Patent Description:
The present disclosure relates to the technical field of cell culture, in particular, to a human Vγ9Vδ2T cell proliferation method and a culture medium.

Immunocyte therapy has become a novel important medical method for treating refractory diseases (such as malignant tumor) in the medical field all around the world, for example, a CAR-T cell is used to treat B-cell lymphoma. For the scientific and medical communities, it is very critical to obtain an immunocyte with stable and reliable quality and good functions for rendering good clinical treatment effect.

Human Vγ9Vδ2T cell which only exists in primates and human beings is a T-cell subset with anti-tumor and anti-infection functions. Human Vγ9Vδ2T cell can be used for immunocyte therapy or immunologic function reconstruction of people who suffers from tumor, refractory infectious diseases or immune function unbalanced diseases.

Currently, there are mainly two types of human Vγ9Vδ2T cell proliferation methods:.

The purpose of the present disclosure comprises, for example, providing a human Vγ9Vδ2T cell proliferation method, which can improve the proliferation efficiency and the cell purity of human Vγ9Vδ2T cells, and the cultured human Vγ9Vδ2T cells have relatively stronger killing ability and suppressive ability to tumor, and have a stronger anti-apoptotic ability and a longer cell survival time.

The purpose of the present disclosure also comprises, for example, providing a human Vγ9Vδ2T cell culture medium, and the culture medium is used to culture the human Vγ9Vδ2T cells and can improve the proliferation efficiency and the cell purity of the human Vγ9Vδ2T cells, and the cultured human Vγ9Vδ2T cells have a relatively stronger killing ability and suppressive ability to tumor, and have a stronger anti-apoptotic ability and a longer cell survival time.

The purpose of the present disclosure also comprises, for example, providing a human Vγ9Vδ2T cell stimulation and proliferation culture medium, which can selectively proliferate the human Vγ9Vδ2T cells from the peripheral blood mononuclear cells, and the proliferated human Vγ9Vδ2T cells have high purity, stronger killing ability and stronger anti-apoptotic ability.

The invention provides a T cell proliferation method as defined in claim <NUM>. The dependent claims define further optional features of the T cell proliferation method.

The present disclosure provides a T cells proliferation method, comprising:.

In one or multiple embodiments, the T cells comprises Vγ9Vδ2T cells.

The first culture medium comprises interleukin-<NUM> and phosphonic acid compounds.

In one or multiple embodiments, the first culture medium comprises interleukin-<NUM>, phosphonic acid compounds, interleukin-<NUM>, and vitamin C or derivatives of vitamin C.

In one or multiple embodiments, the derivatives of vitamin C are selected from vitamin C ethyl ether, vitamin C palmitate, vitamin C glucoside, vitamin C magnesium phosphate and vitamin C sodium phosphate.

In one or multiple embodiments, in the second culture medium, the concentration of the interleukin-<NUM> is <NUM>-1000ng/ml.

In one or multiple embodiments, in the second culture medium, the concentration of the vitamin C or derivatives of vitamin C is <NUM>-<NUM>.

In one or multiple embodiments, the composition containing the T cells comprises peripheral blood mononuclear cells extracted from the peripheral blood of a human body.

In one or multiple embodiments, in step A, the culture duration is <NUM>-<NUM> hours.

In one or multiple embodiments, the phosphonic acid compounds comprise bisphosphoric acid compounds, preferably, selected from the group consisting of zoledronic acid, etidronic acid, ibandronic acid, pamidronic acid, alendronic acid, risedronic acid, minodronic acid and combinations thereof, for example, zoledronic acid.

In one or multiple embodiments, the second culture medium comprises a basic culture medium, interleukin-<NUM>, interleukin-<NUM>, and vitamin C or derivatives of vitamin C.

In one or multiple embodiments, the basic culture medium is selected from the group consisting of RPMI-<NUM> culture medium, D-MEM, MEM, RPMI, Opti-MEM and combinations thereof, for example, RPMI-<NUM> culture medium.

The present disclosure also provides a pharmaceutical composition, comprising the T cells obtained by using the proliferation method according to the present text and a pharmaceutically acceptable carrier.

The present disclosure also provides a pharmaceutical composition, comprising the Vγ9Vδ2T cells obtained by using the proliferation method according to the present text and a pharmaceutically acceptable carrier.

In one or multiple embodiments, the pharmaceutical composition is a cell suspension.

The present disclosure also provides use of the T cells obtained by the proliferation method according to the present disclosure or the pharmaceutical composition of the present disclosure in preparation of medicines for suppressing, preventing or treating infectious diseases, autoimmune diseases or malignant diseases.

In one or multiple embodiments, the malignant disease is cancer.

In one or multiple embodiments, the malignant disease is lung cancer.

The present disclosure also provides a method for suppressing, preventing or treating infectious diseases, autoimmune diseases or malignant diseases, comprising:.

The present disclosure also provides the use of the T cells obtained by the proliferation method according to the present text or the pharmaceutical composition of the present text in suppressing, preventing or treating infectious diseases, autoimmune diseases or malignant diseases.

The present disclosure also provides the use of the culture medium of the present text used for proliferating T cells.

In one or multiple embodiments, the T cells are Vγ9Vδ2T cells.

The present disclosure provides a human Vγ9Vδ2T cell proliferation method, comprising:.

In one or multiple embodiments, the basic culture medium is the RPMI-<NUM> culture medium; and the phosphonic acid compounds comprise one or a plurality of zoledronic acid, etidronic acid, ibandronic acid, pamidronic acid, alendronic acid, risedronic acid, minodronic acid.

In one or multiple embodiments, in step <NUM>, the cell density of the cell suspension is <NUM>~<NUM>×<NUM><NUM> cells/ml.

In one or multiple embodiments, in step <NUM>, in the first culture medium, the concentration of the phosphonic acid compounds is <NUM>-<NUM>; and
In the first culture medium and the second culture medium, the concentration of the interleukin-<NUM> is <NUM>-1000ng/ml, the concentration of the interleukin-<NUM> is <NUM>-1000ng/ml, and the concentration of the vitamin C or derivatives of vitamin C is <NUM>-<NUM>.

In one or multiple embodiments, the cell culture container is a <NUM>-well plate, <NUM>-well plate or <NUM>-well plate; in the step <NUM>, the cell suspension is inoculated into the cell culture container to culture for <NUM> hours; and in the subsequent culture process in the step <NUM>, the medium for cells is changed every <NUM>-<NUM> hours.

In one or multiple embodiments, the basic culture medium is the RPMI-<NUM> culture medium.

In one or multiple embodiments, in the culture medium, the concentration of the interleukin-<NUM> is <NUM>-1000ng/ml, the concentration of the interleukin-<NUM> is <NUM>-1000ng/ml, and the concentration of the vitamin C is <NUM>-<NUM>.

In one or multiple embodiments, in the first culture medium, the concentration of the phosphonic acid compounds is <NUM>-<NUM>.

The present disclosure has the following advantageous effects:.

In order to more clearly illustrate technical solutions of embodiments of the present disclosure, accompanying drawings which need to be used in the embodiments will be introduced briefly below.

The term of "basic culture medium" used in the present text refers to a solution containing nutrients that nourish the growth of mammalian cells. The basic culture medium provides standard inorganic salts such as zinc, iron, magnesium, calcium and potassium, and vitamins, glucose, buffer system and essential amino acids. For example, the basic culture medium is RPMI-<NUM> culture medium, D-MEM, MEM, RPMI, Opti-MEM or the combinations thereof.

The term of "prepared by" has the same meaning with "containing". The terms of "include", "comprise", "have", "contain" or any other variants used herein are intended to cover non-exclusive comprising. For example, the composition, step, method, article or device comprising listed elements is not necessary to be limited to those elements, other elements not explicitly listed or elements inherent in such a composition, step, method, article or device can also be contained.

The conjunction phrase of "consist of" excludes any unspecified elements, steps or components. If used in a claim, this phrase will make the claim closed, so that the claim does not comprise materials other than those described, except for the conventional impurities associated with them. When the phrase of "consist of" appears in a sub-sentence of the subject of the claim rather than immediately after the subject, it only defines the elements described in the sub-sentence; and other elements are not excluded from the claim as an entirety.

When the amount, concentration, or other value or parameter is expressed by a range, a preferred range, or a range defined by a series of preferred upper limit values and preferred lower limit values, it should be understood as the discourse of all ranges formed by any pairing of any upper limit or preferred value of any range and any lower limit or preferred value of any range, regardless of whether the range is separately disclosed. For example, when the range of "<NUM>~<NUM>" is disclosed, the described range should be explained to comprise the ranges of "<NUM>~<NUM>", "<NUM>~<NUM>", "<NUM>~<NUM>", "<NUM>~<NUM> and <NUM>~<NUM>", "<NUM>~<NUM> and <NUM>" and the like. When the value range is described in the present text, unless otherwise described, the range intends to comprise its end values and all integers and fractions within the range.

The term of "parts by mass" refers to the basic unit of measurement that represents the mass ratio relationship of multiple components; <NUM> part can represent any unit mass, for example, it can represent <NUM>, or <NUM> and the like. If the parts by mass of component A is a parts and the parts by mass of component B is b parts, then it shows that the mass ratio of component A to component B is a:b. Or, it shows that the mass of the component A is aK, and the mass of the component B is bK (K is a random number, representing the multiple factor). What should not be misunderstood is that the sum of the parts by mass of all the components is not limited to <NUM> parts, which is different from the mass fraction.

The term of "and/or" is used to refer that one or both of the described conditions may occur, for example, A and/or B comprises (A and B) and (A or B).

In addition, the indefinite articles "a" and "an" before an element or component of the present disclosure have no restriction on the quantity (i.e., the occurrence times) of the element or component. Therefore, "a" and "an" should be understood to comprise one or at least one, and the element or component in the singular form also contains the plural form, unless the number described is clearly intended to refer to the singular form.

The term of "derivatives of vitamin C" generally is a type of compounds with an enediol structure that stabilizes a reduced vitamin C by introducing other groups to the carbon atom at position <NUM> of vitamin C. Examples of derivatives of vitamin C comprise vitamin C phosphate, such as vitamin C magnesium phosphate and vitamin C sodium phosphate; vitamin C palmitate, and vitamin C ethyl ether; and vitamin C carbohydrates, such as vitamin C glucoside and the like.

The terms of "peripheral blood mononuclear cells", "PBMCs" or "mononuclear cell" refer to mononuclear cells separated from the peripheral blood, and are usually used for anti-cancer immunotherapy. The peripheral blood mononuclear cells can be obtained from collected human blood using, for example, Ficoll-Hypaque density gradient method.

The "peripheral blood mononuclear cells" can be obtained from normal people, subjects who are at risk of having disease, or patients. The peripheral blood mononuclear cells used here need not necessarily be derived from the autologous, and allogeneic peripheral blood mononuclear cells can also be used.

The term of "malignant disease" in the present text is used in the broadest sense thereof, and refers to diseases characterized by uncontrolled cell growth. It comprises, but is not limited to, adrenocortical carcinoma, anal cancer, bladder cancer, ependymoma, medulloblastoma, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic cholangiocarcinoma, eye cancer, gallbladder cancer, gastric cancer, germ cell tumors, extragonadal cancer, head and neck cancer, hypopharyngeal cancer, pancreatic islet cell cancer, laryngeal cancer, leukemia, acute lymphoblastic leukemia, oral cancer, liver cancer, lung cancer, and the like.

The term of "autoimmune disease" refers to diseases and disorders caused by the body's immune response to its own tissues, which causes prolonged inflammation and subsequent tissue destruction. Examples of autoimmune diseases comprise, but is not limited to, alopecia areata, type <NUM> diabetes, Guillain-Barré syndrome, multiple sclerosis, rheumatoid arthritis, scleroderma, polymyositis, vitiligo, and systemic lupus erythematosus.

The term of "infectious disease" can be the result of any pathogen. Its examples comprise, but is not limited to, the result of viral infections such as AIDS, hepatitis B and C, cell infections, bacterial infections, parasites and fungal infections.

The present disclosure firstly provides a human Vγ9Vδ2T cell proliferation method, comprising:.

Specifically, in the step <NUM>, the basic culture medium is RPMI-<NUM> culture medium. RPMI is short for Roswell Park Memorial Institute, representing Roswell Park Memorial Institute. RPMI is a type of cell culture medium developed by the institute, and <NUM> refers to the code name of the culture medium. <NUM>% fetal bovine serum is added when using this culture medium.

Optionally, the phosphonic acid compounds comprise one of or a plurality of zoledronic acid (zoledronate, ZOL), etidronic acid, ibandronic acid, pamidronic acid, alendronic acid, risedronic acid, minodronic acid.

In an embodiment of the present disclosure, the phosphonic acid compound is zoledronic acid.

Specifically, in the step <NUM>, the cell density of the cell suspension is <NUM>~<NUM>×<NUM><NUM> cells/ml.

Specifically, the purpose of steps <NUM> and <NUM> lies in culturing the peripheral blood mononuclear cells by using the first culture medium which contains phosphonic acid compounds, selectively proliferating human Vγ9Vδ2T cells in the peripheral blood mononuclear cells, and suppressing the growing of other cells to make other cells apoptotic.

Optionally, the cell culture container is a <NUM>-well plate, <NUM>-well plate or <NUM>-well plate.

The purpose of the step <NUM> is to further proliferate the human Vγ9Vδ2T cells with a relatively high purity obtained by the selective proliferation in step <NUM>, to further add the amount of human Vγ9Vδ2T cells.

Specifically, in the step <NUM>, in the first culture medium, the concentration of the phosphonic acid compound is <NUM>-<NUM>; and
in the first culture medium and the second culture medium, the concentration of the interleukin-<NUM> is <NUM>-1000ng/ml, the concentration of the interleukin-<NUM> is <NUM>-1000ng/ml, and the concentration of the vitamin C is <NUM>-<NUM>.

Preferably, in the step <NUM>, the cell suspension is inoculated into the cell culture container to culture for <NUM> hours.

Specifically, in the subsequent culture process in the step <NUM>, the medium for cells is changed every <NUM>-<NUM> hours.

Specifically, the total culture duration of the steps <NUM> and <NUM> usually is <NUM>-<NUM> days, and the cells at this point reach a sufficient amount and has the strongest killing ability, which are especially suitable for cell therapy.

The present disclosure provides a T cell proliferation method, comprising:.

The present disclosure provides a method for suppressing, preventing or treating infectious diseases, autoimmune diseases or malignant diseases, comprising:.

preferably, the first subject and the second subject can be a same subject. Alternatively, the first subject and the second subject are different subjects.

In the above, the peripheral blood mononuclear cells are compositions containing T cells, and preferably, the malignant disease is cancer, for example, lung cancer.

In the human Vγ9Vδ2T cell proliferation method of the present disclosure, the operating methods without specific conditions are carried out in accordance with the methods described in general conditions (for example, <NPL>).

The critical technical feature of the present disclosure is the use of interleukin-<NUM> and vitamin C in the cell culture process, through adding interleukin-<NUM> and vitamin C in the process of proliferation culture of the human Vγ9Vδ2T cells, compared with the conventional proliferation method (the second type of proliferating technology), the proliferation efficiency and cell purity of the human Vγ9Vδ2T cells can be improved, and the cultured human Vγ9Vδ2T cells have stronger anti-apoptotic ability and longer cell survival time; in addition, the expression level of critical killer molecule NKG2D thereof is higher, and then the killing ability to tumor cells is stronger. Compared with the second type of the existing proliferating technology (referring to the introduction in the background art for the details), the present disclosure solves, for example, the technical problems of low proliferation efficiency and low purity, the problem that the obtained cells do not have a strong killing ability, and the technical problems that the obtained cells do not have enough survival time and the obtained cells are prone to senescence and apoptosis, and the like.

To sum up, the human Vγ9Vδ2T cell proliferation method of the present disclosure has the following advantageous effects:.

Based on the above-mentioned human Vγ9Vδ2T cell proliferation method, the present disclosure also provides a human Vγ9Vδ2T cell culture medium (also referred to as the second culture medium in the present text), which comprises a basic culture medium and interleukin-<NUM>, interleukin-<NUM>, and vitamin C added in the basic culture medium.

In one or multiple embodiments, in the first culture medium and the second culture medium, the concentration of the interleukin-<NUM> is <NUM>-1000ng/ml, <NUM>-500ng/ml, <NUM>-200ng/ml, or <NUM>-130ng/ml. In one or multiple embodiments, in the second culture medium, the concentration of the interleukin-<NUM> is <NUM>-1000ng/ml, <NUM>-500ng/ml, <NUM>-200ng/ml, or <NUM>-130ng/ml. In one or multiple embodiments, in the second culture medium, the concentration of the of the vitamin C is <NUM>-<NUM>, or <NUM>-<NUM>, or <NUM>-<NUM>.

The present disclosure also provides a human Vγ9Vδ2T cell stimulation and proliferation culture medium (also referred to as the first culture medium in the present text), which comprises the above-mentioned second culture medium and phosphonic acid compounds.

Optionally, in the first culture medium, the concentration of the phosphonic acid compound is <NUM>-<NUM>.

Main reagents and materials used in the experiments were provided in the following Table <NUM>.

After the PBMC in fresh blood was separated using the Ficoll separation solution, RPMI-<NUM> complete culture medium with <NUM>% FBS was used to adjust the cell concentration to <NUM>~<NUM>×<NUM><NUM> cells/ml, and inoculated to a <NUM>-well plate, and 40ng/mL IL-<NUM> and <NUM> zoledronic acid were added for stimulating for <NUM> days. Then, the zoledronic acid was removed, the medium was changed every <NUM>~<NUM> days for passage, and the RPMI-<NUM> culture medium containing cytokines of the following concentrations was used: 100IU/mL IL-<NUM>, 100IU/mL IL-<NUM>, and <NUM> vitamin C, to culture in a culture incubator at <NUM>, <NUM>% CO<NUM>, PH=<NUM>-<NUM>, and a humidity of <NUM>% for <NUM>-<NUM> days.

Vγ9Vδ2T cells proliferated in vitro for <NUM>-<NUM> days were collected and placed inside a <NUM> of EP tube, and then centrifuged at 3500rpm for <NUM> in a miniature centrifuge (Eppendorf <NUM>), and after the supernatant was discarded, washing was conducted twice with <NUM> pre-cooled PBS; the cells were transferred into the corresponding test tubes according to the blank control tube, isotype control and experimental group, respectively, and each tube contained about <NUM>×<NUM><NUM> cells. The control tube and the detection tube were respectively equipped with the following fluorescent antibody staining solution according to the antibody specification: anti-human CD3-V500, anti-human TCR-δ2-PE, anti-human CD45-PE-cy5, anti-human CD27-PB and NKG2D-PE-cy7. The cells were resuspended with the prepared fluorescent antibody staining solution, then placed in a refrigerator at <NUM> or on ice, stained in a dark place for <NUM>-<NUM> minutes, washed twice with PBS, and centrifuged at 3500rpm for <NUM> minutes, and the supernatant was discarded, and then the cells were resuspended with <NUM>-300µL PBS, and the purity and phenotype of Vγ9Vδ2T cells were detected using a flow cytometry.

<FIG> was a comparison chart of the proliferation ratios showing that a culture medium containing zoledronic acid was first used for stimulation and then four different cell culture mediums were used to conduct proliferation culture to the human Vγ9Vδ2T cells.

In <FIG>, IL2 indicated what was used in the entire proliferation culture process was the culture medium obtained by adding IL2 based on the basic culture medium;.

In the stage of stimulation and proliferation, the solutions of the above four culture mediums all used the basic culture medium added with ZOL (zoledronic acid) and IL-<NUM> to stimulate the cells, and the amount of the components of each culture medium in <FIG> refers to the concentration as described in the portion of "Vγ9Vδ2T cell proliferation in vitro" of this example.

As shown in <FIG>, after the above four culture mediums were respectively used for conducting proliferation culture to the human Vγ9Vδ2T cells, the obtained data showed that the cell proliferation efficiency of the culture medium (IL2 and IL2+VC), in which IL15 was not added, was relatively low, while the cell proliferation efficiency of the culture medium (IL2+ IL15 and IL2+ IL15+VC), in which IL15 was added, was relatively high, and thus, it could be seen that the adding of IL-<NUM> could obviously improve the proliferation efficiency (rise of Ki67 protein expression) of the human Vγ9Vδ2T cells.

<FIG> was a comparison chart of the apoptosis ratio of the human Vγ9Vδ2 T cells obtained by using four different culture mediums to conduct proliferation culture to the human Vγ9Vδ2T cells, and IL2, IL2+VC, IL2+IL15, IL2+IL15+VC in <FIG> represented the same meanings with those in <FIG>.

Based on <FIG>, it could be seen that adding VC into the culture medium could obviously reduce the apoptosis ratio and apoptosis rate of cells, and in addition, the combination of IL-<NUM> and VC could more significantly enhance the anti-apoptotic ability of cells.

<FIG> was a comparison chart of the expression levels of the critical killer molecule NKG2D of the human Vγ9Vδ2 T cells obtained by using four different culture mediums to conduct proliferation culture to the human Vγ9Vδ2T cells; and IL2, IL2+VC, IL2+IL15, IL2+IL15+VC in <FIG> represented the same meanings with those in <FIG> and <FIG>.

Based on <FIG>, it could be seen that the combination of IL-<NUM> and vitamin C could make the critical killer molecule NKG2D of human Vγ9Vδ2T cells continuously maintain a high level of expression, and the critical killer molecule NKG2D could still maintain a high level of expression at the <NUM>st day of the culturing and after <NUM> days of culturing. In other words, the formula of the combination of IL-<NUM> and VC (i.e., the technical solution of the present disclosure) could maintain a longer time of the high killing ability of human Vγ9Vδ2T cells.

Rag2-/-γc-/- female mice aged <NUM>-<NUM> weeks were purchased from Taconic Company, which were of SPF grade. Immunodeficient mice used independently ventilated IVC cages. <NUM> mice were raised in each cage. Each isolator had independent HEPA air inlet and outlet and had <NUM>-hour temperature and humidity control, and the feed and litter supply of the mice were vacuum-packed and sterilized by γ- radiation, and sterilized water was used as animal drinking water, and the animal raised in this environment was also packaged in a sterile environment; and the animals were transported in a biologically safe transportation box to ensure that the feeding and transportation were maintained in the same microbial state, so as to ensure and maintain the quality of the animals. The animal experiment was approved by the experimental animal ethics committee.

PBMC (huPBMC) were separated by Ficoll liquid density gradient centrifugation to construct humanized mouse models (huPBMCs humanized mice). The HLA type of constructing the humanized mouse-related PBMC was not consistent with the γδ T cells for reinfusion, and generally was HLA-A2+/-. It was convenient for distinguishing the γδ T cells for reinfusion and from the humanized mice themselves during subsequent detection.

Then, normal Rag2-/-γc-/- mice aged <NUM>-<NUM> weeks were selected, after being irradiated with a sublethal dose of <NUM> cGay, <NUM>×<NUM><NUM> huPBMCs were injected intraperitoneally. After <NUM> weeks, the surviving humanized mice could be used for the construction of lung cancer models in the next step. After A549 cells growing adherently were digested, a cell suspension was prepared, and the concentration of A549 cells was adjusted to <NUM>×<NUM><NUM> cells/mL with phosphate buffered saline (PBS). The prepared cell suspension was inoculated into humanized mice by inguinal subcutaneous injection at a dose of <NUM>µL/mouse.

The result of tumor treatment experiment was shown in <FIG>, <FIG> and <FIG>.

<FIG> was a schematic view of the size of tumor after a cell treatment to a humanized mouse with lung cancer using the human Vγ9Vδ2T cells obtained by a conventional proliferation method (IL-<NUM>).

The conventional proliferation method (the second type of proliferating technology) was always to add IL2 into the basic culture medium for culturing and to add ZOL for stimulating proliferation in the early stage of the culturing.

<FIG> was a schematic view of the size of tumor after a cell treatment to a humanized mouse with lung cancer using the human Vγ9Vδ2T cells obtained by the proliferation method (IL2+IL15+VC) of the present disclosure.

<FIG> was a schematic view of the size of tumor of the humanized mouse with lung cancer in the case of not being treated.

The humanized mice with lung cancer used in the experiments as illustrated in <FIG> had the same tumor type and substantively the same tumor volume, and growing time of the tumor as illustrated in <FIG> were the same.

Through the comparison of <FIG>, it could be seen that after the human Vγ9Vδ2T cells obtained by the proliferation method of the present disclosure were used to perform cell treatment on the humanized mice with lung cancer, compared with the human Vγ9Vδ2T cells obtained by a conventional proliferation method, the human Vγ9Vδ2T cells proliferated by the present disclosure could more effectively suppress the growth of lung cancer cells (the size of the tumor was significantly reduced), and could significantly improve the survival rate of humanized mice with lung cancer. On the <NUM>nd day, the humanized mice with lung cancer that were treated with human Vγ9Vδ2T cells obtained by a conventional proliferation method and the untreated humanized lung cancer mice all died, however, the humanized mice with lung cancer that were treated with human Vγ9Vδ2T cells obtained by the proliferation method of the present disclosure all survived with a survival rate of <NUM>%, and the tumor in one mouse completely disappeared (shown in the block of <FIG>).

<FIG> was a schematic view of the variations of the volume of the tumor of the humanized mice with lung cancer under different treating conditions, and <FIG> respectively illustrated the volume changes of the tumor of the humanized mice with lung cancer when the humanized mice with lung cancer were treated with the human Vγ9Vδ2T cells obtained by the conventional proliferation method (IL2) and with the human Vγ9Vδ2T cells obtained by the proliferation method (IL2+IL15+VC) of the present disclosure, and were not treated.

It could be seen that with the increase of time, the volumes of tumors of the humanized mice with lung cancer which were treated with human Vγ9Vδ2T cells obtained by the conventional proliferation method (IL2) and which were not treated increased continuously, and the tumor volumes increased very quickly in the later stage, however, the tumor volume of humanized mice with lung cancer treated with the human Vγ9Vδ2T cells obtained by the proliferation method (IL2+IL15+VC) of the present disclosure increased very slowly, in other words, compared with the human Vγ9Vδ2T cells obtained by the conventional proliferation method (IL2), the human Vγ9Vδ2T cells obtained by the proliferation method (IL2+IL15+VC) of the present disclosure had a stronger suppressing effect on tumor growth in vivo, and rendered smaller tumor volumes.

<FIG> was a schematic view of the variations of the survival rates of the humanized mice with lung cancer under different treating conditions, and <FIG> respectively illustrated the variations of the survival rates of the humanized mice with lung cancer which were treated with the human Vγ9Vδ2T cells obtained by the conventional proliferation method (IL2) and with the human Vγ9Vδ2T cells obtained by the proliferation method (IL2+IL15+VC) of the present disclosure, and which were not treated.

It could be seen that with the increase of time, the survival rates of the humanized mice with lung cancer which were treated with human Vγ9Vδ2T cells obtained by the conventional proliferation method (IL2) and which were not treated reduced very quickly, however, the survival rate of the humanized mice with lung cancer treated with the human Vγ9Vδ2T cells obtained by the proliferation method (IL2+IL15+VC) of the present disclosure was always <NUM>%, in other words, the human Vγ9Vδ2T cells obtained by the proliferation method (IL2+IL15+VC) of the present disclosure could significantly improve the survival rate of the humanized mouse with lung cancer.

The example <NUM> provided a second culture medium, which comprised a RPMI-<NUM> culture medium, and interleukin-<NUM>, interleukin-<NUM> and vitamin C added in the RPMI-<NUM> culture medium.

In the above, in the second culture medium, the concentration of the interleukin-<NUM> was 300ng/ml, the concentration of the interleukin-<NUM> was 500ng/ml, and the concentration of the vitamin C was <NUM>.

In the above, in the second culture medium, the concentration of the interleukin-<NUM> was 500ng/ml, the concentration of the interleukin-<NUM> was 700ng/ml, and the concentration of the vitamin C was <NUM>.

The example <NUM> provided a first culture medium, which comprised the second culture medium as mentioned in the example <NUM> and zoledronic acid.

In the above, in the first culture medium, the concentration of the zoledronic acid was <NUM>.

It is difficult to describe all the numerical ranges of the process parameters involved in the present disclosure in the above-mentioned embodiments, but those skilled in the art can fully imagine that any value falling within the above-mentioned value range can perform the present disclosure, and certainly the present disclosure contains any combination of specific values of several numerical ranges. Here, for the sake of space, the embodiments providing specific values within one or more numerical ranges are omitted, while this should not be regarded as insufficient disclosure of the technical solutions of the present disclosure.

Claim 1:
A T cell proliferation method, characterized by comprising:
step A, culturing a composition containing the T cells, by using a first culture medium which is able to stimulate T cells, so as to stimulate the T cells, wherein the first culture medium comprises interleukin-<NUM> and phosphonic acid compounds, and optionally, also comprises interleukin-<NUM>, and vitamin C or a derivative of vitamin C.; and
step B, using a second culture medium to culture stimulated T cells, wherein the second culture medium contains interleukin-<NUM>, interleukin-<NUM>, and vitamin C or a derivative of vitamin C.