RU2791182C2 - CULTIVATION METHOD AND CULTURAL MEDIUM FOR PROLIFERATION OF HUMAN Vγ9Vδ2 T-CELLS - Google Patents

Abstract

FIELD: cellular biology.

SUBSTANCE: present invention relates to the field of cellular biology, in particular to a method for proliferation of human Vγ9Vδ2 T-cells and a cultural medium for their proliferation. For implementation of the specified method, first, a composition is cultivated, containing T-cells, using a cultural medium capable of stimulating T-cells, which contains a full RPMI-1640 cultural medium with 10% fetal calf serum, interleukin-2, and a phosphonic acid compound, namely zoledronic acid. Next, the second cultural medium is used for cultivation of stimulated T-cells, which contains a full RPMI-1640 cultural medium with 10% fatal calf serum, interleukin-2, interleukin-15, and vitamin C. At the same time, a concentration of interleukin-2 is 40-500 ng/ml, a concentration of interleukin-15 is 3.48-700 ng/ml, a concentration of vitamin C is 70 mcM – 300 mM, a concentration of phosphonic acid compounds is 50-500 mcM.

EFFECT: present invention allows for improvement of the efficiency of proliferation and purity of Vγ9Vδ2 T-cells; human Vγ9Vδ2 T-cell obtained by means of cultivation in this method has a stronger antiapoptotic capacity and a longer period of cell survival, and, in addition, a level of expression of its critical NKG2D killer molecules is higher, which allows it to have a stronger capacity of destruction of tumor cells.

5 cl, 8 dwg, 1 tbl, 6 ex

Description

Cross-reference to related applications

This disclosure claims priority over Chinese Patent Application No. 201811580040.2, filed by the Chinese Patent Office on December 24, 2018, titled "Method and Culture Medium for Proliferating Human Vγ9Vδ2 T Cells", which is incorporated herein by reference in its entirety.

The field of technology to which the invention belongs

The present disclosure relates to the technical field of cell culture, in particular to a method and culture medium for the proliferation of human Vγ9Vδ2 T cells.

Prior Art

Immunocyte-based therapy has become an important new medical treatment for refractory diseases (such as cancer) in the field of medicine throughout the world, for example, CAR-T cell is used to treat B-cell lymphoma. It is very important for the scientific and medical communities to obtain an immunocyte with stable and reliable quality and good functions in order to provide a good clinical treatment effect.

The human Vγ9Vδ2 T cell, which exists only in primates and humans, is a subpopulation of T cells with antitumor and anti-infective functions. The human Vγ9Vδ2 T cell can be used for immunocyte-based therapy or restoration of immunological function in humans who suffer from tumor, refractory infectious diseases, or diseases associated with imbalanced immune function.

At present, there are mainly two types of methods for the proliferation of human Vγ9Vδ2 T cells:

The first type of existing proliferation technology is to add monoclonal antibodies against IL-2 (from the English interleukin - interleukin 12) and TCR (from the English T cell receptor - T-cell receptor) into the culture medium of peripheral blood mononuclear cells (PBMC - from the English peripheral blood mononuclear cell). The main disadvantage of this method is that these two types of γδ T cells (Vδ1 cell subpopulation and Vδ2 cell subpopulation) in peripheral blood are both proliferated, while the Vδ1 cell subpopulation has an immunosuppressive function, and thus cannot be applied. for immunocyte-based therapy; And

the second type of existing proliferation technology is to add IL-2 and a low molecular weight phosphate compound such as zoledronic acid to the PBMC culture medium. The method can render the γδ T cells of the Vδ2 cell subpopulation relatively high purity, and thus can be used in immunocyte-based therapy, and it is a common method currently used to proliferate human Vγ9Vδ2 T cells in human peripheral blood within countries and abroad. However, the method has the following disadvantages: the cell culture proliferation period is about 14 days, proliferated human Vγ9Vδ2 T cells have a relatively short survival period (which can survive for a long time for about 7 days), and thus the anti-apoptotic ability is not strong. , and thus the ability to kill tumor cells is also not strong.

Brief summary of the invention

The purpose of the present disclosure includes, for example, providing a method for the proliferation of human Vγ9Vδ2 T cells that can improve the proliferation efficiency and purity of human Vγ9Vδ2 T cells, and cultured human Vγ9Vδ2 T cells have a relatively stronger killing ability and tumor suppression ability, and have a stronger anti-apoptotic ability and a longer period of cell survival.

The purpose of the present disclosure also includes, for example, providing a culture medium for human Vγ9Vδ2 T cells, and this culture medium is used for culturing human Vγ9Vδ2 T cells, and it can improve the proliferation efficiency and purity of human Vγ9Vδ2 T cells, and cultured human Vγ9Vδ2 T cells. .beta.-cells have a relatively stronger killing ability and a tumor suppression ability, and have a stronger anti-apoptotic ability and a longer cell survival period.

The purpose of the present disclosure also includes, for example, to provide a culture medium for stimulation and proliferation of human Vγ9Vδ2 T cells, which can selectively proliferate human Vγ9Vδ2 T cells from peripheral blood mononuclear cells, and the proliferated human Vγ9Vδ2 T cells are of high purity, stronger killing ability and stronger anti-apoptotic ability.

The present disclosure provides a method for T cell proliferation comprising:

step A, culturing the T cell-containing composition by using a first culture medium that is capable of stimulating T cells to stimulate the T cell; And

step B, using the second culture medium to culture the stimulated T cells; wherein said second culture medium contains interleukin-2, interleukin-15 and vitamin C or vitamin C derivatives.

In one or more embodiments, the T cells include Vγ9Vδ2 T cells.

In one or more embodiments, the first culture medium contains interleukin-2 and phosphonic acid compounds.

In one or more embodiments, the first culture medium contains interleukin-2, phosphonic acid compounds, interleukin-15, and vitamin C or vitamin C derivatives.

In one or more embodiments, the vitamin C derivatives 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 more embodiments, the concentration of interleukin-15 in the second culture medium is 1-1000 ng/ml.

In one or more embodiments, the concentration of vitamin C or vitamin C derivatives in the second culture medium is 10 μM-800 mM.

In one or more embodiments, the concentration of interleukin-2 in the second culture medium is 1-1000 ng/ml.

In one or more embodiments, the T cell composition comprises peripheral blood mononuclear cells isolated from human peripheral blood.

In one or more embodiments in step A, the culture time is 60-100 hours.

In one or more embodiments, the phosphonic acid compounds include 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, e.g., zoledronic acid.

In one or more embodiments, the second culture medium contains the base culture medium, interleukin-2, interleukin-15, and vitamin C or vitamin C derivatives.

In one or more embodiments, the base culture medium is selected from the group consisting of RPMI-1640 culture medium, D-MEM, MEM, RPMI, Opti-MEM, and combinations thereof, eg, RPMI-1640 culture medium.

The present disclosure also provides a culture medium containing a base culture medium, interleukin-2, interleukin-15, and vitamin C or vitamin C derivatives.

In one or more embodiments, the concentration of interleukin-2 in the culture medium is 1-1000 ng/ml, the concentration of interleukin-15 is 1-1000 ng/ml, and the concentration of vitamin C or vitamin C derivatives is 10 μM-800 mM.

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

The present disclosure also provides a pharmaceutical composition comprising Vγ9Vδ2 T cells obtained by using the proliferation method of the present text and a pharmaceutically acceptable carrier.

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

The present disclosure also provides the use of T cells obtained by the proliferation method of the present disclosure or the pharmaceutical composition of the present disclosure in the preparation of medicaments for the suppression, prevention, or treatment of infectious diseases, autoimmune diseases, or malignant diseases.

In one or more embodiments, the cancer is cancer.

In one or more embodiments, the cancer is lung cancer.

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

(1) isolating peripheral blood mononuclear cells from the peripheral blood of a subject in need thereof;

(2) carrying out the proliferation of peripheral blood mononuclear cells by means of the proliferation method of the present disclosure; And

(3) administering the proliferated product to a subject in need thereof.

The present invention also provides the use of T cells obtained by the proliferation method of the present text or a pharmaceutical composition of the present text in the suppression, prevention or treatment of infectious diseases, autoimmune diseases or malignant diseases.

The present disclosure also provides for the use of the culture medium of the present text used for T cell proliferation.

In one or more embodiments, the T cells are Vγ9Vδ2 T cells.

The present disclosure provides a method for the proliferation of human Vγ9Vδ2 T cells, comprising:

step 1, isolating the peripheral blood mononuclear cells from the peripheral blood of the human body;

step 2, providing the first culture medium, resuspending the peripheral blood mononuclear cells by using the first culture medium to obtain a cell suspension,

where the first culture medium contains the second culture medium and phosphonic acid compounds; and the second culture medium contains the base culture medium and interleukin-2, interleukin-15 and vitamin C or vitamin C derivatives added to the base culture medium; And

the order of step 1 and step 2 can be adjusted;

step 3 (or step A), inoculating the cell suspension into the cell culture container and culturing for 60-100 hours; And

step 4 (or step B), using the second culture medium to replace the medium, where the second culture medium is used in all subsequent culture methods.

In one or more embodiments, the base culture medium is RPMI-1640 culture medium; and phosphonic acid compounds include one or more of the following compounds: zoledronic acid, etidronic acid, ibandronic acid, pamidronic acid, alendronic acid, risedronic acid, minodronic acid.

In one or more embodiments, in step 2, the cell density of the cell suspension is 3~4×10 ⁶ cells/ml.

In one or more embodiments in step 2, in the first culture medium, the concentration of phosphonic acid compounds is 1-1000 μM; And

In the first culture medium and the second culture medium, the concentration of interleukin-2 is 1-1000 ng/ml, the concentration of interleukin-15 is 1-1000 ng/ml, and the concentration of vitamin C or vitamin C derivatives is 10 μM-800 mM.

In one or more embodiments, the cell culture container is a 24-well plate, a 48-well plate, or a 96-well plate; in step 3, the cell suspension is inoculated into the cell culture container for culturing for 72 hours; and in the subsequent culture process in step 4, the cell medium is replaced every 48-72 hours.

The present disclosure also provides a culture medium containing a base culture medium and interleukin-2, interleukin-15 and vitamin C added to the base culture medium.

In one or more embodiments, the base culture medium is RPMI-1640 culture medium.

In one or more embodiments, in the culture medium, the concentration of interleukin-2 is 1-1000 ng/ml, the concentration of interleukin-15 is 1-1000 ng/ml, and the concentration of vitamin C is 10 μM-800 mM.

The present disclosure also provides a first culture medium which contains a second culture medium and phosphonic acid compounds.

In one or more embodiments, in the first culture medium, the concentration of phosphonic acid compounds is 1-1000 μM.

The present disclosure has the following advantageous effects:

(1) compared with the conventional proliferation method (second type of proliferation technology), human Vγ9Vδ2 T cells obtained by the proliferation method of the present disclosure have higher proliferation efficiency and higher cell purity; 90% cell purity can be achieved by culturing for 10-12 days (starting from step 3), and the cell proliferation index reaches more than 1000 times (namely, 100,000 human Vγ9Vδ2 T cells can proliferate to at least at least 100 million human Vγ9Vδ2 T cells), which obviously shortens the culture period for the proliferation of human Vγ9Vδ2 T cells;

(2) compared with human Vγ9Vδ2 T cells obtained by conventional proliferation method (second type of proliferation technology), human Vγ9Vδ2 T cells obtained by the proliferation method of the present disclosure have stronger tumor killing and suppression abilities; And

(3) human Vγ9Vδ2 T cells obtained by the proliferation method of the present disclosure have a stronger anti-apoptotic ability and a longer survival period, and after the end of the culture period with cell proliferation (10-12 days from step 3), human Vγ9Vδ2 T β-cells produced by the proliferation method of the present disclosure can continue to survive for about 20 days.

Brief description of graphic materials

To more clearly illustrate the technical solutions of the embodiments of the present disclosure, the accompanying drawings to be used in the embodiments will be briefly introduced below, and it should be understood that these accompanying drawings only demonstrate some embodiments of the present disclosure, thus, they should not be construed as volume limitation.

Fig. 1 is a graph comparing proliferation efficiencies when four different culture media are used to carry out proliferation of a culture of human Vγ9Vδ2 T cells.

Fig. 2 is a graph comparing the apoptosis indices of human Vγ9Vδ2 T cells obtained by using four different culture media to conduct human Vγ9Vδ2 T cell culture proliferation.

Fig. 3 is a graph comparing expression levels of the critical killer molecule NKG2D in human Vγ9Vδ2 T cells obtained by using four different culture media to conduct human Vγ9Vδ2 T cell culture proliferation.

Fig. 4A is a schematic representation of tumor size after treatment with humanized mouse lung cancer cells using human Vγ9Vδ2 T cells obtained by conventional proliferation (IL-2) method.

Fig. 4B is a schematic representation of tumor size after treatment with humanized mouse lung cancer cells using human Vγ9Vδ2 T cells obtained by the (IL2+IL15+VC) proliferation method of the present disclosure.

Fig. 4C is a schematic representation of the tumor size of a humanized mouse with lung cancer in the absence of treatment.

Fig. 5 is a schematic representation of the variation in tumor volume of a humanized mouse with lung cancer under different treatment conditions; And

Fig. 6 is a schematic representation of the variation in survival rate of a humanized mouse with lung cancer under different treatment conditions.

Detailed Description of Embodiments

Terms used in this disclosure

The term "basic culture medium" as used in this text refers to a solution containing nutrients that fuel the growth of mammalian cells. The base culture medium provides standard inorganic salts such as zinc, iron, magnesium, calcium and potassium salts and vitamins, glucose, a buffer system and essential amino acids. For example, the base culture medium is RPMI-1640, D-MEM, MEM, RPMI, Opti-MEM, or combinations thereof.

The term "obtained by" has the same meaning as the term "comprising". The term "include", "comprise", "have" or any other variations used in this document is intended to cover non-exclusive content. For example, a composition, step, method, article, or device containing the listed elements is not necessarily limited to these elements, other elements not explicitly listed, or elements inherent in such a composition, step, method, article, or device, may also be contained.

The connective phrase "consist of" excludes any unspecified elements, steps, or components. When used in a claim, this phrase will close that claim, such that the claim does not contain substances other than the described substances, except for the traditional impurities associated with them. When the phrase "consist of" appears in a subclause of the subject matter of a claim, and not immediately after the subject matter, it only specifies the elements described in the subclause; and other elements are not completely excluded from the claim.

When an amount, concentration, or other quantity or parameter is expressed in terms of a range, a preferred range, or a range defined by a range of preferred upper limits and preferred lower limits, it should be understood to mean the disclosure of all ranges formed by any pairing of any an upper limit or preferred value of any range; and any lower limit or preferred value of any range, whether or not that range is specifically disclosed. For example, when the interval "1~5" is disclosed, it should be explained that the described interval contains the intervals "1~4", "1~3", "1~2", "1~2 and 4~5", "1~ 3 and 5" and the like. When a range of values is described in this text, unless otherwise described, the range is understood to contain its boundary values and all integers and fractions within the range.

The term "parts by weight" refers to a basic unit of measure that represents the ratio of the masses of a plurality of components; 1 part can represent any unit weight, for example, it can represent 1 g or 2.689 g and the like. If the parts by weight of component A are parts a and the parts by weight of component B are parts b, then this indicates that the weight ratio of component A to component B is a:b. Or, it shows that the mass of component A is aK and the mass of component B is bK (K is a random number representing a multiple factor). What should not be misunderstood is that the sum of these mass parts of all these components is not limited to 100 parts, which is different from the mass fraction.

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

In addition, the element or component of the present disclosure in the singular does not have restrictions on the number (namely, the number of times the event occurs) of the element or component. Thus, an element or component in the singular is to be understood to include one or at least one, and an element or component in the singular also includes the plural, unless the number described is expressly intended to refer to the singular.

The term "vitamin C derivatives" is generally a type of compound with an enediol structure that stabilizes reduced vitamin C by introducing other groups to the carbon atom at position 2 of vitamin C. Examples of vitamin C derivatives include vitamin C phosphate, such as vitamin C magnesium phosphate and vitamin C phosphate. sodium vitamin C; vitamin C palmitate and vitamin C ethyl ester; and vitamin C carbohydrates such as vitamin C glucoside and the like.

The terms "peripheral blood mononuclear cells", "PBMC", or "mononuclear cell" refer to mononuclear cells isolated from peripheral blood and are commonly used for cancer immunotherapy. Peripheral blood mononuclear cells can be obtained from collected human blood using, for example, the Ficoll-Hypaque density gradient method.

"Peripheral blood mononuclear cells" can be obtained from normal people, subjects who are at risk of disease, or patients. The peripheral blood mononuclear cells used herein need not be autologous, and allogeneic peripheral blood mononuclear cells may also be used.

The term "malignant disease" in the present text is used in its broadest sense, and it refers to diseases characterized by uncontrolled cell growth. It includes 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 "autoimmune disease" refers to diseases and disorders caused by the body's immune response to its own tissues, which causes long-term inflammation and subsequent tissue destruction. Examples of autoimmune diseases include, but are not limited to, alopecia areata, type 1 diabetes, Guillain-Barré syndrome, multiple sclerosis, rheumatoid arthritis, scleroderma, polymyositis, vitiligo, and systemic lupus erythematosus.

The term "infectious disease" may be the result of any pathogen. Its examples include, but are not limited to, the result of viral infections such as AIDS (acquired immunodeficiency syndrome), hepatitis B and C, cell infections, bacterial infections, parasites, and fungal infections.

First, the present disclosure provides a method for the proliferation of human Vγ9Vδ2 T cells, comprising:

step 1, isolating peripheral blood mononuclear cells (PBMCs) from the peripheral blood of the human body;

step 2, providing the first culture medium, resuspending the peripheral blood mononuclear cells by using the first culture medium to obtain a cell suspension,

where the first culture medium contains the second culture medium and phosphonic acid compounds; and the second culture medium contains the base culture medium and interleukin-2 (IL-2), interleukin-15 (IL-15) and vitamin C added to the base culture medium; And

the order of step 1 and step 2 can be adjusted;

step 3, inoculating the cell suspension into the cell culture container and culturing for 60-100 hours; And

step 4, using the second culture medium to replace the medium, and using the second culture medium in all subsequent culture methods.

In particular, in step 1, the base culture medium is RPMI-1640 culture medium. RPMI stands for Roswell Park Memorial Institute, representing the Roswell Park Memorial Institute. RPMI is a type of cell culture medium developed by this institute, and 1640 refers to the culture medium code name. When using this culture medium, 10% fetal calf serum is added.

Optionally, the phosphonic acid compounds include one or more of the following compounds: zoledronic acid (zoledronic acid, ZOL), etidronic acid, ibandronic acid, pamidronic acid, alendronic acid, risedronic acid, minodronic acid.

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

In particular, in step 2, the cell density of the cell suspension is 3-4×10 ⁶ cells/ml.

In particular, the purpose of steps 2 and 3 is to culture peripheral blood mononuclear cells by using a first culture medium that contains phosphonic acid compounds, selectively proliferation of human Vγ9Vδ2 T cells in peripheral blood mononuclear cells, and suppress the growth of other cells, making other cells apoptotic.

Optionally, the cell culture container is a 24-well plate, a 48-well plate, or a 96-well plate.

The goal of step 4 is to further proliferate relatively high purity human Vγ9Vδ2 T cells obtained by selective proliferation in step 3 with additional addition of human Vγ9Vδ2 T cells.

In particular, in step 2, in the first culture medium, the concentration of the phosphonic acid compound is 1-1000 μM; And

in the first culture medium and the second culture medium, the concentration of interleukin-2 is 1-1000 ng/ml, the concentration of interleukin-15 is 1-1000 ng/ml, and the concentration of vitamin C is 10 μM-800 mM.

Preferably, in step 3, the cell suspension is inoculated into a cell culture container, cultured for 72 hours.

In particular, in the subsequent culture method in step 4, the cell medium is changed every 48 to 72 hours.

In particular, the total culture time of steps 3 and 4 is usually 10-12 days, and the cells at the moment reach a sufficient number and have the strongest killing ability, which are particularly suitable for cell therapy.

The present disclosure provides a method for T cell proliferation comprising:

step A, using a first culture medium that contains interleukin-2 and phosphonic acid compounds, and optionally also contains interleukin-15 and vitamin C or vitamin C derivatives, to culture the composition containing T cells to stimulate T cells; And

step B, using a second culture medium that contains interleukin-2, interleukin-15 and vitamin C or vitamin C derivatives to culture stimulated T cells.

The present disclosure provides a method for the proliferation of human Vγ9Vδ2 T cells, comprising:

Step A, using a culture medium that contains interleukin-2 and phosphonic acid compounds, and optionally also contains interleukin-15 and vitamin C or vitamin C derivatives, to culture a composition containing T cells to stimulate T cells;

step B, selecting and separating human Vγ9Vδ2 T cells from stimulated peripheral blood mononuclear cells; And

step C, using a second culture medium which contains interleukin-2, interleukin-15 and vitamin C or vitamin C derivatives to culture selected and separated human Vγ9Vδ2 T cells.

The present disclosure provides a method for the proliferation of human Vγ9Vδ2 T cells, comprising:

step A, isolating peripheral blood mononuclear cells (PBMCs) from peripheral blood;

step B, using a culture medium that can stimulate T cells to cultivate a composition containing T cells to stimulate T cells;

step C, selecting and separating human Vγ9Vδ2 T cells from stimulated peripheral blood mononuclear cells; And

step D, using a second culture medium that contains interleukin-2, interleukin-15 and vitamin C or vitamin C derivatives to culture selected and separated human Vγ9Vδ2 T cells.

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

(1) isolating peripheral blood mononuclear cells from the peripheral blood of the first subject;

(2) using a culture medium that contains interleukin-2 and phosphonic acid compounds, and optionally also contains interleukin-15 and vitamin C or vitamin C derivatives, to culture peripheral blood mononuclear cells to stimulate human Vγ9Vδ2 T cells;

(3) using a second culture medium that contains interleukin-2, interleukin-15 and vitamin C or vitamin C derivatives to culture stimulated human Vγ9Vδ2 T cells;

(4) introducing the cultured cells to the second subject, where

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

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

In the method for proliferation of human Vγ9Vδ2 T cells of the present disclosure, the methods of operation without specific conditions are carried out in accordance with the methods described in the general conditions (for example, Short Protocols in Molecular Biology, edited by F.M. Ausubel, R.E. Kingston, J.G. Seidman, et al, translated by Ma Xuejun, Shu Yuelong, Beijing: Science Press, 2004).

The extremely important technical feature of the present disclosure is the use of interleukin-15 and vitamin C in the cell culture method, by adding interleukin-15 and vitamin C in the human Vγ9Vδ2 T cell culture proliferation method, compared with the conventional proliferation method (the second type of proliferation technology ), the proliferation efficiency and cell purity of human Vγ9Vδ2 T cells can be improved, and cultured human Vγ9Vδ2 T cells have stronger anti-apoptotic ability and longer cell survival; in addition, the expression level of its critical killer molecule NKG2D is higher, and in such a case, the ability to kill tumor cells is stronger. Compared with the second type of existing proliferation technology (see the introduction to the prior art for details), the present disclosure solves, for example, the technical problems of low proliferation efficiency and low purity, the problem that the resulting cells do not have strong killing ability. , and technical problems that the obtained cells do not have a sufficient survival period and that the obtained cells are prone to senescence and apoptosis, and the like.

In summary, the method for proliferation of human Vγ9Vδ2 T cells of the present disclosure has the following advantageous effects:

(1) compared with the conventional proliferation method (second type of proliferation technology), human Vγ9Vδ2 T cells obtained by the proliferation method of the present disclosure have better proliferation efficiency and higher cell purity; 90% cell purity can be achieved when culture is carried out for 10-12 days (starting from step 3), and the cell proliferation index reaches more than 1000 times (namely, 100,000 human Vγ9Vδ2 T cells can proliferate to at least 100 million human Vγ9Vδ2 T cells), which apparently shortens the period of culture proliferation in the case of human Vγ9Vδ2 T cells;

(2) compared with human Vγ9Vδ2 T cells obtained by conventional proliferation method (second type of proliferation technology), human Vγ9Vδ2 T cells obtained by the proliferation method of the present disclosure have stronger tumor killing and suppression abilities; And

(3) human Vγ9Vδ2 T cells obtained by the proliferation method of the present disclosure have a stronger anti-apoptotic ability and a longer cell survival period, and after the end of the cell proliferation culture period (starting from step 3, for 10-12 days), human Vγ9Vδ2 T cells produced by the proliferation method of the present disclosure can continue to survive for about 20 days.

Based on the above-mentioned method for the proliferation of human Vγ9Vδ2 T cells, the present disclosure also provides a culture medium for human Vγ9Vδ2 T cells (also referred to as the second culture medium in the present text), which contains a base culture medium and interleukin-2, interleukin-15 and vitamin C added to the base culture medium.

In one or more embodiments, the base culture medium is RPMI-1640 culture medium.

In one or more embodiments, in the first culture medium and the second culture medium, the concentration of interleukin-2 is 1-1000 ng/ml, 1-500 ng/ml, 1-200 ng/ml, or 70-130 ng/ml. In one or more embodiments, in the second culture medium, the concentration of interleukin-15 is 1-1000 ng/ml, 1-500 ng/ml, 1-200 ng/ml, or 70-130 ng/ml. In one or more embodiments, the concentration of vitamin C in the second culture medium is 10 µM-800 mM or 20 µM-400 mM or 50 µM-100 µM.

The present disclosure also provides a culture medium for stimulation and proliferation of human Vγ9Vδ2 T cells (also referred to as the first culture medium in the present text), which contains the above-mentioned second culture medium and phosphonic acid compounds.

Perhaps in the first culture medium, the concentration of the phosphonic acid compound is 1-1000 μM.

Examples

Experimental reagents and materials

The main reagents and materials used in the experiments are suggested in the following Table 1.

CFSE - from English. carboxyfluorescein succinimidyl ester - carboxyfluorescein succinimidyl ester

DMEM - from English. Dulbecco modified Eagle's medium

PBS - from English. phosphate buffered saline - phosphate-saline buffer solution

RE - from English. phycoerythrin - phycoerythrin

PerCP / Su5.5 - from English. Peridinin chlorophyll protein-Cyanine5.5 - peridinin-chlorophyll-protein complex-cyanine5.5

Example 1 Proliferation Effects of Vγ9Vδ2 T Cells and Characteristics of Proliferated Vγ9Vδ2 T Cells

Isolation of peripheral blood mononuclear cells

1) 4 ml of human lymphocyte separation solution was taken and added to a 15 ml conical centrifuge tube, 1×PBS buffer was used to dilute the peripheral blood in equal proportions and mixed thoroughly, and then 10 ml of the diluted peripheral blood was slowly spread over the surface of the lymphocyte separation solution along the wall of the analytical tube, and centrifugation at 600 g was carried out for 25 min at 25°C.

2) A sterile Pasteur pipette was used to transfer the middle white flocculent cell layer to another sterile centrifuge tube, and an equal volume of PBS was added and thoroughly mixed, and then centrifugation was performed at 1500 rpm. within 10 min.

3) After discarding the supernatant, 5~10 ml of erythrocyte lysing buffer was added to resuspend the cells for lysing for 3~7 minutes at room temperature. A 5-fold volume of PBS was added to complete the lysis. After filtration through a 40 μm screen, centrifugation was carried out at 1500 rpm. within 10 min.

4) After discarding the supernatant, 10 ml of RPM11640 serum-free culture medium was added to resuspend the cells, and centrifugation was performed at 1500 rpm. within 10 min.

5) After discarding the supernatant, complete culture medium RPM11640 (containing 10% fetal bovine serum) was used to dilute cells sinking to the bottom of the tube to a level of 2 ml, after thorough and slow mixing, 5 µl of cell diluent was taken to dilute to the appropriate fold, and a hemocytometer was used for counting.

In vitro proliferation of Vγ9Vδ2 T cells

After isolating PBMCs in fresh blood using Ficoll-based isolation solution, RPMI-1640 complete culture medium with 10% FBS was used to adjust the cell concentration to 3~4×10 ⁶ cells/mL and inoculated into a 24-well plate, and 40 ng/ml IL-2 and 50 μM zoledronic acid were added to stimulate for 3 days. Then, zoledronic acid was removed, the medium was changed every 2~3 days for passage, and culture medium RPMI-1640 containing cytokines of the following concentrations was used: 100 IU (International Unit)/ml IL-15, 100 IU/ml IL-2 and 70 μM vitamin C, for cultivation in a culture incubator at 37° C., 5% CO ₂ , pH 7.2-7.4, and 95% humidity for 10-14 days.

Purity and Phenotype Identification of Vγ9Vδ2 T Cells

Vγ9Vδ2 T cells proliferating in vitro for 10~14 days were collected and placed inside a 1.5 ml EP tube, and then centrifuged at 3500 rpm. for 5 min in a miniature centrifuge (Eppendorf 5424), and after discarding the supernatant, washing was done twice with pre-chilled PBS, 4°C; cells were transferred to appropriate tubes according to the blank control tube, isotype control and experimental group, respectively, and each tube contained approximately 5×10 ⁵ cells. The control tube and the detection tube respectively were equipped with the following fluorescent antibody staining solution according to the detailed description of the antibody: anti-human CD3-V500 antibody, anti-human TCR-δ2-PE antibody, anti-human CD45-PE-cy5 antibody, anti-human CD27 antibody -PB and NKG2D-PE-su7. The cells were resuspended with the resulting fluorescent antibody staining solution, then placed in a refrigerator at 4°C or on ice, stained in a dark place for 15-20 minutes, washed twice with PBS, and centrifuged at 3500 rpm. for 5 minutes, and the supernatant was discarded, and then the cells were resuspended with 200-300 μl of PBS, and then the purity and phenotype of Vγ9Vδ2 T cells were determined using a flow cytometer.

Analysis of ki67 Vγ9Vδ2 T cells

1) 7~10×10 ⁵ Vγ9Vδ2 T cells proliferating in vitro for 10-14 days were collected, placed inside a 1.5 ml EP tube, centrifuged at 3500 rpm for 5 min in a miniature centrifuge (Eppendorf 5424) and, after discarding the supernatant, washed twice with PBS pre-cooled to 4°C.

2) The cell pellet was resuspended with 100 µl of PBS, a surface-stained anti-CD3-FITC and TCR-δ2-PE antibody (1:200 dilution) was added for flow cytometry, an isotype control antibody of the same color and the same concentration was added to the control tube, and they were incubated at 4°C in the dark for 15 minutes.

3) After staining, the resulting product was washed twice in PBS and centrifuged at 3500 rpm. within 5 min.

4) A set of Foxp3 staining buffers was used to detect the expression level of intranuclear ki67 as follows: preparation of a fixing and permeabilizing buffer, uniform mixing of the F & P concentration (from the English fixation and permeabilization - fixation and permeabilization) with the F & P diluent in a volume ratio of 1: 3; and resuspension of the cells with 500 μl of buffer F & P, settling in a refrigerator at 4°C or on ice in a dark place for 0.5-18 hours, thereby fixing and permeabilizing the cells.

5) 10×Permeabilization Buffer was diluted to 1×Permeabilization Buffer ddH ₂ O (double distilled water). After fixation, 1 ml of 1xpermeabilization buffer was added and the resulting product was centrifuged at 5500 rpm. at 4°C for 5 min and washed twice.

6) Anti-APC-Ki67 fluorescent antibody was diluted in 1x Perm buffer at a volume ratio of 1:200 and mixed evenly; the cell pellet was resuspended with staining solution, placed in a refrigerator at 4°C or on ice, and stained in a dark place for 30 minutes.

7) Buffer 1×Perm was added, the resulting product was centrifuged at 5500 rpm. at 4°C for 5 min and resuspended with 1×Perm buffer and centrifuged again to wash off unbound fluorescent antibody. Cells were resuspended with 200-300 μl PBS, the volume was adjusted, and Ki67 expression level was analyzed by flow cytometry.

Fig. 1 is a comparison graph of proliferation indices showing that culture medium containing zoledronic acid was first used for stimulation and then four different cell culture media were used to carry out the proliferation of a culture of human Vγ9Vδ2 T cells.

On FIG. 1, IL2 indicates that what was used in the entire proliferation culture process was the culture medium resulting from the addition of IL2 based on the base culture medium;

IL2+VC indicates that what was used in the entire proliferation culture process was the culture medium resulting from the addition of IL2+VC based on the base culture medium;

IL2+IL15 indicates that what was used in the entire proliferation culture process was the culture medium resulting from the addition of IL2+IL15 based on the base culture medium; And

IL2+IL15+VC indicates that what was used in the entire proliferation culture process was the culture medium resulting from the addition of IL2+IL15+VC based on the base culture medium.

At the stage of stimulation and proliferation in all solutions of the above culture media, the base culture medium was used with the addition of ZOL (zoledronic acid) and IL-2 to stimulate the cells, and the number of components of each culture medium in Fig. 1 refers to the concentration as described in the "Proliferation of Vγ9Vδ2 T cells in vitro" section of this example.

As shown in FIG. 1, after the above four culture media were respectively used to conduct human Vγ9Vδ2 T cell culture proliferation, the data obtained showed that the cell proliferation efficiency of the culture medium (IL2 and IL2+VC) to which IL15 was not added was relatively low. , while the cell proliferation efficiency of the culture medium (IL2+IL15 and IL2+IL15+VC) to which IL15 was added was relatively high, and thus it can be seen that the addition of IL-15 could obviously improve the proliferation efficiency ( increased expression of the protein Ki67) of human Vγ9Vδ2 T cells.

Fig. 2 is a graph comparing the apoptosis index of human Vγ9Vδ2 T cells obtained by using four different culture media to conduct human Vγ9Vδ2 T cell culture proliferation and IL2, IL2+VC, IL2+IL15, IL2+IL15+VC in FIG. 2 represent the same values as in FIG. 1.

According to Fig. 2, it can be seen that the addition of VC to the culture medium can obviously decrease the apoptosis index and the degree of cell apoptosis, and furthermore, the combination of IL-15 and VC can more significantly enhance the anti-apoptotic ability of the cells.

Fig. 3 is a graph comparing expression levels of the critical killer molecule NKG2D in human Vγ9Vδ2 T cells obtained by using four different culture media to conduct human Vγ9Vδ2 T cell culture proliferation; and IL2, IL2+VC, IL2+IL15, IL2+IL15+VC in FIG. 3 represent the same values as in FIG. 1 and FIG. 2.

According to Fig. 3, it can be seen that the combination of IL-15 and vitamin C can lead to long-term maintenance of a high level of expression by the critical killer molecule NKG2D of human Vγ9Vδ2 T cells, and this critical killer molecule NKG2D can still maintain a high level of expression on the ^21st day. cultivation and after 21 days of cultivation. In other words, the combination formula of IL-15 and VC (namely, the technical solution of the present disclosure) can maintain a high killing ability in human Vγ9Vδ2 T cells for a longer time.

Example 2 Antitumor Effect of Proliferated Vγ9Vδ2 T Cells Humanized Lung Cancer Mouse Model Construction and Experimental Methods

1. Experimental animals

Female Rag2 ^-/- γc ^-/- mice, aged 3-4 weeks, were purchased from Taconic Company, which belonged to the class SPF (from the English. Specified Pathogen Free free from pathogenic flora). For immunodeficient mice, independently ventilated IVCs (individually ventilated cages) were used. 5 mice were kept in each cage. Each isolator had an independent air inlet and outlet of a HEPA (high efficiency particulate air filter) and had a 24-hour temperature and humidity control, and the supply of food and bedding to mice was subjected to vacuum packaging and sterilization by the action of γ- radiation, and sterilized water was used as drinking water for the animal, and the animal contained in this environment was also placed in a sterile environment; and the animals were transported in a biologically safe transport box to ensure that nutrition and transport 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 animal research ethics committee.

2. Method for constructing animal models with tumors

PBMCs (huPBMCs) were isolated by Ficoll fluid density gradient centrifugation for the construction of humanized mouse models (huPBMCs, humanized mice). The type of HLA construct of the PBMCs associated with the humanized mouse did not match the γδ T cells for reinfusion, and was typically HLA-A2+/-. It was convenient to distinguish between γδ T cells for reinfusion and proper humanized mice during subsequent detection.

Then, normal Rag2 ^-/- γc ^-/" mice were selected, at the age of 4-6 weeks, after irradiation with a sublethal dose of 300 cGy, 30×10 ⁶ huPBMC were injected intraperitoneally. After 4 weeks, surviving humanized mice could be used to construct lung cancer models in the next step After splitting A549 cells growing clumped, a cell suspension was prepared and the concentration of A549 cells was adjusted to 1 x 10 ⁷ cells/ml with phosphate buffered saline (PBS).The resulting cell suspension was inoculated into humanized mice by subcutaneous injection into the groin area at a dose of 100 µl/mouse.

3. Application of Proliferated Vγ9Vδ2 T Cells to Process Animal Models

1) After 5-7 days of tumor formation, 5×10 ⁶ γδ T cells cultured with IL-2, IL-2+VC, IL-2+IL-15, IL-2+II-15+VC were injected into humanized mouse model with lung cancer through the mouse tail vein every 3 days, and each group had 5 mice, and the dose is 100 μl/mouse. The PBS injection group was the treatment control group, γδ T cells cultured with IL-2, IL-2+VC, IL-2+IL-15 and IL-2+11-15+VC were obtained by the method and steps described in "in vitro Vγ9Vδ2 T cell proliferation" in Embodiment 1, and IL2, IL2+VC, IL2+IL15 and IL2+IL15+VC represented the same values as in FIG. 1 in embodiment 1.

2) After 3-4 times of treatment, they were observed for more than 60 days. The tumor size of the mice was measured using a caliper twice a week, where the short axis size (A) and the long axis size (B) of the tumor were used to calculate tumor volume according to the formula: V=1/2×A ² ×B.

3) After the end of the reinfusion treatment, peripheral blood was collected to determine the percentage and cellular activity of γδ T cells in mice, and after the end of the reinfusion, peripheral blood was taken every two weeks to determine the percentage and cellular activity of γδ T cells: NKG2D, PDI, CD107a, Fas and FasL.

4) Before killing mice, the colonization situation of γδ T cells in mice was detected. The GFP signal (from the English green fluorescent protein - green fluorescent protein) was detected at the same time, and the number and percentage of A549 tumor cells in the peripheral blood were monitored.

4. The result of the experiment

The result of the tumor treatment experiment is shown in FIG. 4A, 4B, 4C, 5 and 6.

4A is a schematic representation of tumor size after cell treatment in a humanized mouse with lung cancer using human Vγ9Vδ2 T cells obtained by the conventional proliferation method (IL-2).

The conventional proliferation method (the second type of proliferation technology) has always been to add IL2 to the base culture medium for culturing, and to add ZOL to promote proliferation at an early stage of culturing.

Fig. 4B is a schematic representation of tumor size after treatment with humanized mouse lung cancer cells using human Vγ9Vδ2 T cells obtained by the (IL2+IL15+VC) proliferation method of the present disclosure.

Fig. 4C is a schematic representation of the tumor size of a humanized mouse with lung cancer in the absence of treatment.

The humanized lung cancer mice used in the experiments, as illustrated in FIG. 4A, 4B and 4C had the same tumor type and essentially the same tumor volume and tumor growth time as illustrated in FIG. 4A, 4B and 4C was the same.

By comparing Fig. 4A, 4B and 4C, it could be seen that after human Vγ9Vδ2 T cells obtained by the proliferation method of the present disclosure were used to conduct cell treatment in humanized mice with lung cancer, compared to human Vγ9Vδ2 T cells obtained by conventional by proliferation, human Vγ9Vδ2 T cells proliferated by the present disclosure could more effectively suppress the growth of lung cancer cells (tumor size was significantly reduced), and could significantly improve the survival rate of humanized lung cancer mice. At day ⁴² , humanized lung cancer mice that were treated with human Vγ9Vδ2 T cells obtained by conventional proliferation and humanized lung cancer mice that were not treated all died, however, humanized lung cancer mice that were treated with human Vγ9Vδ2 T β-cells obtained by the proliferation method of the present disclosure all survived with a survival rate of 100%, and the tumor completely disappeared in one mouse (shown in the box of Fig. 4B).

Fig. 5 is a schematic representation of tumor volume fluctuations in humanized lung cancer mice under different treatment conditions, and FIG. 5 respectively illustrates changes in tumor volume of humanized lung cancer mice when humanized lung cancer mice were treated with human Vγ9Vδ2 T cells obtained by conventional proliferation method (IL2) and human Vγ9Vδ2 T cells obtained by proliferation method (IL2+IL15+VC). according to the present disclosure, and have not been processed.

It can be seen that, over time, tumor volumes of humanized lung cancer mice that were treated with human Vγ9Vδ2 T cells obtained by the conventional proliferation method (IL2) and were not treated continuously increased, and tumor volumes increased very rapidly at a later stage, however , the tumor volume of humanized mice with lung cancer treated with human Vγ9Vδ2 T cells obtained by the proliferation method (IL2+IL15+VC) of the present disclosure increased very slowly, in other words, compared to human Vγ9Vδ2 T cells obtained by the conventional proliferation method (IL2), human Vγ9Vδ2 T cells obtained by the (IL2+IL15+VC) proliferation method of the present disclosure had a stronger inhibitory effect on tumor growth in vivo and provided smaller tumor volumes.

Fig. 6 is a schematic representation of the variation in survival rates of humanized lung cancer mice under different treatment conditions, and FIG. 6 respectively illustrates fluctuations in survival rates of humanized lung cancer mice treated with human Vγ9Vδ2 T cells obtained by the conventional proliferation method (IL2) and human Vγ9Vδ2 T cells obtained by the proliferation method (IL2+IL15+VC) of the present disclosure, and which were not processed.

It can be seen that over time, the survival rates of humanized lung cancer mice that were treated with human Vγ9Vδ2 T cells obtained by conventional proliferation (IL2) and were not treated decreased very rapidly, however, the survival rate of humanized lung cancer mice treated with human Vγ9Vδ2 T cells obtained by the proliferation method (IL2+IL15+VC) of the present disclosure was always 100%, in other words, human Vγ9Vδ2 T cells obtained by the proliferation method (IL2+IL15+VC) of the present disclosure could significantly improve the survival rate of a humanized mouse with lung cancer.

Example 3

According to Example 1, a second culture medium was provided which contained RPMI-1640 culture medium and interleukin-2, interleukin-15 and vitamin C added to the RPMI-1640 culture medium.

In the above, in the second culture medium, the concentration of interleukin-2 was 300 ng/ml, the concentration of interleukin-15 was 500 ng/ml, and the concentration of vitamin C was 100 mM.

Example 4

According to Example 2, a second culture medium was provided which contained RPMI-1640 culture medium and interleukin-2, interleukin-15 and vitamin C added to the RPMI-1640 culture medium.

In the above, in the second culture medium, the concentration of interleukin-2 was 500 ng/ml, the concentration of interleukin-15 was 700 ng/ml, and the concentration of vitamin C was 300 mM.

Example 5

According to Example 3, a first culture medium was provided which contained a second culture medium as mentioned in Example 1 and zoledronic acid.

In the above, in the first culture medium, the concentration of zoledronic acid was 300 μM.

Example 6

According to Example 4, a first culture medium was provided which contained a second culture medium as mentioned in Example 2 and zoledronic acid.

In the above, in the first culture medium, the concentration of zoledronic acid was 500 μM.

It is difficult to describe all the ranges of numerical values of the process parameters involved in the present disclosure in the above embodiments, but those skilled in the art can fully imagine that any value falling within the above range of values can carry out the present disclosure, and certainly the present the disclosure contains any combination of specific values of several intervals of numerical values. However, to save space, embodiments that provide specific values within one or more ranges of numerical values are omitted, while this should not be considered as an insufficient disclosure of the technical solutions of the present disclosure.

Applicant declares that the above embodiments in the present disclosure are used to describe the specific process equipment and process flow of the present disclosure, but the present disclosure is not limited to the above process equipment and process flow, that is, it does not mean that the present disclosure can only be carried out in depending on the above specific process equipment and process stream. One skilled in the art will appreciate that any improvement to the present disclosure and equivalent substitutions of each product material of the present disclosure and additions, selection of particular methods of auxiliary components, and the like, are all within the scope of this disclosure.

Industrial Applicability

Compared with the conventional proliferation method (second type of proliferation technology), human Vγ9Vδ2 T cells obtained by the proliferation method of the present disclosure have higher proliferation efficiency and higher cell purity, and the proliferation method of the present disclosure apparently shortens the culture period from proliferation of human Vγ9Vδ2 T cells. Compared with human Vγ9Vδ2 T cells obtained by conventional proliferation method (second type of proliferation technology), human Vγ9Vδ2 T cells obtained by the proliferation method of the present disclosure have stronger tumor killing and suppression abilities. Human Vγ9Vδ2 T cells obtained by the proliferation method of the present disclosure have a stronger anti-apoptotic ability and a longer survival period.

Claims (7)

1. Method for T cell proliferation, comprising: stage A, in which the composition containing T cells is cultivated using the first culture medium capable of stimulating T cells, so as to stimulate T cells, where the first culture medium contains a complete culture medium RPMI-1640 with 10% fetal calf serum , interleukin-2 and a phosphonic acid compound, wherein the phosphonic acid compound is zoledronic acid; and stage B, which uses a second culture medium for culturing stimulated T cells, while the specified second culture medium contains a complete culture medium RPMI-1640 with 10% fetal calf serum, interleukin-2, interleukin-15 and vitamin C, where the concentration interleukin-2 is 40-500 ng / ml, the concentration of interleukin-15 is 3.48-700 ng / ml, the concentration of vitamin C is 70 μM - 300 mm, the concentration of phosphonic acid compounds is 50-500 μM, and T cells represent a Vγ9Vδ2 T cell. 2. The method of claim 1 wherein the first culture medium also contains interleukin-15 and vitamin C. 3. The method according to any one of paragraphs. 1, 2, in which the composition containing T cells contains peripheral blood mononuclear cells isolated from the peripheral blood of the human body. 4. The method according to any one of paragraphs. 1-3, in which at stage A the duration of cultivation is from 60 to 100 hours. 5. Culture medium for T cell proliferation containing basic culture medium, interleukin-2, interleukin-15 and vitamin C, where the basic culture medium contains complete culture medium RPMI-1640 with 10% fetal calf serum, the concentration of interleukin-2 is 40 -500 ng/ml, the concentration of interleukin-15 is 3.48-700 ng/ml, the concentration of vitamin C is 70 μM - 300 mm, and the T cells are Vγ9Vδ2 T cells.

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