A method for determining the suitability of an immune checkpoint inhibitor for a tumor patient

Abstract

The present disclosure relates to a method for judging the applicability of an immune checkpoint inhibitor for a tumor patient. The present disclosure provides a reagent for quantitatively detecting the proportion of CD8 + CXCR5 + T cells in peripheral blood of a subject before treatment. Animal models and clinical tumor samples demonstrate that the higher the proportion of CXCR5 + CD8 + T cells, the stronger the killing ability of the CD8 + CD8 + T cells against autologous tumor cells. After receiving checkpoint inhibitor treatment, the tumor growth inhibition of the high proportion group is more significant, the objective remission rate is higher, the overall survival and metastasis-free survival are longer, and the tumor metastasis load is lower. The present disclosure realizes the prediction of therapeutic effect by using peripheral blood samples, can quickly distinguish the population benefiting from immunotherapy, and solves the clinical problems of large individual differences in the efficacy of immune checkpoint inhibitors and the lack of convenient predictive markers.

Description

Technical Field

[0001] This disclosure relates to the field of oncology. Specifically, it relates to the application of a novel T-cell surface marker in determining the suitability of tumor immune checkpoint inhibitors. Background Technology

[0002] CXCR5 (CXC chemokine receptor type 5, CXC-R5), also known as CD185 or Burkitt lymphoma receptor 1 (BLR1), is a G protein-coupled seven-transmembrane receptor belonging to the CXC chemokine family. In tumors, chronic inflammation, and autoimmune diseases, CXCR5 primarily functions by transmitting signals through the CXCL13-CXCR5 axis.

[0003] CXCR5 + T cells are a special subset of T cells discovered in recent years, CXCR5 + CD4 + T cells are called follicular T-helper cells (Tfh). After being stimulated by dendritic cells (DCs), Tfh cells migrate to the T cell boundary region and further enter the B cell region, regulating B cell differentiation and promoting germinal center formation. Similar to Tfh, CXCR5... + CD8 + T cells, also known as follicular cytotoxic T cells (Tfc), can enter B cell follicles under conditions of chronic antigen exposure and inflammation. Recent studies have found that Tfc cells play a crucial role in controlling viral replication in chronic viral infection models, and that Tfc cells, compared to CXCR5-CD8, are more effective at controlling viral replication. + T cells express lower levels of the inhibitory receptor molecules PD-1 and Tim-3, and higher levels of the stimulatory molecule KLRG1. When T cells were used to treat chronically infected mice via adoptive transfer, the Tfc cell subset showed better therapeutic potential than the CXCR5- subset. Furthermore, Tfc cells in tumor patients expressed higher levels of cytokines such as perforin and granzyme; the proportion of Tfc cells was significantly positively correlated with patient prognosis. Some studies have shown that tumor-associated Tfc cells share similar transcriptional characteristics with tumor-associated lymphoid tissue (TILs).

[0004] Tumor immunotherapy utilizes the body's own immune system to recognize and attack cancer cells. In recent years, immunotherapy has become an important branch of cancer treatment and has shown significant efficacy in treating certain types of tumors. Immunotherapy includes immune checkpoint inhibitors, adoptive cell therapy, tumor vaccine therapy, cytokine therapy, and more. While immunotherapy is highly effective, it also faces many challenges. Immune checkpoint inhibitors are a large and important class of drugs in tumor immunotherapy. They work by blocking the binding of immune checkpoints to their ligands, relieving the immunosuppression caused by immune checkpoints, and thus reactivating immune cells to exert their anti-tumor effects. These inhibitors mainly include antibodies targeting cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed death receptor 1 (PD-1), and programmed death receptor ligand 1 (PD-L1). However, immune checkpoint inhibitors also face many difficulties in treatment, such as significant individual differences in efficacy, the occurrence of immune-related adverse events, drug resistance, and high treatment costs. Therefore, assessing whether cancer patients are suitable for immune checkpoint inhibitor therapy is an important clinical problem that urgently needs to be solved. Summary of the Invention

[0005] Currently, when using immune checkpoint inhibitors to treat solid tumors and hematological malignancies in clinical practice, there is a lack of convenient, non-invasive, and highly reproducible peripheral blood biomarkers for rapid screening of suitable treatment populations and early prediction of immunotherapy efficacy. To assess whether cancer patients are suitable for immune checkpoint inhibitor therapy, the applicant has, for the first time, proposed CXCR5 in the peripheral blood of subjects. + CD8 + T cells are significantly associated with tumor prognosis and the suitability of immune checkpoint inhibitors.

[0006] On the one hand, this disclosure provides a method based on peripheral blood CXCR5. + CD8 + T cells account for a significant portion of CD8. + A novel application of reagents for detecting T-cell ratios. Peripheral blood testing can rapidly determine whether a subject is suitable for immune checkpoint inhibitors, or predict the long-term benefits of immunotherapy and the risk of tumor progression, providing objective and convenient in vitro assessment data for personalized clinical immunotherapy.

[0007] In some embodiments, this disclosure provides the use of a detection reagent in the preparation of a kit for determining a subject's therapeutic need for or suitability for an immune checkpoint inhibitor.

[0008] The detection reagent can quantitatively detect CXCR5 in the peripheral blood of the subject. + CD8 + T cells account for a significant portion of total CD8+. + The proportion of T cells.

[0009] In some embodiments, the detection reagent comprises an antibody / antigen binding fragment that specifically binds to CXCR5 and an antibody / antigen binding fragment that specifically binds to CD8.

[0010] In some embodiments, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv, scFv, bispecific antibodies, single-domain antibodies VHH, dual-domain antibodies, and microantibodies. In some embodiments, antibody / antigen-binding fragments that specifically bind to CXCR5 and antibody / antigen-binding fragments that specifically bind to CD8 can be aliquoted into the same container or stored in separate containers.

[0011] The test reagents can take any appropriate form in the field of clinical testing.

[0012] For example, when manifested as a flow cytometry antibody detection reagent, the detection reagent is a fluorescently labeled antibody. For example, but not limited to, the detection reagent is a fluorescently labeled anti-human CXCR5 antibody (or its antigen-binding fragment: Fab / scFv / VHH, etc.); or anti-human CD8 antibodies (or their antigen-binding fragments: Fab / scFv / VHH, etc.) labeled with different fluorescence channels. The fluorescence emission spectra of these two types do not interfere with each other and can be co-stained in the same tube of peripheral blood samples for cluster counting. The detection reagent can be prepared in the following forms: (1) Mixing premixed antibody solution: fluorescently labeled CXCR5 antibody + fluorescently labeled CD8 antibody are premixed in the same buffer; (2) The reagent kit for separately packaged antibodies contains fluorescently labeled CXCR5 antibody and fluorescently labeled CD8 antibody, which are packaged separately in two bottles. Mix them according to the ratio before use. Another example, when manifested as a chemiluminescent immunoassay reagent, the reagent comprises: an antibody conjugated to a chemiluminescent label (acridinium ester, ruthenium tripyridine, etc.); and a magnetic microparticle-conjugated capture reagent (containing magnetic bead-labeled anti-CD8 antibody, capturing all CD8 in the sample). + T cells); luminescent labeling assay reagent (containing chemiluminescent group-labeled anti-CXCR5 antibody). The number of CXCR5-positive cells is quantified by the luminescent signal, and the CXCR5 level is calculated in conjunction with the total CD8 cell signal. + CD8 + T cells account for a significant portion of CD8. + T cell ratio.

[0013] In some implementations, the kit is used to determine a subject's therapeutic need for or suitability for an immune checkpoint inhibitor, using the following determination methods: - If the subject's peripheral blood CXCR5 + CD8 + T cells in CD8 +The subject was deemed suitable for immune checkpoint inhibitor therapy if the percentage of T cells was ≥5%. - If the subject's peripheral blood CXCR5 + CD8 + T cells in CD8 + If the percentage of T cells is ≤2%, the subject is deemed unsuitable for immune checkpoint inhibitor therapy.

[0014] On the other hand, this disclosure provides the use of a detection reagent in the preparation of a kit for predicting treatment outcomes after a subject receives an immune checkpoint inhibitor; the detection reagent can quantitatively detect CXCR5 in the peripheral blood of a subject. + CD8 + T cells account for a significant portion of CD8. + The proportion of T cells. The detection reagent may be in the form described above.

[0015] In some implementations, the kit is used to predict treatment outcomes for subjects receiving immune checkpoint inhibitors, determined by the following criteria: - When peripheral blood CXCR5 + CD8 + T cells / CD8 + When the T cell percentage is ≥5%, predict one or more of the following effects: (1) Compared to peripheral blood CXCR5 + CD8 + T cells / CD8 + When the proportion of T cells is ≤2%, the subjects' autologous T cells have a stronger ability to specifically kill tumor cells after intervention with immune checkpoint inhibitors (with statistically significant differences). (2) Compared to peripheral blood CXCR5 + CD8 + T cells / CD8 + When the proportion of T cells is ≤2%, CD45 in the tumor tissue of the subject + Immune cells and / or tumor infiltration CD8 + Higher T-cell infiltration abundance (with statistically significant difference); (3) Compared to peripheral blood CXCR5 + CD8 + T cells / CD8 + When the proportion of T cells is ≤2%, the inhibitory effect of immune checkpoint inhibitors on tumor growth in the subjects is more significant (with statistical significance). (4) Compared to peripheral blood CXCR5 + CD8 + T cells / CD8 +When the proportion of T cells is ≤2%, the tumor lesion volume of the subjects is smaller after treatment (with statistically significant difference). (5) Compared to peripheral blood CXCR5 + CD8 + T cells / CD8 + When the proportion of T cells is ≤2%, the objective response rate of immunotherapy is higher (with statistically significant difference). (6) Compared to peripheral blood CXCR5 + CD8 + T cells / CD8 + When the proportion of T cells is ≤2%, the overall survival (OS) of the subjects after treatment is significantly prolonged (with statistical significance). (7) Compared to peripheral blood CXCR5 + CD8 + T cells / CD8 + When the proportion of T cells is ≤2%, the risk of tumor metastasis and disease progression is lower after treatment (with statistically significant difference). (8) Compared to peripheral blood CXCR5 + CD8 + T cells / CD8 + When the proportion of T cells is ≤2%, the subjects have a longer metastasis-free survival (MFS) after treatment (with statistical significance).

[0016] - When peripheral blood CXCR5 + CD8 + T cells / CD8 + When the proportion of T cells is ≤2%, the subject is considered to have a poor response to immune checkpoint inhibitors or limited benefit from immunotherapy.

[0017] The phrase "poor response to immune checkpoint inhibitors or limited therapeutic effect of immune checkpoint inhibitors" refers to subjects meeting at least one or more of the following clinical outcomes after treatment with immune checkpoint inhibitors: 1) The objective response rate of tumors is low, making it impossible to achieve complete or partial remission; 2) The body's T cells have a weak ability to specifically kill tumor cells; 3) The level of immune cell infiltration in tumor tissue is low, and the effect of inhibiting tumor growth is weak; 4) The risk of tumor recurrence or distant metastasis is significantly increased after treatment; 5) Shortened metastasis-free survival or progression-free survival; 6) Overall survival was not significantly extended, and no long-term survival benefit could be obtained.

[0018] In some implementations, the immune checkpoint inhibitor is selected from: PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors, TIGIT inhibitors, TIM-3 inhibitors, VISTA inhibitors, BTLA inhibitors, and combinations thereof.

[0019] In a preferred embodiment, the immune checkpoint inhibitor is selected from: PD-1 inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors.

[0020] In some implementation plans, the subjects are individuals diagnosed with cancer, suspected of having cancer, or at risk of developing cancer.

[0021] In some implementations, the tumor is a solid tumor or a hematologic malignancy.

[0022] In some implementations, hematologic malignancies include, but are not limited to (e.g., according to WHO classification): I. Myeloid tumors: 1. Myeloproliferative neoplasms: chronic myeloid leukemia, polycythemia vera, essential thrombocythemia, myelofibrosis, chronic neutrophilic leukemia, chronic eosinophilic leukemia, mastocytosis; 2. Myelodysplastic syndromes - myeloproliferative neoplasms: chronic myelomonocytic leukemia, atypical chronic myelomonocytic leukemia, juvenile myelomonocytic leukemia, MDS / MPN with ring sideroblasts and thrombocytosis; 3. Myelodysplastic syndromes: MDS with unilineage dysplasia, MDS with ring sideroblasts, MDS with multilineage dysplasia, MDS with blast cell proliferation - 1, MDS with blast cell proliferation - 2, and refractory cytopenia in children; 4. Acute myeloid leukemia; 5. Myeloid sarcoma; 6. Myeloid proliferation associated with Down syndrome; 7. Mast cell leukemia; II. Lymphatic tumors: 1. B-cell tumors; 2. T / NK cell tumors; 3. Hodgkin's lymphoma; III. Histiocytic and Dendritic Cell Tumors: Langerhans cell histiocytosis, Langerhans cell sarcoma, finger dendritic cell sarcoma, follicular dendritic cell sarcoma, histiocytic sarcoma, disseminated juvenile xanthogranuloma.

[0023] In some implementation schemes, solid tumors include, but are not limited to: head and neck tumors (intracranial tumors, ocular tumors, nasopharyngeal carcinoma, laryngeal cancer, oral cancer, hypopharyngeal cancer, thyroid tumors, salivary gland tumors); thoracic tumors (lung tumors, mediastinal tumors, breast tumors, pleural tumors); digestive system tumors (esophageal tumors, gastric tumors, intestinal tumors, liver tumors, biliary tract tumors, pancreatic tumors, peritoneal tumors); genitourinary system tumors (kidney cancer, bladder cancer, ureteral cancer, prostate cancer, testicular tumors, penile cancer); cervical cancer, ovarian tumors, endometrial cancer, vulvar / vaginal tumors; bone and soft tissue tumors (primary malignant bone tumors, soft tissue sarcomas); and skin tumors (melanoma, squamous cell carcinoma of the skin, basal cell carcinoma).

[0024] In some implementation schemes, solid tumors are selected from: non-small cell lung cancer, melanoma, colorectal cancer, nasopharyngeal carcinoma, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, prostate cancer, bladder cancer, and kidney cancer.

[0025] In some implementation schemes, hematologic malignancies are selected from: acute leukemia, chronic leukemia, lymphoma, plasma cell tumor, myeloproliferative neoplasm, and myelodysplastic syndrome.

[0026] In some embodiments, the antibody or antigen-binding fragment is conjugated to a detectable marker, which may be selected from one or more of the following classes: (1) Fluorescent labels: FITC, PE, APC, PerCP, PE-Cy5, PE-Cy7, APC-Cy7, Alexa Fluor, Brilliant Violet, Dylight, Cy3 / Cy5 / Cy5.5 / Cy7; (2) Chemiluminescent labeling: acridine ester, luminol, isoluminol, ruthenium terpyridine, AMPPD; (3) Enzyme labeling: horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase; (4) Radioactive isotopes: 125 I, 3 H, 14 C 32 P; (5) Nanoscale tracer labeling: fluorescent quantum dots, colloidal gold, immunomagnetic particles; (6) Colorimetric markers: biotin, digoxin.

[0027] In some implementations, in addition to antibody / antigen-binding fragments that specifically bind to CXCR5 and antibody / antigen-binding fragments that specifically bind to CD8, the kit may also include any one or more of the following reagents: peripheral blood lymphocyte separation medium, antibody or antigen-binding fragment that specifically binds to CD3.

[0028] This disclosure also provides an in vitro detection method for determining whether a subject with a solid tumor is suitable for an immune checkpoint inhibitor, including the following steps: 1) Provide peripheral blood samples from the subjects; 2) Identify CD8 in the peripheral blood sample using CXCR5 antibody or its antigen-binding fragment, and CD8 antibody or its antigen-binding fragment. + CXCR5 in T cells + Cell ratio; 3) Based on CD8 + CXCR5 in T cells + The proportion of cells is used to determine the suitability of the subject for treatment with immune checkpoint inhibitors; - When the peripheral blood CXCR5 of the subject + CD8 + T cells in CD8 + The proportion of T cells ≥5% indicates that the subject is suitable for immune checkpoint inhibitor treatment; - When the peripheral blood CXCR5 of the subject + CD8 + T cells in CD8 + If the proportion of T cells is ≤2%, the subject is deemed not suitable for immune checkpoint inhibitor treatment.

[0029] This disclosure also provides a method for predicting the treatment outcome of immune checkpoint inhibitors in subjects, including the steps of: 1) Provide peripheral blood samples from the subjects; 2) Identify CD8 in the peripheral blood sample using CXCR5 antibody or its antigen-binding fragment, and CD8 antibody or its antigen-binding fragment. + CXCR5 in T cells + Cell ratio; 3) Based on CD8 + CXCR5 in T cells + The proportion of cells is used to determine the suitability of the subject for treatment with immune checkpoint inhibitors; - When the peripheral blood CXCR5 of the subject + CD8 + T cells in CD8 + The proportion of T cells ≥5% is considered to indicate that the subject will benefit from immune checkpoint inhibitor treatment; - When the peripheral blood CXCR5 of the subject + CD8 + T cells in CD8 +If the proportion of T cells is ≤2%, the subject is considered to have a poor response to immune checkpoint inhibitors or limited therapeutic effect of immune checkpoint inhibitors. Attached Figure Description

[0030] Figure 1 Display CXCR5 + CD8 + T cells are tumor-responsive T cells: A: In mouse models, CXCR5 showed different effector-to-target ratios (E:T = 1:2, 1:4, 1:8). + CD8 + T cells and CXCR5-CD8 + Comparison of T cell killing rates against MC38-OVA target cells. Results showed that CXCR5 + The group showed significantly higher levels of CXCR5- at all effect-to-target ratios.

[0031] B: Autologous CXCR5 in tumor patient samples with different effect-to-target ratios + CD8 + T cells and CXCR5-CD8 + A comparison of the killing rate of T cells against autologous tumor cells. The results also showed that CXCR5 + The group has a stronger specific killing ability.

[0032] Figure 2 Display of CD8 in peripheral blood + CXCR5 in T cells + The higher the proportion, the more immune cells, especially CD8, infiltrate the tumor. + T cells: A: CD8+ in peripheral blood of tumor-bearing mice + CXCR5 in T cells + Percentage and CD45 infiltration in tumors + Scatter plot showing the correlation between the proportion of immune cells.

[0033] B: Peripheral blood CD8 of tumor-bearing mice + CXCR5 in T cells + Percentage and CD8 infiltration in tumors + Scatter plot of correlation of T cell proportions.

[0034] C: Peripheral blood CD8 of cancer patients + CXCR5 in T cells + Percentage and CD45 infiltration in tumors + Scatter plot showing the correlation between the proportion of immune cells.

[0035] D: CD8+ in peripheral blood of cancer patients + CXCR5 in T cells +Percentage and CD8 infiltration in tumors + Scatter plot of correlation of T cell proportions.

[0036] Figure 3 Display of CD8 in peripheral blood + CXCR5 in T cells + The higher the proportion, the smaller the tumor size in animals and the greater the degree of tumor remission in humans after treatment with tumor immune checkpoint inhibitors. A: In tumor-bearing mouse models, based on peripheral blood CD8... + CXCR5 in T cells + Tumor growth curves after PD-1 inhibitor treatment were shown for the high-proportion and low-proportion groups. The tumor volume in the high-proportion group was significantly smaller than that in the low-proportion group.

[0037] B: Survival curves (Kaplan-Meier) for the two groups of mice. The median survival time in the high-proportion group was significantly longer than that in the low-proportion group.

[0038] C: Tumor growth curves in a tumor-bearing mouse model, divided into high-proportion and low-proportion groups based on the proportion of CXCR5+ in peripheral blood CD8+ T cells, after treatment with PD-L1 inhibitors. The tumor volume in the high-proportion group was significantly smaller than that in the low-proportion group.

[0039] D: Survival curves (Kaplan-Meier) for the two groups of mice. The median survival time in the high-proportion group was significantly longer than that in the low-proportion group.

[0040] E: Tumor growth curves in a tumor-bearing mouse model, divided into high-proportion and low-proportion groups based on the proportion of CXCR5+ in peripheral blood CD8+ T cells, after treatment with CTLA-4 inhibitors. The tumor volume in the high-proportion group was significantly smaller than that in the low-proportion group.

[0041] F: Survival curves (Kaplan-Meier) for the two groups of mice. The median survival time in the high-proportion group was significantly longer than that in the low-proportion group.

[0042] G: Among cancer patients, the objective response rate (ORR) after PD-1 inhibitor treatment was significantly higher in the high-proportion group of CXCR5+ CD8+ T cells in peripheral blood before treatment compared with that in the low-proportion group.

[0043] H: The overall survival (OS) curves (Kaplan-Meier) of the two groups of patients treated with PD-1 inhibitors showed that the OS of the high-proportion group was significantly longer than that of the low-proportion group.

[0044] I: Comparison of objective response rate (ORR) between high and low proportions of CXCR5+ cells in peripheral blood CD8+ T cells before treatment in cancer patients after PD-L1 inhibitor therapy (stacked bar chart showing CR+PR ratio), the high proportion group was significantly higher than the low proportion group (P < 0.05).

[0045] J: The overall survival (OS) curves (Kaplan-Meier) of the two groups of patients treated with PD-L1 inhibitors showed that the OS of the high proportion group was significantly longer than that of the low proportion group (P < 0.05).

[0046] K: In cancer patients, the objective response rate (ORR) after CTLA-4 inhibitor treatment was compared between the high and low proportions of CXCR5+ cells in peripheral blood CD8+ T cells before treatment (stacked bar chart showing the CR+PR ratio). The high proportion group was significantly higher than the low proportion group (P < 0.05).

[0047] L: The overall survival (OS) curves (Kaplan-Meier) of the two groups of patients treated with CTLA-4 inhibitors showed that the OS of the high proportion group was significantly longer than that of the low proportion group (P < 0.05).

[0048] Figure 4 Display of CD8 in peripheral blood + CXCR5 in T cells + The higher the proportion, the weaker the degree of tumor metastasis after treatment with tumor immune checkpoint inhibitors. A: In a mouse lung metastasis model, peripheral blood CD8 levels before PD-1 inhibitor treatment... + CXCR5 in T cells + Scatter plot showing the correlation between the proportion and the number of metastatic nodules on the lung surface after treatment.

[0049] B: In cancer patients, peripheral blood CD8 levels before treatment + CXCR5 in T cells + A comparison of the incidence of "good metastasis control" and "metastasis progression" after PD-1 inhibitor treatment between the high-proportion group and the low-proportion group (stacked bar chart or percentage comparison between the two groups) showed that the metastasis progression rate in the high-proportion group was significantly lower than that in the low-proportion group.

[0050] C: The metastasis-free survival (MFS) curves (Kaplan-Meier) of the two groups of patients above show that the MFS of the high-proportion group is significantly longer than that of the low-proportion group.

[0051] D: Scatter plot showing the correlation between the proportion of CXCR5+ in peripheral blood CD8+ T cells before PD-L1 inhibitor treatment and the number of metastatic nodules on the lung surface after treatment in a mouse lung metastasis model.

[0052] E: Among cancer patients, the incidence of "good metastasis control" and "metastasis progression" after PD-L1 inhibitor treatment was compared between the high proportion and low proportion of CXCR5+ T cells in peripheral blood before treatment (stacked bar chart or percentage comparison between the two groups). The metastasis progression rate in the high proportion group was significantly lower than that in the low proportion group.

[0053] F: The metastasis-free survival (MFS) curves (Kaplan-Meier) of the two groups of patients above show that the MFS of the high-proportion group was significantly longer than that of the low-proportion group.

[0054] G: Scatter plot showing the correlation between the proportion of CXCR5+ in peripheral blood CD8+ T cells before CTLA-4 inhibitor treatment and the number of metastatic nodules on the lung surface after treatment in a mouse lung metastasis model.

[0055] H: In cancer patients, the incidence of "good metastasis control" and "metastasis progression" after CTLA-4 inhibitor treatment was compared between the high proportion and low proportion of CXCR5+ T cells in peripheral blood before treatment (stacked bar chart or percentage comparison between the two groups). The metastasis progression rate in the high proportion group was significantly lower than that in the low proportion group.

[0056] I: Metastasis-free survival of the two groups of patients mentioned above. Detailed Implementation

[0057] Peripheral blood from cancer patients was obtained from the Cancer Hospital of the Chinese Academy of Medical Sciences; C57BL / 6J was purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.; and the mouse colorectal cancer cell line MC38-OVA was purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. Unless otherwise specified, all raw materials and reagents used in this disclosure are commercially available.

[0058] Example 1. CXCR5 + CD8 + T cells are tumor-responsive T cells. 1. Experimental Procedure (1) Subcutaneous injection of 5*10 saturates into the right axilla of C57 mice 6 A mouse tumor-bearing model was constructed using MC38-OVA cells. One week after inoculation, approximately 1 mL of blood was collected from the mouse eyeballs, and CXCR5 cells were isolated from the blood. + CD8 + T cells and CXCR5-CD8 + T cells. Effector-to-target ratios (E:T) were 1:2, 1:4, 1:8, and 5*10⁻⁶. 4MC38-OVA cells were co-incubated for 24 hours. After 24 hours, the target cell viability was detected using the CellTiter-Glo® Luminescent Cell Viability Assay. The target cell death rate was calculated using the target cell group without effector cells as a control.

[0059] (2) Collect peripheral blood from tumor patients and separate CXCR5. + CD8 + T cells and CXCR5-CD8 + T cells. Simultaneously, tumor tissue samples were taken from the patient, mechanically minced, and digested with collagenase to obtain single-cell suspensions. The two types of CD8 cells were then used at effector-to-target ratios (E:T) of 1:2, 1:4, and 1:8. + T cells were co-incubated with the patient's autologous tumor cells for 24 hours. The target cell viability was detected using CellTiter-Glo® Luminescent Cell Viability, and the target cell mortality rate was calculated.

[0060] 2. Experimental Results ( Figure 1 ) (1) In mouse models, with CXCR5-CD8 + Compared to the T cell co-incubation group, CXCR5 + CD8 + The T cell co-incubation group showed a significantly increased MC38-OVA target cell death rate in an effector-to-target ratio-dependent manner, indicating that CXCR5 + CD8 + T cells have a significantly stronger ability to kill tumor cells than CXCR5-CD8. + T cells.

[0061] (2) In tumor patient samples, compared with autologous CXCR5-CD8 + Compared to T cells, autologous CXCR5 + CD8 + T cells exhibit a stronger specific killing ability against the patient's own tumor cells, and the target cell mortality rate is significantly increased.

[0062] Example 2. CXCR5 in peripheral blood + CD8 + The higher the proportion of T cells, the more immune cells infiltrate the tumor, especially CD8 cells. + T cells 1. Experimental Procedure (1) Take the MC38-OVA tumor-bearing mouse model established in Example 1, collect about 1 mL of peripheral blood from its eyeball, and use flow cytometry to detect CD8.+ CXCR5 in T lymphocytes + The proportion of cells was determined. Mice were then sacrificed, tumor tissue was isolated and weighed, and a single-cell suspension was prepared. CD45+ in tumor-infiltrating lymphocytes was detected by flow cytometry. + The proportion of immune cells, and further analysis of CD8 cells. + The percentage of T cells.

[0063] (2) Peripheral blood was collected from tumor patients and their CD8 count was detected by flow cytometry. + CXCR5 in T lymphocytes + The proportion of cells. Simultaneously, tumor tissue samples were obtained from the same patient, weighed, digested into single-cell suspensions, and tumor infiltration CD45 was detected by flow cytometry. + The proportion of immune cells and CD8 + The percentage of T cells.

[0064] 2. Experimental Results ( Figure 2 ) (1) In tumor-bearing mouse models, peripheral blood CD8 + CXCR5 in T cells + Proportion and total immune cells (CD45) in tumor tissue + The infiltration rate was positively correlated with the tumor infiltration CD8 group, especially with the infiltration rate of tumor CD8. + The proportion of T cells was significantly positively correlated with the tumor microenvironment. A higher proportion of these cells in peripheral blood was associated with a higher proportion of CD8+ cells. + The more abundant the T cell infiltration.

[0065] (2) In tumor patient samples, the following was also observed: CD8+ in the peripheral blood of patients. + CXCR5 in T cells + The higher the percentage, the more CD45 infiltrates in the tumor tissue. + Total proportion of immune cells and CD8 + The higher the proportion of T cell subsets, the more likely it is to be related to peripheral blood CXCR5. + CD8 + The proportion of T cells can reflect the local immune activity status of the tumor.

[0066] Example 3. CXCR5 in peripheral blood + CD8 + A higher proportion of T cells leads to smaller tumor size and longer survival time in animals treated with tumor immune checkpoint inhibitors, and a higher degree of tumor remission and longer survival time in humans. 1. Experimental Procedure (1) Take the MC38-OVA tumor-bearing mouse model established in Example 1, and wait until the tumor volume reaches about 50 to 100 mm. 3Peripheral blood was collected by tail clipping and CD8 was detected using flow cytometry. + CXCR5 in T cells + Cell proportions were defined as follows: ≥5% CXCR5+ in CD8+ T cells was defined as high CXCR5+ group, and ≤2% CXCR5+ in CD8+ T cells was defined as low CXCR5+ group. Subsequently, all mice were injected via tail vein with an immune checkpoint inhibitor (anti-mouse PD-1, PD-L1, or CTLA-4 antibody, 200 μg / mouse), administered every 3 days for a total of 4 doses. Tumor volume was measured every 2 days during treatment, and survival time for each mouse was recorded. Cell proportions were determined based on peripheral blood CXCR5+ levels. + CD8 + The median proportion of T cells was used to divide mice into high-proportion and low-proportion groups. Tumor growth curves and survival curves were plotted for the two groups, and correlation analysis was performed.

[0067] (2) Collect peripheral blood samples from cancer patients (non-small cell lung cancer, melanoma) who received immune checkpoint inhibitor therapy before treatment and use flow cytometry to detect CD8. + CXCR5 in T cells + Cell proportions. Assess the degree of tumor response after treatment (complete response CR, partial response PR, stable disease SD, progressive disease PD) and record overall survival (OS). Plot the CXCR5 value in peripheral blood before treatment. + CD8 + The correlation curve between T cell proportion and OS was used to compare the survival differences between the high-proportion and low-proportion groups.

[0068] 2. Experimental Results (1) CXCR5 in peripheral blood of tumor-bearing mice + CD8 + T cells account for a significant portion of CD8. + A higher proportion of T cells (≥5%) resulted in slower tumor growth and significantly smaller tumor volume after PD-1 inhibitor treatment compared to the low proportion group. Survival analysis showed that the median survival time of mice in the high proportion group was significantly longer than that in the low proportion group, and peripheral blood CXCR5 levels were also higher. + CD8 + The proportion of T cells was positively correlated with mouse survival time (see [reference]). Figure 3 (A and B).

[0069] Similar results were observed with the administration of PD-L1 inhibitors or CTLA-4 inhibitors in animal models. CXCR5 levels in the peripheral blood of tumor-bearing mice... + CD8 + A higher T ratio resulted in significantly smaller tumor volume compared to the low ratio group, and a significantly longer median survival time for mice in the high ratio group compared to the low ratio group (see [link to relevant documentation]). Figure 3 C to Figure 3 (F).

[0070] (2) In cancer patients, peripheral blood CXCR5 levels before treatment + CD8 + Patients with a high proportion of T cells have a higher objective response rate (CR) after receiving immune checkpoint inhibitor therapy. + Higher PR (partial response) and significantly prolonged overall survival (OS) were observed. Correlation analysis showed that peripheral blood CXCR5 levels were higher in patients before treatment. + CD8 + The proportion of T cells is positively correlated with overall survival, suggesting that this indicator can serve as a biomarker for predicting the efficacy and prognosis of immune checkpoint inhibitors (for PD-1 inhibitors). Figure 3 G and H; for PD-L1 inhibitors, Figure 3 I and J; for CTLA-4 inhibitors, Figure 3 (K and L).

[0071] Example 4. CXCR5 in peripheral blood + CD8 + The higher the proportion of T cells, the weaker the degree of tumor metastasis after treatment with tumor immune checkpoint inhibitors. 1. Experimental Procedure (1) Mouse model experiment: 6-8 week old C57BL / 6J mice were injected with 5×10 mmol / L via the tail vein. 5 An experimental lung metastasis model was established using MC38-OVA cells. On day 7 post-inoculation, peripheral blood was collected by tail clipping, and CD8+ levels were detected using flow cytometry. + CXCR5 in T cells + Cell proportions. Mice were then randomly divided into two groups: the treatment group received a tail vein injection of an immune checkpoint inhibitor (anti-mouse PD-1, PD-L1, or CTLA-4 antibody, 200 μg / mouse), and the control group received an equal amount of IgG isotype control antibody. Administration was repeated every 3 days for a total of 4 doses. Seven days after the last administration, mice were sacrificed, lung tissue was removed, fixed with Bouin's fixative, and the number of visible metastatic nodules on the lung surface was counted under a dissecting microscope. Peripheral blood CXCR5 was analyzed. + CD8 + The correlation between T cell proportion and the number of lung metastatic nodules was investigated. The group with CXCR5+ ≥5% in CD8+ T cells was defined as having a high CXCR5 proportion, and the group with CXCR5+ ≤2% in CD8+ T cells was defined as having a low CXCR5 proportion.

[0072] (2) Clinical patient sample experiment: Peripheral blood samples were collected from patients with solid tumors (such as colorectal cancer and melanoma) who were pathologically diagnosed and scheduled to receive immune checkpoint inhibitors (PD-1 inhibitors, PD-L1 inhibitors and CTLA-4 inhibitors) before treatment. CD8 was detected by flow cytometry. + CXCR5 in T cells + Cell proportions. Patients received standard doses of immune checkpoint inhibitors (once every 3 weeks). Tumor burden and metastasis were assessed every 8–12 weeks after treatment initiation by imaging (CT or MRI). Based on imaging results, patients were divided into a "well-controlled metastasis group (no new metastases or shrinking metastases)" and a "metastasis progression group (new metastases or enlarged metastases)". Peripheral blood CXCR5 levels before treatment were compared between the two groups. + CD8 + The study investigated the differences in the proportion of T cells and analyzed the correlation between this proportion and metastasis-free survival (MFS) using the Kaplan-Meier method.

[0073] 2. Experimental Results (1) In the MC38-OVA lung metastasis mouse model, immune checkpoint inhibitor treatment significantly reduced the number of lung metastatic nodules in a high proportion of mice. Correlation analysis showed that peripheral blood CXCR5 levels were significantly reduced before treatment. + CD8 + T cells account for a significant portion of CD8. + The proportion of T cells was significantly negatively correlated with the number of lung metastatic nodules after treatment; that is, the higher the proportion of T cells in peripheral blood (≥5%), the weaker the degree of lung metastasis in mice. Figure 4 (A, D, G).

[0074] (2) In clinical cancer patients, peripheral blood CXCR5 levels before treatment + CD8 + Patients with a higher proportion of T cells had a significantly lower incidence of new metastases or metastatic progression after receiving immune checkpoint inhibitor therapy compared to the low proportion group. Kaplan-Meier survival analysis showed that the metastasis-free survival (MFS) was significantly longer in the high proportion group than in the low proportion group. Figure 4 (B, C; E, F, H, and I). This result suggests that peripheral blood CXCR5... + CD8 + The proportion of T cells can serve as a potential biomarker for predicting the anti-tumor metastasis efficacy of immune checkpoint inhibitors.

[0075] This disclosure provides information on the detection of CXCR5 in peripheral blood. + CD8 + The proportion of T cells is used to determine the suitability of tumor immune checkpoint inhibitor therapy. The inventors discovered through experimental research that CXCR5 in peripheral blood... +CD8 + The proportion of T cells is significantly correlated with the suitability of tumor immune checkpoint inhibitor therapy. Peripheral blood CXCR5 + CD8 + The higher the proportion of T cells, the more CD8 cells infiltrate the tumor. + The more T cells, the higher the CXCR5 level in peripheral blood after treatment with tumor immune checkpoint inhibitors. + CD8 + A higher proportion of T cells is associated with longer patient survival, smaller tumor size, and less metastasis. This is not limited to specific theories; peripheral blood CXCR5... + CD8 + The higher the proportion of T cells, the more T cells migrate to the tumor site, and the greater the degree of tumor infiltration. This indicates that after treatment with immune checkpoint inhibitors, more effector T cells can be enhanced, resulting in a stronger tumor-killing effect and a better treatment outcome.

Claims

1. The use of the detection reagents in the preparation of the kit, including: The kit is used to determine the therapeutic need or suitability of a subject for an immune checkpoint inhibitor; The detection reagent can quantitatively determine the CXCR5 level in the peripheral blood of the subject. + CD8 + T cells in CD8 + The proportion of T cells; - When the peripheral blood CXCR5 of the subject + CD8 + T cells in CD8 + The proportion of T cells ≥5% indicates that the subject is suitable for immune checkpoint inhibitor treatment; - When the peripheral blood CXCR5 of the subject + CD8 + T cells in CD8 + If the proportion of T cells is ≤2%, the subject is deemed not suitable for immune checkpoint inhibitor treatment.

2. The use of the detection reagents in the preparation of the kit, including: The kit is used to predict the treatment outcome of immune checkpoint inhibitors in subjects; The detection reagent can quantitatively determine the CXCR5 level in the peripheral blood of the subject. + CD8 + T cells in CD8 + The proportion of T cells; - When the peripheral blood CXCR5 of the subject + CD8 + T cells in CD8 + The proportion of T cells is ≥5%, and the determination is selected from any one or a combination of the following: 1) It is predicted that the subject's autologous T cells will have a stronger specific killing ability against tumor cells after treatment with immune checkpoint inhibitors; 2) Predicting CD45 in the tumor tissue of the subject + Immune cells and / or tumor infiltration CD8 + The higher the abundance of T cell infiltration; 3) It is predicted that the subjects treated with immune checkpoint inhibitors will experience more significant inhibition of tumor growth; 4) It is predicted that the tumor volume will be smaller after the subjects are treated with immune checkpoint inhibitors; 5) It is predicted that the subjects will have a higher objective response rate after treatment with immune checkpoint inhibitors; 6) It is predicted that the overall survival (OS) of the subjects will be significantly prolonged after treatment with immune checkpoint inhibitors; 7) It is predicted that the subjects will have a lower risk of tumor metastasis and progression after treatment with immune checkpoint inhibitors; 8) It is predicted that the subjects will have longer metastasis-free survival (MFS) after treatment with immune checkpoint inhibitors; - When the peripheral blood CXCR5 of the subject + CD8 + T cells in CD8 + If the proportion of T cells is ≤2%, the subject is considered to have a poor response to immune checkpoint inhibitors or limited therapeutic effect of immune checkpoint inhibitors.

3. The use according to claim 1 or 2, wherein: The immune checkpoint inhibitors are selected from: PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors, TIGIT inhibitors, TIM-3 inhibitors, VISTA inhibitors, BTLA inhibitors, and combinations thereof.

4. The use according to claim 3, wherein: The immune checkpoint inhibitors are selected from: PD-1 inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors.

5. The use according to claim 1 or 2, wherein: The subjects had, were suspected of having, or were susceptible to solid tumors or hematologic malignancies.

6. The use according to claim 5, wherein: The solid tumors are selected from: non-small cell lung cancer, melanoma, colorectal cancer, nasopharyngeal carcinoma, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, prostate cancer, bladder cancer, and kidney cancer. The hematologic malignancies are selected from: acute leukemia, chronic leukemia, lymphoma, plasma cell tumor, myeloproliferative neoplasm, and myelodysplastic syndrome.

7. The use according to claim 1 or 2, wherein the detection reagent comprises: Antibodies or their antigen-binding fragments that specifically recognize CXCR5, and Antibodies or their antigen-binding fragments that specifically recognize CD8; The antigen-binding fragments are selected from: Fab, Fab', F(ab')2, Fv, scFv, bispecific antibodies, single-domain antibodies VHH, dual-domain antibodies, and minibody. The antibody that specifically recognizes CXCR5 or its antigen-binding fragment and the antibody that specifically recognizes CD8 or its antigen-binding fragment are in the same or different containers.

8. The use according to claim 7, wherein: The antibody or its antigen-binding fragment is conjugated with a detectable portion; The detectable portion is selected from: fluorescently labeled molecules, chemiluminescent labels, enzyme labels, radioactive isotopes, nanoparticle tracer labels, and chromogenic substrate labels; Preferably, the fluorescent labeling molecule is selected from: fluorescein isothiocyanate; phycoerythrin, allophycocyanin, PerCP, PE-Cy5, PE-Cy7, APC-Cy7; Alexa Fluor; Brilliant Violet; Dylight fluorescent dye; Cy3, Cy5, Cy5.5, Cy7; Preferably, the chemiluminescent label is selected from: acridine ester, luminol, isoluminol, ruthenium terpyridine, and AMPPD; Preferably, the enzyme marker is selected from: horseradish peroxidase, alkaline phosphatase, β-galactosidase, and glucose oxidase; Preferably, the radioactive isotope is selected from: 125 I, 3 H, 14 C 32 P; Preferably, the nanoparticle tracer label is selected from: fluorescent quantum dots, colloidal gold nanoparticles, and magnetic particles; Preferably, the chromogenic substrate marker is selected from biotin and digoxin.

9. The use according to claim 1 or 2, wherein the kit further comprises any one or a combination of the following: Peripheral blood lymphocyte separation reagent, CD3 antibodies or their antigen-binding fragments.

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