Culture medium for culturing long-life iNKT cells and application thereof
The problem of difficulty in amplification and short duration of action in iNKT cell culture medium was solved by adding IL-2, IL-7, IL-15, α-GalCer, glutamine, TWS119 and vitamin C in iNKT cell culture medium was solved. , achieving efficient and long-term iNKT cell culture and anti-tumor effects.
Patent Information
- Application Number
- CN202411889643.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-20
- Publication Date
- 2025-02-14
AI Technical Summary
The difficulty in amplification, low purity and short duration of cell action in the in vitro culture of existing iNKT cells limit their effective application in clinical anti-tumor treatment.
Provided is a culture medium including IL-2, IL-7, IL-15, α-GalCer, glutamine, TWS119 and vitamin C for the culture of iNKT cells, through initial culture and amplification culture, without the need for magnetism. Bead enrichment obtains a large number of high-purity long-lived iNKT cells.
The efficient amplification and optimization of iNKT cells has been achieved. The obtained cells have stronger amplification ability, longer in vivo survival life and greater anti-tumor potential. They are suitable for a wide range of applications based on NKT/iNKT cell immunotherapy.
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Figure CN119432736A_ABST
Abstract
Description
Technical Field
[0001] The present application belongs to the technical field of immune cell culture, and specifically relates to a culture medium for culturing long-lived iNKT cells and its application. Background Art
[0002] Natural killer T cells (NKT) are a type of lymphocyte subpopulation that expresses both T cell receptors and NK cell receptors, and have dual characteristics of T cells and NK cells. They participate in a variety of immune responses such as anti-tumor, the onset of autoimmune diseases, the regulation of allergic reactions, and the inhibition of parasitic infections, and play an important role in both innate and adaptive immune responses (Annu. Rev. Immunol. 25.1 (2007): 297-336.). NKT cells are mainly divided into three subtypes: type I NKT cells (i.e., iNKT cells), which express constant TCR chains, recognize lipid antigens presented by CD1d molecules, and mainly play anti-tumor and anti-infection roles; type II NKT cells, which are associated with immunosuppression; and type III NKT cells, which neither rely on CD1d molecules to recognize antigens nor respond to lipid antigens, and their characteristics and functions have not yet been clarified.
[0003] iNKT cell targeted therapy is considered to be the most ideal treatment for the treatment and prevention of tumor recurrence and metastasis due to its strong and broad-spectrum anti-tumor activity, superior safety and allogeneic universal potential (Current opinion inimmunology 20.3 (2008): 358-368.): (1) iNKT cells can directly dissolve tumor cells expressing CD1d, and the amount of tumor killing factors they rapidly release is much higher than that of NK cells and T cells; (2) iNKT cells secrete a large number of pro-inflammatory factors that can indirectly and widely activate DC cells, macrophages, NK cells, T cells and B cells, etc., and recruit and induce these cells to exert anti-tumor effects; (3) After activation, iNKT cells can lyse TAM / MDSC expressing CD1d, reprogram tumor-promoting M2 into anti-tumor M1, reduce the number of type II NKT cells, reverse the exhausted T cells and NK cells, and thus reverse the entire tumor microenvironment (TME); (4) iNKT cells can promote CD8α +DC cells cross-present, induce tumor immune memory formation, prevent tumor recurrence or new tumor cells; (5) iNKT cells have no MHC / HLA restriction, do not cause allogeneic immune rejection, and can be used allogeneically without matching, overcoming the limitation that tumor patients can only use autologous cell transfusion in the case of immunodeficiency; (6) iNKT cells can migrate to non-lymphoid tissues and are suitable for various malignant solid tumors. In addition, no serious adverse reactions such as iNKT-related inflammatory cytokine storm have been reported in clinical practice, and the safety is high. The latest research has confirmed that iNKT cells play a key role in clinical anti-tumor treatment. Many top foreign pharmaceutical companies (such as MiNK, Therapeutic, Athenex / Kuur, AppiaBio / Kite, Arovella, etc.) have made iNKT cell anti-tumor layout as the core R&D pipeline and entered the clinical trial stage (Central European Journal of Immunology 42.2(2017):181-195.).
[0004] In related technologies, Harvard Medical School reported a method for amplifying autologous iNKT cells in peripheral blood mononuclear cells by magnetic bead separation and IL-2 and CD3 monoclonal antibodies (Clinical Cancer Research 23.14 (2017): 3510-3519.); Cancer Research recently reported a method for in vitro amplification of iNKT by α-GalCer (α-galactosylceramide) and IL-2 (Cancer research 83.4 (2023): 582-594.). However, the existing iNKT culture scheme still has obvious shortcomings, which seriously limits its effective application in clinical anti-tumor treatment, specifically:
[0005] (1) iNKT cells are few in number, accounting for about 0.1-1% of T cells in the peripheral blood of healthy people. In vitro screening, amplification, and culture are very difficult, requiring high-cost equipment and experienced operators;
[0006] (2) iNKT cells acquire exhaustion and senescent phenotypes during traditional culture, such as increased expression of PD-1 and TIM-3, rapid apoptosis of the cells after entering the body, and a short duration of action.
[0007] Therefore, it is urgent to develop new iNKT culture programs to effectively expand and activate iNKT cells in large quantities and to durably enhance the anti-tumor activity of iNKT cells, so as to truly unleash the huge clinical application potential of iNKT cells and open up new avenues for the clinical treatment of malignant solid tumors with iNKT. Summary of the invention
[0008] 1. Problem to be solved
[0009] In view of one of the limitations of the prior art in the process of in vitro culture of iNKT cells, such as difficulty in amplification, low iNKT purity, and short duration of iNKT action after culture, the present application provides a new culture medium for culturing long-lived iNKT cells and its application. The culture medium includes additives IL-2, IL-7, IL-15, α-GalCer, vitamin C, glutamine and TWS119. When used for the culture of iNKT cells, mononuclear cells isolated from peripheral blood or umbilical cord blood can be used as a source. After in vitro amplification and optimized culture, a large number of high-purity long-lived iNKT cells can be obtained without magnetic bead enrichment. The cells have strong proliferation ability and strong anti-tumor effect, laying a foundation for the widespread application of NKT / iNKT cell immunotherapy.
[0010] 2. Technical solution
[0011] In order to solve the above problems, the technical solutions adopted in this application are as follows:
[0012] The present application provides a culture medium for culturing long-lived iNKT cells, which culture medium includes: GT-T551 H3 complete culture medium, and also includes additives: human interleukin 2 (IL-2), human interleukin 7 (IL-7), human interleukin 15 (IL-15), α-galactosylceramide (α-GalCer), glutamine, TWS119 and vitamin C, wherein: IL-2, IL-7 and IL-15 are used to expand iNKT cells; α-galactosylceramide (α-GalCer) is used to activate iNKT cells; glutamine is an important additive for maintaining iNKT expansion and its function; TWS119 is used to inhibit the occurrence of iNKT excessive differentiation and exhaustion and prolong cell life; vitamin C is a reducing agent, which is used to remove peroxide free radicals generated during cell metabolism and prevent excessive accumulation of peroxide free radicals from inducing iNKT cell aging and apoptosis.
[0013] Furthermore, in the above-mentioned medium for culturing long-lived iNKT cells, the concentration of each additive is:
[0014] Human interleukin 2 (IL-2), 100-200 U / mL;
[0015] Human interleukin-7 (IL-7), 20-40 ng / mL;
[0016] Human interleukin 15 (IL-15), 20-40 ng / mL;
[0017] α-galactosylceramide (α-GalCer), 50-100 ng / mL;
[0018] Glutamine, 0.1-3 mM;
[0019] TWS119, 1-3 μM;
[0020] Vitamin C, 20-50 μg / mL.
[0021] Furthermore, in the above-mentioned culture medium for culturing long-lived iNKT cells, the concentration of glutamine is 0.3-3 mM.
[0022] Furthermore, in the above-mentioned culture medium for culturing long-lived iNKT cells, the concentration of glutamine is 1 to 3 mM.
[0023] Furthermore, in the above-mentioned culture medium for culturing long-lived iNKT cells, the concentration of glutamine is 1 mM, 2 mM or 3 mM.
[0024] Furthermore, in the above-mentioned culture medium for culturing long-lived iNKT cells, the concentration of TWS119 is 2 to 3 μM.
[0025] Furthermore, in the above-mentioned culture medium for culturing long-lived iNKT cells, the concentration of TWS119 is 2 μM or 3 μM.
[0026] Furthermore, in the above-mentioned medium for culturing long-lived iNKT cells, the concentration of each additive is:
[0027] Human interleukin 2 (IL-2), 100 U / mL;
[0028] Human interleukin-7 (IL-7), 40 ng / mL;
[0029] Human interleukin 15 (IL-15), 40 ng / mL;
[0030] α-galactosylceramide (α-GalCer), 100 ng / mL;
[0031] Glutamine, 1 mM;
[0032] TWS119, 3 μM;
[0033] Vitamin C, 50 μg / mL.
[0034] Furthermore, in the above-mentioned culture medium for culturing long-lived iNKT cells, the GT-T551 H3 complete culture medium comprises: GT-T551 H3 serum-free culture medium, 2-3% inactivated plasma, 100 U / mL penicillin, and 100 μg / mL streptomycin.
[0035] Furthermore, the above-mentioned inactivated plasma is autologous inactivated plasma.
[0036] This application also provides the use of the above-mentioned culture medium for culturing long-lived iNKT cells in the preparation of long-lived iNKT cells, which uses mononuclear cells separated from peripheral blood or umbilical cord blood as a source, and collects and obtains long-lived iNKT cells after initial culture and expansion culture; wherein:
[0037] The initial culture includes: the mononuclear cells are statically cultured in the initial culture medium for 1 to 2 days, and the initial culture medium includes: GT-T551H3 complete culture medium and additives such as human interleukin 2 (IL-2), human interleukin 15 (IL-15), human interleukin 18 (IL-18), CD3 monoclonal antibody (αCD3), and human granulocyte macrophage colony stimulating factor (GM-CSF); IL-2, IL-15, IL-18, αCD3 and GM-CSF are cytokines necessary for the growth of NK cells and T cells, and are also the most common basic additives for traditional iNKT cell culture, and can effectively amplify NK and NKT cells in lymphocytes in the initial culture system;
[0038] The expansion culture includes: supplementing the culture medium for culturing long-lived iNKT cells after the initial culture, and continuing static culture for 14 to 21 days; when the culture medium changes to orange or yellow, or the cell density reaches 70% to 80%, supplementing the culture medium for culturing long-lived iNKT cells.
[0039] Furthermore, in the above application, the iNKT cells are expanded and cultured until the number reaches ≥10 8 , ≥10 8 It has reached the clinical treatment level.
[0040] Furthermore, in the above application, collecting and obtaining long-lived iNKT cells includes: harvesting the cells by centrifugation at 1400 rpm for 10 min, washing the cells twice with physiological saline, and obtaining long-lived iNKT cells.
[0041] Furthermore, the above-mentioned culture medium for supplementing the culture of long-lived iNKT cells includes: culture medium for supplementing the culture of long-lived iNKT cells in a volume of 1:1.
[0042] Furthermore, in the above application, the cell density reaching 70% to 80% refers to the cell density observed under a microscope.
[0043] Furthermore, in the above initial culture medium, the concentration of each additive is:
[0044] Human interleukin 2 (IL-2), 10-50 U / mL;
[0045] Human interleukin 15 (IL-15), 40-100 ng / mL;
[0046] Human interleukin 18 (IL-18), 20-50 ng / mL;
[0047] CD3 monoclonal antibody (αCD3), 3-10 ng / mL;
[0048] Human granulocyte macrophage colony-stimulating factor (GM-CSF), 200-500 U / mL;
[0049] The IL-2 dosage is reduced by 50 to 90% compared with the conventional T lymphocyte culture dosage in order to inhibit the excessive proliferation of T lymphocytes.
[0050] Furthermore, in the above initial culture medium, the concentration of each additive is:
[0051] Human interleukin 2 (IL-2), 10 U / mL;
[0052] Human interleukin 15 (IL-15), 40 ng / mL;
[0053] Human interleukin 18 (IL-18), 50 ng / mL;
[0054] CD3 monoclonal antibody (αCD3), 10 ng / mL;
[0055] Human granulocyte macrophage colony-stimulating factor (GM-CSF), 500 U / mL.
[0056] Furthermore, in the above application, static culture includes: static culture in an incubator at 37° C., 5% CO 2 , and saturated humidity.
[0057] The present application also provides a method for culturing long-lived iNKT cells, the method comprising: using mononuclear cells separated from peripheral blood or umbilical cord blood as a source, and collecting and obtaining long-lived iNKT cells after initial culture and expansion culture; wherein:
[0058] The initial culture includes: the mononuclear cells are statically cultured in the initial culture medium for 1 to 2 days, and the initial culture medium includes: GT-T551H3 complete culture medium and additives such as human interleukin 2 (IL-2), human interleukin 15 (IL-15), human interleukin 18 (IL-18), CD3 monoclonal antibody (αCD3), and human granulocyte macrophage colony stimulating factor (GM-CSF); IL-2, IL-15, IL-18, αCD3 and GM-CSF are cytokines necessary for the growth of NK cells and T cells, and are also the most common basic additives for traditional iNKT cell culture, and can effectively amplify NK and NKT cells in lymphocytes in the initial culture system;
[0059] The expansion culture includes: supplementing the culture medium for culturing long-lived iNKT cells after the initial culture, and continuing static culture for 14 to 21 days; when the culture medium changes to orange or yellow, or the cell density reaches 70% to 80%, supplementing the culture medium for culturing long-lived iNKT cells.
[0060] Furthermore, in the above method, the iNKT cells are expanded and cultured until the number of iNKT cells reaches ≥10 8 , ≥10 8 It has reached the clinical treatment level.
[0061] Furthermore, in the above method, collecting and obtaining long-lived iNKT cells includes: harvesting the cells by centrifugation at 1400 rpm for 10 min, washing the cells twice with physiological saline, and obtaining long-lived iNKT cells.
[0062] Furthermore, the above-mentioned culture medium for supplementing the culture of long-lived iNKT cells includes: culture medium for supplementing the culture of long-lived iNKT cells in a volume of 1:1.
[0063] Furthermore, in the above method, the cell density reaching 70% to 80% refers to the cell density observed under a microscope.
[0064] Furthermore, in the above initial culture medium, the concentration of each additive is:
[0065] Human interleukin 2 (IL-2), 10-50 U / mL;
[0066] Human interleukin 15 (IL-15), 40-100 ng / mL;
[0067] Human interleukin 18 (IL-18), 20-50 ng / mL;
[0068] CD3 monoclonal antibody (αCD3), 3-10 ng / mL;
[0069] Human granulocyte macrophage colony-stimulating factor (GM-CSF), 200-500 U / mL.
[0070] Furthermore, in the above initial culture medium, the concentration of each additive is:
[0071] Human interleukin 2 (IL-2), 10 U / mL;
[0072] Human interleukin 15 (IL-15), 40 ng / mL;
[0073] Human interleukin 18 (IL-18), 50 ng / mL;
[0074] CD3 monoclonal antibody (αCD3), 10 ng / mL;
[0075] Human granulocyte macrophage colony-stimulating factor (GM-CSF), 500 U / mL.
[0076] Furthermore, in the above method, static culturing includes: static culturing in an incubator at 37° C., 5% CO 2 , and saturated humidity.
[0077] The present application also provides long-lived iNKT cells cultured by the above method for culturing long-lived iNKT cells.
[0078] Furthermore, the above-mentioned long-lived iNKT cells have highly expressed longevity marker CD62L, and downregulated expression of exhaustion markers PD-1 and TIM-3, and have stronger cell proliferation ability, longer in vivo survival life and greater anti-tumor potential.
[0079] 3. Beneficial effects
[0080] Compared with the prior art, the present application has the following beneficial effects:
[0081] (1) The present application provides a culture medium for culturing long-lived iNKT cells and its application, the culture medium comprising additives such as human interleukin 2 (IL-2), human interleukin 7 (IL-7), human interleukin 15 (IL-15), α-galactosylceramide (α-GalCer), glutamine, TWS119 and vitamin C, wherein: IL-2, IL-7 and IL-15 are used to expand iNKT cells; α-galactosylceramide (α-GalCer) is used to activate iNKT cells; glutamine is an important additive for maintaining iNKT expansion and its function; TWS119 is used to inhibit the occurrence of iNKT over-differentiation and exhaustion and prolong cell life; vitamin C is a reducing agent, which is used to remove peroxide free radicals produced during cell metabolism and prevent excessive accumulation of peroxide free radicals from inducing iNKT cell aging and apoptosis.
[0082] (2) The present application provides a culture medium for culturing long-lived iNKT cells and its application, which is used to culture long-lived iNKT cells, and can use mononuclear cells separated from peripheral blood or umbilical cord blood as a source, and after initial culture and expansion culture, long-lived iNKT cells are collected; the application (culture method) can obtain a large number of high-purity long-lived iNKT cells without magnetic bead enrichment, and compared with the traditional iNKT culture scheme, the iNKT cell expansion multiple is further improved, and high-purity iNKT cells are harvested; at the same time, the cultured iNKT cells highly express the longevity marker CD62L, downregulate the expression of exhaustion markers PD-1 and TIM-3, reduce the expression of exhaustion and aging phenotypes, have a longer lifespan, and a long duration of action; the high purity and longer in vivo survival lifespan further enhance the anti-tumor potential of iNKT cells, and have a strong ability to kill tumor cells, laying the foundation for the widespread application of NKT / iNKT cell immunotherapy, so that more tumor patients can benefit. BRIEF DESCRIPTION OF THE DRAWINGS
[0083] Figure 1 It is the ratio and number of iNKT cells cultured in culture medium containing different combinations of additives.
[0084] Figure 2 iNKT cells CD62L cultured in medium containing different additive combinations + ratio and expression levels of PD-1 and TIM-3.
[0085] Figure 3 It is the result of long-lived iNKT cells killing target cells.
[0086] Figure 4 The effect of different concentrations of glutamine on the expansion level of iNKT cells.
[0087] Figure 5 The figure shows the effect of different concentrations of TWS119 on the expansion level of iNKT cells. DETAILED DESCRIPTION
[0088] The present application is further described below in conjunction with specific embodiments.
[0089] It should be noted that the terms such as "upper", "lower", "left", "right", "middle", etc. cited in this specification are only for the convenience of description and are not used to limit the scope of implementation. Changes or adjustments to their relative relationships should be regarded as the scope of implementation of this application without substantially changing the technical content.
[0090] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.
[0091] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.
[0092] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement or value. The degree of flexibility for a particular variable can be easily determined by one skilled in the art.
[0093] As used herein, the term "at least one of" is intended to be synonymous with "one or more of." For example, "at least one of A, B, and C" expressly includes only A, only B, only C, and combinations of each thereof.
[0094] Concentration, amount and other numerical data can be presented in range format herein.It should be understood that such range format is only used for convenience and simplicity, and should be flexibly interpreted as not only including the numerical value clearly described as range limit, but also including all single numerical values or sub-ranges contained in the range, just as each numerical value and sub-range are clearly described.For example, the numerical range of about 1 to about 4.5 should be interpreted as not only including the limit value of 1 to about 4.5 clearly described, but also including single numerals (such as 2,3,4) and sub-ranges (such as 1 to 3,2 to 4, etc.).The same principle is applicable to the scope of only narrating a numerical value, such as "less than about 4.5", which should be interpreted as including all the above-mentioned values and ranges.In addition, no matter how the breadth of the described range or feature is, this explanation should be applicable.
[0095] As used herein, the preparation of inactivated plasma or autologous inactivated plasma is a conventional technical means in the art. As an example, the present application provides one of the preparation methods, including the following steps:
[0096] Take plasma or autologous plasma, seal the centrifuge tube containing plasma with sealing film, and place it in a water bath preheated at 56℃ to inactivate complement for 30 minutes. After inactivation, freeze the plasma in a -18 to -20℃ refrigerator overnight, thaw the frozen plasma in a 4℃ refrigerator, centrifuge at 3000rpm for 20 minutes after thawing, aspirate the upper plasma as autologous inactivated plasma, and discard the lower precipitate.
[0097] As used herein, plasma or autologous plasma is derived from peripheral blood or umbilical cord blood, and its preparation is a conventional technical means in the art. As an example, the present application provides one of the preparation methods, comprising the following steps:
[0098] Aseptically collect 20-100 mL of peripheral venous blood using a heparin anticoagulation blood collection tube, or collect 20-100 mL of umbilical cord venous blood using umbilical vein puncture; immediately inject into the heparin anticoagulation blood collection tube (try to ensure aseptic operation during the collection process), invert and mix several times to allow the anticoagulant to fully contact the blood; centrifuge at 1400 rpm for 10 minutes. After the centrifugation is completed, aspirate the upper layer of plasma into a new sterile tube in a biosafety cabinet and mark it as plasma.
[0099] As used herein, peripheral blood or cord blood mononuclear cells are obtained by separation from peripheral blood or cord blood using Ficoll density gradient centrifugation, and the preparation thereof is a conventional technical means in the art. As an example, the present application provides one of the preparation methods, comprising the following steps:
[0100] Use a heparin anticoagulation blood collection tube to aseptically collect 20-100 mL of peripheral venous blood, or use umbilical vein puncture to collect 20-100 mL of umbilical cord venous blood; immediately inject into the heparin anticoagulation blood collection tube (try to ensure aseptic operation during the collection process), invert and mix several times to allow the anticoagulant to fully contact the blood; centrifuge at 1400 rpm for 10 minutes. After the centrifugation is completed, add an equal volume of sterile saline to the lower layer of blood cell sediment to dilute it and gently mix it with a Pasteur pipette to obtain a blood cell dilution solution;
[0101] Take a 50mL centrifuge tube and add an equal volume of peripheral blood mononuclear cell separation solution (Dayou) according to the volume of the blood cell diluent. Use a Pasteur pipette to slowly add the blood cell diluent along the wall of the centrifuge tube at 1 cm above the separation solution. Do not break the liquid surface of the separation solution. After completion, centrifuge at room temperature 2000rpm for 30min (the centrifuge speed increase and decrease are both set to 3); after the centrifugation of the blood cell diluent, aspirate the white film layer (about 2-5mL) into a new 50mL centrifuge tube, add physiological saline to the 40mL mark, blow and aspirate the cells with a Pasteur pipette to mix them thoroughly, centrifuge at 1400rpm for 10min, discard the supernatant, obtain the mononuclear cell precipitate, wash the cells twice with physiological saline and count them.
[0102] Example 1
[0103] This embodiment provides a culture medium for culturing long-lived iNKT cells and its application.
[0104] In this embodiment, the culture medium includes GT-T551 H3 complete culture medium and additives, wherein:
[0105] GT-T551 H3 complete medium includes: GT-T551 H3 serum-free medium, 2-3% inactivated plasma, 100 U / mL penicillin, 100 μg / mL streptomycin;
[0106] Additives include:
[0107] Human interleukin 2 (IL-2), 100-200 U / mL; human interleukin 7 (IL-7), 20-40 ng / mL; human interleukin 15 (IL-15), 20-40 ng / mL; IL-2, IL-7 and IL-15 are used to expand iNKT cells;
[0108] α-Galactosylceramide (α-GalCer), 50-100 ng / mL; α-Galactosylceramide (α-GalCer) is used to activate iNKT cells;
[0109] Glutamine, 1-3 mM; Glutamine is an important additive to maintain iNKT expansion and function;
[0110] TWS119, 2-3 μM; TWS119 is used to inhibit iNKT overdifferentiation and exhaustion, and prolong cell lifespan;
[0111] Vitamin C, 20-50 μg / mL; Vitamin C is a reducing agent used to remove peroxide free radicals produced during cell metabolism and prevent excessive accumulation of peroxide free radicals from inducing iNKT cell aging and apoptosis.
[0112] In this embodiment, the use of the culture medium for culturing long-lived iNKT cells refers to its use in preparing long-lived iNKT cells, using mononuclear cells separated from peripheral blood or umbilical cord blood as a source, and collecting and obtaining long-lived iNKT cells after initial culture and expansion culture; wherein:
[0113] Initial culture includes: mononuclear cells are inoculated into T75 cell culture flasks filled with initial culture medium at a cell density of 1×10 6 / mL, and cultured in a 37°C, 5% CO2, saturated humidity incubator for 1 to 2 days (observe the cells under a microscope and record the cell growth status. According to the cell growth status, if the culture medium color turns orange or yellow or cell colonies are observed under a microscope, it is about the first to second day of initial culture). The initial culture medium includes: GT-T551 H3 complete medium (Takara GT-T551 H3 serum-free medium was added with 2-3% autologous inactivated plasma, 100 U / mL penicillin, 100 μg / mL streptomycin) and additives human interleukin 2 (IL-2), human interleukin 15 (IL-15), human interleukin 18 (IL-18), CD3 monoclonal antibody (αCD3), and human granulocyte macrophage colony stimulating factor (GM-CSF). The concentrations of each additive were as follows: human interleukin 2 (IL-2), 10-50 U / mL; human interleukin 15 (IL-15), 40-100 ng / mL; human interleukin 18 (IL-18), 20-50 ng / mL; CD3 monoclonal antibody (αCD3), 3-10 ng / mL; and human granulocyte macrophage colony stimulating factor (GM-CSF), 200-500 U / mL.
[0114] The expansion culture includes: supplementing the culture medium for culturing long-lived iNKT cells at a volume ratio of 1:1 after the initial culture, gently shaking the culture bottle to mix the culture medium, and continuing to place it in a 37°C, 5% CO2, saturated humidity incubator for static culture for 14 to 21 days; observing the cells under a microscope every day and recording the growth status of the cells. According to the cell growth status, if the color of the culture medium changes to orange or yellow, or the cell density under the microscope reaches 70% to 80%, supplementing the culture medium for culturing long-lived iNKT cells at a volume ratio of 1:1, and the cells can be transferred to a T225 culture bottle for continued expansion culture;
[0115] iNKT cell collection: After 14 to 21 days of expansion culture, when the number of iNKT cells reaches the clinical therapeutic level (≥10 8 When the number of cells was 400, the cells were harvested by centrifugation at 1400 rpm for 10 min, and the cells were washed twice with physiological saline to obtain long-lived iNKT cells.
[0116] Example 2
[0117] This example provides a study on the number and purity of long-lived iNKT cells.
[0118] Specifically include:
[0119] After collecting peripheral blood from healthy donors and isolating mononuclear cells, 10 8 The mononuclear cells were divided into 5 equal parts (2×10 7), mononuclear cells were initially cultured and expanded with different additive combinations in Table 1. The initial culture and expansion culture methods were referred to Example 1, and a control group was also set up:
[0120] Comparative method (Study on in vitro culture of iNKT cells derived from umbilical cord blood and their killing effect on breast cancer cells [D]. Liaoning: China Medical University, 2017.): The cell pellet was adjusted to 1×10 6 / mL of cell suspension, and add α-GalCer 100ng / mL, and culture it in a 37°C, 5% CO2, saturated humidity incubator. After 16 hours, add IL-2 500U / mL and GM-CSF 1000U / mL; replace the culture medium every 3 days and supplement cytokines.
[0121] Table 1
[0122]
[0123] Result analysis: (1)
[0125] On the 7th and 14th days of culture, cells were counted using a cell counter, an appropriate amount of cell suspension was harvested and centrifuged at 1400 rpm for 10 min, the supernatant was discarded, 100 μL PBS was added to each tube of pellet to resuspend the cells, 1 μL FC receptor blocking agent (Miltenyi) was added to each tube, and the cells were blocked at 4°C for 15 min, anti-human TCR Vα24Jα18-APC and anti-human CD3-FITC fluorescent antibodies (Biolegend) were added and mixed, and the cells were stained at room temperature in the dark for 10 min, PBS was added and centrifuged at 1400 rpm for 10 min, and the supernatant was discarded. 400 μL PBS was added to each tube of pellet to resuspend the cells, and CD3 was detected by flow cytometry. + Vα24Jα18 + Levels of double-positive iNKT cells.
[0126] The results are as follows Figure 1 The results showed that the purity of iNKT cells expanded using the culture medium containing the additive combination of combination 3 and combination 4 was higher than that of other groups, while the number of cells expanded using the culture medium containing the additive combination of combination 2 and combination 4 was significantly increased compared with other groups. (2)
[0128] On the 14th day of culture, an appropriate amount of cells were harvested and centrifuged at 1400 rpm for 10 min, and the supernatant was discarded. 100 μL PBS was added to each tube of pellet to resuspend the cells, and 1 μL FC receptor blocking agent was added to each tube, and the cells were blocked at 4°C for 15 min. Anti-human TCR Vα24Jα18-APC and anti-human CD3-FITC fluorescent antibodies (Biolegend) were added to mark iNKT cells, and anti-human CD62L-PE, anti-human PD-1-PE, and anti-human TIM-3-PE fluorescent antibodies (Biolegend) were added and mixed. The cells were stained at room temperature in the dark for 10 min, and PBS was added to centrifuge at 1400 rpm for 10 min to wash twice. The supernatant was discarded, and 400 μL PBS was added to each tube of pellet to resuspend the cells. The exhaustion status of iNKT cells was detected by flow cytometry.
[0129] The results are as follows Figure 2 Shown: CD62L in iNKT cells expanded using medium containing the additive combinations of combination 3 and combination 4 + iNKT ratio is high, scientists have confirmed CD62L + iNKT has excellent proliferation activity and persistent anti-tumor function in vivo (CD62L + NKT cells have prolonged persistence and antitumor activity in vivo. The Journal of clinical investigation 126.6 (2016): 2341-2355.). At the same time, iNKT cells expanded using a culture medium containing the additive combination of combination 3 and combination 4 significantly downregulated the expression of cell exhaustion markers PD-1 and TIM-3, and had a longer cell lifespan.
[0130] In summary, compared with the traditional iNKT culture (expansion) method, the iNKT cells cultured using the culture medium and expansion method containing the additive combination of combination 4 have stronger cell expansion ability, longer in vivo survival life and greater anti-tumor potential.
[0131] Example 3
[0132] This example provides a study on the killing function of long-lived iNKT cells on target cells.
[0133] Specifically include:
[0134] After collecting peripheral blood from healthy donors and isolating mononuclear cells, the cells were divided into two equal parts (2×10 7The cells were cultured by conventional iNKT cell culture method (comparative method in Example 2) and long-lived iNKT cell culture method of the present application (culture method using culture medium containing additive combination 4 in Example 2), respectively. After 14 days of culture, the cells were harvested and counted, centrifuged at 1400 rpm for 10 min, and the supernatant was discarded. The cell concentration was adjusted to 10 6 / mL.
[0135] Jurkat cells were used as target cells and inoculated in 96-well plates. The effector-target ratio was set to 5:1, 10:1, and 20:1. The plates were placed in a 37°C, 5% CO2, and saturated humidity incubator for 6 hours. The cells were harvested and centrifuged at 1400rpm for 10 minutes, and the supernatant was discarded. 100 μL PBS was added to each tube of precipitate to resuspend the cells. 1 μL FC receptor blocking agent was added to each tube and blocked at 4°C for 15 minutes. Then 10 μL Annexin V-FITC dye (Miltenyi) was added to each tube and mixed. The plates were incubated at room temperature in the dark for 15 minutes. 1×Binding buffer was added and centrifuged at 300g for 10 minutes to wash twice. The supernatant was discarded, and 500 μL 1×Binding buffer was added to resuspend the cells. 5 μL PI dye was added and mixed 5 minutes before the detection on the machine. The killing effect of iNKT cells on tumor cells was detected by flow cytometry. The effector-target ratio was set to 5:1. The plates were placed in a 37°C, 5% CO2 incubator for 6 hours. The supernatant was discarded, and the cells were resuspended in 100 μL PBS. 1 μL FC receptor blocking agent was added to each tube and blocked at 4°C for 15 minutes. Then 10 μL Annexin V-FITC dye (Miltenyi) was added to each tube and mixed. The plates were incubated at room temperature in the dark for 15 minutes. 1×Binding buffer was added and centrifuged at 300g for 10 minutes to wash twice. The supernatant was discarded, and 500 μL 1×Binding buffer was added to resuspend the cells. 5 μL After 24 h of static culture in a CO2 and saturated humidity incubator, the culture supernatant was collected and the levels of IFN-γ and IL-4 in the supernatant were detected using an enzyme-linked immunosorbent assay kit (Biolegend).
[0136] Result analysis:
[0137] The results are as follows Figure 3 As shown, the iNKT cells cultured by the long-lived iNKT cell culture method of the present application have stronger killing function on target cells, higher secretion level of anti-tumor effector molecules, and the anti-tumor function of long-lived iNKT cells is significantly improved.
[0138] Example 4
[0139] This example provides the effect of different concentrations of glutamine in the supplement on the expansion level of iNKT cells.
[0140] Specifically include:
[0141] After collecting peripheral blood from healthy donors and isolating mononuclear cells, 10 8 The mononuclear cells were divided into 5 equal parts (2×10 7), refer to the culture method of Example 2 using a culture medium containing the additive combination of combination 4, other conditions remain unchanged, and different doses of glutamine (0, 0.1, 0.3, 1, 3 mM) are added.
[0142] On the 7th and 14th days of culture, cells were counted using a cell counter, an appropriate amount of cell suspension was harvested and centrifuged at 1400 rpm for 10 min, the supernatant was discarded, 100 μL PBS was added to each tube of pellet to resuspend the cells, 1 μL FC receptor blocking agent was added to each tube, the cells were blocked at 4°C for 15 min, anti-human TCR Vα24Jα18-APC and anti-human CD3-FITC fluorescent antibodies were added and mixed, the cells were stained at room temperature in the dark for 10 min, PBS was added and centrifuged at 1400 rpm for 10 min to wash twice, the supernatant was discarded, 400 μL PBS was added to each tube of pellet to resuspend the cells, and CD3 was detected by flow cytometry. + Vα24Jα18 + The levels of double-positive iNKT cells were compared to compare the effects of glutamine dose on the number of iNKT cell expansion.
[0143] Result analysis:
[0144] The results are as follows Figure 4 As shown, iNKT cell expansion is dose-dependent on glutamine, and the iNKT cell expansion level is higher when the glutamine concentration is 1-3 mM.
[0145] Example 5
[0146] This example provides the effects of different concentrations of TWS119 in the additive on the expansion level of iNKT cells.
[0147] Specifically include:
[0148] After collecting peripheral blood from healthy donors and isolating mononuclear cells, 10 8 The mononuclear cells were divided into 5 equal parts (2×10 7 ), refer to the culture method of Example 2 using a culture medium containing the additive combination of combination 4, other conditions remain unchanged, and different doses of TWS119 (0, 1, 2, 3, 4 μM) are added.
[0149] On the 14th day of culture, an appropriate amount of cells were harvested and centrifuged at 1400rpm for 10min, and the supernatant was discarded. 100μL PBS was added to each tube of pellet to resuspend the cells. 1μL FC receptor blocking agent was added to each tube, and the cells were blocked at 4°C for 15min. Anti-human TCR Vα24Jα18-APC and anti-human CD3-FITC fluorescent antibodies were added to label iNKT cells, and anti-human CD62L-PE fluorescent antibodies were added to mix well. The cells were stained at room temperature in the dark for 10min, and PBS was added to centrifuge at 1400rpm for 10min to wash twice. The supernatant was discarded, and 400μL PBS was added to each tube of pellet to resuspend the cells. The expression level of CD62L in iNKT cells was detected by flow cytometry, and the effects of TWS119 on the purity of iNKT cells and their expression of longevity phenotype (CD62L + )’s impact.
[0150] Result analysis:
[0151] The results are as follows Figure 5 As shown in the figure, the purity of iNKT cells and their expression of longevity phenotypes are dose-dependent on TWS119. When TWS119 is 2-3 μM, the purity of induced iNKT cells is high and the cell longevity phenotype is significantly improved. However, when the concentration of TWS119 is ≥4 μM, the cells die. Therefore, the optimal addition dose of TWS119 is 2-3 μM.
Claims
1. A culture medium for culturing long-lived iNKT cells, characterized in that: The culture medium comprises: a complete culture medium and additives, wherein the additives comprise: human interleukin 2, human interleukin 7, human interleukin 15, α-galactosylceramide, glutamine, TWS119 and vitamin C.
2. A culture medium for culturing long-lived iNKT cells according to claim 1, characterized in that: The concentration of human interleukin 2 is 100-200 U / mL; and / or The concentration of human interleukin 7 is 20-40 ng / mL; and / or The concentration of human interleukin 15 is 20-40 ng / mL; and / or The concentration of α-galactosylceramide is 50-100 ng / mL; and / or The concentration of glutamine is 0.1-3 mM; and / or The concentration of TWS119 is 1-3 μM; and / or The concentration of the vitamin C is 20-50 μg / mL.
3. A culture medium for culturing long-lived iNKT cells according to claim 2, characterized in that: The concentration of the glutamine is 0.3-3 mM; and / or the concentration of the TWS119 is 2-3 μM.
4. A culture medium for culturing long-lived iNKT cells according to claim 3, characterized in that: The concentration of glutamine is 1-3 mM.
5. A culture medium for culturing long-lived iNKT cells according to any one of claims 1 to 4, characterized in that: The complete culture medium is GT-T551 H3 complete culture medium, which includes: GT-T551 H3 serum-free culture medium, 2-3% inactivated plasma, 100 U / mL penicillin, and 100 μg / mL streptomycin.
6. Use of a culture medium for culturing long-lived iNKT cells according to any one of claims 1 to 5 in preparing long-lived iNKT cells, characterized in that: The application is to use mononuclear cells separated from peripheral blood or umbilical cord blood as a source, collect and obtain long-lived iNKT cells after initial culture and expansion culture; wherein: The initial culture comprises: static culture of mononuclear cells in an initial culture medium for 1 to 2 days, wherein the initial culture medium comprises: GT-T551 H3 complete culture medium and additives, wherein the additives comprise: human interleukin 2, human interleukin 15, human interleukin 18, CD3 monoclonal antibody, and human granulocyte macrophage colony stimulating factor; the concentration of each additive is: human interleukin 2, 10 to 50 U / mL; human interleukin 15, 40 to 100 ng / mL; human interleukin 18, 20 to 50 ng / mL; CD3 monoclonal antibody, 3 to 10 ng / mL; and human granulocyte macrophage colony stimulating factor, 200 to 500 U / mL; The expansion culture includes: supplementing the culture medium for culturing long-lived iNKT cells after the initial culture, and continuing static culture for 14 to 21 days; when the culture medium changes color to orange or yellow, or the cell density reaches 70% to 80%, supplementing the culture medium for culturing long-lived iNKT cells.
7. A method for culturing long-lived iNKT cells, characterized in that: The method comprises: using mononuclear cells separated from peripheral blood or umbilical cord blood as a source, collecting and obtaining long-lived iNKT cells after initial culture and expansion culture; wherein: The initial culture comprises: static culture of mononuclear cells in an initial culture medium for 1 to 2 days, wherein the initial culture medium comprises: GT-T551 H3 complete culture medium and additives, wherein the additives comprise: human interleukin 2, human interleukin 15, human interleukin 18, CD3 monoclonal antibody, and human granulocyte macrophage colony stimulating factor; the concentration of each additive is: human interleukin 2, 10 to 50 U / mL; human interleukin 15, 40 to 100 ng / mL; human interleukin 18, 20 to 50 ng / mL; CD3 monoclonal antibody, 3 to 10 ng / mL; and human granulocyte macrophage colony stimulating factor, 200 to 500 U / mL; The expansion culture comprises: supplementing the culture medium for culturing long-lived iNKT cells according to any one of claims 1 to 5 after the initial culture, and continuing static culture for 14 to 21 days; when the color of the culture medium turns orange or yellow, or the cell density reaches 70% to 80%, supplementing the culture medium for culturing long-lived iNKT cells.
8. The use according to claim 6 or the method according to claim 7, characterized in that: The culture medium for supplementing the culture of long-lived iNKT cells comprises: a culture medium for supplementing the culture of long-lived iNKT cells in a volume of 1:
1. 9 . Long-lived iNKT cells cultured according to the method for culturing long-lived iNKT cells according to claim 7 or 8 .
10. The long-lived iNKT cell according to claim 9, characterized in that The long-lived iNKT cells have highly expressed longevity marker CD62L, and downregulated expression of exhaustion markers PD-1 and TIM-3.