In-vitro induction and amplification method for dry memory T cells based on metal ion regulation

Abstract

The invention discloses a dry memory T cell in-vitro induction and amplification method based on metal ion regulation and application thereof. According to the method, Mn < 2 + > is added in the cell culture process to effectively induce the T cells to differentiate to the dry memory phenotype, so that the production cost of the dry memory T cells is greatly reduced while the use of other cell factors and chemical inducers is reduced. The dry memory T cell obtained by adopting the method has good multiplication capacity and functional activity, is suitable for clinical treatment, and particularly shows a wide application prospect in anti-tumor immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and particularly to a method for in vitro induction and amplification of stem cell-like memory T cells regulated by metal ions and its application. Background Art

[0002] The therapeutic effect of adoptive cell therapy (ACT) largely depends on the persistence of T cells in vivo and their differentiation status. A large number of clinical studies have shown that T cells in a low-differentiation state exhibit superior anti-tumor activity and more persistent immune effects during treatment. In particular, low-differentiation memory T cells with stem cell-like characteristics not only possess self-renewal ability but also have strong pluripotency, thus becoming one of the most promising cell types in ACT treatment. However, stem cell-like memory T cells are difficult to amplify under conventional in vitro culture conditions. How to efficiently and stably induce and amplify such T cells in vitro remains a major technical bottleneck at present. Currently, some studies have attempted to co-stimulate with CD3 / CD28 transiently combined with cytokines such as IL-7, IL-15, and IL-21 to jointly induce stem cell-like memory T cells, but this method has problems such as high cost and complex operation in large-scale production, limiting the feasibility of its clinical application. Therefore, it is of great significance to develop a rapid, efficient, low-cost, and highly safe induction strategy for stem cell-like memory T cells.

[0003] As essential elements of life activities, metal ions play key roles in various physiological processes. Nearly half of enzyme activities rely on the participation of metal ions. In particular, transition metals such as iron (Fe), zinc (Zn), manganese (Mn), nickel (Ni), copper (Cu), and cobalt (Co), due to their unique redox properties, have irreplaceable functions in cell metabolism and signal transduction. In recent years, more and more studies have focused on the key role of metal ion homeostasis in the tumor microenvironment in regulating T cell immune function. Existing studies have shown that a higher concentration of potassium ions (K + ) can promote T cell metabolic reprogramming, maintain its stemness, and significantly enhance its persistence in vivo; calcium ions (Ca 2+ ) promote the phosphorylation of the TCR-CD3 complex after TCR binds to the antigen and activate relevant transcription factors in the T cell nucleus as a second messenger; sodium ions (Na + ) change the membrane potential by enhancing the activity of the sodium-potassium pump on the membrane, thereby improving the metabolic adaptability of T cells and enhancing their effector functions. In addition, K + , Na + and Ca 2+ also play a synergistic role in regulating the activation process of the NLRP3 inflammasome. It is worth noting that zinc ions (Zn 2+) plays a key role in the process of cGAS recognizing cytoplasmic DNA, but its excess may induce the production of ROS, activate the classical pyroptosis pathway dependent on caspase-1 / GSDMD or the alternative pathway dependent on caspase-3 / GSDME, and then trigger the pyroptosis reaction of tumor cells. In addition, platinum-based chemotherapeutic drugs have been proven to synergistically induce immunogenic cell death with immune checkpoint inhibitors, further enhancing the anti-tumor immune effect. Among many metal ions, manganese ion (Mn 2+ ) has received extensive attention because it can effectively activate the cGAS-STING signaling pathway. Research shows that Mn 2+ can not only enhance the infiltration and specific killing ability of cytotoxic T cells in tumor tissues, but also be used in combination with PD-1 antibodies, significantly enhancing the therapeutic effect in various tumor models, while reducing the dosage of PD-1 antibodies and alleviating the treatment burden. Although Mn 2+ has shown great potential in enhancing T cell function, there is currently no study systematically exploring the role of Mn 2+ in inducing the differentiation of stem memory T cells. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for in vitro induction and amplification of stem memory T cells based on metal ion regulation. The stem memory T cells obtained by this method can be applied clinically, especially in the process of anti-tumor immunotherapy.

[0005] To achieve the above purpose, the present invention provides the following technical solution: A method for in vitro induction and amplification of stem memory T cells based on metal ion regulation, comprising the following steps:

[0006] 1) Activate and amplify T cells;

[0007] 2) Add Mn 2+ to the culture medium when or after activating T cells for culture.

[0008] Preferably, the step 1) includes: After coating the cell culture plate with antibodies, activate and amplify T cells in the culture medium containing cytokines.

[0009] Preferably, the antibodies are CD3 antibody and CD28 antibody.

[0010] Preferably, the cytokines are selected from one or more of IL-2, IL-7, IL-15, IL-17 and IL-21.

[0011] Preferably, the cytokine is IL-2.

[0012] Preferably, the concentration of the CD3 antibody is 1-2 μg / mL.

[0013] Preferably, the concentration of the CD28 antibody is 1-2 μg / mL.

[0014] Preferably, the concentration of the Mn 2+ is 1.0-20.0 μM.

[0015] Preferably, the concentration of the Mn 2+ is 2.0-10.0 μM.

[0016] Preferably, the method further comprises the following steps:

[0017] 3) Replace the culture medium containing cytokines and Mn 2+ and culture to obtain a lymphocyte population mainly composed of stem memory T cells.

[0018] Preferably, the time for replacing the culture medium is 1-3 days.

[0019] Preferably, the time for replacing the culture medium is 2 days.

[0020] Preferably, the lymphocyte population includes CD4 T cells, CD8 T cells, γδT, TILs, TCR-T, CAR-T, STAR-T.

[0021] Preferably, the species source of the T cells includes human-derived T cells or mouse-derived T cells.

[0022] On the other hand, the present invention provides a lymphocyte population prepared by the above method.

[0023] On the other hand, the present invention provides a pharmaceutical composition, and the pharmaceutical composition comprises the above lymphocyte population.

[0024] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0025] Preferably, the pharmaceutical composition further comprises an additional therapeutic agent.

[0026] On the other hand, the present invention provides the use of the lymphocyte population prepared by the above method, the above lymphocyte population or the above composition in the preparation of adoptive immunotherapy drugs.

[0027] Preferably, the adoptive immunotherapy includes anti-tumor immunotherapy, anti-infection immunotherapy and autoimmune disease treatment.

[0028] On the other hand, the present invention provides the use of Mn 2+ in the in vitro induction and amplification of stem memory T cells.

[0029] In summary, the present invention has the following beneficial effects:

[0030] 1. The present invention uses a simple cell culture to obtain a large number of stem memory T cells under basic T cell culture conditions. By adding Mn to the culture conditions 2+ , and using Mn 2+ to induce the differentiation of stem memory cells, the use of other cytokines and compounds is reduced. Thus, the toxic and side effects caused by the application of other cytokines are reduced. Moreover, the reserves of manganese in nature are huge, and the preparation, transportation, and storage of divalent manganese solution are very convenient, greatly reducing the production cost of stem memory T cells. That is, this method has the characteristics of low price and high production safety.

[0031] 2. The stem memory T cells obtained by the present invention show longer persistence in vivo, can significantly prolong the therapeutic effect of adoptive T cell therapy, and thus achieve a more persistent immune response and protective effect. Therefore, such cells have broad clinical application potential in adoptive immunotherapy for various indications such as tumor treatment and autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a schematic diagram of the induction and amplification of stem memory T cells in PBMC;

[0033] Figure 2 It is that Mn 2+ treatment significantly promotes the formation of stem memory T cells in PBMC. Among them, in the figure, A is the proportion of CD62L + CCR7 + (T SCM ) and CD45RO - CD27 + (T SCM ) T cells under the CD8 gate; B in the figure is the mean fluorescence intensity of the T cell stemness transcription factor TCF1 under the CD8 gate;

[0034] Figure 3 It is that Mn 2+ treatment significantly promotes the formation of mouse stem memory T cells. Among them, in the figure, A is the proportion of CD44 + CD62L + (T CM ) T cells under the CD8 gate; B in the figure is the mean fluorescence intensity of the T cell stemness transcription factor TCF1 under the CD8 gate;

[0035] Figure 4 It is that the T cells treated with Mn 2+ show strong in vivo persistence; A in the figure is a schematic diagram of the in vivo experiment in mice; B and C in the figure are the adoptive transfer of different treatment groups (control group and Mn 2+The proportion of adoptive T cells in the blood and spleen of mice after adoptive transfer of OT-I T cells in the treatment group;

[0036] Figure 5 is Mn 2+ The treated T cells showed strong in vivo anti-tumor activity and persistence; among them, Figure A is a schematic diagram of the in vivo experiment in B16-OVA tumor-bearing mice; Figure B is the tumor growth curve after adoptive transfer of OT-I T cells in different treatment groups (control group and Mn 2+ treatment group); Figures C and D are the proportions of adoptive T cells in the tumors of mice after adoptive transfer of different treatment groups (control group and Mn 2+ treatment group); Figure E is the proportion of the stem cell subset of TCF1 + and TIM3 + in tumor-infiltrating CD8 - T cells and the proportion of the effector subset of IFN-γ + and TNF-α + . Detailed implementation method

[0037] The present invention mainly uses Mn 2+ to induce the differentiation of stem memory cells to obtain a lymphocyte population mainly composed of stem memory T cells, and the lymphocyte population includes CD4 T cells, CD8 T cells, γδT, TILs, TCR-T, CAR-T cells, STAR-T and other in vitro expanded T cells.

[0038] The in vitro expansion method of stem memory T cells provided by the embodiment of the present invention includes the following steps:

[0039] 1) Activate and expand T cells;

[0040] 2) Add Mn 2+ to the culture medium when or after activating the T cells and culture.

[0041] Among them, step 1) includes: after coating the cell culture plate with antibodies, activate and expand T cells in the culture medium containing cytokines. Among them, the antibodies are CD3 antibody and CD28 antibody. The cytokines are selected from one or more of IL-2, IL-7, IL-15, IL-17 and IL-21; preferably IL-2. The concentration of the CD3 antibody is 1-2 μg / mL. The concentration of the CD28 antibody is 1-2 μg / mL.

[0042] In some preferred embodiments, the following steps are further included:

[0043] 3) Replace with the culture medium containing cytokines and Mn 2+Cultured in the medium to obtain a lymphocyte population mainly composed of stem memory T cells. Among them, the time for changing the medium is 1 to 3 days; preferably 2 days. The lymphocyte population includes CD4 T cells, CD8 T cells, γδT, TILs, TCR-T, CAR-T, STAR-T.

[0044] In some preferred embodiments, the concentration of Mn 2+ is 1.0 to 20.0 μM. Preferably, the concentration of Mn 2+ is 2.0 to 10.0 μM.

[0045] The embodiment of the present invention provides a lymphocyte population prepared by the above in vitro expansion method of stem memory T cells.

[0046] The embodiment of the present invention provides a pharmaceutical composition, including the above lymphocyte population. Preferably, it further includes a pharmaceutically acceptable excipient. The excipient is a conventional excipient well-known to those skilled in the art, and will not be specifically described and limited herein, and those skilled in the art can easily know it. Further preferably, it further includes an additional therapeutic agent for achieving combination therapy. The therapeutic agent can be selected as T cell therapy or immune checkpoint inhibitor therapy, etc.

[0047] The embodiment of the present invention also provides the application of the lymphocyte population prepared by the above preparation method, the above lymphocyte population, and the above pharmaceutical composition in the preparation of a drug for adoptive immunotherapy. The adoptive immunotherapy described above includes anti-tumor immunotherapy, anti-infection immunotherapy, and autoimmunity therapy.

[0048] The embodiment of the present invention also provides the application of Mn 2+ in the in vitro expansion of stem memory T cells. The preparation raw materials and methods of Mn 2+ in the embodiment of the present invention are not specifically described and limited, and are the existing conventional preparation raw materials and methods of divalent manganese solution.

[0049] The raw materials and reagents used in the present invention are all commercially available.

[0050] The term "T cell" refers to a cell with TCR or TCR genetically edited or modified on the cell surface. In this specification, "T cell" can be a cell population containing other cell types in addition to T cells themselves. For example, it can be a cell population containing more than 60%, more than 70%, more than 80%, more than 90%, or more than 95% of T cells. In addition, T cells include T cells at all differentiation stages and can be T cells with antigens or markers on the cell surface.

[0051] The terms "naive memory T cells" and "memory stem-like T cells" generally refer to cells (T memory stem cells, TSCM) at the early differentiation stage of memory T cells, which have stem cell characteristics and strong multi-directional differentiation potential. After being stimulated by antigens, TSCM cells can differentiate into central memory T cells (central memory T cells, TCM), effector memory T cells (effector memory T cells, TEM), and effector T cells (effector T cells, TEF), and then produce a large amount of effectors such as IFN-γ, generating more powerful killing ability. While having the potential for differentiation, they also have strong self-renewal ability.

[0052] The term "CD3 antibody" generally refers to an antigen-binding molecule that specifically binds to CD3. For example, a CD3 antibody can include T3, I, eu4, and HCHT1. The CD3 molecule is connected to the T cell antigen receptor through a salt bridge and participates in the signal transduction of T cells.

[0053] The term "CD28 antibody" generally refers to an antigen-binding molecule that specifically binds to CD28. Human CD28 is located at 2q33 and has similar exons and introns to CTLA4. The ligands of both are the B7 family, including B7-1 (CD80) and B7-2 (CD86). The binding affinity between CD28 and CD80 is high, and it can interact with various signal molecules or kinases, such as PI-3K, GRB-2 / s, and the tyrosine kinase ITK.

[0054] The term "pharmaceutical composition" refers to a lymphocyte population that has been formulated for administration to an individual and contains the naive memory T cells described herein or is mainly composed of naive memory T cells. Preferably, the pharmaceutical composition is sterile. In one embodiment, the pharmaceutical composition is pyrogen-free.

[0055] In the present invention, the acquisition of the PBMCs includes, but is not limited to, obtaining them by density gradient centrifugation. Specific examples:

[0057] Example 1 In vitro amplification method using human naive memory T cells as the main cell population

[0058] The method of this example includes the following steps

[0059] 1. Coat a cell culture plate with human CD3 antibody at a final concentration of 1 μg / mL;

[0060] 2. Collect 10 mL of peripheral blood from healthy individuals into a centrifuge tube, dilute the peripheral blood 1:1 with phosphate-buffered saline (PBS), and mix well. Take another new 50 mL centrifuge tube, add 20 mL of lymphocyte separation medium (ficoll), and then slowly add the mixed blood dilution along the tube wall to the upper layer of the lymphocyte separation medium according to the volume ratio of ficoll to blood dilution liquid of 1:1, so that the two form a clear layer. Centrifuge at 800 g with an acceleration of 0 and a deceleration of 0 for 20 min.

[0061] 3. After centrifugation, aspirate the cells in the monocyte layer into a 50 mL centrifuge tube, add 20 mL of PBS to wash once, centrifuge at 800 g for 5 min, and discard the supernatant. Subsequently, resuspend and count the cells with T cell medium and dilute the cells to 1 million / ml.

[0062] 4. Then, add human CD28 antibody at a final concentration of 1 μg / ml and rhIL-2 at 300 U / ml to the T cell suspension, mix evenly, seed the cells in a pre-coated 24-well plate, and add the corresponding concentration of Mn 2+ (2.5 μM), pipette evenly, and then place it in an incubator for culture. According to the growth status of T cells, replenish the medium containing rhIL-2 and Mn 2+ every 2 days to maintain the T cell density at 0.5 - 2×10 6 / ml. Culture for 12 days, and use flow cytometry to detect the proportion of stem cell memory T cells in the culture and the expression level of the transcription factor TCF-1 that regulates the differentiation of stem cell memory T cells.

[0063] The results are as Figure 2 shown: On the 12th day of culture, in the Mn 2+ treatment group, the proportion of stem cell memory T cells of CCR7 + CD62L + and CD45RO - CD27 + was significantly higher than that of the control group under normal culture conditions. At the same time, the expression of the transcription factor TCF1 that regulates T cell stemness in T cells treated with Mn 2+ was also significantly higher than that of the control group.

[0064] The control group in this example is only different from the Mn 2+ treatment group in that Mn 2+ is not added in step 4.

[0065] In this example, the addition concentration range of Mn 2+ can be between 1 - 20.0 μM, and the recommended concentration range is between 2.5 - 10 μM.

[0066] In vitro amplification method with murine stem memory T cells as the main cell population in Example 2

[0067] The method of this example includes the following steps:

[0068] 1. First, coat a cell culture plate with mouse CD3 antibody at a final concentration of 2 μg / mL;

[0069] 2. Isolate mouse spleen cells, obtain mouse T cells by nylon wool column or magnetic bead sorting, and dilute them to 1 million / ml with T cell medium;

[0070] 3. Add mouse CD28 antibody at a final concentration of 1 μg / ml and 20 - 50 U / ml of rmIL-2 to the T cells, and add Mn at the corresponding concentration (10.0 μM) 2+ , pipette evenly, inoculate the cells in the cell culture plate coated with mouse CD3 antibody, and place it in an incubator for culture;

[0071] 4. Then, according to the growth status of the T cells, replenish an equal amount of medium containing mouse IL-2 recombinant protein and Mn 2+ every 24 hours to maintain the cell density at 0.5 - 2×10 6 / ml. When the T cells are cultured to the 6th day, use flow cytometry to detect the proportion of stem memory T cells in the culture and the expression level of the transcription factor TCF1 that regulates the differentiation of stem memory T cells.

[0072] The results are as Figure 3 shown: On the 6th day of culture, in the Mn 2+ treatment group, the proportion of CD44 + CD62L + stem memory T cells increased from about 60.7% to about 72.7%, which was significantly higher than that of T cells under normal culture conditions; at the same time, in the T cells treated with Mn 2+ , the expression of the transcription factor TCF1 that regulates T cell stemness and the transcription level of transcription factors related to T cell stemness were significantly higher than those of the control group.

[0073] The control group of this example is only different from the Mn 2+ treatment group in that Mn 2+ is not added in step 3.

[0074] In this example, the addition concentration range of Mn 2+ can be between 1.0 - 20 μM, and the recommended concentration range is between 2.5 - 10.0 μM.

[0075] Example 3 Detection of the persistence in vivo of mouse stem memory T cells induced by Mn 2+ ​

[0076] To prove that Mn 2+ The memory stem T cells obtained by treatment have better anti-tumor function and persistence compared to normally treated T cells. This invention intends to use the OT-I T cell model to explore the in vivo persistence of the stem memory T cells induced by Mn 2+ induced by.

[0077] The operating steps of this example are as follows: As Figure 4 shown, first, 3×10 6 Mn-treated or untreated 45.1 2+ OT-I T cells were injected into the tail vein of 7- to 8-week-old C57BL6 / N mice. After 7 days of adoptive transfer, the mice were euthanized, and blood and spleen were taken for flow cytometry analysis to detect the proportion of adoptive transferred 45.1 + T cells. + T cell ratio.

[0078] The results are as Figure 5 shown. It can be seen from the flow cytometry staining results that the infiltration ratio of Mn 2+ -treated OT-I T cells in the mouse blood increased from about 0.023% to about 0.042%. At the same time, the proportion of infiltrating T cells in the spleen also increased significantly, indicating that Mn 2+ -treated OT-I T cells showed stronger persistence in the mouse adoptive transfer model.

[0079] Example 4 Detection of the anti-tumor function in vivo of the mouse stem memory T cells induced by Mn 2+ The anti-tumor experiment in vivo of the stem memory T cells induced by Mn

[0080] Mn 2+ induced stem memory T cells was carried out in 7- to 8-week-old C57BL6 / N mice. To prove that Mn 2+ The memory stem T cells obtained by treatment have better anti-tumor function and persistence compared to normally treated T cells. This example intends to use the OT-I T cell model to explore the in vivo anti-tumor function of the stem memory T cells induced by Mn 2+ induced by.

[0081] The specific operation steps are as follows: First, 2×10 5 B16-OVA cells were subcutaneously inoculated at the right axilla of the hind limb of C57BL6 / N mice. After the tumor cells grew subcutaneously in the mice for about 8-10 days, a vernier caliper was used to detect the tumor volume; when the tumor volume was between 75 mm 3 -150 mm 3 , 3×10 6 Mn-treated 2+For treated or untreated OT-IT cells, the tumor volume was measured every 2 days thereafter. At around 14 days (the specific operation time is determined by the experimental phenomena), the spleens and tumor tissues of C57BL6 / N mice were obtained. After grinding the tumor tissues of the mice, Percoll density gradient centrifugation was performed, and analysis was carried out after flow cytometry staining.

[0082] The results are as Figure 5 shown. First, through the statistics of the tumor growth volume, we found that, compared with the control group, in the experimental group of adoptively transferred T cells treated with Mn 2+ , the tumor growth rate of the mice was significantly slowed down. In addition, it can be seen from the results of flow cytometry staining that: the infiltration ratio of OT-I T cells treated with Mn 2+ in the tumor tissues of tumor-bearing mice increased from about 7.34% to about 27.3%, the proportion of effector T cell subsets increased from about 22.4% to about 46.7%, and the proportion of TCF1 + TIM3 - stem cell-like T cell subsets increased from about 22.2% to about 33.6%. This indicates that OT-I T cells treated with Mn 2+ exhibit stronger antitumor effects and persistence in the mouse adoptive transfer model.

[0083] The above are only the preferred embodiments of the present invention. The protection scope of the present invention is not limited to the above embodiments. All technical solutions falling within the idea of the present invention belong to the protection scope of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements should also be regarded as within the protection scope of the present invention.

Claims

1. An in vitro induction and amplification method of memory T cells with stemness regulated by metal ions, characterized in that, The method described above comprises the following steps: 1) Activating and expanding T cells; 2) Add Mn to the culture medium during or after T cell activation 2+ .

2. The in vitro amplification method of memory T cells according to claim 1, characterized in that, Step 1) includes: after coating a cell culture plate with an antibody, activating and expanding T cells in a culture medium containing cytokines; Preferably, the antibody is a CD3 antibody and a CD28 antibody; Preferably, the cytokine is selected from one or more of IL-2, IL-7, IL-15, IL-17, and IL-21.

3. The method according to claim 2, wherein The concentration of the CD3 antibody is 1 - 2 μg / mL; and / or The concentration of the CD28 antibody is 1 - 2 μg / mL; and / or The cytokine is IL-2.

4. The method according to any one of claims 1-2, characterized in that The concentration of said Mn 2+ is 1.0 - 20.0 μM; preferably 2.0 - 10.0 μM.

5. The method according to any one of claims 1 to 4, characterized in that, The method further comprises the following steps: 3) Replace the culture medium containing cytokines and Mn 2+ and culture to obtain a lymphocyte population mainly composed of stem memory T cells; preferably, the time for replacing the culture medium is 1 to 3 days, preferably 2 days; Preferably, the lymphocyte population includes CD4 T cells, CD8 T cells, γδT, TILs, TCR-T, CAR-T, STAR-T.

6. The method according to any one of claims 1-5, characterized in that, The species source of the T cells includes human-derived T cells or mouse-derived T cells.

7. A lymphocyte population, characterized in that, Prepared by the method according to any one of claims 1 - 6.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition described above contains the lymphocyte population according to claim 7; Preferably, the pharmaceutical composition further contains a pharmaceutically acceptable excipient; Preferably, the pharmaceutical composition further contains an additional therapeutic agent.

9. Use of the lymphocyte population prepared by the method according to any one of claims 1 - 6, the lymphocyte population according to claim 7, or the pharmaceutical composition according to claim 8 in the preparation of adoptive immunotherapy drugs; Preferably, the adoptive immunotherapy is anti-tumor immunotherapy, anti-infection immunotherapy, and autoimmune disease treatment.

10. Mn 2+ Application in in vitro induction and expansion of stem memory T cells.

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