention patent application No. 202010475465.8 filed on 29/5/2020, entitled "a method for in vitro expansion and activation of γ δ -T cells", the entire contents of which are incorporated herein by reference.

Technical Field

The invention relates to a method for amplifying and activating gamma delta-T cells in vitro. The invention also relates to a pharmaceutical composition containing the gamma delta-T cells.

Background

Immune cell therapy is a treatment mode in which the immune cells of a patient are proliferated and activated in vitro and then returned to the body. At present, immune cell therapy is gradually applied to cancer therapy, and the key point of success is to understand the characteristics and functions of various immune cells and select the most suitable immune cell types, such as NK cells, T cells and the like, according to the conditions and gene characteristics of cancer patients.

T cells in the human immune system are largely divided into two main classes, alpha-Beta T cells and Gamma-Delta T (γ δ -T) cells. The gamma delta-T cells are present in peripheral blood in an amount of only 1% to 5% of all T cells in peripheral blood. It distinguishes normal from abnormal cells by recognizing isopentenyl pyrophosphate (IPP) molecules on the cell surface. IPP is an intermediate product of cellular metabolism and its production in cancer cells is increased, especially when the p53 gene of cancer cells is mutated. The γ δ -T cells recognizing IPP proliferate and activate and enhance the force to attack tumor cells, which is a special function of γ δ -T cells, which can find and attack cancer cells if other immune cells do not detect cancer cell markers. In general, abnormal cells will possess molecules that are different from other normal cells, and γ δ -T cells will recognize and attack these as cancer cell markers. In addition to IPP molecules, γ δ -T cells can also be identified by markers such as MIC A/b, HMB-PP, intercellular adhesion molecule-1, CD166, etc. Thus, γ δ -T cells can recognize and kill a variety of cancer cells.

One important feature of γ δ -T cells is that they do not require HLA in identifying abnormal cells and are therefore independent of the individual's HLA type. This feature allows γ δ -T cells to be used in any human without producing graft-versus-host disease (GVHD). Many of the clinical applications of cell therapy in humans, which have been carried out in the united states, europe and japan, are currently carried out using γ δ -T cells, and these clinical applications have confirmed that the use of γ δ -T cells is safe and safe.

Since γ δ -T cells are scarce in blood and their proliferative capacity varies from person to person, the challenge of cell therapy with γ δ -T cells is whether it is possible to expand and activate γ δ -T cells in vitro in large quantities and rapidly. In recent years, the use of Zoledronic acid (Zoledronic acid) has been found to proliferate γ δ -T cells in large quantities, and its technology has also been established. In addition to using zoledronic acid in culturing γ δ -T cells, the injection of zoledronic acid into cancer patients can find that cancer cells exhibit IPP in greater amounts, thereby increasing the sensitivity of γ δ -T cells to cancer cells. However, zoledronic acid is a drug for osteoporosis, and there is still a concern about the use of zoledronic acid in cell culture or injection into patients, and therefore there is still a great need to develop a convenient, effective and safe method for in vitro expansion and activation of γ δ -T cells.

Disclosure of Invention

In one aspect, the present invention provides a method for in vitro amplification and activation of γ δ -T cells, comprising the steps of: separating peripheral blood mononuclear cells from a blood sample; isolating γ δ -T cells from the peripheral blood mononuclear cells; suspending the gamma delta-T cells in a culture medium and culturing in a cell culture tray; and continuously applying a visible light wave stimulation or an audible sound wave stimulation to the gamma delta T cells in the cell culture plate, and culturing for 12 to 16 days.

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

In one embodiment, the waveform of the audible sound wave is a sine wave, a triangular wave or a square wave; in a preferred embodiment, the audible sound has a frequency of 110Hz and an intensity of 70dB.

In some embodiments of the present invention, the culture medium comprises an autologous dendritic cell exosome (autologous dendritic cell exosome); in a preferred embodiment, the concentration of the autologous dendritic cell exosomes is 25 μ g/ml. In a preferred embodiment, the culture medium further comprises zoledronic acid (zoledronic acid); in another preferred embodiment, the culture medium further comprises a basal medium and a cytokine.

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

In a further aspect, the present invention provides a pharmaceutical composition for inhibiting tumor cell proliferation, 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 appended drawings. For the purpose of illustrating the invention, the 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. The accompanying drawings, which are incorporated in and constitute a part of this specification, help explain the principles of the invention.

Drawings

FIG. 1 is a flow chart of the method for in vitro expansion and activation of γ δ -T cells according to the present invention.

FIG. 2A is a cell expansion fold analysis of gamma delta-T cells in a cell culture dish after continuous stimulation with different wavelengths of visible light waves.

FIG. 2B is an analysis of the killing efficiency of the gamma delta T cells to Daudi cells after the stimulation of the gamma delta T cells in the cell culture dish with different wavelengths of visible light waves.

Fig. 2C is an analysis of the poisoning efficiency of γ δ -T cells on a549 cells after the present invention continuously applies stimulation of γ δ -T cells in cell culture plates with visible light waves of different wavelengths.

FIG. 2D shows the results of the flow cytometry analysis of the γ δ -T cells in the cell culture dish after the stimulation with visible light waves of different wavelengths.

FIG. 3A is a cell fold expansion analysis of the invention following continuous application of different waveform audible sonic stimuli to γ δ -T cells in a cell culture dish.

FIG. 3B is an analysis of the killing efficiency of the γ δ -T cells on Daudi cells after different waveform audible sonic stimuli were continuously applied to the γ δ -T cells in the cell culture tray according to the present invention.

Fig. 3C is an analysis of the poisoning efficiency of γ δ -T cells on a549 cells after the present invention continues to apply audible sound wave stimuli of different waveforms to γ δ -T cells in cell culture plates.

FIG. 4A is a gamma delta-T cell fold expansion assay of the invention after addition of autologous dendritic cell exosomes to the culture medium.

FIG. 4B is an analysis of the killing efficiency of γ δ -T cells on Daudi cells after adding autologous dendritic cell exosomes to the culture medium according to the present invention.

FIG. 4C is the analysis of the poisoning efficiency of gamma delta T cells on A549 cells after the autologous dendritic cell exosomes are added into the culture medium according to the present invention.

Detailed Description

In view of the above problems, the present invention provides a method for in vitro amplification and activation of γ δ -T cells, which applies visible light wave stimulation, audible sound wave stimulation and autologous dendritic cell exosomes to γ δ -T cell in vitro amplification, efficiently increases the amplification factor of γ δ -T cells, and produces γ δ -T cells with high purity and high cancer cytotoxic activity.

Definition of

Unless defined otherwise, 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, the present document, including definitions, will control.

As used herein, "about," "about," or "approximately" shall generally mean within 20%, preferably within 10%, and more preferably within 5% of a particular value or range. As used herein, the terms "about", "approximately", or "approximately", unless expressly stated otherwise, are intended to be open-ended and are intended to encompass any number of degrees of certainty.

As used herein, "gamma delta T cell" refers to a cell surface antigen presenting CD3 ⁺ And expressing TCR V gamma 9 and TCR V delta 2.

In the present invention, the "cell amplification factor" is determined in the following manner: "cell number after 12 days in vitro culture" was divided by "initial γ δ -T cell number isolated from peripheral blood mononuclear cells".

In the present invention, the "cancer cell killing efficiency" is obtained by performing a killing test using γ δ -T cells as effector cells (effector cells) and Daudi cell lines or a549 cell lines as target cells (target cells) at a ratio (E: T ratio) of the effector cells to the target cells of 0.5, 1 or 5, and using the ratio of the target cells to the effector cells as the killing efficiency.

Materials and methods

The peripheral blood sample is prepared by collecting whole blood from the arm of a subject according to the plan passed by the ethical committee, placing the whole blood in a sterile blood collection tube, and storing the whole blood at room temperature for subsequent treatment.

The basic culture medium used in the invention can be selected from: commercially available basal media such as CellGro SCGM (CellGenix corporation), KBM 501 (Kohjin Bio corporation), AIM-V (Thermo Fisher corporation), X-VIV015 (Lonza corporation), DMEM, and RPM 1-1640.

The medium of the present invention may contain suitable components such as proteins, cytokines, antibodies, serum, and compounds. 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

Extracting 7.5-8ml of bloodThe blood collecting tube contains anticoagulant-heparin, ficoll-Hypaque reagent, and a polyester gel interlayer for separating the two liquids. Blood collection tubes were centrifuged at 1800g for 20 min at room temperature. After centrifugation is complete, plasma fractions are collected for subsequent cell culture and 5 to 10mm plasma layers are left on the interface, operating without disturbing the cell layer. Then, a layer of Peripheral Blood Mononuclear Cells (PBMC) from the interface was collected by a pipette into a 15ml conical tube, the PBMC was washed with 10ml of Phosphate Buffered Saline (PBS) and the conical tube was inverted 5 times, and centrifuged at 400g for 5 minutes. After repeating the washing step twice, the cells were resuspended in 5ml of PBS. Counting the number of cells, 1.3x10 can be isolated as a rule from 1ml of whole blood ⁶ The PBMC of (1). Finally, the proportion and the phenotype of the gamma delta-T cells in the PBMCs are confirmed by a flow cytometer.

Method for amplifying and activating gamma delta-T cells in vitro

After centrifugation of the peripheral blood mononuclear cell suspension in a 15ml conical tube at 400g for 5 minutes at room temperature, the supernatant was discarded. A cell culture medium was prepared, to which interleukin-2 (IL-2) and zoledronic acid (Zometa) were added to final concentrations of 1000IU/ml and 5. Mu.M, respectively. Wherein the zoledronic acid is added in liquid form, and 50. Mu.l of zoledronic acid (concentration 4mg/5 ml) is added to each 30ml of the culture medium. The cell pellet was then resuspended in medium and adjusted to 1X10 per ml of medium ⁶ A cell. 24-well cell culture plates were used, 1X10 added per well ⁶ The cells are cultured. If a large amount of culture is needed, the culture can be 0.5x10 per square centimeter ⁶ The cell density was adjusted in accordance with the surface area of the culture dish or the culture flask used as a principle. Autologous plasma, human AB serum, fetal bovine serum, or autologous dendritic cell exosomes were then added to make up about 10% of the total culture volume (equivalent to a 24-well cell culture plate with 100 μ l cells per well). The cell culture plate is placed in a cell culture box with 37 ℃ and 5% CO2 for 24-48 hours. Maintain cell density at 0.5X10 per ml ⁶ ～2x 10 ⁶ Adding fresh culture medium containing 1000IU/ml interleukin-2 to each cell every 2-3 days, if necessaryDegree of cell expansion the cells are transferred to a new culture dish or flask for continued culture. Serum concentration in the medium was maintained at least at 1% during the culture. Cells were harvested on day 12 and the number, phenotype and function of γ δ -T cells were confirmed by flow cytometry.

Analysis of cell surface antigens by flow cytometry

At 2x10 ⁵ Cells/200. Mu.l the amplified and activated cells were placed in a 96-well plate, and 3. Mu.l of a fluorescence-labeled antibody was added to react at 4 ℃ for 15 minutes, followed by washing 3 times with PBS, adding 400. Mu.l of PBS to suspend the cells, and analyzing the fluorescence-labeling on the cell surface by flow cytometry. The fluorescence labeled antibody comprises an anti-CD3 antibody, an anti-TCR V gamma 9 antibody and an anti-TCR V delta 2 antibody.

Gamma delta-T cell virus cancer cell killing capability test mode

The amplified and activated gamma delta-T cells are used as action cells (effector cells), and Daudi cell strains (lymph cancer cell strains) or A549 cell strains (lung cancer cell strains) are used as target cells (target cells). After the affected cells and the target cells were cultured in a mixture of 0.5.

Method for stimulating gamma delta-T cells by visible light waves

To create a stable light source in the same culture environment, a standard 8 watt fluorescent tube was placed in the same incubator, 15 cm above the plate, to ensure that all cells in culture received the same light stimulus. The intensity of light on the culture dish was set to 1,000 lumens with a photometer, and the wavelength was set to 400nm, 550nm or 700nm. The control plates were also placed in the same environment at a distance of 15 cm from the light source, but covered with a white paper card to completely block the light.

Method for stimulating gamma delta T cells with audible sound waves

Sine waves, triangular waves and square waves with the acoustic frequency of 110Hz used in the invention are all generated by NCH audio generator software, the wave pattern and the frequency spectrum are analyzed by SP4Win software, a Fostex 6301NB full-frequency horn is used to output the acoustic waves 15 cm above the culture dish, the RION NL-31 sound level meter is used to detect the sound pressure level beside the cell culture dish, and the sound pressure level is set to 70dB.

Preparation and purification of autologous dendritic cell exosomes

Autologous dendritic cell exosomes (Autologous dendritic cell exosomes) were prepared by replacing the dendritic cell culture medium cultured up to the fifth day with fresh cell culture medium, and adding Granulocyte-colony stimulating growth factor (GM-CSF) and interleukin-4 and continuing to culture the dendritic cells for 24 hours. The cultured medium was collected and centrifuged at 300g and 1000g for 10 minutes, respectively, followed by filtration through a filter having a pore size of 0.45 μm to remove cells and debris. The filtered medium was concentrated by centrifugation through a Centricon Plus-70 Millipore filter at 1000g for 45 minutes, then ultracentrifugation at 100,000g for one hour was carried out to separate the dendritic cell exosomes from the medium, washed twice with PBS, centrifuged at 1000g through an Amicon Ultra-15 filter for 25 minutes, and finally the dendritic cell exosomes were resuspended in 200. Mu.L of PBS. Quantification of dendritic cell exosomes was performed using BCA protein assay kit (Thermo Scientific).

The amplified and activated gamma delta-T cells obtained by the method can be mixed with a proper excipient for storage, and the excipient can be a phosphate buffer solution, and finally, the pharmaceutical composition is prepared.

Examples

Example 1 culture results of γ δ -T cells stimulated with visible light waves

FIG. 2A shows the results of fold amplification analysis of cells after constant application of visible light wave stimuli of different wavelengths to γ δ -T cells in cell culture plates. As seen from FIG. 2A, on the 12 th day of culture, the amplification factor of the cells irradiated with the 700nm visible light wave group was 4,989 times; the amplification factor of the cells irradiated by a 550nm visible light wave group is 3,433 times; the amplification factor of the cells irradiated by a 400nm visible light wave group is 2,335 times; the control group (no visible light wave stimulation) had a cell expansion factor of 2,750-fold. FIG. 2B is an analysis of the killing efficiency of γ δ -T cells on Daudi cells after continuous application of visible light wave stimuli of different wavelengths to γ δ -T cells in cell culture plates. From FIG. 2B, it is found that the cytotoxic efficiency of the 700nm visible light wave irradiated group is 62.1% in the case where the E: T ratio is 5; the cytotoxic efficiency of the 550nm visible light wave group is 66.8%; the cytotoxic efficiency of irradiating 400nm visible light wave group is 60.5%; the cytotoxic efficiency of the control group (no visible light wave stimulation) was 63.4%. Fig. 2C is an analysis of the poisoning efficiency of γ δ -T cells on a549 cells after continuous application of visible light wave stimuli of different wavelengths to γ δ -T cells in cell culture plates. From FIG. 2C, it is found that the cytotoxic efficiency of the visible light wave group irradiated at 700nm is 48.9% in the case where the E: T ratio is 5 and zoledronic acid is added; the cytotoxic efficiency of the 550nm visible light wave group is 52.3%; the cell poisoning efficiency of irradiating 400nm visible light wave group is 45.1%; the cytotoxic efficiency of the control group (no visible light wave stimulation) was 42.7%. Fig. 2D shows the results of cell analysis performed by flow cytometry after the gamma delta T cells in the cell culture dish were continuously stimulated with visible light waves of different wavelengths. As shown in FIG. 2D, on the 12 th day of culture, gamma.delta.T cells irradiated with a visible light wave group of 700nm had a cell purity of 92.1%; irradiating gamma delta-T cells of a 550nm visible light wave group, wherein the cell purity is 91.7%; irradiating gamma delta-T cells of a 400nm visible light wave group, wherein the cell purity is 90.7%; the cell purity of the gamma delta-T cells of the control group is 73.3 percent. In conclusion, the visible light wave stimulation with specific wavelength is continuously applied during the cell culture, so that the amplification rate, the cell purity and the cancer cell poisoning activity of the gamma delta-T cells can be effectively improved.

Example 2 results of stimulating γ δ -T cell culture with audible Sound waves

FIG. 3A shows the results of fold amplification analysis of cells following continuous application of different waveform audible sound stimuli to γ δ -T cells in cell culture dishes. As seen from FIG. 3A, the cell expansion was 3,006-fold at day 12 of culture by continuous stimulation with audible sound waves of 110Hz and 70dB sine waves; continuously stimulating with audible sound wave of 110Hz and 70dB triangle wave, wherein the cell expansion multiple on the 12 th day of culture is 3,226 times; continuously stimulating by audible sound wave of 110Hz and 70dB square wave, wherein the cell expansion multiple on the 12 th day of culture is 2,864 times; the control group (without audible sonic stimulation) had a 3,108 fold expansion of cells. FIG. 3B is a graph showing the poisoning efficiency of γ δ -T cells on Daudi cells after different waveform audible sound wave stimuli were continuously applied to γ δ -T cells in the cell culture tray. From FIG. 3B, it is found that the γ δ -T cell killing efficiency after continuous stimulation with audible sound waves of 110Hz and 70dB sine waves is 75.6% at an E: T ratio of 5; the poisoning efficiency of the gamma delta-T cells after continuous stimulation by audible sound waves of 110Hz and 70 decibel triangular waves is 78.1 percent; the gamma delta-T cell poisoning efficiency after continuous stimulation by audible sound waves of 110Hz and 70dB square waves is 72.6 percent; the cytotoxic efficiency of the control group (no audible sonic stimulation) was 61.9%. Fig. 3C is an analysis of the poisoning efficiency of γ δ -T cells on a549 cells after continuous application of different waveform audible sound wave stimuli to γ δ -T cells in cell culture plates. From fig. 3C, it is found that the γ δ -T cell killing efficiency after continuous stimulation with audible sound waves of 110Hz, 70db sine waves is 55.1% with the E: T ratio of 5 and the addition of zoledronic acid; the gamma delta-T cell poisoning efficiency after continuous stimulation by audible sound waves of 110Hz and 70dB triangular waves is 58.0 percent; the gamma delta-T cell poisoning efficiency after continuous stimulation by audible sound waves of 110Hz and 70dB square waves is 60.4 percent; the cytotoxic efficiency of the control group (no audible sonic stimulation) was 43.3%. In conclusion, the cell purity and the cancer cell poisoning activity of the gamma delta-T cells can be effectively improved by continuously applying the audible sound wave stimulation with a specific waveform during the cell culture.

Example 3 culture results of addition of autologous dendritic cell exosomes to γ δ -T cell culture Medium

FIG. 4A is a γ δ -T cell fold expansion analysis after addition of autologous dendritic cell exosomes to the culture medium. From FIG. 4A, it was found that the cell expansion ratio was 6,236 times at the 12 th day of culture using the medium containing 25. Mu.l/ml of the autologous dendritic cell exosomes; the control group (without autologous dendritic cell exosomes) had a 3,754 fold cell expansion. FIG. 4B is an analysis of the killing efficiency of γ δ -T cells on Daudi cells after addition of autologous dendritic cell exosomes to the culture medium. It was found from FIG. 4B that the γ δ -T cell killing efficiency at an E: T ratio of 5 was 78.3% when cultured using a medium containing 25 μ l/ml autologous dendritic cell exosomes; the control group (without autologous dendritic cell exosomes) had a γ δ -T cell cytotoxic efficiency of 64.4%. Fig. 4C is an analysis of the poisoning efficiency of γ δ -T cells on a549 cells after addition of autologous dendritic cell exosomes to the culture medium. It was found from fig. 4C that the γ δ -T cell killing efficiency in the case of an E: T ratio of 5 with the addition of zoledronic acid was 58.9% using a medium containing 25 μ l/ml autologous dendritic cell exosomes; the control group (without autologous dendritic cell exosomes) had a γ δ -T cell cytotoxic efficiency of 43.7%. In conclusion, the autologous dendritic cell exosomes are added into the gamma delta T cell culture medium, so that the amplification rate of the gamma delta T cells and the cancer cell poisoning activity can be effectively improved.

The following table summarizes the data of the gamma delta-T cells, such as amplification times, cell purity, and cytotoxic activity, under various culture conditions.

TABLE I summary of the proliferation fold, cell purity, and cytotoxic activity of γ δ -T cells under various culture conditions.

Claims

1. A method for in vitro expansion and activation of γ δ -T cells, comprising the steps of:

(a) Separating peripheral blood mononuclear cells from a blood sample;

(b) Isolating γ δ -T cells from the peripheral blood mononuclear cells;

(c) Suspending the gamma delta-T cells in a culture medium and culturing in a cell culture tray; and

(d) An audible sonic stimulus is continuously applied to the gamma delta-T cells in the cell culture dish, and the cells are cultured for 12 to 16 days.

2. The method of claim 1, wherein the audible sound wave has a sine wave, a triangular wave, or a square wave.

3. The method of claim 4, wherein the audible sound waves have a frequency of 110Hz and an intensity of 70dB.

4. The method of any one of claims 1-3, wherein the culture medium comprises an autologous dendritic cell exosome.

5. The method of claim 4, wherein the culture medium further comprises zoledronic acid.

6. The method of claim 4, wherein the culture medium further comprises a basal medium and a cytokine.

7. A method for in vitro expansion and activation of γ δ -T cells, comprising the steps of:

(a) Separating peripheral blood mononuclear cells from a blood sample;

(b) Isolating γ δ -T cells from the peripheral blood mononuclear cells; and

(c) Suspending the gamma delta-T cells in a culture medium and placing the cells in a cell culture dish for 12 to 16 days; wherein the culture medium comprises an autologous dendritic cell exosome.

8. The method of claim 7, wherein the concentration of the autologous dendritic cell exosomes is 25 μ g/ml.

9. The method of any one of claims 7-8, wherein step (c) of the method further comprises continuing to apply an audible sonic stimulus to the γ δ -T cells in the cell culture dish.

10. The method of claim 9, wherein the audible sound wave has a sine wave, a triangular wave or a square wave with a frequency of 110Hz and an intensity of 70dB.

11. A γ δ -T cell produced according to the method of any one of claims 1 to 10.

12. A pharmaceutical composition for inhibiting tumor cell proliferation comprising the γ δ -T cell of claim 11 and a pharmaceutically acceptable excipient.