Method for detecting triple negative breast cancer immunotherapy efficacy prediction marker ccl19

Abstract

The application discloses a detection method of a triple negative breast cancer immunotherapy efficacy prediction marker CCL19. The application provides application of CCL19 as a marker in preparation of a breast cancer immunotherapy efficacy and / or prognosis prediction product. A triple negative breast cancer immunotherapy efficacy and prognosis prediction kit comprising a CCL19 protein detection reagent and optionally an instruction is also provided. Application of a CCL19 molecule in combination with a second therapeutic agent in preparation of an antitumor drug is also provided. The application greatly fills the vacancy of triple negative breast cancer immunotherapy efficacy prediction liquid biopsy, and realizes precise prediction of immunotherapy efficacy in a noninvasive, rapid and low-cost manner.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of CCL19 as a biomarker in the preparation of products for predicting the efficacy and / or prognosis of breast cancer immunotherapy. Background Technology

[0002] Triple-negative breast cancer (TNBC) refers to a type of breast cancer that is negative for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), accounting for 15%-20% of all breast cancers. Compared to patients with hormone receptor-positive breast cancer, TNBC patients often have a younger age of onset, are more prone to visceral metastasis, have a higher risk of postoperative recurrence, and a poorer prognosis, making it a challenge for clinical treatment.

[0003] Because TNBC lacks the expression of key receptors, patients cannot benefit from targeted therapy. The advent of immunotherapy has brought a breakthrough in TNBC treatment. Immunotherapy mainly includes vaccines, immune checkpoint inhibitors (ICIs), adoptive immune cell therapy, and cytokine therapy, among which ICI therapy has attracted much attention due to its significant clinical efficacy. The IMpassion130 trial demonstrated that the anti-PD-L1 monoclonal antibody atezolizumab combined with albumin-bound paclitaxel as first-line treatment can significantly prolong metastasis-free survival in patients with unresectable, locally advanced, or metastatic TNBC. Subsequently, the KERNOTE-355 trial also showed that the anti-PD-1 monoclonal antibody pembrolizumab in combination with chemotherapy can be used as first-line treatment for patients with locally recurrent unresectable or metastatic TNBC, significantly reducing the risk of disease progression or death.

[0004] Intravascular coagulation (ICI) therapy has benefited an increasing number of patients, but only a subset of patients are sensitive to ICI treatment. Screening for biomarkers that can predict the efficacy of ICI therapy remains a significant clinical challenge. Currently, commonly used biomarkers for predicting ICI efficacy include PD-1 / PD-L1 expression, microsatellite instability, loss of mismatch gene repair, tumor mutational burden, and tumor-infiltrating lymphocytes (TILs). However, the predictive power of currently proposed immunotherapy efficacy biomarkers is unsatisfactory, and most are based on high-throughput sequencing, hindering their widespread clinical application. Existing PD-1 and PD-L1 kits are convenient and rapid, but their accuracy is low and they cannot effectively identify patients who benefit from immunotherapy. Therefore, there is an urgent need for a clinically applicable biomarker for predicting the efficacy of immunotherapy in breast cancer, capable of providing a simple, efficient, and accurate prediction of immunotherapy efficacy for any breast cancer patient, thus supporting precision treatment decisions. Summary of the Invention

[0005] To address the aforementioned technical problems, the first aspect of this invention provides the application of CCL19 as a biomarker in the preparation of products for predicting the efficacy and / or prognosis of breast cancer immunotherapy.

[0006] In some implementations, the breast cancer is triple-negative breast cancer.

[0007] In some implementations, the efficacy and / or prognosis prediction product predicts efficacy by detecting CCL19 expression in the subject's blood or tumor tissue.

[0008] In some embodiments, the detection is performed by ELISA to detect the expression level of CCL19 protein in serum; patients with high levels of CCL19 in their serum are sensitive to immunotherapy, while patients with low levels are resistant to immunotherapy; preferably, 29.84 pg / ml is used as the cutoff point for CCL19 levels. When CCL19 > 29.84 pg / ml, it is defined as a high level, indicating sensitivity to immunotherapy; when CCL19 ≤ 29.84 pg / ml, it is defined as a low level, indicating resistance to immunotherapy.

[0009] In some embodiments, the detection is performed by immunohistochemistry to detect the expression level of CCL19 protein in the tissue; a positive CCL19 staining indicates sensitivity to immunotherapy; a negative CCL19 staining indicates resistance to immunotherapy.

[0010] A second aspect of the present invention provides a kit for predicting the efficacy and / or prognosis of breast cancer immunotherapy, the kit comprising a CCL19 protein detection reagent and optionally instructions. Preferably, the breast cancer is triple-negative breast cancer.

[0011] In some embodiments, the detection reagent is a CCL19 antibody.

[0012] The third aspect of this invention provides the use of CCL19 molecules in combination with a second therapeutic agent in the preparation of antitumor drugs.

[0013] In some embodiments, the tumor is breast cancer, preferably triple-negative breast cancer.

[0014] In some embodiments, the second therapeutic agent includes an anti-PD-1 monoclonal antibody and an anti-PD-L1 monoclonal antibody.

[0015] A fourth aspect of the present invention provides a pharmaceutical composition comprising a CCL19 molecule and a second therapeutic agent.

[0016] In some embodiments, the composition is used for tumor treatment, preferably for breast cancer, and more preferably for triple-negative breast cancer.

[0017] In some embodiments, the second therapeutic agent includes a PD-1 monoclonal antibody and a PD-L1 monoclonal antibody.

[0018] In some embodiments, the CCL19 and the second therapeutic agent may be administered simultaneously or sequentially.

[0019] The beneficial effects of this invention are:

[0020] This invention is the first to discover a novel ICI (intracytoplasmic sperm injection) biomarker, CCL19. CCL19 levels were characterized using both serum level detection and immunohistochemical detection, and validated across multiple dimensions and external cohorts. This invention significantly fills the gap in liquid biopsy for predicting the efficacy of TNBC immunotherapy, achieving accurate prediction of immunotherapy efficacy non-invasively, rapidly, and at low cost. This invention provides a clinical foundation for the further development and advancement of TNBC immunotherapy. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 This shows the results of identifying DC cell subsets using a single-cell atlas of breast cancer dendritic cells (DCs). Figure 1 A. Based on single-cell data from the Biokey breast cancer immunotherapy cohort, dendritic cells (DCs) were divided into four major subpopulations. Figure 1 B, DC subgroup clustering heatmap and molecular markers, in which subgroup 1 is mainly marked by CLEC9A, subgroup 2 is mainly marked by CLEC10A, subgroup 3 is mainly marked by CCL19, and subgroup 4 is mainly marked by LILRA4.

[0023] Figure 2 The results show that single-cell atlases reveal DC subsets that are highly correlated with immunotherapy response. Figure 2 A. The relationship between the infiltration rate of each DC subgroup and the patient's treatment response; Figure 2 B, DC subset 3 infiltration and CD8, which has been identified in previous studies as a potential benefit of immunotherapy. + The functional subsets of T cells were significantly positively correlated.

[0024] Figure 3 Display external independent queue verification CCL19 + DC subsets are highly correlated with immunotherapy response. Figure 3A, The relationship between the level of DC subgroup infiltration in cohort A, GSE169246 and patient treatment response; Figure 3 B, DC subset 3 infiltration and CD4, which have been identified in previous studies as suggesting benefit from immunotherapy. + T cells and CD8 + The major functional subsets of T cells were significantly positively correlated.

[0025] Figure 4 The CCL19 was validated using Biokey cohort single-cell analysis. + DC characteristics exist. Figure 4 A. Based on single-cell data from the Biokey breast cancer immunotherapy cohort, immune cells were divided into four major subsets, and it was found that CCL19 was mainly expressed in myeloid cells. Figure 4 B, CCL19 + DC is a distinct cell subpopulation that can be validated in whole cells.

[0026] Figure 5 Single-cell analysis of the GSE169246 cohort validated CCL19 + DC characteristics exist. Figure 5 A. Based on single-cell data from the GSE169246 breast cancer immunotherapy cohort, immune cells were divided into four major subsets, and it was found that CCL19 was mainly expressed in myeloid cells. Figure 5 B, CCL19 + DC is a distinct cell subpopulation that can be validated in whole cells.

[0027] Figure 6 Display CCL19 + DCs are mainly enriched in patients with TNBC. Figure 6 A, Immunoflow cytometry analysis of 87 fresh paired surgical samples from the FUSCC cohort; Figure 6 B, Transcriptome analysis of the TCGA cohort; Figure 6 C, FUSCC cohort transcriptome analysis.

[0028] Figure 7 CCL19 was visualized using TNBC tissue microarray dual immunohistochemical staining. + DC infiltration was assessed and visualized using dual immunohistochemical staining on 186 TNBC tissue microarrays. + DC infiltration status.

[0029] Figure 8 Kaplan-Meier survival analysis indicated CCL19. + DC suggests a better prognosis for patients with TNBC. Figure 8 A, in the FUSCC queue, CCL19 +High expression of DC characteristic or CCL19 + High DC infiltration rates generally indicate better overall survival and relapse-free survival in patients. Figure 8 B, in the TCGA queue, CCL19 + High expression of DC features suggests better overall survival and relapse-free survival in patients.

[0030] Figure 9 Cox analysis indicated CCL19. + Dendritic cell count (DC) is an independent prognostic factor for patients with total nephrotic syndrome (TNBC). CCL19 was assessed using dual immunohistochemical staining on 186 TNBC tissue microarrays. + DC invasion was analyzed using univariate and multivariate Cox regression analysis, including age, tumor size, number of metastatic lymph nodes, Ki67 proliferation index, and CCL19. + DC infiltration revealed CCL19 + DC (denaturation-related lesions) indicates better relapse-free survival and is an independent prognostic factor for TNBC patients.

[0031] Figure 10 Display CCL19 + High dendritic cell (DC) infiltration in TNBC tissue suggests high infiltration of activated immune cells. Flow cytometry analysis of different CCL19 values ​​was performed on fresh surgical tissue from 10 TNBC patients. + The infiltration of T cells, NK cells, and macrophage subsets in dendritic cell-infiltrating tissue revealed CCL19 in the tumor tissue. + Patients with higher dendritic cell (DC) infiltration also have higher levels of activated immune cell infiltration.

[0032] Figure 11 Display CCL19 + TNBC patients with higher DC scores showed upregulation of immune-related molecules. Using the FUSCC cohort RNA-seq cohort, immune activation and immune checkpoint-related molecules were included, and correlation analysis revealed CCL19. + Patients with high DC expression showed significant upregulation of immune-related molecules.

[0033] Figure 12 CCL19 was shown to be enriched through gene set analysis. + Patients with high DC expression have upregulated immune activation-related pathways. Gene set enrichment analysis using the FUSCC cohort RNA-seq cohort revealed CCL19. + Patients with high DC expression showed significant upregulation of immune activation-related pathways.

[0034] Figure 13 The correlation between CCL19 and different immune cells and the expression ratio in different immune cells. Figure 13A. Using the FUSCC cohort RNA-seq cohort, the CIBERSORTx algorithm was used to calculate the infiltration status of 22 immune cells. Correlation analysis suggested a correlation between CCL19 molecules and CCL19. + DC expression is similarly correlated with various immune cells; Figure 13 B. Flow cytometry analysis of immune cells from 87 fresh breast cancer tissue samples showed that dendritic cells (DCs) were the main cell subpopulation expressing CCL19.

[0035] Figure 14 Survival analysis of patients with high CCL19 expression. Figure 14 In the FUSCC cohort, high CCL19 expression suggests better overall survival and relapse-free survival. Figure 14 B. In the PostgreSQL queue, high CCL19 expression suggests better overall survival and relapse-free survival in patients.

[0036] Figure 15 Cox regression analysis showed that CCL19 is an independent prognostic factor for TNBC patients. Using tissue microarrays from 186 TNBC patients, CCL19 expression was assessed by immunohistochemical staining. Univariate and multivariate Cox regression analyses, including age, tumor size, number of metastatic lymph nodes, Ki67 proliferation index, and CCL19 expression, revealed that CCL19 indicates better recurrence-free survival and is an independent prognostic factor for TNBC patients.

[0037] Figure 16 The results showed that CCL19 expression was positively correlated with immunotherapy biomarkers, and suggested that the mouse model of in situ breast cancer tumorigenesis was sensitive to immunotherapy. Figure 16 A. Correlation between CCL19 and sTIL, PD-1, PD-L1, CD8A, CXCL9, and CD68 in the FUSCC cohort. The numerical values ​​represent the R-values ​​of the correlation analysis. Figure 16 In cohort B, GSE124821, in vivo experiments using immunotherapy showed that the Ccl19 level was significantly higher in the treatment-sensitive group compared to the non-sensitive group.

[0038] Figure 17 This study demonstrated the efficacy of CCL19-sensitized PD-1 antibody in an orthotopic tumorigenesis model of breast cancer in mice. Orthotopic tumors were induced in BALB / c mice using the 4T1 mouse TNBC cell line. The mice were divided into four groups, receiving control, anti-PD-1 monoclonal antibody, CCL19, and combination therapy, respectively. Tumor growth curves were observed, and tumor weight was recorded after the experiment.

[0039] Figure 18 This shows a comparison of the proportion of immune cells in each experimental group of the mouse model. Flow cytometry analysis was used to analyze CD4+ in tumors of each group. + T, CD8 +T cell infiltration, and its functional molecules (CD4+) + T-bet in T cells, CD8 + Expression of Granzyme B and Perforin in T cells.

[0040] Figure 19 Higher serum CCL19 levels in patients suggest benefit from immunotherapy. In the FUTURE clinical trial, 18 patients who received immunotherapy were included, and baseline serum CCL19 concentrations and efficacy data were measured. The baseline CCL19 levels were compared between patients with clinical remission and those without clinical remission, along with the relationship between baseline CCL19 levels and objective response rate, and changes in serum CCL19 concentration before and after immunotherapy.

[0041] Figure 20 This demonstrates the accuracy of using the patient's serum CCL19 threshold concentration to predict the efficacy of immunotherapy. Figure 20 In the A, FUTURE cohort, the sensitivity, specificity, and Youden index of different serum CCL19 levels as thresholds for predicting the efficacy of immunotherapy were analyzed. Figure 20 B. The receiver operating characteristic curve and the corresponding area under the curve (AUC) of serum CCL19 level for predicting the efficacy of immunotherapy are used to indicate the accuracy of efficacy prediction.

[0042] Figure 21 High CCL19 expression in patients suggests benefit from immunotherapy. In the FUTURE-C-Plus clinical trial, 29 patients who received immunotherapy and had both CCL19 immunohistochemical staining results and efficacy data were included. The association between CCL19 and objective response rate, tumor regression rate, PD-L1 expression, and progression-free survival was compared. Detailed Implementation

[0043] The present invention is further described below. The embodiments described herein are merely exemplary and do not constitute a limitation on the scope of the invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the principles and methods of the invention, but such modifications or substitutions shall all fall within the protection scope of the present invention.

[0044] Terminology Definition

[0045] As used in this article, the term "triple-negative breast cancer" refers to breast cancer that is negative for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Clinically, ER and PR are collectively referred to as hormone receptors (HR).

[0046] As used herein, the basic principle of the term "immune checkpoint inhibitor (ICI)" is based on the activation mechanism of immune cells, specifically T cells. Programmed death receptors (PDRs) are expressed on the surface of T cells, and their ligands are expressed on the surface of tumor cells and myeloid-derived suppressor cells. The binding of PDRs to their ligands can exhaust T cells, preventing them from normally killing tumor cells, thus allowing tumor cells to evade the host's immune surveillance. Therefore, PDRs and their ligands are referred to as "immune checkpoints." Immune checkpoint inhibitors based on PDRs and their ligands enhance the host's immune system's attack on tumor cells by inhibiting their binding.

[0047] The present patent will be further described in detail below with reference to the accompanying drawings and specific experiments. Unless otherwise specified, the reagents, instruments, equipment and methods used in this patent are all commercially available reagents, instruments, equipment and methods conventional in this technical field.

[0048] Example 1: Discovery and Functional Verification of the CCL19 Molecule

[0049] Dendritic cells (DCs) are the primary antigen-presenting cells capable of initiating T-cell-mediated immune responses, but their role in tumor immunotherapy remains unclear. Based on single-cell sequencing, multi-omics analysis, in vivo experiments, and clinical trials, this invention identifies a key DC subset, CCL19, that can predict response to TNBC immunotherapy. + DC (Examples 1.1 and 1.2). After verification by multiple external queues, CCL19 + DCs were shown to be widely present in breast cancer tissues, enriched in TNBC, and suggested anti-tumor immune activation (Examples 1.3 and 1.4). More importantly, we characterized CCL19 using CCL19. + DC aims to simplify clinical applications and has found that CCL19 is mainly composed of CCL19 + DC secretion, and by promoting CD8 + T cell function plays an anti-tumor immune role (Examples 1.5 and 1.6). Therefore, the CCL19 molecule can serve as a novel immunotherapeutic biomarker to predict the efficacy of TNBC immunotherapy.

[0050] 1.1 Analysis of DC single-cell atlas in a cohort of breast cancer patients receiving immunotherapy

[0051] To systematically reveal the relationship between dendritic cell (DC) subgroups and intracranial pressure incision cytokinesis (ICI) in human breast cancer, we collected single-cell sequencing (scRNA-seq) data (Biokey clinical trial cohort, https: / / ega-archive.org / studies / EGAS00001004809) from 29 breast cancer patients treated with pembrolizumab (PD-1 monoclonal antibody), including a total of 1021 DCs. Cell dimensionality reduction clustering was performed using the Seurat and PCA algorithms in R, and the t-SNE algorithm was used to represent the data. This identified four main subgroups of DCs. The "FindAll Markers" function in the MAST algorithm was used to define characteristic markers for each subgroup, and the most significant marker was used as the naming convention, designated as CLEC9A. + DC, CLEC10A + DC, CCL19 + DC and LILRA4 + DC( Figure 1 A and Figure 1 B).

[0052] 1.2CCL19 + DC invasion suggests that breast cancer patients benefit from ICI treatment.

[0053] Patients exhibiting T-cell clonal expansion are considered to benefit from immunotherapy, and the published data in this cohort also include T-cell clonal expansion data from these 29 patients. Single-cell clustering in Example 1.1 shows that although the overall DC and CLEC9A... + DC, CLEC10A + DC and LILRA4 + DC subsets have no predictive value for T cell clonal expansion, but CCL19 + The proportion of patients with high DC subset infiltration is increased to have T cell clonal expansion. Figure 2 A), the proportion of T cell subsets associated with the efficacy of immunotherapy also increased significantly. Figure 2 B) suggests that the high infiltration of the DC subset in this tumor tissue may indicate that the patient is sensitive to immunotherapy.

[0054] Treatment response can also be defined through clinical assessment. Complete response (CR) is defined as the disappearance of all target lesions, no new lesions appearing, and normal tumor markers, maintained for at least 4 weeks. Partial response (PR) is defined as a reduction of ≥30% in the sum of the largest diameters of target lesions, maintained for at least 4 weeks. Disease progression (PD) is defined as an increase of at least ≥20% in the sum of the largest diameters of target lesions, or the appearance of new lesions. Stable disease (SD) is defined as a reduction in the sum of the largest diameters of target lesions that does not meet PR, or an increase that does not meet PD. To validate CCL19... +To further investigate the predictive role of dendritic cells (DCs) in immunotherapy benefit, we performed the same analysis on a single-cell sequencing cohort from another breast cancer immunotherapy clinical trial (GSE169246, https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE169246). This cohort included 11 patients who received atezolizumab (a PD-L1 monoclonal antibody) and were matched for clinical efficacy data assessment, comprising a total of 702 DCs. In this cohort, we also found CCL19... + Patients with high DC infiltration showed better immunotherapy benefit and had more PR patients, while there was no significant difference in the proportion of PR and SD patients among overall DC or other DC subgroups. Figure 3 A) Similarly, we also found that CCL19 + In patients with high DC subset infiltration, the proportion of T cell subsets associated with immunotherapy efficacy was significantly increased. Figure 3 B).

[0055] In summary, CCL19 + High invasiveness of DC subsets in breast tumors suggests that patients benefit from immunotherapy and may serve as an effective biomarker for the efficacy of immunotherapy.

[0056] 1.3 CCL19 in breast cancer + DC is widely present

[0057] To further confirm CCL19 + With the presence of DCs, we performed a comprehensive analysis of scRNA-seq data in the Biokey clinical trial cohorts in Examples 1.1 and 1.2. Figure 4 A and Figure 4 B), GSE169246 Figure 5 A and Figure 5 B) In both independent datasets, we were able to define independent CCL19 in immune cells within the breast tumor microenvironment. + The DC subgroup suggests that this subgroup is prevalent in breast tumor tissue.

[0058] Furthermore, we used flow cytometry (…) Figure 6 A) Omics analysis ( Figure 6 BC) and immunohistochemical staining ( Figure 7 CCL19 was verified. + Dendritic cells (DCs) are prevalent in breast cancer and have value for widespread application. First, we collected matched breast tumors and surrounding tissues from 87 patients who underwent breast surgery at Fudan University Cancer Center (FUSCC), including 41 high-risk (HR) lesions. + HER2 -Tumor, 36 HR + / - HER2 + Tumor and 10 HR - HER2 - Tumors, analyzed by flow cytometry using fresh tissue, confirmed CCL19. + The DC subgroup is present in all breast cancer subtypes and is more abundant in tumor tissue than in adjacent normal tissue. Figure 6 A). Second, we conducted our analysis in two separate cohorts (TCGA cohort, with a total sample size of 1070 cases). Figure 6 B; FUSCC cohort, with a total sample size of 360 cases. Figure 6 In C), transcriptome sequencing (RNA-seq) data was used, employing CCL19. + DC marker gene set calculation of cell enrichment score revealed CCL19 + DCs were expressed in all samples, and their expression levels were higher in tumors than in adjacent normal tissues. Third, we also collected tissue microarrays from 186 patients at FUSCC and used dual immunohistochemical staining with CD11c (a universal DC marker, Proteintech, catalog number 60258-1-Ig, dilution 1 / 200) and CCL19 (Sigma, catalog number HPA067758, dilution 1 / 200) to visually visualize CCL19. + DCs are widely present in breast cancer tissue. Figure 7 ).

[0059] 1.4CCL19 + DC is enriched in TNBC and is associated with long-term prognosis.

[0060] In the above 87 fresh breast cancer samples ( Figure 6 A) and TCGA queue ( Figure 6 In B), we observed CCL19 in all cases. + DC has the highest percentage on TNBC. High CCL19 + Patients with dendritic cell (DC) infiltration had smaller tumors and more stromal tumor-infiltrating lymphocytes. These results were validated in the TCGA cohort (Table 1), further demonstrating the clinical significance of this cell subset. Furthermore, high CCL19 levels... + TNBC patients with DC infiltration have longer overall survival and recurrence-free survival. Figure 8 After excluding confounding variables such as age, tumor size, lymph node status, and Ki67 score through multivariate Cox analysis, CCL19 was found to be the most significant factor. + DC is an independent predictor of a better prognosis for patients with TNBC. Figure 9 ).

[0061] Table 1 CCL19 + Relationship between DC proportion and clinicopathological features of breast cancer patients

[0062]

[0063]

[0064] We further discovered that CCL19 + TNBC with high dendritic cell infiltration showed higher levels of anti-tumor immune activation, suggesting the presence of CCL19. + DC's predictive ability for ICI efficacy. At the cellular level, using... Figure 6 Further analysis of 10 cases of TNBC in group A revealed CCL19 + Tumor tissues with high dendritic cell infiltration exhibit a higher phenotype of activated immune cells, including higher levels of M1 pro-inflammatory macrophages and CD3+. + T cells, CD8 + T cells, NK cells, and cytotoxic Granzyme B + T cells ( Figure 10 At the molecular level, using the FUSCC cohort RNA-seq cohort, we found CCL19. + Highly invasive dendritic cells (DCs) tumors exhibit upregulation of immunomodulatory and immune-activating molecules, including PD-1 and PD-L1. Figure 11 At the functional level, gene set enrichment analysis (GSEA) showed that CCL19 + Tumors with high dendritic cell infiltration show a significant enrichment of immune activation pathways. Figure 12 ).

[0065] In summary, we found that CCL19 + DC is enriched in TNBC, and CCL19 + Patients with TNBC who have high dendritic cell infiltration have a better prognosis and an activated tumor immune microenvironment, suggesting potential predictive value for immunotherapy.

[0066] 1.5CCL19 suggests a better prognosis and benefit from ICI treatment for TNBC patients.

[0067] In revealing CCL19 + Following the presence of dendritic cells (DCs) in the TNBC immune-activated microenvironment, to facilitate clinical use, we further explored whether CCL19 could represent this cell subset. CCL19 is a chemokine that plays a crucial role in lymphocyte maturation. In the two single-cell sequencing datasets described in Examples 1.1 and 1.2, we confirmed that CCL19 was characteristically expressed in both datasets. + DCs, rather than other immune cells ( Figure 4 , Figure 5Furthermore, we used RNA-seq data from TNBC and employed the CIBERSORTx algorithm to calculate the infiltration fractions of 22 immune cells, comparing them with CCL19 and CCL19... + Correlation analysis of DCs showed that CCL19 exhibited similar characteristics to CCL19. + DC mode ( Figure 13 A), and flow cytometry analysis of breast tumor tissues from 87 patients indicated that dendritic cells (DCs) were the main cells expressing CCL19. Figure 13 B).

[0068] Furthermore, we explored the relationship between CCL19 levels and clinical characteristics of TNBC at the patient level. Patients with high CCL19 expression had smaller tumors and higher TIL infiltration (Table 2). Meanwhile, high CCL19 expression suggested better overall survival and recurrence-free survival in TNBC patients. Figure 14 After excluding confounding variables such as age, tumor size, lymph node status, and Ki67 score through multivariate Cox analysis, CCL19 was found to be the most significant factor. + DC is an independent predictor of a better prognosis for patients with TNBC. Figure 15 ).

[0069] Table 2. Relationship between CCL19 expression level and clinicopathological features of breast cancer patients.

[0070]

[0071] To better explore the predictive ability of CCL19 for the efficacy of immunotherapy, we used RNA-seq data (360 cases) and immunohistochemical staining (186 cases) from the FUSCC cohort to perform Spearman correlation analysis on CCL19 and previously reported effective predictive biomarkers for ICI efficacy. We found that, at both the mRNA and protein levels, CCL19 showed significant positive correlations with indicators including intertumor TIL (sTIL), PD-1, PD-L1, CD8A, CXCL9, and CD68. Figure 16 A). Furthermore, we collected RNA-seq data from 47 in vivo TNBC models treated with immunotherapy (GSE124821, https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE124821), and found that CCL19 expression was significantly higher in immunotherapy-sensitive samples than in immunotherapy-insensitive samples. Figure 16 B).

[0072] In summary, the CCL19 molecule can be used as CCL19 +Alternative biomarkers for dendritic cells (DCs) suggest better prognosis and efficacy of immunotherapy for TNBC.

[0073] 1.6CCL19 may serve as a target for combination immunotherapy in TNBC patients.

[0074] To further confirm the immune-activating effect of CCL19, we examined the therapeutic effect of CCL19 combined with an immune checkpoint inhibitor on a mouse TNBC orthotopic tumor model. Animal experiments were conducted according to the experimental protocol approved by the FUSCC Research Ethics Committee. Experimental mice were purchased from Shanghai Jihui Laboratory Animal Co., Ltd., and subsequent experiments were conducted under conditions that ensured animal ethics. We used 3 × 10 5 4T1 cells (mouse-derived TNBC cell line) were injected subcutaneously into the fourth mammary fat pad region of 6-8 week old female BALB / c mice. After the tumor became palpable, the mice were randomly divided into a control group, a PD-1 monoclonal antibody treatment group, a mouse-derived CCL19 treatment group, and a PD-1 monoclonal antibody combined with mouse-derived CCL19 treatment group. Figure 17 The drugs used included murine CCL19 (R&D, catalog number 400-M3, 0.5 μg intratumorally injected three times a week), PD-1 monoclonal antibody (BioXCell, catalog number BE0146, 200 μg intraperitoneally injected twice a week), and PD-1 isotype antibody control (BioXCell, catalog number BE0089, 200 μg intraperitoneally injected twice a week). The experimental endpoint was set at a maximum tumor diameter ≥20 mm, tumor necrosis, or mouse death. Tumor volume was measured using calipers and calculated using the formula: 0.5 × length × width × width.

[0075] Experiments have confirmed that combination therapy can significantly improve the efficacy of TNBC immunotherapy. Injection of CCL19 alone can significantly inhibit tumor growth, while the combined use of CCL19 and PD-1 monoclonal antibodies can further inhibit tumor growth. Figure 17 Furthermore, combined treatment with murine CCL19 and PD-1 monoclonal antibodies significantly induced an immune response and promoted CD4+. + T immune cells and CD8 + T cell infiltration, further analysis revealed that T-bet plays a major role in the immune response. + CD4 + T cells and cytotoxic Granzyme B + CD8 + T cells and Perforin + CD8 + The proportion of T cells was significantly increased ( Figure 18 ).

[0076] In summary, CCL19 supplementation can effectively enhance the efficacy of PD-1 monoclonal antibody therapy.

[0077] Example 2: Serum CCL19 levels in TNBC patients before treatment as a predictive biomarker for ICI efficacy.

[0078] The CCL19 level in the serum of patients with metastatic TNBC before immunotherapy was detected using an ELISA kit. The results showed that the pre-treatment serum CCL19 expression level could predict the patient's benefit from immunotherapy. The specific validation process included the following steps:

[0079] 1) Research Cohort

[0080] We collected serum samples from 18 patients with metastatic TNBC who received a regimen containing camrelizumab (a PD-1 monoclonal antibody) in the FUTURE clinical trial (international registration number NCT03805399) before and after treatment. Serum CCL19 levels were measured using an ELISA kit (Abcam, catalog number ab100601). Figure 19 Immune response assessment can be divided into clinical remission (the sum of CR and PR) and clinical non-remission (the sum of PD and SD). Patients achieving clinical remission had significantly higher pre-treatment serum CCL19 concentrations than those in patients with clinical non-remission, and patients with higher pre-treatment serum CCL19 levels had a higher probability of achieving clinical remission. Furthermore, we also found a significant increase in serum CCL19 levels after immunotherapy, demonstrating an immune-activated phenotype. Notably, patients with more prior treatments showed a trend towards higher CCL19 levels, indicating that immunotherapy may also be applied to patients with metastatic breast cancer and has broad application potential.

[0081] 2) Define the cutoff value of serum CCL19

[0082] We calculated the Youden index (sensitivity + specificity - 1) of serum CCL19 to predict the efficacy of immunotherapy, and then selected the optimal CCL19 cutoff value. Higher sensitivity results in fewer missed diagnoses, while lower specificity (1 - 1) leads to a lower false positive rate. Therefore, the value at which the Youden index is maximized is the optimal cutoff value. Figure 20 A). In the receiver operating characteristic curve, the optimal cutoff point is the point closest to the top left corner. Calculations show that CCL19 has the best predictive effect at a cutoff point of 29.84 pg / ml. Figure 20 B). When CCL19 > 29.84 pg / ml, it is defined as a high level, indicating sensitivity to immunotherapy; when CCL19 ≤ 29.84 pg / ml, it is defined as a low level, indicating resistance to immunotherapy. In the FUTURE clinical trial, when CCL19 < 29.84 pg / ml, none of the patients achieved remission; when CCL19 > 29.84 pg / ml, 93.3% of the patients achieved disease remission, indicating good predictive efficacy. Figure 19 ).

[0083] These results validate that peripheral blood CCL19 levels can serve as a biomarker to predict the efficacy of ICI in patients with metastatic TNBC, demonstrating clinical application value.

[0084] Example 3: Immunohistochemical staining of paraffin sections of tumor tissue from TNBC patients before treatment to detect CCL19 as a predictive biomarker for ICI efficacy.

[0085] Immunohistochemistry was used to detect CCL19 levels in tissues of metastatic TNBC patients before immunotherapy, and it was found that the expression level of CCL19 in tumor tissues before treatment could predict the patient's benefit from immunotherapy.

[0086] We collected pretreatment paraffin sections from 30 patients with advanced TNBC treated with camrelizumab as a base regimen in the FUTURE-C-Plus clinical trial (international registration number NCT04129996). Immunohistochemical staining was performed using CCL19 antibody (Sigma, catalog number HPA067758, dilution 1:200) (using the Ventana Benchmark ULTRA assay system). CCL19 positivity was defined as ≥1% of CCL19-positive cells / total cells. We assessed its relationship with patient efficacy. In 29 patients with response data, we observed that CCL19-positive patients had more disease remissions, higher PD-L1 ratios, and longer progression-free survival. Figure 21 When CCL19 was negative, only 73.3% of patients achieved disease remission; when CCL19 was positive, all patients achieved disease remission. Figure 21 ).

[0087] These results validate that CCL19 levels in tumor tissue can serve as a biomarker to predict the efficacy of ICI in patients with metastatic TNBC, demonstrating clinical application value.

[0088] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any simple modifications, alterations, or equivalent structural changes made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. Use of CCL19 as a marker in the preparation of a triple negative breast cancer immunotherapy efficacy prediction product, wherein, The immunotherapy is immune checkpoint inhibitor therapy.

2. The use according to claim 1, wherein the therapeutic effect prediction product is used to predict the therapeutic effect by detecting the expression of CCL19 in the blood or tumor tissue of the subject.

3. The use according to claim 2, wherein the detection is the detection of the expression level of CCL19 protein in serum by ELISA; patients with high level of CCL19 in serum are sensitive to immunotherapy, and patients with low level are resistant to immunotherapy.

4. The use according to claim 2, wherein the detection is the detection of the expression level of CCL19 protein in tissue by immunohistochemistry; when CCL19 is positively stained, the immunotherapy is sensitive; when CCL19 is negatively stained, the immunotherapy is resistant.

5. Use of CCL19 in combination with a second therapeutic agent in the manufacture of a medicament for the treatment of a tumor, wherein, The tumor is triple-negative breast cancer, and the second therapeutic agent is an anti-PD-1 monoclonal antibody.

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