Application of CCL5+CD4 + T cell in preparation of product for predicting treatment effect of bladder cancer patient

Abstract

The invention relates to application of CCL5 + CD4 + T cells in preparation of products for predicting the treatment effect of bladder cancer patients, bioinformatics analysis shows that the abundance of the CCL5 + CD4 + T is higher in CR / PR patients, and the high abundance of the CCL5 + CD4 + T and the CCL5 + CD4 + T indicates that the overall survival prognosis of immunotherapy is better. The invention further relates to application of the CCL5 + CD4 + T cell and the combination of the CCL5 + CD4 + T cell and the immune checkpoint blocking agent in preparation of the medicine for treating bladder cancer, the immune checkpoint blocking agent is anti-PD-1, the CCL5 + CD4 + T cell is adoptively transfused back in vivo to inhibit growth of tumors, and the CCL5 + CD4 + T cell and the anti-PD-1 are combined for treatment to significantly inhibit growth of the tumors; transwell co-culture experiments prove that the CCL5 + CD4 + T cell population plays an anti-tumor role through the CCL5 / CCR1 polarized M1 type macrophages, and a new hope is brought for accurately selecting ICBs to treat patients.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to CCL5 + CD4 + The application of T cells in the preparation of products to predict the treatment effect of bladder cancer patients. Background Art

[0002] Bladder cancer is one of the most common malignancies of the urinary system, with urothelial carcinoma of the bladder (UCB) being the most common pathological type. Approximately 70% of bladder cancer patients have non-muscle invasive bladder cancer (NMIBC), while 30% have muscle invasive bladder cancer (MIBC). In recent years, immune checkpoint blockers (ICBs) have demonstrated promising efficacy in the treatment of advanced metastatic urothelial bladder cancer. However, their use in clinical practice remains challenging. Firstly, the objective response rate (ORR) of ICBs in patients with advanced bladder cancer is only 14.8%-21%, indicating that approximately 80% of patients exhibit primary resistance to ICB therapy. Secondly, some patients may initially respond well to ICBs but develop acquired resistance with treatment progression. Therefore, in-depth research on the primary and acquired resistance mechanisms of immunotherapy is of great clinical significance for improving the effectiveness of bladder cancer immunotherapy.

[0003] Recent studies have shown that CD4 + T cells play an important role in maintaining the anti-tumor activity of CD8 T cells. + There is a close interaction between T cells and immune cells such as dendritic cells, B cells and macrophages, suggesting that CD4 + T cells have a unique role in immunotherapy. However, currently in bladder cancer, CD4 + Research on T cells is still relatively limited, especially CD4 + The relationship between T cell subsets and immunotherapy responsiveness and its specific mechanism are still unclear. Summary of the Invention

[0004] In response to the deficiencies in the prior art, the present invention proposes a pharmaceutical composition of an adoptive cell therapy product and an immune checkpoint blocker and its application.

[0005] The technical solution of the present invention is: CCL5 + CD4 + The application of T cells in the preparation of products to predict the treatment effect of bladder cancer patients.

[0006] Furthermore, the CCL5 + CD4 + T cells are lowly expressed in bladder cancer patients.

[0007] Furthermore, the CCL5 + CD4 + The T cell sorting method is to isolate bladder cancer tumors with CD4 + CD25 - CXCR6 + It marks logistics sorting.

[0008] CCL5 + CD4 + The use of T cells in the preparation of drugs for treating bladder cancer patients.

[0009] CCL5 + CD4 + The application of T cells and immune checkpoint blockers in combination in the preparation of drugs for the treatment of bladder cancer.

[0010] Furthermore, the immune checkpoint blocker is anti-PD-1.

[0011] Furthermore, the CCL5 + CD4 + The effective dose of T cells is 1×10 5 The effective dose of the immune checkpoint blocker is 10 mg / kg.

[0012] Furthermore, the CCL5 + CD4 + T cells exert anti-tumor immune function through CCL5 / CCR1 polarization to M1 macrophages.

[0013] Furthermore, the bladder cancer is urothelial carcinoma.

[0014] Furthermore, the CCL5 + CD4 + The T cells are administered by intratumoral injection, and the immune checkpoint blocker is administered by intraperitoneal injection.

[0015] Compared with the prior art, the present invention has at least the following advantages: 1. The present invention relates to CCL5 + CD4 +The application of T cells in the preparation of products for predicting the treatment effect of bladder cancer patients was found through bioinformatics analysis. + CD4 + T abundance was higher in CR / PR patients, and CCL5 + CD4 + High abundance of T indicates better overall survival prognosis after immunotherapy.

[0016] 2. The present invention relates to CCL5 + CD4 + T cells, CCL5 + CD4 + The invention relates to the application of T cells and immune checkpoint blockers in the preparation of drugs for treating bladder cancer, wherein the immune checkpoint blocker is anti-PD-1. + CD4 + T cells can inhibit tumor growth, CCL5 + CD4 + T cells combined with anti-PD-1 treatment significantly inhibited tumor growth; Transwell co-culture experiments showed that CCL5 + CD4 + T cell populations exert anti-tumor effects by polarizing M1 macrophages through CCL5 / CCR1, bringing new hope for the precise selection of ICBs for treatment of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the specific embodiments of the present invention, the following briefly introduces the drawings required for the specific embodiments or the description of the prior art.

[0018] Figure 1 This is the first embodiment of the present invention, CCL5 + CD4 + The relationship between T cells and bladder urothelial carcinoma immunotherapy response; A is the CD4 + T cell subset dimensionality reduction diagram; B is the CCL5 in bladder cancer patient specimens before immunotherapy in the public database IMvigor210, which achieved CR / PR (complete remission / incomplete remission) and SD / PD (stable disease / progressive disease). + CD4 + T is the comparison of infiltration degree; C is CCL5 in the public database + CD4 + The relationship between T infiltration and overall survival after immunotherapy for bladder urothelial carcinoma; D is a multiple immunohistochemical comparison of CCL5 in patients who achieved CR (complete remission) and non-CR (not achieved complete remission) + CD4 +Double positive cells in CD4 + The proportion in cells; Figure 2 Example 2 of the present invention adoptively infused CCL5 into mice + CD4 + T combined with ICBs treatment to observe the tumor size of tumor-bearing mice; A is the analysis of CXCR in bladder cancer + CD25 - CD4 + T cells relative to CXCR - CD25 - CD4 + CCL5 expression in T cells; B is CXCR expression by flow cytometry + CD25 - CD4 + The effect of T cells on ICBs treatment after being reinfused into mice; Figure 3 This is the third embodiment of the present invention CCL5 + CD4 + T cells mainly promote macrophage M1 polarization through CCL5 / CCR1 to exert anti-tumor immune function; Figure A is CCL5 in bladder cancer single-cell sequencing analysis + CD4 + Interaction analysis between T cells and all populations of the microenvironment; Figure B is CCL5 + CD4 + Analysis of cell interactions between T cells and myeloid cell subsets; Figure C is CD4 + The possibility that T cell subsets can affect macrophages through the CCL5 / CCR1 ligand pair; D is CCL5 + CD4 + Schematic diagram of co-culture of T cells and mouse macrophage cell line RAW264.7; E is the analysis of changes in M1 macrophage-related markers by qPCR after macrophage co-culture; E is the observation of whether the addition of CCR1 inhibitor (BX471) can inhibit CCL5 + CD4 + The promoting effect of T cells on ICBs treatment. DETAILED DESCRIPTION

[0019] The present invention is described in further detail below. It is necessary to point out that the following specific implementation methods are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0020] This document provides general and / or specific descriptions of the materials and experimental methods used in the experiments. Unless otherwise specified, all experimental or testing methods are conventional methods. All reagents and instruments used, unless the manufacturer is specified, are commercially available, conventional products and were prepared or used using conventional methods.

[0021] Example 1 CCL5 + CD4 + T cells linked to better outcomes with immunotherapy in urothelial bladder cancer This example uses single-cell sequencing analysis, Cibersort deconvolution, and multiple immunohistochemical staining to compare CD4 T cells in patients with CR / PR (Complete Response / Partial Response) and SD / PD (Stable Disease / Progressive Disease). + Differences in different T cell subsets and analysis of CCL5 + CD4 + The relationship between T cells and prognosis. Single-cell sequencing samples were collected from five specimens of bladder cancer patients in the Department of Urology at the Army Specialty Medical Center from 2022 to 2024, plus eight tumor samples from the public dataset PRJNA662018. All participants voluntarily participated in the study and signed informed consent.

[0022] For single-cell sequencing analysis, single-cell RNA-seq libraries were generated using the 10X Genomics ChromiumController instrument and the Chromium Single Cell 3′ V3 kit (10X Genomics, Pleasanton, CA). During the assay, cells were concentrated to approximately 1000 cells / μL and loaded into each lane to generate single-cell gel-bead emulsions (GEMs). After a reverse transcription step, the GEMs were fragmented, and the barcoded cDNA was purified and amplified. The amplified barcoded cDNA was fragmented, A-tailed, adapter-ligated, and subjected to indexed PCR amplification. The final libraries were quantified using the Qubit High Sensitivity DNA Kit (Thermo Fisher Scientific), and the library size distribution was determined using a high-sensitivity DNA chip on a Bioanalyzer 2200 (Agilent). All libraries were sequenced using an Illumina sequencer (Illumina, San Diego, CA) using 150-bp paired-end sequencing. The raw data were processed using Cell Ranger (version V7.2.0) to generate a raw unique molecular identifier (UMI) count matrix, which was converted into a Seurat object using the R package Seurat (version: V4.1.1). Cells with less than 500 UMIs or more than 15% of mitochondrial-derived UMI counts were identified as low-quality cells and removed. Doublets were removed from each sample using DoubletFinder. Batch effects between samples were removed using Harmony, and the top 30 PCA results were selected for analysis. Seurat's FindNeighbors function was used to construct a shared nearest neighbor graph, and an unsupervised clustering analysis was performed based on this graph using the FindClusters function in Seurat with the parameter set to "resolution = 0.1". It was visualized using a UMAP graph and further clustered into subgroups. Using the above methods, human bladder cancer CD4 T cell subpopulations were obtained, as shown in the following figure. Figure 1 As shown in A.

[0023] For the deconvolution method selection, this embodiment uses the Cibersortx deconvolution method (https: / / cibersortx.stanford.edu / ) to calculate the different CD4 + T cell abundance, the results are as follows Figure 1 As shown in B, CCL5 + CD4 + T cells were more abundant in CR / PR patients, and CCL5 + CD4 +High abundance of T indicates better overall survival prognosis after immunotherapy ( Figure 1 C).

[0024] For multiplex immunohistochemistry, in this example, the patient tissues before immunotherapy were washed with PBS, fixed with formaldehyde, and embedded in paraffin. Multiplex immunohistochemistry was then performed on serial tissue sections cut from these paraffin-embedded samples. All primary antibodies were incubated overnight in a frozen laboratory to achieve more specific antigen-antibody binding. The sections were then blocked with antifade mounting medium and scanned using a phenoImager Fusion. The results showed that CCL5 + CD4 + The proportion of double positive cells was higher in the CR group ( Figure 1 D) The patients were 40 patients who received neoadjuvant immunotherapy + bladder cancer at the Army Specialty Medical Center from 2022 to 2024. 70% of the patients received 3 cycles of treatment. All participants voluntarily participated in the study and signed informed consent.

[0025] Example 2: Adoptive transfer of CCL5 + CD4 + T cells can promote responsiveness to immunotherapy Subcutaneous tumor mouse model: The mouse model was sourced from Beijing SPF Biotechnology, 6-8 week old C57BL / 6J mice, using 8 × 10 MB49 cells. 5 The tumor volume was calculated as follows: the longest diameter of the tumor × the widest diameter of the tumor 2 .

[0026] In this example, flow cytometry was used to separate CCL5 infiltrating tumor cells through specific surface markers. + CD4 + T cells were then injected intratumorally into mice and combined with ICBs for treatment, and the differences in tumor volume between different groups were compared.

[0027] For flow cytometry, CD4 + CD25 - CXCR6 + As a sorting marker, CCL5 + CD4 + T cells were purified from Figure 2 As shown in A, after flow cytometry sorting, the CXCR6 + CD25 - CD4 + T cells relative to CXCR6 - CD25 - CD4 +T cell CCL5 gene expression was significantly different, indicating that CCL5 was successfully sorted by flow sorting + CD4 + T cells.

[0028] For treatment, when the tumor volume reached 100-200mm 3 (7th day), tumor-bearing mice were randomly divided into 4 groups: the first group was the control group (Ctrl group), which received 100 μl PBS intraperitoneal injection and 50 μl PBS intratumoral injection; the second group received 10 mg / kg anti-PD-1 treatment intraperitoneally, while 50 μl PBS was injected intratumorally (Anti-PD-1 group); the third group received 100 μl PBS intraperitoneally, while CCL5 + CD4 + T cells suspended in 50 μl PBS (CCL5 hi -CD4 group); the fourth group received 10 mg / kg anti-PD-1 treatment intraperitoneally, while CCL5 + CD4 + T cells suspended in 50 μl PBS (Anti-PD-1+ CCL5 hi -CD4 group). 1×10 3 CCL5 5 CD4 + T cells were purified from subcutaneous ectopic transplanted tumors (tumor volume greater than 400mm + 2) were injected intratumorally, 5 days at a time, a total of 2 times, and the treatment effects of the four groups are shown in Figure 2 B, as can be seen from the figure, the adoptive transfer of CCL5 + CD4 + T cells can inhibit tumor growth, and significantly inhibit tumor growth after combined ICB treatment, which is significantly different from the Anti-PD-1 group.

[0029] Example Three CCL5 + CD4 + T cells mainly polarize M1 type macrophages through CCL5 / CCR1 This example found that CCL5 + CD4 + T cells mainly interact with macrophages, and found that CCL5 / CCR1 is one of the main ligands of CCL5 + CD4 + T cells affecting macrophages, proving that CCL5 + CD4 +The abundance of T and M1 macrophages was positively correlated, and finally, CCL5 was demonstrated in in vitro co-culture experiments. + CD4 + T cells can promote M1 macrophage polarization.

[0030] In this example, the CellChat.R package (a single cell data analysis tool) was used to analyze the microenvironment cell interaction and compare CCL5 + CD4 + The possibility of crosstalk between T and microenvironmental immune cells. Figure 3 A is CCL5 in single-cell sequencing analysis of bladder cancer + CD4 + Interaction analysis of T cells and microenvironmental populations, Figure 3 B is CCL5 + CD4 + Analysis of cellular interactions between T cells and myeloid cell subsets; Figure 3 C is CD4 + The possibility that T cell subsets can affect macrophages through CCL5 / CCR1 ligand pairs. + CD4 + T cells mainly interact with macrophages, and it was also found that CCL5 / CCR1 is CCL5 + CD4 + One of the main ligands that T cells use to affect macrophages.

[0031] To evaluate CCL5 + CD4 + Effects of T cells on macrophage polarization, RAW264.7 mouse macrophages (1×10 4 ) were seeded in 24-well flat-bottom culture plates. + CD4 + T cells (5 × 10 4 ) were isolated from the mouse tumor tissue of Example 2 by flow cytometry and co-cultured with RAW264.7 cells in 0.4 μm pore size Transwell inserts. The co-culture schematic is shown in FIG. Figure 3 D. The co-culture medium was RPMI 1640 medium containing 10% FBS, and the culture conditions were 37°C, 5% CO2, with or without the addition of CCR1 inhibitor BX471. After 24 hours, RAW264.7 cells were collected for qPCR analysis to study the changes in M1 macrophage-related markers. The qPCR analysis of the changes in M1 macrophage-related markers is shown in the figure. Figure 3 As shown in E, it can be seen from the figure that + CD4 +After co-culture with T cells, the levels of M1 macrophage markers Nos2, IL-1β, Mcp-1, and TNF-α increased significantly. After the use of the CCR1 inhibitor BX471, the M1 macrophage markers decreased significantly, indicating that CCL5 + CD4 + T cells mainly promote macrophage M1 polarization through CCL5 / CCR1 to exert anti-tumor immune function.

[0032] For in vivo experiments in mice, CCL5 + CD4 + Mice with T cells were treated with BX471 (50 mg / kg, daily, intraperitoneal injection) to observe the effect of CCR1 inhibitor on CCL5 + CD4 + The effect on T cell function Figure 3 F is Anti-PD-1 group, Anti-PD-1+CCL5 hi -CD4 group, Anti-PD-1+CCL5 hi -Tumor size in the CD4+BX471 group. The figure shows that BX471 can inhibit CCL5 + CD4 + T cells promote the anti-tumor effects of immunotherapy.

[0033] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and description of the present invention.

Claims

1. CCL5 + CD4 + The application of T cells in the preparation of products to predict the treatment effect of bladder cancer patients.

2. The use according to claim 1, characterized in that The CCL5 + CD4 + T cells are lowly expressed in bladder cancer patients.

3. The use according to claim 2, characterized in that The CCL5 + CD4 + The T cell sorting method is to isolate bladder cancer tumors with CD4 + CD25 - CXCR6 + It marks logistics sorting. 4.CCL5 + CD4 + The use of T cells in the preparation of drugs for treating bladder cancer patients. 5.CCL5 + CD4 + The application of T cells and immune checkpoint blockers in combination in the preparation of drugs for the treatment of bladder cancer.

6. The use according to claim 5, characterized in that The immune checkpoint blocker is anti-PD-1.

7. The use according to claim 5, characterized in that The CCL5 + CD4 + The effective dose of T cells is 1×10 5 The effective dose of the immune checkpoint blocker is 10 mg / kg.

8. The use according to claim 7, characterized in that The CCL5 + CD4 + T cells exert anti-tumor immune function through CCL5 / CCR1 polarization to M1 macrophages.

9. The use according to claim 8, characterized in that The bladder cancer is urothelial carcinoma.

10. The use according to claim 1, characterized in that The CCL5 + CD4 + The T cells are administered by intratumoral injection, and the immune checkpoint blocker is administered by intraperitoneal injection.

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