A method for preparing iNKT cells and applications

Abstract

The invention relates to a method for preparing iNKT cells, the method comprising the steps of dividing human peripheral blood mononuclear cells (PBMCs) into two parts, culturing one part in a culture medium containing one or more iNKT cell agonists, and then mixing the two parts of cells.

Description

A method for preparing iNKT cells and its application Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a method for preparing iNKT cells, a culture medium for culturing iNKT cells, and applications thereof. Background Art

[0002] Invariant natural killer T cells (iNKT cells) are innate-like CD1d-restricted T cells that express the invariant T cell receptor (iTCR). They undergo a unique differentiation program in the thymus, acquiring NK-like properties, including potent cytotoxicity, rapid cytokine secretion, and efficient tissue trafficking. iNKT cells are among the first cell types to respond to pathogenic stimuli, and their cytokine production helps determine the course of the immune response. Due to their anti-tumor effects, iNKT cells have recently emerged as a potential target for immunotherapy.

[0003] However, the content of iNKT cells in the human body is very small. In human peripheral blood, iNKT cells account for about 1% of circulating T cells; in human adipose tissue, iNKT cells can be as high as about 10-25%; in human liver, iNKT cells are very rare. Therefore, it is urgent to develop a method to prepare iNKT cells to quickly obtain a large number of iNKT cells for the treatment of related diseases. Summary of the Invention

[0004] The present application provides a method for preparing iNKT cells, which can significantly increase the expansion multiple of iNKT cells. After about 24 days of culture, the expansion multiple reaches about 80,000 times, and the cell survival rate is above 80%.

[0005] In one aspect, the present application provides a method for preparing iNKT cells, comprising the following steps:

[0006] (1) Obtain human peripheral blood mononuclear cells (PBMCs);

[0007] (2) PBMCs were divided into two parts, the first part was Res cells and the second part was Sti cells;

[0008] (3) culturing Sti cells using a medium containing one or more iNKT cell agonists; and

[0009] (4) Res cells were added to Sti cells for culture.

[0010] In certain embodiments, the method is performed in the absence of serum and exogenous feeder cells.

[0011] In certain embodiments, in the method, the iNKT cell agonist is selected from α-Galcer or 7DW8-5.

[0012] In certain embodiments, in the method, the concentration of the iNKT cell agonist is 100 ng / ml-5000 ng / ml.

[0013] In certain embodiments, in the method, the iNKT cell agonist is α-Galcer.

[0014] In certain embodiments, in the method, the concentration of α-Galcer is 100 ng / ml-5000 ng / ml.

[0015] In certain embodiments, the culturing time of step (3) is 0.5-3 h.

[0016] In certain embodiments, the culturing time of step (4) is 10-40 days.

[0017] In certain embodiments, step (4) further comprises culturing the mixed Res cells and Sti cells in a culture medium containing one or more factors selected from the following group: IL-2, IL-4, IL-7, IL-15, IL-21, TNF-α, IFN-γ and IGF.

[0018] In certain embodiments, the step (4) further comprises culturing the mixed Res cells and Sti cells in a culture medium containing IGF, IL-7 and IL-15.

[0019] In certain embodiments, the step (4) further comprises culturing the mixed Res cells and Sti cells in a culture medium containing IL-7 and IL-15.

[0020] In certain embodiments, the step (4) further comprises culturing the mixed Res cells and Sti cells in a culture medium containing IL-15.

[0021] In certain embodiments, in the method, the concentration of the factor is 5 ng / ml-100 ng / ml.

[0022] In certain embodiments, in the method, the concentration of the factor IGF is 10 ng / ml-1000 ng / ml.

[0023] In certain embodiments, in the method, the concentration of the factor IL-7 is 5 ng / ml-100 ng / ml.

[0024] In certain embodiments, in the method, the concentration of the factor IL-15 is 5 ng / ml-100 ng / ml.

[0025] In certain embodiments, in the method, the culture medium further comprises Serum replacement.

[0026] In certain embodiments, in the method, the culture medium comprises a basal medium selected from one or more of the following groups: TexMACS medium, X-VIVO media, CTS OpTmizer T Cell Expansion SFM, CTS AIM V SFM, SCGM medium, OptiVitro T Cell Serum-free Medium, OptiVitro UniEx T Cell Serum-free Medium, ImmunoCult TM -XF T Cell Expansion Medium, PRIME-XV medium.

[0027] In certain embodiments, in the method, the basal culture medium is TexMACS medium.

[0028] In certain embodiments, in the method, the ratio of Res cells to Sti cells is 1:0.01-1:100.

[0029] In certain embodiments, the method is performed under culture conditions of 37°C ± 1°C.

[0030] In certain embodiments, the method is performed under culture conditions of 5±0.5% CO2.

[0031] On the other hand, the present application provides a culture medium, which is a single-component culture medium comprising components (1), (2) and (3);

[0032] The components are:

[0033] Components (1) basal culture medium;

[0034] Component (2) one or more iNKT cell agonists; and

[0035] Component (3) is one or more factors selected from the group consisting of IL-2, IL-4, IL-7, IL-15, IL-21, TNF-α, IFN-γ and IGF.

[0036] On the other hand, the present application provides a culture medium, which is a combination of multiple step-by-step culture media, wherein the step-by-step culture media comprises the following culture media and / or combinations thereof:

[0037] (a): components (1) and (2),

[0038] (b): components (1) and (3), and

[0039] (c): components (1), (2) and (3);

[0040] The components are:

[0041] Components (1) basal culture medium;

[0042] Component (2) one or more iNKT cell agonists; and

[0043] Component (3) is one or more factors selected from the group consisting of IL-2, IL-4, IL-7, IL-15, IL-21, TNF-α, IFN-γ and IGF.

[0044] In certain embodiments, the basal medium in the culture medium is selected from one or more of the following groups: TexMACS medium, X-VIVO media, CTS OpTmizer T Cell Expansion SFM, CTS AIM V SFM, SCGM medium, OptiVitro T Cell Serum-free Medium, OptiVitro UniEx T Cell Serum-free Medium, ImmunoCult TM -XF T Cell Expansion Medium, PRIME-XV medium.

[0045] In certain embodiments, the basal medium in the culture medium is TexMACS medium.

[0046] In certain embodiments, in the culture medium, the iNKT cell agonist is selected from α-Galcer or 7DW8-5.

[0047] In certain embodiments, in the culture medium, the iNKT cell agonist is α-Galcer.

[0048] In certain embodiments, the concentration of α-Galcer in the culture medium is 100 ng / ml-5000 ng / ml.

[0049] In certain embodiments, the concentration of the factor in the culture medium is 5 ng / ml-100 ng / ml.

[0050] In certain embodiments, in the culture medium, the factors are IGF, IL-7 and IL-15.

[0051] In certain embodiments, in the culture medium, the factors are IL-7 and IL-15.

[0052] In certain embodiments, in the culture medium, the factor is IL-15.

[0053] In certain embodiments, the concentration of IGF in the culture medium is 10 ng / ml-1000 ng / ml.

[0054] In certain embodiments, the concentration of IL-7 in the culture medium is 5 ng / ml-100 ng / ml.

[0055] In certain embodiments, the concentration of IL-15 in the culture medium is 5 ng / ml-100 ng / ml.

[0056] In certain embodiments, the culture medium may further comprise Serum replacement.

[0057] In certain embodiments, the culture medium does not comprise serum.

[0058] In another aspect, the present application provides a method for expanding and culturing iNKT cells, comprising using the method described herein and / or using the culture medium described herein.

[0059] In another aspect, the present application provides a composition comprising human peripheral blood mononuclear cells and the culture medium described herein.

[0060] In another aspect, the present application provides a composition comprising iNKT cells and the culture medium described herein.

[0061] In another aspect, the present application provides a composition comprising human peripheral blood mononuclear cells and iNKT cells, and the culture medium described herein.

[0062] On the other hand, the present application provides the use of the composition in preparing a medicament for preventing, alleviating and / or treating a disease and / or condition.

[0063] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0065] FIG1 shows the initial iNKT cell ratio and the iNKT cell ratio after culture in PBMCs described in the present application.

[0066] FIG2A shows the expansion fold of iNKT cells at different culture times after using the current method and the control method described in this application.

[0067] FIG2B shows the survival rate of iNKT cells at different culture times after using the current method and the control method described in this application.

[0068] FIG2C shows the proportion of iNKT cells at different culture times after using the current method and the control method described in this application.

[0069] FIG3A shows the proportion of iNKT cells after cells were cultured with the added cytokines IL-7 and IL-15 as described in the present application.

[0070] FIG3B shows the proportion of iNKT cells after cells were cultured with the added cytokines IL-7, IL-15, and IGF as described in the present application.

[0071] FIG4 shows the evaluation of the killing effect of iNKT cells described in the present application.

[0072] FIG5A and FIG5B show the detection results of the proportions of CD4 and CD8 cell subsets in iNKT cells cultured with different factors in the present application.

[0073] FIG5C shows the evaluation of the killing effect of iNKT cells cultured with different factors in the present application. DETAILED DESCRIPTION

[0074] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0075] Definition of terms

[0076] In this application, the term "iNKT cells" generally refers to a special T cell subset involved in innate immunity, which has a constant TCRα receptor. iNKT cells can recognize antigens in the presence of CD1d molecules, can specifically recognize α-GalCer, and iNKT cells can be labeled with CD1d / α-GalCer tetramers. iNKT cells can differentiate into NKT1, NKT2 and NKT17 subsets, which are similar to Th1, Th2 and Th17 subsets. iNKT cells contain a large amount of cytotoxic cytokines (such as IL-4, IL-10, IFN-γ, etc.), which are much higher than NK cells and T cells. When iNKT cells are stimulated, they can quickly release cytokines to kill tumor cells.

[0077] In this application, the term "cell agonist" generally refers to a natural or artificial molecule that can bind to a receptor on a cell to produce a reaction. Natural cell agonists can be hormones or neurotransmitters. Artificial cell agonists can be drugs similar to natural agonists. In this application, an "iNKT cell agonist" can bind to a receptor on a cell. For example, the receptor can be a receptor on an iNKT cell, such as CD1d. Use of an iNKT cell agonist can promote the proliferation / differentiation / activation of iNKT cells. For example, an iNKT cell agonist can be α-GalCer or 7DW8-5.

[0078] In this application, the term "human peripheral blood mononuclear cells" or "PBMCs" generally refers to cells in peripheral blood that have a single nucleus. For example, any blood cell with a round nucleus (i.e., lymphocytes, monocytes, or macrophages). These blood cells are key components of the immune system that fights infection and adapts to invaders. The lymphocyte population consists of CD4+ and CD8+ T cells, B cells and natural killer cells, CD14+ monocytes, and basophils / neutrophils / eosinophils / dendritic cells. Typically, FICOLL is used. TM (a hydrophilic polysaccharide that separates blood), these cells are separated from whole blood, where monocytes and lymphocytes form the buffy coat beneath the plasma layer.

[0079] As used herein, the term "diagnosis" generally refers to the detection of a disease or condition, or the determination of the state or extent of a disease or condition, and also includes the detection of the cause of a disease or condition, the determination of the therapeutic effect of a drug therapy, or the prediction of a response pattern to a drug therapy or a xenobiotic. The diagnostic methods described herein can be used independently or in combination with other diagnostic and / or staging methods known in the medical field for a particular disease or condition.

[0080] As used herein, the term "prevent" generally refers to methods used to prevent, inhibit, or reduce the likelihood of the occurrence or recurrence of a disease or condition (e.g., cancer). It also refers to delaying the onset or recurrence of a disease or condition or delaying the onset or recurrence of symptoms of a disease or condition. "Prevent" and similar terms also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition prior to its onset or recurrence.

[0081] As used herein, the term "treat" generally refers to any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated (e.g., cancer). Treatment may optionally involve a reduction or amelioration of the symptoms of the disease or condition, or a delay in the progression of the disease or condition. "Treatment" does not necessarily indicate complete eradication or cure of the disease or condition or its associated symptoms.

[0082] In this application, the term "proliferation" generally refers to the generation of multiple individual cells by division of a starting cell. The multiple individual cells can be of the same type or of different types. The starting cells used for proliferation need not be the same as the cells produced by proliferation. For example, the proliferated cells can be generated from the growth and differentiation of a starting cell population.

[0083] In this application, the term "differentiation" generally refers to the process by which a non-specific or less specific cell acquires specific cell characteristics. A differentiated or differentiation-induced cell is a cell that occupies a more specific position in a cell lineage.

[0084] In this application, the term "composition" generally refers to a product comprising a specified amount of a specified ingredient, as well as any product produced directly or indirectly by a combination of the specified amounts of the specified ingredients. In this application, the composition may also include other inactive ingredients, for example, carriers, excipients, adjuvants, stabilizers, etc.

[0085] In this application, the term "ex vivo" generally refers to operations performed on cells, tissues and / or organs that have been removed from an organism. In some embodiments, the cells, tissues and / or organs can be returned to the organism or introduced into another organism by certain methods.

[0086] In this application, the term "in vitro" generally refers to removing or releasing a part of an organism from the organism.

[0087] In this application, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "to be", "to be composed of..."

[0088] In this application, the term "and / or" should be understood to mean any one, two, more than one or any combination of the alternatives.

[0089] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0090] Detailed Description of the Invention

[0091] iNKT cell preparation method

[0092] In one aspect, the present application provides a method for preparing iNKT cells, comprising the following steps:

[0093] (1) Obtain human peripheral blood mononuclear cells (PBMCs);

[0094] (2) PBMCs were divided into two parts, the first part was Res cells and the second part was Sti cells;

[0095] (3) culturing Sti cells using a medium containing one or more iNKT cell agonists; and

[0096] (4) Res cells were added to Sti cells for culture.

[0097] In the present application, the method can contact and culture PBMCs with an iNKT cell agonist to promote the preparation / expansion / differentiation / culture of iNKT cells.

[0098] In the present application, the iNKT cell agonist can bind to receptors on cells to generate a response.

[0099] For example, the preparation / expansion / differentiation / culture of iNKTs can be promoted.

[0100] In the present application, the iNKT cell agonist may be natural or artificial. Natural agonists may be hormones or neurotransmitters, and artificial agonists may be drugs similar to natural agonists.

[0101] For example, iNKT cell agonists can be full agonists, also known as direct-binding agonist drugs, which bind directly to the receptor at the same binding site where the natural ligand binds. For example, iNKT cell agonists can be partial agonists, also known as indirect-binding agonist drugs, which promote the binding of the natural ligand to the receptor site. These drugs can produce a delayed response; in some cases, partial agonists can act as antagonists by competing for the same receptor as the full agonist.

[0102] For example, the iNKT cell agonist may bind to CD1d, for example, the iNKT cell agonist may be α-GalCer, for example, the iNKT cell agonist may be 7DW8-5.

[0103] In the present application, the concentration of the iNKT cell agonist may be 100 ng / ml-5000 ng / ml.

[0104] For example, it can be 100 ng / mL, about 150 ng / mL, about 200 ng / mL, about 250 ng / mL, about 300 ng / mL, about 350 ng / mL, about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, about 550 ng / mL, about 600 ng / mL, about 650 ng / mL, about 700 ng / mL, about 750 ng / mL, about 800 ng / mL, about 850 ng / mL, about 900 ng / mL, about 950 ng / mL, about 1000 ng / mL.

[0105] In the present application, the Sti cells may be cultured in the iNKT cell agonist for 0.5-3 h.

[0106] For example, it can be about 0.5h, about 0.55h, about 0.6h, about 0.65h, about 0.7h, about 0.75h, about 0.8h, about 0.85h, about 0.9h, about 0.95h, about 1h, about 1.1h, about 1.15, about 1.2h, about 1.25h, about 1.3h, about 1.35h, about 1.4h, about 1.45h, about 1.5h, about 1.55, about 1.6h, about 1.65h, about 1.7h , about 1.75h, about 1.8h, about 1.85h, about 1.9h, about 1.95h, about 2h, about 2.1h, about 2.15, about 2.2h, about 2.25h, about 2.3h, about 2.35h, about 2.4h, about 2.45habout 2.5h, about 2.55, about 2.6h, about 2.65h, about 2.7h, about 2.75h, about 2.8h, about 2.85h, about 2.9h, about 2.95h, about 3h.

[0107] In the present application, the time for adding Res cells to Sti cells for culture may be 10-40 days.

[0108] For example, it can be about 5-10 days, about 7-10 days, about 7-15 days, about 7-20 days, about 10-21 days, about 10-30 days, about 15-20 days, about 20-25 days, about 20-30 days, about 25-30 days, about 30-40 days.

[0109] In the present application, when Res cells are added to Sti cells for culture, one or more substances may be added to the culture system, including but not limited to: nutrients / extracts, growth factors, hormones, cytokines and / or culture medium additives.

[0110] For example, one or more of the following substances may be added, including but not limited to serum replacement, glutamine, NEAA (non-essential amino acids), ascorbic acid, epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), bone morphogenetic protein (BMP4), vascular endothelial growth factor (VEGF), transferrin, insulin, selenium, various interleukins (e.g., IL-1 to IL-18), various colony stimulating factors (e.g., granulocyte / macrophage colony stimulating factor (GM-CSF)), various interferons (e.g., IFN-γ), stem cell factor (SCF), thrombopoietin (TPO), erythropoietin (EPO), N2 supplement, B27 supplement, Fms-related tyrosine kinase 3 ligand (FLt3). The additives are not limited to their sources and can be commercially available, natural, or recombinant.

[0111] For example, IL-2, IL-4, IL-7, IL-15, IL-21, TNF-α, IFN-γ, and IGF can be added. For example, IGF, IL-7, and IL-15 can be added. For example, Serum replacement can be further added.

[0112] In the present application, the concentration of the added substance(s) may be 5 ng / mL-100 ng / mL.

[0113] For example, it can be about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL.

[0114] For example, the concentration of IL-2 can be about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL.

[0115] For example, the concentration of IL-4 can be about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL.

[0116] For example, the concentration of IL-7 can be about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL.

[0117] For example, the concentration of IL-15 can be about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL.

[0118] For example, the concentration of IL-21 can be about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL.

[0119] For example, the concentration of TNF-α can be about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL.

[0120] For example, the concentration of IFN-γ can be about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL.

[0121] For example, the concentration of IGF can be about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL.

[0122] In the present application, when culturing cells using the method, a basal culture medium may be added. The basal culture medium may comprise any culture medium known in the art.

[0123] For example, the basal culture medium may comprise one or more of the following: TexMACS medium, X-VIVO media, CTS OpTmizer T Cell Expansion SFM, CTS AIM V SFM, SCGM medium, OptiVitro T Cell Serum-free Medium, OptiVitro UniEx T Cell Serum-free Medium, ImmunoCult TM -XF T Cell Expansion Medium, PRIME-XV medium. For example, the basal culture medium may be TexMACS medium.

[0124] In the present application, the Res cells and Sti cells can be mixed in a suitable ratio to achieve the purpose of preparing iNKT cells.

[0125] For example, the ratio of Res cells to Sti cells is about 1:0.01-1:100, for example, about 1:0.5, about 1:1.25, about 1:1.5, about 1:1.75, about 1:2, about 1:2.25, about 1:2.5, about 1:2.75, about 1:3, about 1:5, about 1:10, about 1:15, or about 1:30.

[0126] In the present application, the method is carried out under culture conditions of 37°C ± 1°C.

[0127] For example, about 34.5°C, about 35°C, about 35.5°C, about 36.°C, about 36.5°C, about 37°C, about 37.5°C, about 38°C, about 38.5°C, about 39°C, about 39.5°C.

[0128] In the present application, the method can be performed under culture conditions of 5±0.5% CO 2 .

[0129] For example, about 3% CO2, about 3.5% CO2, about 4% CO2, about 4.5% CO2, about 5% CO2, about 5.5% CO2, about 6% CO2, about 6.5% CO2, about 7% CO, about 7.5% CO2.

[0130] In the present application, the method can be performed under serum-free conditions.

[0131] In the present application, the method can be performed in the presence of serum.

[0132] In the present application, the method can be performed in the absence of exogenous trophoblast cells.

[0133] In the present application, the method can be performed with or without exogenous trophoblast cells.

[0134] culture medium

[0135] On the other hand, the present application provides a single-component culture medium for culturing cells, comprising components (1), (2) and (3); wherein the components are:

[0136] Components (1) basal culture medium;

[0137] Component (2) one or more iNKT cell agonists; and

[0138] Component (3) is one or more factors selected from the group consisting of IL-2, IL-4, IL-7, IL-15, IL-21, TNF-α, IFN-γ and IGF.

[0139] On the other hand, the present application provides a culture medium that is a combination of multiple step-by-step culture media, wherein the step-by-step culture media comprises the following culture media and / or combinations thereof:

[0140] (a): components (1) and (2),

[0141] (b): components (1) and (3), and

[0142] (c): components (1), (2) and (3);

[0143] The components are:

[0144] Components (1) basal culture medium;

[0145] Component (2) one or more iNKT cell agonists; and

[0146] Component (3) is one or more factors selected from the group consisting of IL-2, IL-4, IL-7, IL-15, IL-21, TNF-α, IFN-γ and IGF.

[0147] For example, the factors may be IGF, IL-7, and IL-15. For example, the factors may be IL-7 and IL-15. For example, the factors may be IL-15. For example, Serum replacement may be further added to the culture medium.

[0148] In the present application, the culture medium can be used alone or in combination with other culture media, and the ratio of each culture medium can be adjusted arbitrarily during use.

[0149] For example, the single-component culture medium can be used with other culture media. For example, multiple step-by-step culture media can be used alone, one or more of the multiple step-by-step culture media can be used in combination, or one or more of the multiple step-by-step culture media can be used in combination with other culture media.

[0150] In the present application, the types and concentrations of the iNKT cell agonist, the one or more factors, the iNKT cell agonist, and the basal culture medium in the culture medium can be adaptively adjusted as described in the iNKT cell preparation method of the present application to achieve the purpose of preparing / expanding / differentiating / culturing iNKT cells.

[0151] In the present application, one or more substances may be added to the culture medium, including but not limited to: nutrients / extracts, growth factors, hormones, cytokines and / or culture medium additives.

[0152] For example, one or more of the following substances may be added, including but not limited to serum replacement, glutamine, NEAA (non-essential amino acids), ascorbic acid, epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), bone morphogenetic protein (BMP4), vascular endothelial growth factor (VEGF), transferrin, insulin, selenium, various interleukins (e.g., IL-1 to IL-18), various colony stimulating factors (e.g., granulocyte / macrophage colony stimulating factor (GM-CSF)), various interferons (e.g., IFN-γ), stem cell factor (SCF), thrombopoietin (TPO), erythropoietin (EPO), N2 supplement, B27 supplement, Fms-related tyrosine kinase 3 ligand (FLt3). The additives are not limited to their sources and can be commercially available, natural, or recombinant.

[0153] In the present application, the culture medium may be further supplemented with serum.

[0154] In the present application, the types and sources of cells that can be cultured in the culture medium are not limited.

[0155] For example, the cells may be of natural origin or modified. For example, the cells may be of mammalian origin or non-mammalian origin. For example, the cells may be a single component or a mixture of multiple cells. For example, the cells may be PBMCs, iNKT cells, or a mixture of PBMCs and iNKT cells.

[0156] Composition

[0157] In another aspect, the present application provides a composition comprising PBMC and the culture medium described herein.

[0158] In another aspect, the present application provides a composition comprising iNKT cells and the culture medium described herein.

[0159] In another aspect, the present application provides a composition comprising PBMC and the culture medium of iNKT cells described herein.

[0160] In the present application, the composition can be used to prepare / expand / differentiate / cultivate iNKT cells.

[0161] use

[0162] In another aspect, the present application provides use of the composition in preparing a medicament for preventing, alleviating and / or treating a disease and / or condition.

[0163] On the other hand, the present application also provides a culture platform for obtaining iNKT cells, which comprises the method and the culture medium.

[0164] On the other hand, the present application also provides a method for preventing and / or treating a disease, comprising administering iNKT cells to a subject in need thereof, wherein the iNKT cells are obtained by using the method, the culture medium, the composition, and the culture platform.

[0165] For example, the iNKT cells of described administration can be prepared pharmaceutically according to any conventional method.For example, carrier, excipient or diluent can be used to mix or dilute active ingredient.The example of suitable carrier, excipient or diluent is lactose, dextrose, sucrose, sorbitol, mannitol, glycine, polyethylene glycol, starch, gum arabic, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.Described preparation can comprise filler, anti-agglomeration agent, lubricant, wetting agent, flavoring agent, emulsifier, preservative etc. additionally.Prepare the composition of the present invention to provide quick, continuous or delayed release active ingredient after giving patient by using any method known in the art.

[0166] The cells herein can be administered by injection (e.g., intramuscular, intravenous, intraperitoneal, subcutaneous), or by other methods such as infusion to ensure that they enter the bloodstream in an effective form. The cells can also be administered via intratumoral, peritumoral, intralesional, or perilesional routes to exert local and systemic therapeutic effects. For example, they can be administered topically or intravenously.

[0167] In the present application, the administration dosage of the cells may be a single dose or multiple doses. For example, the actual administration amount of the cells may be determined based on a variety of relevant factors, such as the type of disease; the route of administration; the age, sex, and / or weight of the patient; and the severity of the patient's symptoms.

[0168] Without intending to be bound by any theory, the following examples are merely intended to illustrate the preparation method and application of the present application, and are not intended to limit the scope of the present invention.

[0169] Experimental Materials

[0170] Example 1 Preparation of iNKT cells from human peripheral blood mononuclear cells (PBMC)

[0171] 1.1 Obtaining peripheral blood mononuclear cells (PBMCs)

[0172] For frozen PBMC samples, thaw the cells in a 37°C water bath before use.

[0173] For fresh blood samples, PBMCs were isolated from the blood samples by Ficoll density gradient centrifugation as follows:

[0174] 1) Measure the blood sample volume and dilute it with 2-4 times the volume of isotonic buffer (PBS / normal saline);

[0175] 2) Prepare centrifuge tubes and add Ficoll equal to the volume of the diluted blood sample to each tube;

[0176] 3) Gently layer equal volumes of diluted single-collected blood onto Ficoll in each tube and centrifuge at 400g for 30 minutes at room temperature using a 1:1 density gradient.

[0177] 4) After centrifugation, collect the buffy coat cells into a centrifuge tube, add buffer, and centrifuge at 500g for 10 minutes at room temperature (9°C to 9°C) to collect the cell pellet.

[0178] 5) Resuspend the cell pellet in buffer, centrifuge at 500g for 10 min at room temperature, and set aside.

[0179] 1.2 PBMC Enumeration and Detection

[0180] 1) Count the cells in the PBMC sample using the trypan blue counting method;

[0181] 2) Flow cytometry was used to detect the proportion of iNKT cells in PBMCs.

[0182] 1.3 Preparation of RES cells

[0183] 1) Based on the PBMC count results, separate two-thirds of the total PBMCs into a centrifuge tube and centrifuge at 350g for 10 min to remove the supernatant;

[0184] 2) Resuspend the cell pellet with TexMACS medium, transfer the cells into a suitable culture flask and culture at 37°C in a 5% CO2 environment until ready for use.

[0185] 1.4 Preparation of STI cells

[0186] 1) Prepare TexMACS medium containing α-Galcer;

[0187] 2) Based on the PBMC count results, separate one-third of the total PBMCs into a centrifuge tube and centrifuge at 350g for 10 minutes to remove the supernatant;

[0188] 3) Resuspend the cell pellet in the aforementioned TexMACS medium containing α-Galcer and transfer to a suitable culture flask and culture at 37°C in a 5% CO2 environment for 0.5-3 hours;

[0189] 4) After the culture is completed, the cells are collected into a centrifuge tube and centrifuged at 350g for 10 minutes to collect the cell pellet;

[0190] 5) Resuspend the cell pellet in TexMACS medium and wash the cells twice by centrifugation at 350g for 10 min.

[0191] 6) Resuspend the cell pellet in TexMACS medium and set aside.

[0192] 1.5 Mixed culture of RES cells and STI cells

[0193] 1) Mix the prepared RES cells and STI cells and transfer them to a suitable culture container for static culture;

[0194] 2) adding IGF to the culture system to a final concentration of about 100 ng / ml; adding IL-7 and IL-15 to the culture system to a final concentration of about 5 ng / ml;

[0195] On the third and fifth days of culture, serum replacement was added at 5% of the total volume.

[0196] 1.6 Cell centrifugation and medium replacement

[0197] 1) Prepare TexMACS complete medium containing 5% serum replacement, 10 ng / ml IL-7, and 10 ng / ml IL-15;

[0198] 2) On the seventh day of culture, collect all the cell suspension into a centrifuge tube and centrifuge at 350g for 10 min to collect the cell pellet;

[0199] 3) Resuspend the cell pellet in the above complete medium and culture at 37°C in a 5% CO2 environment.

[0200] 1.7 Cell subculture

[0201] 1) Starting from the seventh day, cells were sampled and counted every 2-3 days, and the proportion of iNKT cells was detected by flow cytometry;

[0202] 2) Starting from the seventh day, the cell density was adjusted to 1E6 / ml-2E6 / ml every 2-3 days using TexMACS complete medium containing 5% serum replacement, 10 ng / ml IL-7, and 10 ng / ml IL-15 for subculture.

[0203] The experimental results are shown in Figure 1:

[0204] The initial iNKT cell ratio in PBMC was 0.43%. After 13 days of culture using the method of Example 1, the iNKT cell ratio was 77.9%.

[0205] The above results indicate that the method described in this application can promote the preparation / expansion / differentiation / culture of iNKT cells.

[0206] Example 2 Evaluation of the effects of different iNKT cell culture methods

[0207] 2.1 Evaluation of the effects of different culture methods for preparing iNKT cells (I)

[0208] Current method: the method described in Example 1

[0209] Control method:

[0210] 1) Starting directly with PBMC (without dividing the PBMC into two parts; excluding steps 1.3, 1.4, and 1.5 in Example 1), culturing in a medium containing α-Galcer, IL-7, and IL-15;

[0211] 2) Other culture steps are the same as in Example 1.

[0212] The experimental results are shown in Figures 2A-2C:

[0213] In Figure 2A, in the current method (the method in Example 1), the expansion fold of iNKT cells increased rapidly about 16-24 days after culture, reaching about 80,000-fold around 24 days after culture. In the control method (the method in Example 2.1), the expansion fold remained essentially unchanged around 24 days after culture.

[0214] In Figure 2B, in the current method (the method in Example 1), the survival rate of iNKT cells remained above 80% around day 12 after culture, which was significantly higher than that of the control method (the method in Example 2.1). The survival rate of iNKT cells was still maintained above 80% around day 23 of culture.

[0215] In Figure 2C, in the current method (the method in Example 1), the proportion of iNKT cells continued to increase. After 12 days of culture, the proportion of iNKT cells was above 50%, and even reached 70% after 16-24 days of culture, which was significantly higher than the 20% of the control method (the method in Example 2.1).

[0216] These results demonstrate that the methods described herein can promote the preparation, expansion, differentiation, and cultivation of iNKT cells. Approximately 24 days after culture, the methods described herein can significantly increase the expansion multiple, survival rate, and cell ratio of iNKT cells, resulting in the production of large numbers of iNKT cells in a short period of time.

[0217] 2.2 Evaluation of the effects of different culture methods for preparing iNKT cells (Part 2)

[0218] Other culture conditions were the same, except that different cytokines were added to the culture system in step 1.5 of Example 1. The remaining steps were as described in Example 1. Ten days after culture, the proportion of iNKT cells was detected.

[0219] The experimental results are shown in Figures 3A-3B:

[0220] In FIG3A , the added cytokines were IL-7 and IL15, and the proportion of iNKT cells was 27.8%.

[0221] In FIG3B , the added cytokines were IL-7, IL15, and IGF, and the proportion of iNKT cells was 40.8%.

[0222] The above results indicate that the method described in Example 1 of the present application can promote the preparation / expansion / differentiation / culture of iNKT cells.

[0223] Example 3 Verification of iNKT cell killing function

[0224] Prepare target cells and iNKT cells, and fluorescently label target cells using Cell Trace dye. Count labeled target cells and iNKT cells, mix them in a 1:1 ratio, and culture in a 37°C 5% CO2 incubator. After culturing for 4 hours, collect all cells, wash them by centrifugation, and then stain them with Annexin V. After staining, use flow cytometry to measure the proportion of Annexin V-positive cells in the Cell Trace fluorescent signal-positive cell population, which is the killing rate of iNKT against target cells.

[0225] The experimental results are shown in Figure 4:

[0226] iNKT cells cultured using the method described in this application can effectively kill target cells. After being co-cultured with target cells for 4 hours, they can kill about 73.3% of target cells.

[0227] The above results indicate that the iNKT cells prepared by the method described in this application have good functions and can be used for treatment.

[0228] Example 4 Evaluation of CD4 and CD8 cell subsets of iNKT cells cultured with different cytokines and cell killing effects

[0229] 1) Other culture conditions were the same as in Example 1. Different cytokines were added to the culture system in steps 1.5, 1.6, and 1.7, and the proportions of CD4+, CD8+, CD4-CD8-, and CD4+CD8+ cell subsets in iNKT cells were detected at different time points of culture.

[0230] The experimental results are shown in Figures 5A and 5B:

[0231] On day 20, with the addition of IL15 or IL7+IL15 cytokine combination, the CD4+ subpopulation accounted for about 20%, and the corresponding other non-CD4+ subpopulations (CD8+, CD4-CD8-, CD4+CD8+ cell subpopulations) accounted for about 80%; with the addition of IL2 or IL2+IL15 factor combination, the CD4+ subpopulation accounted for about 40%, and the corresponding other non-CD4+ subpopulations (CD8+, CD4-CD8-, CD4+CD8+ cell subpopulations) accounted for about 60%.

[0232] 2) Using the iNKT cell killing assay method described in Example 3, iNKT cells cultured with different cytokines were co-cultured with target cells at an effector-target ratio of 1:5 for 4 hours, and the killing rate of target cells was then detected.

[0233] The experimental results are shown in Figure 5C:

[0234] The target cell killing rates of iNKT cells cultured with IL15 or IL-7+IL15 cytokine combination were 62.2% and 59.6%, respectively, which were higher than the target cell killing rates of iNKT cells cultured with IL2 or IL2+IL15 factor combination of 32.2% and 46.7%.

Claims

1. A method for preparing iNKT cells, the method comprising the following steps: (1) Obtaining human peripheral blood mononuclear cells (PBMC); (2) Dividing the PBMC into two parts, wherein the first part is Res cells and the second part is Sti cells; (3) Culturing the Sti cells using a medium containing one or more iNKT cell agonists; and (4) Adding the Res cells to the Sti cells for culturing.

2. The method according to claim 1, wherein the method is carried out under conditions of serum-free and exogenous trophoblast cells.

3. The method according to any one of claims 1-2, wherein the iNKT cell agonist is selected from α-Galcer or 7DW8-5.

4. The method according to any one of claims 1-3, wherein the concentration of the iNKT cell agonist is 100 ng / ml - 5000 ng / ml.

5. The method according to any one of claims 1-4, wherein the iNKT cell agonist is α-Galcer.

6. The method according to any one of claims 1-5, wherein the concentration of the α-Galcer is 100 ng / ml - 5000 ng / ml.

7. The method according to any one of claims 1-6, wherein the culture time of step (3) is 0.5 - 3 h.

8. The method according to any one of claims 1-7, wherein the culture time of step (4) is 10 - 40 days.

9. The method according to any one of claims 1-8, wherein step (4) further comprises culturing the mixed Res cells and Sti cells in a medium containing one or more factors selected from the group consisting of: IL-2, IL-4, IL-7, IL-15, IL-21, TNF-α, IFN-γ and IGF.

10. The method according to any one of claims 1-9, wherein step (4) further comprises culturing the mixed Res cells and Sti cells in a medium containing IGF, IL-7 and IL-15.

11. The method according to any one of claims 1-9, wherein step (4) further comprises culturing the mixed Res cells and Sti cells in a medium containing IL-7 and IL-15.

12. The method according to any one of claims 1-9, wherein step (4) further comprises culturing the mixed Res cells and Sti cells in a medium containing IL-15.

13. The method according to any one of claims 9-12, wherein the concentration of the factor is 5 ng / ml - 100 ng / ml.

14. The method according to any one of claims 9-10, wherein the concentration of the factor IGF is 10 ng / ml - 1000 ng / ml.

15. The method according to any one of claims 9-11, wherein the concentration of the factor IL-7 is 5 ng / ml - 100 ng / ml.

16. The method according to any one of claims 9 - 12, wherein the concentration of the factor IL-15 is 5 ng / ml - 100 ng / ml.

17. The method according to any one of claims 9 - 16, wherein the culture medium further comprises Serum replacement.

18. The method according to any one of claims 9-17, wherein the culture medium comprises a basal medium selected from one or more of the following groups: TexMACS medium, X-VIVO media, CTS OpTmizer T Cell Expansion SFM, CTS AIM V SFM, SCGM medium, OptiVitro T Cell Serum-free Medium, OptiVitro UniEx T Cell Serum-free Medium, ImmunoCult TM -XF T Cell Expansion Medium, PRIME-XV medium.

19. The method according to claim 18, wherein the basal medium is TexMACS medium.

20. The method according to any one of claims 1 - 19, wherein the ratio of the Res cells to the Sti cells is 1:0.01 - 1:

100.

21. The method according to any one of claims 1 - 20, wherein the method is carried out under culture conditions of 37°C ± 1°C.

22. The method according to any one of claims 1 - 21, wherein the method is carried out under culture conditions of 5 ± 0.5% CO2.

23. A culture medium, wherein the culture medium is a single-component culture medium, comprising components (1), (2), and (3); wherein the components are: Component (1) basal medium; Component (2) one or more iNKT cell agonists; and Component (3) one or more factors selected from the group consisting of IL-2, IL-4, IL-7, IL-15, IL-21, TNF-α, IFN-γ, and IGF.

24. A culture medium, wherein the culture medium is a combination of multiple stepwise culture media, and the stepwise culture media comprise the following culture media and / or their combinations: (a): Components (1) and (2), (b): Components (1) and (3), and (c): Components (1), (2), and (3); wherein the components are: Component (1) basal medium; Component (2) one or more iNKT cell agonists; and Component (3) one or more factors selected from the group consisting of IL-2, IL-4, IL-7, IL-15, IL-21, TNF-α, IFN-γ, and IGF.

25. The culture medium according to any one of claims 23-24, wherein the basal medium is selected from one or more of the following groups: TexMACS medium, X-VIVO media, CTS OpTmizer T Cell Expansion SFM, CTS AIM V SFM, SCGM medium, OptiVitro T Cell Serum-free Medium, OptiVitro UniEx T Cell Serum-free Medium, ImmunoCult TM -XF T Cell Expansion Medium, PRIME-XV medium.

26. The culture medium according to any one of claims 23 - 25, wherein the basal medium is TexMACS medium.

27. The culture medium according to any one of claims 23 - 26, wherein the iNKT cell agonist is selected from α-Galcer or 7DW8-5.

28. The culture medium according to any one of claims 23 - 27, wherein the iNKT cell agonist is α-Galcer.

29. The culture medium according to any one of claims 27 - 28, wherein the concentration of the α-Galcer is 100 ng / ml - 5000 ng / ml.

30. The culture medium according to any one of claims 23 - 29, wherein the concentration of the factor is 5 ng / ml - 100 ng / ml.

31. The culture medium according to any one of claims 23 - 30, wherein the factors are IGF, IL-7, and IL-15.

32. The culture medium according to any one of claims 23 - 31, wherein the factors are IL-7 and IL-15.

33. The culture medium according to any one of claims 23 - 32, wherein the factor is IL-15.

34. The culture medium according to any one of claims 23 - 33, wherein the concentration of the IGF is 10 ng / ml - 1000 ng / ml.

35. The culture medium according to any one of claims 23 - 34, wherein the concentration of the IL-7 is 5 ng / ml - 100 ng / ml.

36. The culture medium according to any one of claims 23 - 35, wherein the concentration of the IL-15 is 5 ng / ml - 100 ng / ml.

37. The culture medium according to any one of claims 23 - 36, wherein the culture medium may further comprise Serum replacement.

38. The culture medium according to any one of claims 23 - 37, wherein the culture medium does not contain serum.

39. The culture medium according to any one of claims 23 - 38, wherein the culture medium is used for preparing iNKT cells.

40. A method for amplifying and culturing iNKT cells, which comprises the method according to any one of claims 1 - 22, and / or uses the culture medium according to any one of claims 23 - 39.

41. A composition, which comprises human peripheral blood mononuclear cells and the culture medium according to any one of claims 23 - 39.

42. A composition, which comprises iNKT cells and the culture medium according to any one of claims 23 - 39.

43. A composition, which comprises human peripheral blood mononuclear cells and iNKT cells and the culture medium according to any one of claims 23 - 39.

44. Use of the composition according to any one of claims 41 - 43 in the preparation of a drug for preventing, alleviating, and / or treating a disease and / or disorder.