Application of alpinetin in preparation of CD8 < + > memory T cell accelerant, cellular immune drug and anti-tumor immunotherapy drug
By using samcinol to promote the formation of Tscm and/or Tcm cells and the secretion of IFN-γ expression in CD8+ memory T cells, the problem of CD8+ memory T cell exhaustion was solved, and the long-term anti-tumor function of T cells was improved, and the tumor was improved. Immunotherapy provides sustained and efficient immune response support.
Patent Information
- Application Number
- CN202411880874.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-19
- Publication Date
- 2025-02-14
AI Technical Summary
CD8+ memory T cells are prone to depletion in tumor immunotherapy, resulting in a decrease in their proliferation ability and loss of killing function, affecting the effectiveness of tumor treatment.
The immune response function of Tscm and/or Tcm cells in CD8+ memory T cells is enhanced by using Alpinenetin (ALP).
By promoting the formation of CD8+ memory T cells and the secretion of IFN-γ expression, sarcosin enhances the long-term anti-tumor function of T cells, providing continuous and efficient immune response support for tumor immunotherapy.
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Figure CN119424408A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of tumor immunotherapy, in particular to the use of shanjiangsu in the preparation of CD8 + Application of memory T cell promoters, cellular immune drugs and anti-tumor immunotherapy drugs. Background Art
[0002] Currently, the incidence of liver cancer ranks sixth among tumors in the world, and the mortality rate ranks second. The main reason is that liver cancer patients are usually diagnosed in the late stage of cancer, resulting in a poor prognosis. Therefore, it is very necessary to develop new ways to prevent and treat liver cancer.
[0003] At present, tumor immunotherapy has been widely used in clinical practice as the fourth treatment method after surgery, radiotherapy and chemotherapy. + Memory T cells are the most powerful effectors in tumor immunotherapy and the most important cells in tumor immunotherapy. They can directly kill and eliminate target cells. However, T cells are prone to functional abnormalities such as exhaustion, losing the long-term anti-tumor function and affecting the effect of tumor treatment. + After memory T cells are exhausted, their proliferation capacity is reduced, their killing function is lost, and the CD8 + The long-lasting anti-tumor effect of memory T cells is thus achieved. + Memory T cells, prevent CD8 + Memory T cell exhaustion is one of the difficult problems that needs to be overcome urgently in the clinical application of tumor immunotherapy.
[0004] Chronic inflammation is an important cause of T cell exhaustion, and its mechanism of action mainly includes the following aspects: 1) Continuous antigen stimulation from viruses or tumor cells leads to overactivation of T cells, which gradually loses their effector function and causes exhaustion before terminal differentiation; 2) Proinflammatory cytokines and inhibitory cytokines released by virus-infected cells, immunosuppressive cells, tumor cells, etc. play an important role in causing T cell exhaustion. To a certain extent, the combination of different cytokines can affect the specific response of T cells; 3) Inflammatory cytokines can not only cause T cells to express inhibitory receptors, but also activate tumorigenic signaling pathways and induce the expression of inhibitory ligands, thereby preventing T cell activation and making it impossible for T cells to produce a strong immune response.
[0005] T cells can be divided into effector T cells (T cells) according to their differentiation and fate. EFF ) and memory T cells (T M ), which can be divided into T memory stem cells (T SCM ), central memory T cells (T CM ) and effector memory T cells (TEM ) and other subgroups. M It has the advantages of lower antigen reactivation conditions, stronger effector function, and rapid immune response, and plays an important role in the anti-tumor immune response process.
[0006] Researchers have found that Alpinetin (ALP) plays an important role in preventing tumors, inflammatory diseases, bacterial infections, viral infections, liver damage, cardiovascular diseases, nerve damage, etc. The reason for this is that Alpinetin can inhibit the release of inflammatory factors by immune cells and relieve inflammation. Alpinetin (MW: 270.28) is a natural small molecule flavonoid compound isolated from the seeds and rhizomes of Alpinia plants. Alpinetin is easily available and has broad application prospects.
[0007] This application is based on the existing technology to further develop the pharmacological effects of shanjiangsu, so that it can be used to prepare CD8 + Memory T cell promoters, cellular immunity drugs and anti-tumor immunotherapy drugs provide favorable support for the development and clinical application of galangin. Summary of the invention
[0008] The technical problem to be solved by the present invention is to provide a method for preparing CD8 + Application of memory T cell promoter to promote CD8 + Memory T cells scm and / or T cm Cell formation, promoting CD8 + The expression and secretion of IFN-γ by memory T cells enhances the immune response function of T cells, providing favorable support for further exerting the clinical efficacy of shanpin.
[0009] In order to solve the above technical problems, the present invention provides a method for preparing CD8 + Application in memory T cell promoter.
[0010] Further improvement, Alpinia officinalis promotes CD8 + Memory T cells scm and / or T cm Cell formation.
[0011] Further improvement, Alpinia officinalis promotes CD8 + Expression and secretion of IFN-γ by memory T cells.
[0012] The present invention also provides an application of alpinia officinalis in preparing cellular immunity medicine.
[0013] Further improvement, the cellular immune drug is CD8 + Memory T cell promoter, Alpinia officinalis promotes CD8 +Memory T cells scm and / or T cm Cell formation.
[0014] Alpinia officinalis promotes CD8 + Expression and secretion of IFN-γ by memory T cells.
[0015] The present invention also provides an application of shanjiangsu in preparing anti-tumor immunotherapy drugs.
[0016] Further improvement, the anti-tumor immunotherapy drug is CD8 + Memory T cell promoter, Alpinia officinalis promotes CD8 + Memory T cells scm and / or T cm Cell formation.
[0017] Alpinia officinalis promotes CD8 + Expression and secretion of IFN-γ by memory T cells.
[0018] The anti-tumor immunotherapy drug is a long-acting anti-tumor drug.
[0019] After adopting such a design, the present invention has at least the following advantages:
[0020] The present invention can increase T scm and / or T cm Cell formation, promoting CD8 + The expression and secretion of IFN-γ by memory T cells can enhance the immune response function of T cells, rather than just relying on the relieving effect of inflammation brought about by inhibiting the release of inflammatory factors by T cells. + Memory T cell promoters, cellular immunity drugs or anti-tumor immunotherapy drugs provide favorable support for further exerting the clinical efficacy of galangin.
[0021] The present invention promotes CD8 + Memory T cells scm and / or T cm The generation of cells, thereby enhancing the long-term anti-tumor function of T cells. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The above is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.
[0023] Figure 1 and Figure 2 This is a graph showing the apoptosis ratio of T cells after 24, 48, and 72 hours of ALP treatment in the experiment of the effect of ALP on T cell apoptosis in the embodiment of the present invention.
[0024] Figure 3 and Figure 4 The ALP of the present invention promotes CD8 + In the memory T cell formation experiment, CD8 + T CM and CD8 + T SCM A graph showing the change in the proportion of .
[0025] Figure 5 and 6 This is a graph showing the killing ability of T cells against target mouse Hepa1-6 liver cancer cells at different effector-target ratios after T cells were treated with ALP for 24 hours in the ALP short-term anti-tumor immune function regulation experiment of the present invention.
[0026] Figure 7 This is a graph showing changes in IFN-γ concentration in the cell supernatant of T cells at different effector-target ratios after T cells were treated with ALP for 24 hours in the ALP short-term anti-tumor immune function regulation experiment of the present invention.
[0027] Figure 8 and 9 This is a graph showing the killing ability of T cells against target mouse Hepa1-6 liver cancer cells at different effector-target ratios after T cells were treated with ALP for 7 days in the ALP long-term anti-tumor immune function regulation experiment of the present invention.
[0028] Fig.10 This is a graph showing changes in IFN-γ concentration in the cell supernatant of T cells at different effector-target ratios after T cells were treated with ALP for 7 days in the ALP long-term anti-tumor immune function regulation experiment of the present invention.
[0029] Fig.11 The ALP of the present invention promotes CD8 + T CM Flow chart of mouse model establishment and drug administration in the formation and long-acting anti-tumor effect experiment.
[0030] Fig.12 The ALP of the present invention promotes CD8 + T CM In the experiment of formation and long-term anti-tumor effect, after mouse modeling and drug treatment, the mice in the control group were killed at the 4th, 6th and 8th months to detect the CD8 + The proportion of memory T cells.
[0031] Fig.13 The ALP of the present invention promotes CD8 + T CMIn the experiment of formation and long-term anti-tumor effect, after mouse modeling and drug treatment, the mice in the ALP group were killed at the 8th month to detect CD8 + Memory T cells and CD8 + T CM Proportion ratio chart.
[0032] Fig.14 The ALP of the present invention promotes CD8 + T CM The occurrence of primary liver tumors and pathological changes of liver tissue in mice in the ALP group and control group in the formation and long-term anti-tumor effect experiment. DETAILED DESCRIPTION
[0033] 1. Materials and Methods
[0034] 1. Experimental Materials
[0035] Mouse hepatocarcinoma Hepa1-6 cell line and virus packaging cell line 293T were purchased from the American Type Culture Collection (ATCC). All cells were stored in DMEM (Lonza) containing 10% fetal bovine serum (FBS) (Biosera) and 10,000 IU / ml penicillin / 100 micrograms / ml streptomycin (EallBio Life Sciences). All cells were cultured in a humidified incubator at 37°C with 5% CO2 and 95% air.
[0036] Alpinetin (ALP) (AB6697) was purchased from Acima, and N-nitrosodiethylamine (N0258) was purchased from Sigma.
[0037] C57BL / 6 mice (2 weeks old, 6-8 weeks old) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.
[0038] 2. Experimental Methods
[0039] 2.1 T cell culture
[0040] Normal C57BL / 6 mice were killed by spinal dislocation, and spleen tissue was separated. Splenocytes were obtained by grinding and cultured in RPMI-1640 (Lonza) medium containing IL-2 (20 ng / ml) (Beijing Shuanglu Pharmaceutical), PHA (4 μg / ml) (Solarbio), and 10% FBS (Biosera) for 7 days. The purity of T cells was detected by flow cytometry.
[0041] 2.2 Flow cytometry
[0042] Flow cytometry was performed using a FACSCanto Plus instrument (BD Biosciences), and data were analyzed using FlowJo V.10 (FlowJo, LLC).
[0043] Mouse T cells were stained with Percp-labeled CD3 antibody (Biolegend), APC-Cy7-labeled CD8 antibody (Biolegend), FITC-labeled CD4 antibody (Biolegend), APC-labeled CD62L antibody (Biolegend), PE-labeled CD44 antibody (Biolegend), and BV421-labeled Sca-1 antibody (Biolegend). Flow cytometry was used to detect CD4, CD8, and T CM and T SCM proportion.
[0044] 2.3 Apoptosis detection
[0045] Mouse T cells were stained with antibody FITC-Annexin V (4 μl / tube) (Biyuntian, C1062L) and dye 7-AAD (1 μl / tube) (eBioscience, 00-6993-50), and the proportion of apoptotic cells was analyzed using FACSCanto Plus instrument (BD Biosciences) and software FlowJo V.10 (FlowJo, LLC).
[0046] 2.4 Cytotoxicity assay
[0047] In a 96-well plate, mouse T cells were co-cultured with target cells (Hepa1-6-luc) at different effector-target ratios (E:T) for 24 hours, and then 20 μl of substrate D-luciferin potassium salt (1.51 mg / ml) (Sigma) was added. Luciferase activity was detected using a fluorescent imager (IVIS, Xenogen) to analyze T cell toxicity.
[0048] 2.5 ELISA for IFN-γ
[0049] In a 96-well plate, mouse T cells were co-cultured with target cells (Hepa1-6-luc) at different effector-target ratios (E:T) for 24 hours, and the supernatant was collected. The IFN-γ concentration in the cell supernatant was detected using an IFN-γ ELISA detection kit (Thermo, 88-7314-77).
[0050] 2.6 Construction of mouse induced hepatocellular carcinoma tumor model
[0051] Two-week-old normal C57BL / 6 male mice were taken, and N-nitrosodiethylamine (25 mg / kg, 50 μL) was intraperitoneally injected into the liver of the mice to induce primary liver cancer in mice. One month later, the mice were divided into a control group and an experimental group. The mice in the control group and the experimental group were intraperitoneally injected with normal saline and ALP (10 mg / kg) solution twice a week, 100 μl each time. The mice were killed at the 4th, 6th, and 8th month after administration, and liver tissues and spleen T cells were obtained to analyze tumor occurrence and the proportion of T cell subtypes.
[0052] The above animal experimental procedures were approved by the Ethics Committee of Beijing Shijitan Hospital Affiliated to Capital Medical University (Ethics No. 2023-KY-0009-001).
[0053] 2.7 Statistical methods
[0054] In this example, Graphpad Prism 8.0.2 was used to analyze the experimental data. The data were analyzed by t-test, with p value < 0.05 as significant; * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001; **** represents P < 0.0001. All experiments were repeated at least three times.
[0055] 2. Results and Analysis
[0056] 1. Effect of ALP on T cell apoptosis
[0057] After culturing mouse spleen T cells for 7 days using the above 2.1 T cell culture method, they were divided into a control group or an alpinia galanga group, and treated with equal doses of solvent dimethyl sulfoxide (DMSO) and ALP (100 μM) compounds, respectively, and cultured for 24, 48, and 72 h, respectively.
[0058] Then, the cells were collected and the apoptosis of T cells in the control group or the shanjiangsu group was detected using the cell apoptosis detection kit in the above 2.3 cell apoptosis detection method and the flow cytometer in the above 2.2 flow cytometry.
[0059] The results are attached Figure 1 and Figure 2 As shown, there was no significant difference between the ALP group and the control group at 24h, 48h, and 72h.
[0060] The results showed that ALP did not induce T cell apoptosis.
[0061] 2.ALP promotes CD8 + Memory T cells (T SCM 、T CM )
[0062] After culturing mouse spleen T cells for 7 days using the above 2.1 T cell culture method, they were divided into a control group or an alpinia galanga group, and treated with equal doses of solvent dimethyl sulfoxide (DMSO) and ALP (100 μM) compounds, respectively, and cultured for 1 day and 7 days, respectively.
[0063] After treating T cells for 1 day and 7 days, the cells were collected and stained with CD3, CD4, CD8, CD44, CD62L, and Sca-1 flow cytometry antibodies as described in 2.2 to detect CD4, CD8, and T cells. CM and T SCM proportion.
[0064] The results are shown in Table 1 and Appendix Figure 3 and 4 As shown, when T cells were treated for 1 day, the ALP group did not promote CD8 + T CM However, after 7 days of T cell treatment, the ALP group significantly promoted CD8 + T CM The formation of CD8 T cells increased from 15.5% to 18.4%. After 1 day and 7 days of treatment, the ALP group promoted CD8 + T SCM The formation of CD8 + T SCM has dropped to 0, while the ALP group CD8 + T SCM The proportion reaches 7.69%.
[0065] Table 1 ALP promotes memory T cells (T SCM 、T CM )
[0066]
[0067] The results showed that ALP has the ability to promote CD8 + Memory T cells (T SCM 、T CM ) formed.
[0068] 3. ALP regulates the short-term anti-tumor immune function of T cells
[0069] After culturing mouse spleen T cells for 7 days using the above 2.1 T cell culture method, they were divided into a control group or an alpinia galanga group, and equal doses of solvent dimethyl sulfoxide (DMSO) and ALP (100 μM) compounds were added to treat the cells and cultured for 1 day respectively.
[0070] After 1 day of T cell treatment, the above 2.4 cytotoxicity assay was used, and T cells were co-cultured with target Hepa1-6-luc cells at effector-target ratios (E:T) of 10:1, 5:1, and 2:1. After 24 hours, D-luciferin potassium salt (20 μl / well) was added, and cell activity was detected by fluorescence imaging to analyze the killing ability of T cells on target cells. At the same time, the cell supernatant was collected, and the concentration of IFN-γ in the cell supernatant was detected by the ELISA detection kit described in 2.5 above.
[0071] The results are attached Figure 5 , 6 As shown in Figures 7 and 7 , the IFN-γ concentration and cytotoxicity in the ALP group were reduced compared with the control group.
[0072] The results showed that ALP had the effect of inhibiting T cell activity when it was treated with T cells for 1 day, indicating that ALP can inhibit chronic inflammation in the short term, but cannot enhance the anti-tumor function of T cells.
[0073] 4. ALP regulates the long-term anti-tumor immune function of T cells
[0074] After culturing mouse spleen T cells for 7 days using the above 2.1 T cell culture method, they were divided into a control group or an alpinia galanga group, and treated with equal doses of solvent dimethyl sulfoxide (DMSO) and ALP (100 μM) compounds, respectively, and cultured for 7 days.
[0075] After 7 days of treatment of T cells, the above 2.4 cytotoxicity assay was used, and T cells were co-cultured with target Hepa1-6-luc cells at effector-target ratios (E:T) of 10:1, 5:1, and 1:1. After 24 hours, D-luciferin potassium salt (20 μl / well) was added, and cell activity was detected by fluorescence imaging to analyze the killing ability of T cells on target cells. At the same time, the cell supernatant was collected and the concentration of IFN-γ in the cell supernatant was detected by the ELISA detection kit described in 2.5 above.
[0076] The results are attached Figure 8 , 9 As shown in Figures 10, compared with the control group, the IFN-γ concentration in the ALP group was significantly increased and the cytotoxicity was also significantly enhanced.
[0077] The results showed that, in contrast to treatment for 1 day, ALP treatment of T cells for 7 days significantly enhanced the tumor killing function of T cells, indicating that ALP has the ability to enhance the long-term anti-tumor function of T cells.
[0078] 5.ALP promotes CD8 in induced liver cancer mice + T CM The formation and long-term anti-tumor effect
[0079] The mouse induced hepatocellular carcinoma tumor model and administration method established in 2.6 above are as follows: Fig.11 As shown, mice were killed at 4, 6, and 8 months after administration, and spleen T cells were obtained to analyze the CD8 + The proportion of memory T cells and T CM ratio, and observed the formation of primary tumors in the mouse liver.
[0080] The results are attached Fig.12 , 13 , 14, Fig.12 The results show that when the mice in the control group were killed at 4, 6, and 8 months, the number of CD8 + The proportion of memory T cells, from Fig.12 It can be seen that when the severity of liver cancer in the control group continued to increase, CD8 + The proportion of memory T cells gradually decreased. Fig.13 The results show that when the mice in the ALP group were sacrificed at 8 months, the number of CD8 + Memory T cells and CD8 + T CM The proportion of Fig.13 It can be seen that compared with the control group mice, the CD8 + Memory T cells and CD8 + T CM There was a significant increase. Fig.14 The figure shows the occurrence of primary liver tumors and pathological changes in liver tissues in the ALP group and the control group when the mice were killed at the 8th month. Fig.14 It can be seen that no tumor foci were observed in the mice in the ALP group, while obvious primary tumor foci were observed in the mice in the control group. The results of tissue HE staining showed that the liver cells of the mice in the ALP group were uniform in texture and no tumor foci were observed, while vesicles and tumor foci appeared in the livers of the mice in the control group.
[0081] The results showed that ALP can promote CD8 + The formation of memory T cells with long-lasting anti-tumor function.
[0082] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Those skilled in the art may make some simple modifications, equivalent changes or modifications using the technical contents disclosed above, which all fall within the protection scope of the present invention.
Claims
1. Preparation of Alpinia officinalis in CD8 + Application in memory T cell promoter.
2. The method of preparing CD8 + The use of the invention in a memory T cell promoter is characterized in that: Alpinia officinalis promotes CD8 + Memory T cells scm and / or T cm Cell formation.
3. The method of preparing CD8 + The use of the invention in a memory T cell promoter is characterized in that: Alpinia officinalis promotes CD8 + Expression and secretion of IFN-γ by memory T cells.
4. Application of Alpinia officinalis in the preparation of cellular immunity drugs.
5. The use of alpinia officinalis according to claim 4 in the preparation of cellular immunity drugs, characterized in that: The cellular immune drug is CD8 + Memory T cell promoter, Alpinia officinalis promotes CD8 + Memory T cells scm and / or T cm Cell formation.
6. The use of alpinia officinalis according to claim 5 in the preparation of cellular immunity drugs, characterized in that: Alpinia officinalis promotes CD8 + Expression and secretion of IFN-γ by memory T cells.
7. Application of alpinia officinalis in the preparation of anti-tumor immunotherapy drugs.
8. The use of alpinia officinalis according to claim 7 in the preparation of anti-tumor immunotherapy drugs, characterized in that: The anti-tumor immunotherapy drug is CD8 + Memory T cell promoter, Alpinia officinalis promotes CD8 + Memory T cells scm and / or T cm Cell formation.
9. The use of alpinia officinalis according to claim 8 in the preparation of anti-tumor immunotherapy drugs, characterized in that: Alpinia officinalis promotes CD8 + Expression and secretion of IFN-γ by memory T cells.
10. The use of alpinia officinalis according to claim 9 in the preparation of anti-tumor immunotherapy drugs, characterized in that: The anti-tumor immunotherapy drug is a long-acting anti-tumor drug.