Application of vimogib in preparation of product for delaying and / or reversing T cell senescence

Abstract

The invention discloses application of vimogib in preparation of a product for delaying and / or reversing T cell senescence, and belongs to the field of biological medicine. Experiments prove that the vimogii can enhance the lysosome function of T cells by enhancing the expression of TFE3 protein, so that the expression of anti-tumor and anti-virus related cytokines is enhanced, the lysosome function of senescent T cells of old people is improved, and the anti-tumor and anti-virus infection capability of the old people is enhanced. It is found for the first time that vimogii can improve the lysosome function of T cells by enhancing TFE3 protein expression, and the blank in the field is filled. According to the application, a new treatment thought is provided for improving immune aging related diseases of old people, the T cell lysosome function is enhanced by enhancing TFE3 protein expression through the medicine, then T cell aging is delayed and / or reversed, and an innovative application direction is provided for intervention of an aging immune system.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to the use of Vimodegib in the preparation of products for delaying and / or reversing T cell aging. Background Art

[0002] As people age, the function of the body's immune system gradually declines, among which the functional decline of T cells is an important sign of immune aging. T cell aging not only affects the efficacy of immune response, but also may lead to the occurrence and progression of aging-related diseases. Especially in tumor immune surveillance, the function of aging T cells is impaired, which manifests as immune escape and decreased ability to recognize and eliminate tumor cells. The cytokine expression level of aged T cells decreases, especially key anti-tumor cytokines such as GZMB (Granzyme B), IFN-γ (Interferon-γ), TNF-α (Tumour necrosis factor-alpha), and IL-2 (Interleukin-2), which makes the immune system unable to effectively respond to the invasion of tumor cells. In addition, aging T cells usually show weakened proliferation ability, decreased effector function, and enhanced immune tolerance. This decline in immune function not only leads to insufficient anti-tumor response, but also makes individuals more susceptible to viral infection and other immune-related diseases. Therefore, reversing T cell aging and restoring its immune function have become the key to improving the immune health level of the elderly.

[0003] Lysosomes are important degradation and metabolic organs in cells, mainly responsible for the removal of intracellular waste, cell autophagy and the decomposition of exogenous substances. It plays a vital role in maintaining intracellular homeostasis, regulating immune responses and responding to stress responses. In T cells, lysosomes not only play an important role in cell autophagy, antigen presentation and cytokine release, but also participate in the metabolic regulation of T cells. With the increase of age, lysosomal function gradually declines, resulting in the weakening of T cell metabolic capacity and autophagy, which makes it impossible for T cells to effectively remove damaged organelles or molecules, thereby affecting their function. TFE3 (Transcription Factor E3) is one of the key transcription factors for lysosomal biogenesis and functional regulation. It can enhance the cell's autophagy and lysosomal function by regulating the expression of lysosome-related genes. Enhancing TFE3 protein expression not only helps to remove damaged cell components, but also improves the responsiveness of T cells to external stimuli. Therefore, enhancing TFE3 expression is expected to reverse T cell aging and improve the decline of immune function in the elderly.

[0004] There are currently no existing drugs that enhance TFE3 protein expression to improve T cell lysosomal function, and existing immunosenescence intervention measures focus on aspects such as immune regulation, while ignoring the key role of lysosomal function. Vimodegib is a small molecule drug that is mainly used to treat basal cell carcinoma (BCC). Vimodegib works by inhibiting the Hedgehog signaling pathway. The Hedgehog signaling pathway plays an important role in the proliferation, survival and metastasis of tumor cells, especially in tumors such as basal cell carcinoma, where this pathway is often abnormally activated. Vimodegib blocks the activation of this signaling pathway by inhibiting the Smoothened (SMO) protein in the Hedgehog signaling pathway, thereby inhibiting the growth and spread of tumor cells. Vimodegib is approved by the FDA for the treatment of metastatic or advanced localized basal cell carcinoma, especially in cases that are ineffective for surgery or radiotherapy. It is also used to treat patients with basal cell carcinoma who cannot undergo surgery or radiotherapy. In addition, Vimodegib is also being explored in clinical studies in the treatment of other tumors, especially those tumors associated with abnormal activation of the Hedgehog signaling pathway. The molecular formula of Vimodegib is C 19 H 14 Cl2N2O3S, molecular weight is 421.3. Summary of the invention

[0005] At present, the research on Vimodegib in the field of T cell aging is still insufficient, and the research on this type of drug in T cell aging-related diseases is the focus of the present invention. In view of the problems existing in the above background technology, the present invention provides the use of Vimodegib in the preparation of products for delaying and / or reversing T cell aging.

[0006] The present invention provides the use of Vimodegib in the preparation of products for delaying and / or reversing T cell aging. Through experiments, it was found that Vimodegib can significantly enhance the expression of TFE3 protein, thereby enhancing the lysosomal function of T cells, and enhancing the expression of anti-tumor and anti-viral related cytokines, including GZMB, IFN-γ, TNF-α, IL-2, etc., thereby improving the lysosomal function of T cells in the elderly and enhancing the ability of the elderly to resist tumors and viral infections.

[0007] The present invention discovered for the first time that Vimodegib can improve the lysosomal function of T cells by enhancing the expression of TFE3 protein, filling the gap in this field. The present invention not only provides a new treatment idea for improving immune aging-related diseases in the elderly, but also provides an innovative application direction for the intervention of the aging immune system. The present invention relates to a method for improving T cell aging-related diseases such as immune aging, tumor immune escape, and viral infection by enhancing the expression of TFE3 protein and then enhancing the lysosomal function of T cells. Therefore, the new drug use of Vimodegib has significant clinical application prospects and scientific value.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The present invention provides the use of Vimodegib in preparing a product for delaying and / or reversing T cell aging.

[0010] Furthermore, the product is a medicine.

[0011] Furthermore, Vimodegib delays and / or reverses T cell aging by improving T cell lysosomal function; the improvement of lysosomal function is achieved by enhancing TFE3 protein expression.

[0012] Furthermore, the delay and / or reversal of T cell senescence is manifested in the enhancement of the expression of GZMB, IFN-γ, TNF-α and IL-2 cytokines.

[0013] Furthermore, the drug contains one or more pharmaceutically acceptable carriers.

[0014] Furthermore, the drug dosage form is selected from injection, solution, emulsion, suspension, suppository, ointment, cream, spray, drop, powder, granule, granule, capsule, pill, tablet, patch or sustained-release preparation.

[0015] The present invention also provides an application of a composition containing Vimodegib in the preparation of a product for delaying and / or reversing T cell aging.

[0016] Furthermore, the composition is a pharmaceutical composition.

[0017] The present invention also provides the use of Vismodegib in the preparation of CAR-T cell products.

[0018] In addition, the present invention also provides the use of Vimodegib in the combined use of CAR-T in vivo.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The present invention proposes for the first time that Vismodegib enhances T cell lysosomal function by enhancing TFE3 protein expression, and provides a new application direction for delaying and / or reversing T cell aging. Existing studies have mostly focused on the role of Vismodegib in inhibiting the Hedgehog signaling pathway, but the present invention reveals its potential in immune aging and T cell function recovery, especially in the treatment of immune aging-related diseases (such as tumors, viral infections, etc.), which has innovative value.

[0021] (2) The present invention systematically verifies the effects of Vimodegib in enhancing TFE3 expression, improving T cell lysosomal function, and enhancing the expression of GZMB, IFN-γ, TNF-α, and IL-2 cytokines in T cells through various technical means such as high-throughput flow cytometry, Western blot, and flow cytometry detection. Through these experiments, the effectiveness of Vimodegib in delaying and / or reversing T cell aging is further confirmed, providing a strong experimental basis for its potential therapeutic drug for T cell aging-related diseases.

[0022] (3) The present invention constructs a novel small molecule drug screening platform that can efficiently screen candidate drug molecules that can enhance TFE3 protein expression and improve T cell lysosomal function. This screening system is not only applicable to Vimodegib, but can also be extended to other potential therapeutic drugs, providing new ideas and methods for the development of drugs for related immune aging diseases.

[0023] (4) By enhancing TFE3 protein expression, Vimodegib can effectively improve the lysosomal function of elderly T cells, enhance immune response, and is expected to enhance their anti-tumor and anti-viral capabilities. The present invention provides an innovative drug application scheme for the treatment of T cell aging-related immune diseases, and has broad clinical application prospects, especially in the elderly population. It has significant potential to delay immune decline, enhance immune function, and improve the quality of life of the elderly population.

[0024] (5) Compared with traditional immunotherapy drugs, Vimodegib, as an FDA-approved drug, has sufficient clinical data to support its safety and tolerability. The present invention opens up a new therapeutic area for the application of this drug, and is expected to have a better effect in enhancing TFE3 protein expression and restoring immune function, while having lower side effects, and can provide patients with a safer and more effective treatment plan. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The following is a flow chart of the entire drug screening experiment involved in Examples 1-3;

[0026] Figure 2 These are the 16 drug molecules screened in Example 1;

[0027] Figure 3 The Western Blot test results in Example 2 (Figure a is a protein expression map; Figure b is a quantitative statistical graph of protein expression);

[0028] Figure 4 This is a graph showing the cytokine expression results in Example 3; DETAILED DESCRIPTION

[0029] In order to better illustrate the present invention, the following embodiments are listed. Obviously, the described embodiments are only a part of the present invention, not all embodiments. Based on the embodiments in the present invention, other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0030] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0031] Example 1 High-throughput flow cytometry to evaluate the effect of drugs on the ability of lysosomes to degrade DQ-BSA

[0032] DQ-BSA (fluorescent dye-labeled bovine serum albumin) is a substrate commonly used in lysosomal function assessment, which can release fluorescent signals after being degraded in lysosomes. In an embodiment, cells were treated with drugs for 48 hours, and DQ-BSA was added to treat for 4 hours, and then a high-throughput flow cytometer was used to detect the changes in the fluorescence intensity of DQ in the cells. The fluorescence signal intensity of DQ is proportional to the lysosomal degradation ability, and the drug can significantly improve the fluorescence signal of DQ-BSA by enhancing the degradation function of lysosomes. This method can efficiently and quantitatively evaluate the impact of drugs on lysosomal function, and screen out candidate drug molecules with the potential to improve lysosomal function. Through this technology, important experimental basis can be provided for subsequent drug screening and mechanism research.

[0033] 1. Materials and Methods

[0034] 1.1 Drugs and reagents

[0035] MCE drug molecule library (No.: HY-L0001, manufacturer: MCE); DQ TM BSAs (Cat. No.: D-12050) were purchased from Thermofisher; Jurkat (human T lymphocyte tumor cells) (Cat. No.: C6456) were purchased from Bio-Technology Co., Ltd.; PBS (phosphate buffered saline containing calcium and magnesium) (Cat. No.: 14040133), FBS (Cat. No.: A5670701), RPMI-1640 culture medium, GlutaMAX TMSupplement (Cat. No.: 61870036) and penicillin-streptomycin (10000U / mL) (Cat. No.: 15140122) were purchased from Gibco TM company.

[0036] 1.2 Experimental methods

[0037] (1) Jurkat cells were inoculated into culture medium and cultured to a cell density of 80%-95%. After the culture was completed, the cells were inoculated into a 96-well plate, with 0.15 million cells in each well. The experimental group was treated with 10 mM of different drugs for 48 hours, and the control group was not treated with any drugs.

[0038] (2) After 48 hours, DQ-BSA (10 ug / mL) was added and the cells were cultured for another 4 hours to ensure that DQ-BSA was degraded by lysosomes.

[0039] (3) High-throughput flow cytometry was used to detect the fluorescence intensity of DQ in cells.

[0040] The intensity of DQ fluorescence signal reflects the degradation ability of lysosomes. The fluorescence signal of each drug-treated group was recorded and compared with the control group to screen out drug molecules that can significantly enhance the degradation function of lysosomes.

[0041] 1.3 Detection indicators

[0042] DQ fluorescence signal intensity: Flow cytometry was used to detect the DQ fluorescence intensity in cells after drug treatment to evaluate the degradation capacity of lysosomes. The intensity of the fluorescence signal is proportional to the degradation capacity of lysosomes. The higher the fluorescence intensity, the stronger the lysosomal function.

[0043] 1.4 Experimental Results

[0044] After detecting the effect of drugs on the ability of lysosomes to degrade DQ-BSA by high-throughput flow cytometry, the present invention screened out the top 16 drug molecules with the best performance. These drug molecules showed strong DQ fluorescence signals. In the experiment, after the cells were treated with drugs for 48 hours, DQ-BSA was added as a substrate for lysosomal degradation. Subsequently, the fluorescence intensity in the cells was detected using a high-throughput flow cytometer. According to the intensity change of the fluorescence signal, we can quantitatively evaluate the degree of improvement of the drug on lysosomal function. The results showed that the top 16 drug molecules performed outstandingly in enhancing the lysosomal degradation function, and the intensity of the fluorescence signal was significantly increased compared with the control group. Further analysis showed that these drug molecules can effectively activate the degradation activity of lysosomes, promote the degradation of DQ-BSA in lysosomes, and significantly improve lysosomal function. Based on these results, the present invention selected 16 candidate drug molecules as the focus of subsequent research. Next, the present invention will further explore the mechanism of action of these molecules and conduct in-depth verification of their potential in the restoration of T cell lysosomal function.

[0045] Example 2 Western Blot Detection of the Effect of Drugs on TFE3 Protein Expression

[0046] Western Blot detection of the effect of drugs on TFE3 protein expression is a common protein analysis technique for evaluating the effect of drugs in cells. TFE3, as a key transcription factor for lysosomal function regulation, participates in regulating the expression of lysosomal related genes and plays an important role in cell autophagy and metabolic function. In the present embodiment, by comparing the relative expression intensity of TFE3 protein in the drug-treated group and the control group, the effect of drugs on TFE3 protein expression is evaluated. If the drug can significantly increase the expression of TFE3, it means that the drug may enhance lysosomal function and cell autophagy ability through TFE3 mediation. This experiment provides a reliable experimental basis for further exploring the effect of drugs on lysosomal function and TFE3 regulatory mechanism, and lays a foundation for drug screening and its functional research.

[0047] 1 Materials and methods

[0048] 1.1 Drugs and reagents

[0049] Candidate drug molecule library (the drugs in the molecule library are the 16 drugs screened in Example 1 above); PBS (phosphate buffer containing calcium and magnesium), FBS, RPMI-1640 culture medium, GlutaMAX TM Supplements, Penicillin-Streptomycin (10000U / mL), Dynabeads TM Human T activator CD3 / CD28 (Cat. No. 11131D) were purchased from Gibco TM Company: EasySep TM Human CD8 + T Cell Isolation Kit (Cat. No. 17968) was purchased from Stemcell; Anti-rabbit IgG, HRP-linked Antibody (Cat. No. 7074P2) was purchased from CST; Clarity Max Western ECL Substrate (Cat. No. 1705062) and Trans-Blot Turbo 5x Transfer Buffer, 1L (Cat. No. 10026938) were purchased from Bio-Rad; Bis-Tris 4-20% high-resolution precast gel (Bis-Tris) (15 wells) (Cat. No. 36256ES10), 10×Tris-Glycine-SDS electrophoresis buffer (Cat. No. 20319ES76), and 5×SDS-PAGE protein loading buffer (Cat. No. 20315ES) were purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; Amersham Protran 0.45NC nitrocellulose protein blotting membrane (Cat. No.: 10600002) was purchased from Cytiva; β-Actin (13E5) Rabbit mAb (Cat. No.: 4970S) and TFE3 (E6T9M) Rabbit mAb (Cat. No.: 81744T) were purchased from CST; 75% alcohol (Cat. No.: 64-17-5) was purchased from Nanshi Company; protease phosphatase inhibitor cocktail (universal type, 50X) (Cat. No.: P1045), colored pre-stained protein molecular weight standard (10-180kD) (Cat. No.: P0068), and skimmed milk powder (Blotting Grade) (Cat. No.: P0216-300g) were purchased from Biyuntian Biotechnology Co., Ltd.; BSA (Cat. No.: 126A028) was purchased from ABSIN; PBMC.

[0050] 1.2 Experimental methods

[0051] (1) Cell culture and drug treatment: Peripheral blood mononuclear cell samples of healthy adults were purchased from Shanghai Saili Bio-Pharmaceutical Co., Ltd., and naive CD8 + T cells were isolated by magnetic bead sorting. + T cells. The cells were seeded into 96-well plates and naive CD8 + T cells and Dynabeads TMHuman T activator CD3 / CD28 was activated with a ratio of 5 magnetic beads to each cell, using RPMI-1640 medium, adding 10% FBS and 1% double antibody (penicillin-streptomycin (10000U / mL)), cultured in a 37°C, 5% CO2 incubator, and added 10mM of drugs after 24 hours for 48 hours. No drugs were added to the control group;

[0052] (2) Protein extraction: The treated T cells in each well were lysed with 50 μL of RIPA buffer and 1:50 of phosphatase inhibitor and 1:50 of protease inhibitor were added;

[0053] (3) SDS-PAGE electrophoresis: Equal amounts of protein (20 μg) were loaded onto a high-resolution precast gel and separated by electrophoresis according to protein size at 90 V for 60 min.

[0054] (4) Protein transfer: The separated proteins were transferred to the NC membrane under the following conditions: using a transfer spectrometer at 2.5A for 7 min;

[0055] (5) Blocking and antibody incubation: 5% skim milk powder was used for blocking and incubation at room temperature for 1 hour. The cells were incubated with a specific primary antibody against TFE3 (1:1000 diluted with 5% BSA antibody diluent) at 4°C overnight.

[0056] (6) Secondary antibody incubation and color development: Incubate with HRP-labeled secondary antibody at room temperature for 1 hour. Perform chemiluminescence detection using ECL color development reagent, and capture images using a gel imaging system.

[0057] (7) Data analysis: The intensity of the TFE3 protein band was quantitatively analyzed using the image analysis software ImageJ V1.8.0.112. The effect of the drug on TFE3 protein expression was evaluated by comparing the relative expression levels of TFE3 in the drug-treated group and the control group.

[0058] 1.3 Detection indicators

[0059] The expression of TFE3 protein was quantitatively analyzed by Western Blot band intensity, and the difference between the drug-treated group and the control group was compared.

[0060] 1.4 Experimental Results

[0061] The experimental results showed that both vismodegib and seroquel significantly enhanced the expression of TFE3 protein after treatment. Western Blot analysis showed that Figure 3. After treatment with vismodegib, the expression of TFE3 protein increased significantly, and the band intensity was significantly higher than that of the control group, indicating that vismodegib can effectively enhance the expression of TFE3. In addition, seroquel also showed the effect of enhancing the expression of TFE3 protein. Although its effect was slightly weaker than that of vismodegib, it was still significantly higher than that of the control group. This shows that seroquel also has the potential to improve lysosomal function by enhancing the expression of TFE3 protein. Comprehensive analysis shows that both vismodegib and seroquel can increase the expression of TFE3 protein under experimental conditions, providing a strong molecular basis for the enhancement of lysosomal function, and laying the foundation for further research on the application of these drugs in immunosenescence or aging-related diseases.

[0062] Example 3 Flow cytometry detection of the effects of drugs on the expression of multiple cytokines

[0063] The experiment aims to detect the effect of drugs on the expression of various cytokines by flow cytometry, and to evaluate whether the drugs can enhance the immune function of T cells by enhancing the expression of TFE3 protein. Specifically, we focused on detecting cytokines closely related to T cell function, including GZMB, IFN-γ, TNF-α and IL-2. These cytokines play a vital role in immune response, especially in anti-tumor and antiviral responses. Through flow cytometry, we can quantitatively analyze the levels of various cytokines secreted by T cells after drug treatment, so as to understand the specific regulatory effects of drugs on T cell effector function. The experimental results will help us reveal whether drugs can improve the immune function of T cells by affecting the expression of TFE3, especially in the context of decreased immune function caused by aging, and provide important basis for subsequent drug screening and clinical application.

[0064] 1 Materials and methods

[0065] 1.1 Drugs and reagents

[0066] Vimodegib; Sertrolast; PBS (phosphate buffered saline containing calcium and magnesium), FBS, RPMI-1640 medium, GlutaMAX TM Supplements, Penicillin-Streptomycin (10000U / mL); EasySep TM Human CD8 + T CellIsolation Kit, Dynabeads TMHuman T activator CD3 / CD28; Cell Activation Cocktail (with Brefeldin A) (Cat. No.: 423303), PE anti-human / mouse Granzyme B Recombinant Antibody (Cat. No.: 372207), APC anti-human TNF-α Antibody (Cat. No.: 502913), Pacific Blue TM anti-human IFN-γAntibody (Cat. No. 502521), Brilliant Violet 605 TM anti-human IL-2Antibody(Cat. No.: 500331), PE / Dazzle TM 594anti-human CD8 Antibody (Cat. No. 344743) were purchased from Biolegend; eBioscience TM Fixable Viability Dye eFluor TM 780 (Cat. No.: 65-0865-14) was purchased from Thermo Fisher Scientific; Fixation and Permeabilization Solution (Cat. No.: 554714) was purchased from BD Cytofix / Cytoperm TM Company; PBMC.

[0067] 1.2 Detection indicators

[0068] In this example, flow cytometry was used to detect the fluorescence intensity of cytokines such as GZMB, IFN-γ, TNF-α, and IL-2.

[0069] 1.3 Experimental Results

[0070] The effects of drugs on the expression of cytokines such as GZMB, IFN-γ, TNF-α and IL-2 were detected by flow cytometry. Figure 4 As shown. Both Vimodegib and Setralast significantly upregulated the production of these immune-related cytokines. In the Vimodegib and Setralast treatment groups, the expression levels of GZMB, IFN-γ, TNF-α and IL-2 were significantly increased, especially in the Vimodegib treatment group, the expression levels of all detected cytokines showed a significant increase. These results indicate that Vimodegib and Setralast can enhance CD8 + The immune function of T cells promotes anti-tumor and anti-viral immune responses.

[0071] Our research shows that Vimodegib can enhance TFE3 protein expression through a new mechanism of action, thereby promoting the enhancement of lysosomal function. Vimodegib's enhancement of TFE3 protein expression can reverse the decline of lysosomal function of T cells in the elderly and enhance the expression of cytokines by T cells when they encounter infection or tumor stimulation, providing a new drug option for the treatment of immune aging-related diseases. This discovery provides a new direction for the development of drugs based on enhancing TFE3 protein expression.

[0072] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. The use of Vimodegib in the preparation of products for delaying and / or reversing T cell aging.

2. The use according to claim 1, characterized in that The product described is a drug.

3. The use according to claim 1, characterized in that Vimodegib delays and / or reverses T cell aging by improving T cell lysosomal function; the improvement of lysosomal function is achieved by enhancing TFE3 protein expression.

4. The use according to claim 1, characterized in that The delay and / or reversal of T cell senescence is manifested in the enhancement of the expression of GZMB, IFN-γ, TNF-α and IL-2 cytokines.

5. The use according to claim 2, characterized in that The medicament comprises one or more pharmaceutically acceptable carriers.

6. The use according to claim 5, characterized in that The pharmaceutical dosage form is selected from injection, solution, emulsion, suspension, suppository, ointment, cream, spray, drop, powder, granule, granule, capsule, pill, tablet, patch or sustained-release preparation.

7. Use of a composition containing Vimodegib in the preparation of a product for delaying and / or reversing T cell aging.

8. The use according to claim 7, characterized in that The composition is a pharmaceutical composition.

9. Application of Vimodegib in the preparation of CAR-T cell products.

10. Application of Vimodegib in combination therapy of CAR-T in vivo.

Images