Application of vitamin B12 in preparation of adjuvant therapy agent for preventing or treating colorectal cancer precancerous lesions

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of vitamin B12 in preparation of an auxiliary therapeutic agent for preventing or treating colorectal cancer precancerous lesions. The invention aims to solve the technical problem of providing a novel prevention and intervention means for colorectal cancer (CAC) caused by the cancerization process of colorectal adenoma and inflammatory atypical hyperplasia. The invention further provides a preparation method of the novel prevention and intervention means for the colorectal cancer caused by the cancerization process of the colorectal adenoma and the inflammatory atypical hyperplasia of the colorectal adenoma and the colorectal cancer caused by the inflammatory atypical hyperplasia of the colorectal adenoma. In a colorectal adenoma canceration process, the level of vitamin B12 in a circulatory system is reduced along with the development of diseases. In inflammatory and hereditary colorectal cancer mouse models, MHC-II signals can be enhanced by supplementing medicine vitamin B12, and CD4 effect memory T cell immunity can be reactivated. The invention provides a new thought for further researching the immune recovery strategy in the colorectal cancer precancerous lesion process and the medical application of the vitamin B12.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to vitamin B. 12 Application in the preparation of adjuvant therapy agents for the prevention or treatment of precancerous lesions of colorectal cancer. Background Technology

[0002] High Risk of Colorectal Adenomas Developing into Colorectal Cancer: Early disruption of immune surveillance is a key event in the evolution of colorectal tumors and a major obstacle to effectively intercepting cancer. Although advances in screening and treatment have improved the prognosis of colorectal cancer patients, most are diagnosed after malignant transformation, at which point treatment response is limited and immune escape has already occurred. Precancerous lesions, including colorectal adenomas and inflammation-associated dysplasia, are frequently detected in other healthy individuals. Therefore, rapidly distinguishing between a healthy state and a precancerous lesion requires not only early detection but also mechanistic risk stratification to identify lesions most likely to progress to malignancy, a pressing technical challenge in this field. Furthermore, the mechanism by which immune surveillance is disrupted in the earliest stages of colorectal tumorigenesis remains unclear, resulting in a lack of therapeutic means to prevent early colorectal cancer development. Summary of the Invention

[0003] Purpose of the invention: The technical problem this invention aims to solve is to provide a drug capable of restoring immune surveillance in precancerous lesions of colorectal cancer. Vitamin B1 12 The serum concentration of vitamin B in patients at all stages of precancerous lesions of colorectal cancer showed a decreasing trend, while vitamin B supplementation... 12 It can effectively inhibit precancerous lesions of colorectal cancer. This invention further investigates immune recovery strategies and vitamin B during the process of precancerous colorectal lesions. 12 This provides new insights into its medicinal applications.

[0004] This invention aims to provide vitamin B. 12 Treatment and prevention of precancerous lesions of colorectal cancer, in order to address the lack of drugs targeting immune surveillance recovery therapy among existing treatments for colorectal cancer.

[0005] Technical solution Vitamin B 12 Application in the preparation of adjuvant therapy agents for the prevention or treatment of precancerous lesions of colorectal cancer.

[0006] Vitamin B 12 Application in the preparation of drugs to restore precancerous immune function in colorectal cancer.

[0007] The application is characterized in that the restoration of precancerous immune function in colorectal cancer is achieved by restoring the immune surveillance function of the tertiary lymphoid structure.

[0008] The application is characterized in that the immune surveillance function of the tertiary lymphoid structure leads to an increase in CD74 molecule expression, enhances MHC-II signal transduction, and reactivates effector memory T cells, restoring the adaptive immune response.

[0009] The application is characterized in that the vitamin B 12 It is at least one of the active forms of methylcobalamin, 5'-deoxyadenosylcobalamin, and cyanocobalamin.

[0010] The application is characterized in that the adjuvant therapeutic agent is any one of the following dosage forms: oral preparation, injectable preparation, or intestinal-targeted preparation.

[0011] The application is characterized in that the reactivated effector memory T cells increase CD4+. + Effector memory cells and CD127 + KLRG1 - Memory-like T cell population achieved.

[0012] Treatment of colorectal adenomas: For high-risk patients with a history of colorectal adenomas, vitamin B... 12 It can be used as an adjunct therapy to restore immune function and reduce the risk of cancer.

[0013] Treatment of inflammation-related dysplasia: For patients with inflammatory dysplasia, vitamin B... 12 Supplementation can restore immune surveillance function, inhibit the progression of inflammation, and slow down the carcinogenesis process.

[0014] Monitoring and assessment: During treatment, serum vitamin B levels should be monitored regularly. 12 Vitamin B levels, immune function (such as activation of CD4-positive T cells and MHC-II signaling pathway), and colonoscopy results were used to assess vitamin B. 12 The effectiveness and safety of the treatment.

[0015] Beneficial effects Vitamin B 12 Vitamin B1 is a water-soluble vitamin involved in DNA synthesis, cell division, and methylation reactions, playing a crucial role in maintaining genome stability and regulating homocysteine ​​metabolism. Since the occurrence of precancerous lesions of colorectal cancer is closely related to DNA damage, abnormal methylation, and chronic inflammation, vitamin B1 is essential. 12 It improves DNA repair, maintains normal methylation levels, and reduces oxidative stress, but there are no reports of it restoring precancerous immune function.

[0016] This invention discovers and verifies vitamin B through analysis of clinical samples and animal models. 12Serum vitamin B levels decrease at all stages of precancerous lesions in colorectal cancer, and as the disease progresses, the immune surveillance function of the tertiary lymphoid structures is gradually lost. Supplementation with exogenous vitamin B... 12 It can restore some immune function of the tertiary lymphoid structure, increase the expression of CD74 molecules, enhance MHC-II signal transduction, and reactivate effector memory T cells. Attached Figure Description

[0017] Figure 1 The spatial transcriptomics analysis data presented in Example 1 show that the tertiary lymphoid structure score decreases with cancer progression. A is the H&E staining map of the tissue adjacent to the slides measured by spatial transcriptomics; B is the spatial niche embedding of all spatial points, stained by the annotated niche cell type; C is the tertiary lymphoid structure score of each patient group; D is the analysis results of spatial transcriptomics verified by immunohistochemistry (IHC); E is the pathway enrichment of the tertiary lymphoid structure region; F is a dot plot of representative chemokine expression; G is the spatial tertiary lymphoid structure score and expression of key chemokines of representative patients.

[0018] Figure 2 The following figures illustrate the various pathological staining patterns of clinical sample sections from Example 2, accompanied by relevant statistical bar charts. Figures AB show the structural and functional staining of the tertiary lymphoid structures in each group of patients; Figure C shows the statistical bar charts of immunostaining of relevant functional molecules; Figure D shows the changes in tertiary lymphoid structure components and antigen-presenting markers at each stage.

[0019] Figure 3 Example 3: Vitamin B 12 The selection criteria and their improvement on animal models of chronic enteritis are presented, with A being an experimental design flowchart; B is a heatmap showing the top 10 drugs predicting sentinel niche reversal based on OCTAD analysis, highlighting cyanocobalamin (vitamin B12). 12 ) is a leading candidate drug; C represents the gene ontology (GO) enrichment analysis of vitamin B. 12 The gene involved in the treatment regulation; D is a schematic diagram of the chronic enteritis animal model; E shows the intestinal length, tumor, and spleen morphology of mice in each group; F shows the length of the colon and rectum; G shows the spleen weight of each group; H shows the serum vitamin B level of mice in each group. 12 Concentration; I represents serum vitamin B in patients with different diseases. 12 Concentration; JK represents representative H&E images and histological scores of colon tissue from mice in the treatment group; L represents vitamin B in the AOM-DSS model. 12 It restored antigen processing and presentation, the MHC class II mediated pathway, and the procedures related to lymphocyte differentiation. MN is vitamin B. 12Representative immunofluorescence images and statistical graphs of CD74 recovery; O represents the gating strategy for T cell population flow cytometry analysis; P represents the flow cytometry results for vitamin B12. 12 Increased CD4 in the spleen of AOM-DSS model mice + Effector memory T cells and CD127 + KLRG1 - Memory-like T cell population; Q is a statistical graph of the P plot.

[0020] Figure 4 Example 4: Vitamin B 12 The therapeutic effect in animal models of intestinal adenoma. A is a schematic diagram of the intestinal adenoma animal model; B shows the intestinal length, tumor, and spleen morphology of mice in each group; CD shows representative H&E images and histological scores of colon tissue in the treatment group mice; E is a UMAP map integrating single-cell transcriptional atlases from the three groups; F is a UMAP map showing the annotation of each cell cluster; G shows the subpopulation classification annotation of each cell cluster; H is a heatmap of marker gene expression in major cell subpopulations for cell type annotation; I shows vitamin B... 12 Changes in cell abundance in each group; J represents the enhanced receptor-ligand communication intensity after treatment; K is a heatmap of the overall signaling patterns of the three key pathways; LM shows representative immunofluorescence images and statistical plots in a larger cohort; N is flow cytometry evidence of vitamin B12. 12 Increased Apc min / + CD4 in mouse spleen + Effector memory T cells and CD127 + KLRG1 - Memory-like T cell population; O plot is a statistical plot of N plot in a larger sample. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used are all commercially available.

[0022] Example 1: Spatial transcriptome analysis identifies the loss of tertiary lymphoid structures as the disease progresses. 1. Experimental Methods 1.1 Spatial transcriptomic data of clinical samples from patients with adenomas and colorectal cancer To investigate whether the tertiary lymphoid structure changes during the progression of colorectal cancer, we performed spatial transcriptome sequencing on clinically obtained adenoma borders (AD_Border), adenoma centers (AD_Center), colorectal cancer borders (CRC_Adacent), and colorectal cancer centers (CRC_Center), and scored the tertiary lymphoid structure (TLS_Score).

[0023] 1.2 Immunohistochemical experiments on pathological sections Intestinal tissue was fixed and dehydrated in 4% paraformaldehyde solution and then embedded in paraffin. Sequence sections (6 μm) were excised, antigen retrieval was performed using sodium citrate, the membrane was perforated with 0.2% Triton X-100, blocked with an endogenous peroxidase inhibitor, and then blocked with 5% BSA for 1 hour. The sections were then incubated overnight with primary antibody at 4°C. After washing following overnight incubation with primary antibody, the sections were incubated with the appropriate secondary antibody, and finally, DAB staining and hematoxylin staining were performed.

[0024] 1.3 H&E staining experiment on pathological sections After dewaxing and hydration of paraffin sections of tissue samples, hematoxylin staining was performed first. The sections were then placed in 1% hydrochloric acid alcohol for differentiation until the cell nuclei were clearly visible. Eosin staining was then performed, and finally the sections were dehydrated and mounted with neutral resin.

[0025] 2. Experimental Results Spatial transcriptomic analysis suggests that the development of colorectal tumors is accompanied by a shift from an ordered immune microenvironment at the adenoma periphery to an immune-rejecting cancerous core. Figure 1 AD). Precancerous lesions possess functional tumor suppressor mechanisms, including antigen presentation and chemokine-mediated lymphocyte recruitment. Figure 1 EG), and malignant progression is accompanied by the disintegration of this immune structure ( Figure 1 These results indicate that tertiary lymphoid structures still possess immune surveillance functions in the early stages of cancer. However, as colorectal cancer progresses and the tertiary lymphoid structures become spatially closer to the tumor parenchyma, they gradually collapse or fail to develop into structurally and functionally mature tertiary lymphoid structures, leading to further tumor progression and deterioration.

[0026] The tertiary lymphoid structure is composed of immune cells such as T cells and B cells that aggregate in non-lymphoid tissues, and can directly initiate and maintain anti-tumor immune responses in the local tumor microenvironment.

[0027] Example 2: Pathological section staining verifies the loss of tertiary lymphoid structures as the lesion deepens and the disease progresses. 1. Experimental Methods 1.1 HE staining experiment After dewaxing and hydration of paraffin sections of tissue samples, hematoxylin staining was performed first. The sections were then placed in 1% hydrochloric acid alcohol for differentiation until the cell nuclei were clearly visible. Eosin staining was then performed, and finally the sections were dehydrated and mounted with neutral resin.

[0028] 1.2 Immunohistochemical experiments on pathological sections Intestinal tissue was fixed and dehydrated in 4% paraformaldehyde solution and then embedded in paraffin. Sequence sections (6 μm) were excised, antigen retrieval was performed using sodium citrate, the membrane was perforated with 0.2% Triton X-100, blocked with an endogenous peroxidase inhibitor, and then blocked with 5% BSA for 1 hour. The sections were then incubated overnight with primary antibody at 4°C. After washing following overnight incubation with primary antibody, the sections were incubated with the appropriate secondary antibody, and finally, DAB staining and hematoxylin staining were performed.

[0029] 1.3 Immunofluorescence assay Dewaxing and hydration of paraffin sections: Sections were sequentially immersed in xylene for 30 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% ethanol for 5 min, 85% ethanol for 5 min, and 75% ethanol for 5 min, followed by washing with distilled water. Antigen retrieval was performed using citrate retrieval buffer, endogenous peroxidase was blocked with 3% hydrogen peroxide solution, and 3% goat serum was blocked at room temperature for 30 min. Primary antibody incubation was performed by adding diluted primary antibody to the sections and incubating overnight at 4°C. The next day, secondary antibody (poly-HRP) was incubated. The ready-to-use poly-HRP goat anti-mouse / rabbit universal secondary antibody (AFIHC001) was used. Concentrated fluorescent dye and TSA buffer were mixed evenly at a ratio of 1:50-1:200. The prepared TSA fluorescent dye reaction solution was added to the sections and evenly covered the tissue, reacting at room temperature for 1-15 min. Cell nuclei were counterstained with DAPI, and the sections were mounted with an anti-fluorescence quencher. Nail polish was then applied around the edges.

[0030] 2. Experimental Results Pathological examination revealed that as the lesion progressed deeper, significant loss of CD3 and CD20 molecules, representing the tertiary lymphoid structure, occurred. Furthermore, markers symbolizing follicular dendritic cells (CD21), activated / germinal center B cells (CD23, AID, BCL6), and the antigen-presenting microenvironment (LAMP3, HLA-DR, CD86) were also lost. Figure 2 A). This change was also observed in tissue samples from patients at different disease stages, including healthy tissue (HC), ulcerative colitis patients (UC), adenoma patients (AD), and colorectal cancer patients (CRC). Figure 2 B). The figure shows the pathological tissue staining results of representative individuals; a larger patient cohort is represented by [data missing]. Figure 2C, D. The above results show that as early lesions progress to cancer, these immune microenvironments undergo synergistic structural decomposition and functional weakening, leading to decreased lymphocyte density, impaired antigen-presenting capacity, and loss of follicular structure. Therefore, the occurrence of colorectal tumors is accompanied by a transformation from an orderly, adaptively immune microenvironment to a fragmented and immunosuppressive state.

[0031] Example 3: Vitamin B 12 Screening and Vitamin B 12 Improvement effect of AOM-DSS on colorectal cancer mice 1. Experimental Methods 1.1 Drug Prediction Analysis Based on OCTAD Previous drug analyses were performed using the OCTAD framework. Differential gene expression analysis was conducted on epithelial cell clusters identified from single-cell RNA sequencing data. Differentially expressed genes (DEGs) were defined as having an absolute value of log2-adjusted fold change > 1 and an adjusted p-value < 0.05. Significantly upregulated and downregulated genes were screened to construct a disease gene tag representing the transcriptional state of precancerous epithelial cell clusters. The resulting gene tags were input into the OCTAD program, which integrates the disease signature with a drug-induced transcriptional perturbation profile from the LINCS L1000 database to calculate a reversal score. For each compound, the degree of inverse correlation between the drug-induced expression profile and the disease tag was quantified by calculating the reversal score based on enrichment. Compounds were ranked according to their predicted reversal potential.

[0032] 1.2 Constructing an AOM-DSS-induced mouse colitis model The AOM (azomethane)-DSS (dextran sulfate sodium)-induced chronic colorectal inflammation model in mice is a commonly used animal model for the inflammatory transformation of colorectal cancer. Healthy adult male mice (C57BL / 6J) were selected. In the first week, they were injected with 7.5 mg / kg of AOM. A 2% concentration of DSS was prepared by dissolving DSS powder in drinking water, ensuring free access to the water. The normal drinking water was replaced with the 2% DSS solution at weeks 2, 5, and 8. Vitamin B was administered starting at week 5. 12 Aqueous solution.

[0033] 1.3 H&E staining tissue score After dewaxing and hydration of paraffin sections of tissue samples, hematoxylin staining was performed first. The sections were then placed in 1% hydrochloric acid alcohol for differentiation until the cell nuclei were clearly visible. Eosin staining was then performed, and finally the sections were dehydrated and mounted with neutral resin.

[0034] 1.4 Flow cytometry Single-cell suspensions were prepared from mouse spleens and lymph nodes using a 70-micron cell filter, followed by erythrocyte lysis. Cells were washed and resuspended in staining buffer (PBS with 2% fetal bovine serum and 2 mmol EDTA). For cell viability assessment, cells were stained with cell-fixing viability dye 545 (BV510; Starter, S0D0014-100T) for 15 minutes at room temperature in the dark, followed by washing. Fc receptors were blocked for 10 minutes at 4°C using anti-mouse CD16 / 32 (Fc blocker) prior to surface antibody staining.

[0035] Then, the cells were stained in the dark at 4°C for 30 minutes with the following fluorescently labeled antibodies: anti-mouse CD45 (APC-Cy7; BioLegend, 103116), anti-mouse CD3ε (BV711; BD, 563123), anti-mouse CD4 (BV421; Elabscience, E-AB-F1097Q), anti-mouse CD8a (RB705; BD, 570255), anti-mouse CD44 (FITC; BD, 553133), anti-mouse CD62L (PE-Cy7; Elabscience, E-AB-F1011H), anti-mouse CD127 (PE; BD, 552543), and anti-mouse KLRG1 (APC; BD, 561620).

[0036] After staining, cells were washed and resuspended in staining buffer for collection. Data were acquired using an Antoon NxT flow cytometer (Thermo Fisher Scientific) and analyzed using FlowJo software (v10.8; BD Biosciences). Compensation was performed using single staining compensation beads or cells for each fluorescent dye.

[0037] T cells were set to live cells (Zombie cells) - ) and marked as CD45 + CD3 + Cells. Further analysis of CD4... + and CD8 + The expression of CD44 and CD62L in T cell subsets was used to determine the initial expression of CD44. - CD62L + Central Memory (CD44) + CD62L + ) and effect memory (CD44) + CD62L -Cell populations. Effector and memory differentiation status were further described by the expression of CD127 and KLRG1.

[0038] 2. Experimental Results Figure 3 A and B demonstrate vitamin B. 12 As the drug that disrupts the top 2, vitamin B 12 Functional annotations of regulatory genes highlight pathways associated with protein modification, membrane transport, calcium signaling, and chromatin regulation. Figure 3 C). In the AOM-DSS model, vitamin B was administered. 12 Significantly reduced the incidence of colitis-related tumors, manifested by improved colon length, reduced splenomegaly, and reduced histological tumor burden. Figure 3 In addition to validation in mice, we also examined circulating vitamin B at various stages of human disease. 12 Levels. Serum vitamin B levels were observed in 441 healthy controls (HC) to 442 patients with ulcerative colitis (UC), adenoma (AD), and colorectal cancer (CRC). 12 The concentration gradually decreased ( Figure 3 I). These data indicate that systemic vitamin B... 12 Insufficiency may be a factor contributing to the collapse of the immune microenvironment and failure of immune surveillance during the evolution of colorectal cancer.

[0039] CD74 expression was significantly reduced in AOM-DSS mice, and the tertiary lymphoid structure was significantly restored after vitamin B12 treatment. Figure 3 Flow cytometry analysis further showed that vitamin B12 treatment increased CD4+. + Effector memory cells (Tem) and CD127 + KLRG1 - Memory-like T cell population ( Figure 3 These results (OQ) indicate that vitamin B12 can restore adaptive immune responses.

[0040] Example 4: Vitamin B 12 For APC min / + Improvement effect in adenomatous mice 1. Experimental Methods 1.1 Construction of a spontaneous mouse adenoma model 2 Select healthy adult male mice C57BL / 6J-APC min / + Feeding patients with a high-fat diet (D12492) to promote adenoma development, and starting vitamin B supplementation in the fifth week. 12 Treatment (see) Figure 4 (As shown in A).

[0041] 1.2 Single-cell analysis The obtained 10×Chromium single-cell gene expression data were aligned, barcoded, and counted using the accompanying software CellRanger (v3.1.0) (using the reference set GRCh38-3.0.0). The filtered count matrix was converted to a sparse matrix using the Seurat package (v4.3.0) in R, and cells with fewer than 200 or more than 4000 expressed genes, as well as cells with more than 20% mitochondrial reads, were excluded from subsequent analysis. The filtered data were then log-normalized and scaled, while removing intercellular variations caused by UMI counts and percentage mitochondrial reads.

[0042] To avoid batch effects between samples and experiments, single-cell data were integrated using the Harmony fusion tool. Cell clustering was performed at a resolution of 0.8 using the "FindClusters" function, and cell identity was defined using the top 20 principal components (PCs). Dimensionality reduction was performed using the "RunUMAP" function, and visualization was performed using uniform manifold approximation projection (UMAP). For subpopulation cell clustering, different cell types were extracted and clustered at different resolutions based on their respective top 20 principal components according to visual inspection. The marker genes for each cluster were determined using the Wilcoxon rank-sum test ("FindAllMarkers" function, using default parameters).

[0043] 2. Experimental Results Similar to the AOM-DSS model, Apc min / + Mice exhibited increased tumor burden and splenomegaly, both of which were induced by vitamin B12. 12 Treatment significantly reduced ( Figure 4 BD). Histological analysis confirmed the application of vitamin B. 12 Subsequently, the severity of dysplasia decreased, and the mucosal structure improved. Integrated single-cell and spatial analyses showed that Apc min / + The immune microenvironment in mice was significantly remodeled. Figure 4 EH). Ro / e analysis showed that untreated Apc min / + The tumor showed a decrease in the lymphatic system and antigen-presenting cell populations, accompanied by an expansion of tumorigenic stromal subsets. Figure 4 I). Consistent with our previous observations in human samples and the AOM–DSS model, Apc min / + The overall ligand-receptor communication strength in mice was significantly reduced. Figure 4J–K), pathways related to antigen presentation and MHC-II signaling are specifically inhibited. Immunofluorescence analysis confirmed that Apc min / + CD74 expression was significantly reduced in tertiary lymphoid structures within tumors, but decreased in vitamin B12. 12 It was restored after treatment. Figure 4 L–M). These data further confirm the conclusion that CD74 deficiency is the key molecular event leading to impaired antigen-presenting microenvironment function during tumor development, and that vitamin B supplementation... 12 Afterward, the loss of CD74 will be reversed, and the expression of this key protein molecule will be restored.

[0044] Flow cytometry analysis showed that Apc min / + Tumors have the following characteristics: CD4 + The number of effector memory (Tem) cells was reduced, and CD127 was also reduced. + The number of KLRG1-like memory T cells is reduced, and these characteristics are observed when using vitamin B1. 12 All patients showed significant recovery after treatment. Figure 4 N). In summary, Vitamin B 12 It can enhance the immune surveillance function of tertiary lymphoid structures in inflammation-driven and gene-driven colorectal tumor models, delay the development of precancerous stages, and may improve the quality of life of patients with early-stage colorectal cancer.

Claims

1. Vitamin B 12 Application in the preparation of adjuvant therapy agents for the prevention or treatment of precancerous lesions of colorectal cancer.

2. Vitamin B 12 Application in the preparation of drugs to restore precancerous immune function in colorectal cancer.

3. The application according to claim 2, characterized in that, The restoration of precancerous immune function in colorectal cancer is achieved by restoring the immune surveillance function of the tertiary lymphoid structure.

4. The application according to claim 3, characterized in that, The immune surveillance function of the tertiary lymphoid structure leads to increased expression of CD74 molecules, enhanced MHC-II signal transduction, and reactivation of effector memory T cells, restoring adaptive immune responses.

5. The application according to claim 1, characterized in that, The vitamin B 12 It is at least one of the active forms of methylcobalamin, 5'-deoxyadenosylcobalamin, and cyanocobalamin.

6. The application according to claim 1, characterized in that, The adjuvant therapy agent can be any of the following dosage forms: oral preparation, injectable preparation, or intestinal-targeted preparation.

7. The application according to claim 4, characterized in that, The reactivated effector memory T cells increased CD4+ + Effector memory cells and CD127 + KLRG1 - Memory-like T cell population achieved.

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