Application of peripheral blood lymphocyte subpopulation in preparation of detection kit for evaluating anti-PD-1 treatment prognosis of advanced non-small cell lung cancer patient

Abstract

The invention discloses application of a peripheral blood lymphocyte subpopulation in preparation of a detection kit for evaluating prognosis of anti-PD-1 treatment of a patient with advanced non-small cell lung cancer. The kit is used for measuring the proportion of lymphocyte subgroups in peripheral blood of an advanced non-small cell lung cancer patient subjected to anti-PD-1 treatment so as to evaluate the prognosis of the advanced non-small cell lung cancer patient. According to the embodiment of the invention, the curative effect of anti-PD-1 treatment of the NSCLC patient can be known in time by detecting the change of the peripheral blood lymphocyte subpopulation of the late NSCLC patient.

Description

Technical Field

[0001] The present application belongs to biomedicine, and in particular, relates to the use of a peripheral blood lymphocyte subset in the preparation of a detection kit for evaluating the prognosis of anti-PD-1 treatment in patients with advanced non-small cell lung cancer. Background Art

[0002] Among malignant tumors, the incidence and mortality of lung cancer in my country are both at the forefront, among which non-small cell lung cancer (NSCLC) is the main type of lung cancer, accounting for about 85% of the total lung cancer. Because the onset of NSCLC is relatively hidden, it is often diagnosed in the late stage. In addition to maintenance therapy, chemotherapy, and targeted therapy, immunotherapy, especially PD-1 / L1 inhibitors, are the first-line / second-line standard treatments for advanced NSCLC patients. For people with high PD-L1 expression, immune monotherapy can bring significant clinical benefits, with a 5-year survival rate of up to 32%, which has changed the treatment concept of advanced lung cancer. However, the clinical benefit of immune monotherapy in people with low or no PD-L1 expression is not significant, and patients with PD-L1 ≥ 50% only account for 29.8% of the total NSCLC population.

[0003] However, lung cancer tissue specimens are not easy to obtain and it is difficult to overcome the temporal and spatial heterogeneity of tumors. Therefore, the efficacy of PD-1 / L1 inhibitors cannot be known in a timely manner. Summary of the invention

[0004] The embodiment of the present application provides an application of a peripheral blood lymphocyte subset in the preparation of a detection kit for evaluating the prognosis of anti-PD-1 treatment in patients with advanced non-small cell lung cancer, which can timely understand the efficacy of anti-PD-1 treatment for NSCLC patients by detecting changes in peripheral blood lymphocyte subsets in patients with advanced NSCLC.

[0005] An embodiment of the present application provides an application of a peripheral blood lymphocyte subset in the preparation of a detection kit for evaluating the prognosis of anti-PD-1 treatment in patients with advanced non-small cell lung cancer. The kit is used to determine the proportion of lymphocyte subsets in the peripheral blood of patients with advanced non-small cell lung cancer after anti-PD-1 treatment to evaluate the prognosis of anti-PD-1 treatment in patients with advanced non-small cell lung cancer; the lymphocyte subsets include at least one of CD3+T cells, CD4+T cells, CD8+T cells, CD3+CD8+PD-1+T cells, CD3+CD8+TIM-3+T cells, CD3+γδT+Vδ2+PD-1+T cells and CD3+γδT+Vδ2+TIM-3+T cells.

[0006] In any embodiment of the present application, the prognosis of anti-PD-1 treatment in patients with advanced non-small cell lung cancer is evaluated by at least one of the ratio of CD3+T cells, the ratio of CD4+T cells to CD8+T cells, the ratio of CD3+CD8+PD-1+T cells, the ratio of CD3+CD8+TIM-3+T cells, the ratio of CD3+γδT+Vδ2+PD-1+T cells and the ratio of CD3+γδT+Vδ2+TIM-3+T cells.

[0007] In any embodiment of the present application, when a patient with advanced non-small cell lung cancer obtains clinical benefit after receiving anti-PD-1 treatment, the proportion of CD3+T cells in the baseline peripheral blood is higher than 49.15%.

[0008] In any embodiment of the present application, when a patient with advanced non-small cell lung cancer obtains clinical benefit after receiving anti-PD-1 treatment, the ratio of CD4+T cells to CD8+T cells in the baseline peripheral blood is higher than 1.06.

[0009] In any embodiment of the present application, when a patient with advanced non-small cell lung cancer obtains clinical benefit after receiving anti-PD-1 treatment, the proportion of CD3+CD8+PD-1+T cells in the baseline peripheral blood is higher than 0.43%.

[0010] In any embodiment of the present application, when patients with advanced non-small cell lung cancer receive anti-PD-1 treatment and obtain clinical benefits, the proportion of CD3+CD8+TIM-3+T cells in the baseline peripheral blood is higher than 3.41%.

[0011] In any embodiment of the present application, when a patient with advanced non-small cell lung cancer obtains clinical benefit after receiving anti-PD-1 treatment, the proportion of CD3+γδT+Vδ2+PD-1+T cells in the baseline peripheral blood is higher than 0.81%.

[0012] In any embodiment of the present application, when patients with advanced non-small cell lung cancer receive anti-PD-1 treatment and obtain clinical benefits, the proportion of CD3+γδT+Vδ2+TIM-3+T cells in the baseline peripheral blood is higher than 1.26%.

[0013] In any embodiment of the present application, the prognosis of anti-PD-1 treatment in patients with advanced non-small cell lung cancer is jointly evaluated by the ratio of CD3+T cells, the ratio of CD4+T cells to CD8+T cells, the ratio of CD3+CD8+PD-1+T cells, the ratio of CD3+CD8+TIM-3+T cells, the ratio of CD3+γδT+Vδ2+PD-1+T cells and the ratio of CD3+γδT+Vδ2+TIM-3+T cells.

[0014] In any embodiment of the present application, the kit includes fluorescently labeled monoclonal antibodies, and the monoclonal antibodies include at least one of anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-TCRγδ antibody, anti-TCRVδ2 antibody, anti-PD-1 antibody and anti-TIM-3 antibody.

[0015] In any embodiment of the present application, the monoclonal antibody consists of anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-TCRγδ antibody, anti-TCRVδ2 antibody, anti-PD-1 antibody and anti-TIM-3 antibody.

[0016] The use of the peripheral blood lymphocyte subsets of the embodiments of the present application in the preparation of a detection kit for evaluating the prognosis of anti-PD-1 treatment in patients with advanced non-small cell lung cancer can detect the CD3+T cell ratio, the ratio of CD4+T cells to CD8+T cells, the CD3+CD8+PD-1+T cell ratio, the CD3+CD8+TIM-3+T cell ratio, the CD3+γδT+Vδ2+PD-1+T cell ratio and the CD3+γδT+Vδ2+TIM-3+T cell ratio in the peripheral blood of patients with advanced NSCLC through the kit, thereby conveniently, non-invasively and repeatedly knowing the immune status of the tumor and the host in the body of NSCLC patients after anti-PD-1 treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solution of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0018] Figure 1 Changes in key immune indicators in patients with non-small cell lung cancer before and after PD-1 monoclonal antibody treatment;

[0019] Figure 2 A prediction model for the efficacy of PD-1 monoclonal antibody therapy for non-small cell lung cancer based on key immune indicators;

[0020] Figure 3-Figure 25 is the flow gating strategy for lymphocytes; Figure 3 In the peripheral blood whole cell population, circle the lymphocytes; Figure 4 Circle the CD3-positive cells in the lymphocyte population, i.e., CD3+ T cells; Figure 5 In the figure, circle the γδ-positive cells in the CD3+T cell population, i.e., γδT cells; Figure 6 In the CD3+T cell population, circle the CD4-positive cells and CD8-positive cells, i.e., CD3+CD4+T cells and CD3+CD8+T cells; Figure 7In the γδT cell population, Vδ1-positive cells and Vδ2-positive cells were circled, i.e., CD3+γδ+Vδ1+T cells and CD3+γδ+Vδ2+T cells; Figure 8-Figure 10 In the figure, three subpopulations were circled in the CD3+γδ+Vδ2+T cell population, namely, CD3+γδ+Vδ2+PD-1+T cells, CD3+γδ+Vδ2+TIM-3+T cells, and CD3+γδ+Vδ2+PD-1+TIM-3+T cells; Figure 11-13 In the figure, three subpopulations were circled in the CD3+γδ+Vδ1+T cell population, namely, CD3+γδ+Vδ1+PD-1+T cells, CD3+γδ+Vδ1+TIM-3+T cells, and CD3+γδ+Vδ1+PD-1+TIM-3+T cells; Figure 14-16 In the γδT cell population, three subpopulations were circled, namely, CD3+γδ+PD-1+T cells, CD3+γδ+TIM-3+T cells, and CD3+γδ+PD-1+TIM-3+T cells; Figure 17-Figure 19 In the figure, three subpopulations were circled in the CD3+T cell population, namely, CD3+PD-1+T cells, CD3+TIM-3+T cells, and CD3+PD-1+TIM-3+T cells; Figure 20-Figure 22 In the CD3+CD8+T cell population, three subpopulations were circled, namely, CD3+CD8+PD-1+T cells, CD3+CD8+TIM-3+T cells, and CD3+CD8+PD-1+TIM-3+T cells; Figure 23-Figure 25 In the figure, three subpopulations were circled in the CD3+CD4+T cell population, namely CD3+CD4+PD-1+T cells, CD3+CD4+TIM-3+T cells and CD3+CD4+PD-1+TIM-3+T cells. DETAILED DESCRIPTION

[0021] In order to more clearly understand the above-mentioned purposes, features and advantages of the present application, the scheme of the present application will be further described below. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0022] In the following description, many specific details are set forth to facilitate a full understanding of the present disclosure, but the present application may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present application, rather than all of the embodiments.

[0023] Hereinafter, the use of the peripheral blood lymphocyte subsets of the present application in the preparation of a detection kit for evaluating the prognosis of anti-PD-1 treatment in patients with advanced non-small cell lung cancer and the implementation method of the kit will be specifically disclosed with appropriate reference to the accompanying drawings. However, there may be cases where unnecessary detailed descriptions are omitted. For example, there are cases where detailed descriptions of well-known matters and repeated descriptions of actually the same structure are omitted. This is to avoid the following description from becoming unnecessarily lengthy and to facilitate the understanding of those skilled in the art. In addition, the drawings and the following descriptions are provided for those skilled in the art to fully understand the present application and are not intended to limit the subject matter described in the claims.

[0024] The "range" disclosed in the present application is defined in the form of a lower limit and an upper limit, and a given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of the particular range. The range defined in this way can be inclusive or exclusive of the end values, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a range. For example, if a range of 60 to 120 and 80 to 110 is listed for a particular parameter, it is understood that a range of 60 to 110 and 80 to 120 is also expected. In addition, if the minimum range values ​​1 and 2 are listed, and if the maximum range values ​​3, 4 and 5 are listed, the following ranges can all be expected: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4 and 2 to 5. In the present application, unless otherwise specified, the numerical range "a to b" represents an abbreviation of any real number combination between a and b, where a and b are both real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" are listed in this document, and "0-5" is just an abbreviation of these numerical combinations. In addition, when a parameter is expressed as an integer ≥ 2, it is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0025] If not otherwise specified, all embodiments and optional embodiments of the present application may be combined with each other to form new technical solutions, and such technical solutions should be deemed to be included in the disclosure of the present application.

[0026] Unless otherwise specified, all technical features and optional technical features of the present application may be combined with each other to form new technical solutions, and such technical solutions should be deemed to be included in the disclosure of the present application.

[0027] If there is no special explanation, all steps of the present application can be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), which means that step (c) may be added to the method in any order. For example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0028] Unless otherwise specified, the raw materials in the examples of this application are purchased through commercial channels. Unless otherwise specified, this application adopts conventional test methods or test methods recommended by instruments.

[0029] Clinical benefit means that all target lesions disappear, no new lesions appear, and tumor markers are normal, which lasts for at least 4 weeks, or the sum of the maximum diameters of target lesions decreases by ≥30% and lasts for at least 4 weeks.

[0030] In the existing technology, the prognosis of advanced NSCLC is mainly based on lung cancer tissue specimens. However, lung cancer tissue specimens are not easy to obtain and it is difficult to overcome the temporal and spatial heterogeneity of tumors. As a supplement to tissue testing, peripheral blood markers have the advantages of convenient sampling, non-invasiveness, and repeated sampling, and can cover information on both tumor and host immune status.

[0031] PD-1 is a membrane receptor protein that is mainly found on the surface of immune cells, such as activated T cells, B cells, and NK cells. PD-L1 is the ligand protein of PD-1 and is a type I transmembrane protein with a size of 40KDa. It is mainly found on the surface of tumor cells and certain antigen-presenting cells. PD-L1 on the surface of tumors can bind to PD-1 on the surface of activated T lymphocytes, reduce T lymphocyte activity through the PD-1 / PD-L1 pathway, and inhibit lymphocyte proliferation, thereby inhibiting the function of T lymphocytes in the local microenvironment of the tumor, reducing the immune killing function of the tumor, and making the tumor prone to immune escape.

[0032] TIM-3 is T lymphocyte immunoglobulin mucin 3, which is expressed in T cells, macrophages and dendritic cells, and mainly functions as an inhibitory receptor. TIM3 can play an immunosuppressive role in the tumor microenvironment by inhibiting Th1 and Th17, inducing CD8+T cell exhaustion, promoting Treg cells to become a highly immunosuppressive cell population, promoting the massive expansion of bone marrow-derived suppressor cells (MDSCs) with strong T cell immune response inhibition function, promoting innate immune suppression and tumor immune escape. TIM-3 is expressed in many types of cancer, including sarcoma, cervical cancer and gastric cancer, myeloma, melanoma and lung cancer, and its expression is associated with poor prognosis.

[0033] γδT cells are major histocompatibility complex (MHC)-unrestricted innate-like lymphocytes that bridge the innate and adaptive immune systems. According to the expression of the γ and δ chains of the TCR, γδT cells are divided into three subtypes: Vδ1T cells, Vδ2T cells, and Vδ3T cells. Among them, the Vδ2 type mainly kills tumor cells and plays an anti-tumor role through the following mechanisms: directly killing tumor cells through NK cell receptors; inducing apoptosis of tumor cells through apoptosis-related factor ligands (TRAIL / FASL); killing tumor cells through secreted Granzyme / Perforin; killing tumor cells through ADCC effects; acting as antigen-presenting cells, activating αβT cells; promoting the maturation of DC cells; promoting B cells to produce antibodies and enhance humoral immune responses; activating NK cells through the expression of 4-1BBL; γδT cells can sense the early changes of cell transformation into tumor cells, which puts γδT cells at the forefront of cancer immune monitoring.

[0034] In a study of patients with non-small cell lung cancer (NSCLC) treated with PD-L1 inhibitors, the number of peripheral blood CD8+T cells was negatively correlated with durable clinical benefit (DCB) (accuracy = 70%). In a study of patients with non-small cell lung cancer (NSCLC) treated with PD-L1 inhibitors, fewer circulating CD8+T cells were associated with durable clinical benefit (DCB) (accuracy = 70%). In patients with non-small cell lung cancer treated with PD-1 / PD-L1 inhibitors, an increase in senescent T cells is associated with a poor prognosis. We found that after NSCLC patients received anti-PD-1 treatment, the peripheral blood immune cells of both effective and ineffective patients underwent varying degrees of change, and the functions performed by different subpopulations may be different. At present, there is no unified laboratory standard for effectively evaluating the clinical efficacy and status of NSCLC patients related to peripheral blood lymphocyte subsets; the industry has not explored and studied γδT cells in NSCLC patients. This application is a new solution proposed based on the current technical problems.

[0035] [Application of peripheral blood lymphocyte subsets in the preparation of a detection kit for evaluating the prognosis of anti-PD-1 treatment in patients with advanced non-small cell lung cancer]

[0036] A use of peripheral blood lymphocyte subsets in the preparation of a detection kit for evaluating the prognosis of anti-PD-1 treatment in patients with advanced non-small cell lung cancer. The kit is used to measure the proportion of lymphocyte subsets in the peripheral blood of patients with advanced non-small cell lung cancer after anti-PD-1 treatment to evaluate the prognosis of anti-PD-1 treatment in patients with advanced non-small cell lung cancer; the lymphocyte subsets include at least one of CD3+T cells, CD4+T cells, CD8+T cells, CD3+CD8+PD-1+T cells, CD3+CD8+TIM-3+T cells, CD3+γδT+Vδ2+PD-1+T cells and CD3+γδT+Vδ2+TIM-3+T cells.

[0037] In some embodiments, the prognosis of anti-PD-1 treatment in patients with advanced non-small cell lung cancer is evaluated by at least one of the ratio of CD3+T cells, the ratio of CD4+T cells to CD8+T cells, the ratio of CD3+CD8+PD-1+T cells, the ratio of CD3+CD8+TIM-3+T cells, the ratio of CD3+γδT+Vδ2+PD-1+T cells, and the ratio of CD3+γδT+Vδ2+TIM-3+T cells. Figure 1 As shown, on the 42nd day after PD-1 monoclonal antibody treatment, compared with the baseline data, the key immune indicator data of patients in group R showed an increase in the proportion of CD3+T cells, a decrease in the proportion of CD3+CD8+PD-1+T cells, an increase in the proportion of CD3+CD8+TIM-3+T cells, and a decrease in CD3+γδT+Vδ2+PD-1+T cells. The proportion of CD3+CD8+PD-1+T cells in group NR decreased, and the results were significant.

[0038] In some embodiments, when patients with advanced non-small cell lung cancer receive anti-PD-1 treatment and obtain clinical benefit, the proportion of CD3+T cells in baseline peripheral blood is higher than 49.15%. The clinical benefit means that all target lesions disappear, no new lesions appear, and tumor markers are normal, which lasts for at least 4 weeks, or the sum of the maximum diameters of target lesions decreases by ≥30% and lasts for at least 4 weeks.

[0039] In some embodiments, when patients with advanced non-small cell lung cancer receive anti-PD-1 treatment and obtain clinical benefit, the ratio of CD4+T cells to CD8+T cells in the baseline peripheral blood is higher than 1.06. The clinical benefit means that all target lesions disappear, no new lesions appear, and tumor markers are normal, which lasts for at least 4 weeks, or the sum of the maximum diameters of target lesions decreases by ≥30% and lasts for at least 4 weeks.

[0040] In some embodiments, when patients with advanced non-small cell lung cancer receive anti-PD-1 treatment and obtain clinical benefit, the ratio of CD3+CD8+PD-1+T cells in the baseline peripheral blood is higher than 0.43%. The clinical benefit means that all target lesions disappear, no new lesions appear, and tumor markers are normal, which lasts for at least 4 weeks, or the sum of the maximum diameters of target lesions decreases by ≥30% and lasts for at least 4 weeks.

[0041] In some embodiments, when patients with advanced non-small cell lung cancer receive anti-PD-1 treatment and obtain clinical benefits, the proportion of CD3+CD8+TIM-3+T cells in the baseline peripheral blood is higher than 3.41%. The clinical benefit refers to the disappearance of all target lesions, the appearance of no new lesions, and normal tumor markers, which last for at least 4 weeks, or the reduction of the sum of the maximum diameters of target lesions by ≥30%, which lasts for at least 4 weeks.

[0042] In some embodiments, when patients with advanced non-small cell lung cancer receive anti-PD-1 treatment and obtain clinical benefits, the proportion of CD3+γδT+Vδ2+PD-1+T cells in peripheral blood is higher than 0.81%. The clinical benefit means that all target lesions disappear, no new lesions appear, and tumor markers are normal, which lasts for at least 4 weeks, or the sum of the maximum diameters of target lesions decreases by ≥30% and lasts for at least 4 weeks.

[0043] In some embodiments, when patients with advanced non-small cell lung cancer receive anti-PD-1 treatment and obtain clinical benefits, the proportion of CD3+γδT+Vδ2+TIM-3+T cells in peripheral blood is higher than 1.26%. The clinical benefit means that all target lesions disappear, no new lesions appear, and tumor markers are normal, which lasts for at least 4 weeks, or the sum of the maximum diameters of target lesions decreases by ≥30% and lasts for at least 4 weeks.

[0044] In some embodiments, the prognosis of anti-PD-1 treatment in patients with advanced non-small cell lung cancer is jointly evaluated by the ratio of CD3+T cells, the ratio of CD4+T cells to CD8+T cells, the ratio of CD3+CD8+PD-1+T cells, the ratio of CD3+CD8+TIM-3+T cells, the ratio of CD3+γδT+Vδ2+PD-1+T cells and the ratio of CD3+γδT+Vδ2+TIM-3+T cells. When detecting the proportions of peripheral blood lymphocyte subsets of patients, if the following conditions are met at the same time: the proportion of CD3+T cells is higher than 49.15%; the ratio of CD4+T cells to CD8+T cells is higher than 1.06; the proportion of CD3+CD8+PD-1+T cells is higher than 0.43%; the proportion of CD3+CD8+TIM-3+T cells is higher than 3.41%; the proportion of CD3+γδT+Vδ2+PD-1+T cells is higher than 0.81%; and the proportion of CD3+γδT+Vδ2+TIM-3+T cells is higher than 1.26%, it can be judged that patients with advanced non-small cell lung cancer have obtained clinical benefits after anti-PD-1 treatment.

[0045] like Figure 2 As shown, through the proportion of CD3+T cells, the ratio of CD4+T cells to CD8+T cells, the proportion of CD3+CD8+PD-1+T cells, the proportion of CD3+CD8+TIM-3+T cells, the proportion of CD3+γδT+Vδ2+PD-1+T cells and the proportion of CD3+γδT+Vδ2+TIM-3+T cells, a total of 6 indicators, the accuracy of jointly evaluating the efficacy of anti-PD-1 treatment in NSCLC patients by these 6 indicators reached 81.7%.

[0046] In some embodiments, the kit includes a fluorescently labeled monoclonal antibody, the monoclonal antibody including at least one of an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8 antibody, an anti-TCRγδ antibody, an anti-TCRVδ2 antibody, an anti-PD-1 antibody, and an anti-TIM-3 antibody;

[0047] In some embodiments, the monoclonal antibody consists of anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-TCRγδ antibody, anti-TCRVδ2 antibody, anti-PD-1 antibody and anti-TIM-3 antibody.

[0048] In some embodiments, the kit further comprises a lysing solution and / or a PBS buffer. The lysing solution may be a FACS Lysing Solution.

[0049] Example 1 Relationship between the expression of lymphocyte subsets in peripheral blood of patients with advanced NSCLC and the therapeutic effect of NSCLC patients

[0050] The following steps were all carried out under light-proof conditions.

[0051] 1. Preparation of mixed antibody working solution: After taking out the antibody from the refrigerator at 4°C, put it into the centrifuge, centrifuge briefly at 3000rpm, and prepare the antibody working solution in a 1.5ml eppendorf tube according to the antibody manual in Table 1 at a ratio of 1:10 (the antibody ratio in the total system is 1:100), then gently blow to mix, and place on ice for use. A blank tube must be set up each time as a control.

[0052] Table 1

[0053]

[0054] 2. Sample incubation: Take 100 μl of whole blood from patients with advanced NSCLC who received anti-PD-1 therapy and put it into a marked flow tube. Mix the prepared antibody working solution and take 10 μl to add to the whole blood (or take 10 μl of mixed antibody to the flow tube and then add 100 μl of whole blood). Gently shake and mix, and incubate at 4°C in the dark for 30 minutes. Among them, patients with advanced NSCLC who received anti-PD-1 treatment were divided into a response group (R) and a non-response group (NR) as shown in Table 2. The response group (R) included a complete remission group (PR) and a partial remission group CR. Complete remission means that all target lesions disappear, no new lesions appear, and tumor markers are normal, which lasts for at least 4 weeks; partial remission means that the sum of the maximum diameters of target lesions decreases by ≥30% and lasts for at least 4 weeks. The non-response group (NR) includes a stable disease group (PD) and a progressive disease group (SD). Stable disease means that the sum of the maximum diameters of target lesions decreases but does not reach PR, or increases but does not reach PD. Disease progression refers to an increase of at least 20% in the sum of the maximum diameters of target lesions or the appearance of new lesions.

[0055] Table 2

[0056]

[0057]

[0058] 3. Sample lysis: After the incubation is completed, add 2 ml of red blood cell lysis buffer to the flow tube, immediately shake and mix, and lyse at room temperature (20-30°C) for 10 minutes. Observe whether the liquid is clear. If it is clear, centrifuge it at 1000 rpm for 5 minutes. If the lysis is not complete, add 200 μL of lysis buffer, mix, lyse at room temperature for 3 minutes, and centrifuge at 1000 rpm for 5 minutes.

[0059] Take out the flow tube from the centrifuge, discard the liquid in the tube into the waste bottle, and gently invert it on paper to absorb the liquid at the tube mouth;

[0060] Add 2ml 1×PBS for washing, centrifuge at 1500rpm for 5min, remove the supernatant, add 200ul 1×PBS for resuspending, mix well for testing, and test on the flow cytometer within 3 hours. The flow cytometer model is BD FACSlyric. The test results are as follows: Figure 3-Figure 25 shown.

[0061] 4. According to Figure 3-Figure 25The key immune indicators of patients in group R and group NR before and after PD-1 monoclonal antibody treatment, namely, CD3+T cell ratio, CD4+T cell to CD8+T cell ratio, CD3+TIM-3+T cell ratio, CD3+CD8+PD-1+T cell ratio, CD3+CD8+TIM-3+T cell ratio, CD3+γδT+T cell ratio, CD3+γδT+Vδ2+PD-1+T cell ratio, and CD3+γδT+Vδ2+TIM-3+T cell ratio, were analyzed using paired T test. The results are shown in the figure. Figure 1 As shown, on the 42nd day after PD-1 monoclonal antibody treatment, compared with the baseline data, the key immune indicator data of patients in group R showed an increase in the proportion of CD3+T cells, a decrease in the proportion of CD3+CD8+PD-1+T cells, an increase in the proportion of CD3+CD8+TIM-3+T cells, and a decrease in CD3+γδT+Vδ2+PD-1+T cells. The proportion of CD3+CD8+PD-1+T cells in group NR decreased, and the results were significant.

[0062] 5. Perform univariate logistic regression analysis on the key immune indicators in step 4 and draw receiver operating characteristic curves (ROC curves). Perform multivariate logistic regression analysis on the key immune indicators and the therapeutic effects of PD-1 monoclonal antibodies and draw ROC curves. The results are as follows: Figure 2 shown.

[0063] Depend on Figure 2 From Part A, we can see that when patients with advanced non-small cell lung cancer receive anti-PD-1 treatment and obtain clinical benefits, the optimal cutoff values ​​of the ROC curve for a single key immune indicator to predict the treatment effect are as follows: the proportion of CD3+T cells in peripheral blood is higher than 49.15%; the ratio of CD3+CD4+T cells to CD3+CD8+T cells is higher than 1.06; the proportion of CD3+CD8+PD-1+T cells is higher than 0.43%, the proportion of CD3+CD8+TIM-3+T cells is higher than 3.41%, the proportion of CD3+γδT+Vδ2+PD-1+T cells is higher than 0.81%, and the proportion of CD3+γδT+Vδ2+TIM-3+T cells is higher than 1.26%.

[0064] Depend on Figure 2As shown in Part B, the ROC model of multi-indicator joint prediction is better than the single indicator prediction model. Among them, the combination of CD3+T cells, CD4+T cells and CD8+T cells, CD3+CD8+PD-1+T cells, CD3+CD8+TIM-3+T cells, CD3+γδT+Vδ2+PD-1+T cells, and CD3+γδT+Vδ2+TIM-3+T cells has the highest accuracy of 81.7% in evaluating the efficacy of anti-PD-1 treatment in NSCLC patients, with a sensitivity of 95.8% and a specificity of 60.0%. The accuracy of combined prediction of prognosis by these six indicators is higher than that of a single indicator, and is also higher than that of the combined prediction of prognosis by eight indicators including CD3+T cell ratio, CD4+T cell to CD8+T cell ratio, CD3+TIM-3+T cell ratio, CD3+CD8+PD-1+T cell ratio, CD3+CD8+TIM-3+T cell ratio, CD3+γδT+T cell ratio, CD3+γδT+Vδ2+PD-1+T cell ratio, and CD3+γδT+Vδ2+TIM-3+T cell ratio.

[0065] Example 2

[0066] 1. Obtain the sample set shown in Table 3 for verification

[0067] A total of 19 patients diagnosed with non-small cell lung cancer were included in the validation cohort and received PD-1 monoclonal antibody treatment. According to the results of the efficacy evaluation, they were divided into a response group (R group) and a non-response group (NR group). There was no statistical difference in age and gender between the two groups.

[0068] Table 3

[0069]

[0070] 2. Test of lymphocyte subset expression

[0071] The sample set in Table 3 was tested for the expression of lymphocyte subsets in peripheral blood. The experimental method was the same as steps 1-4 in Example 1. The final measured results were as follows:

[0072]

[0073]

[0074] In the table, A: CD3+ T cells (%);

[0075] B: ratio of CD4+T cells to CD8+T cells;

[0076] C: CD3+CD8+PD-1+T cells (%);

[0077] D: CD3+γδT+Vδ2+PD-1+T cells (%);

[0078] E: CD3+CD8+TIM-3+T cells (%);

[0079] F: CD3+γδT+Vδ2+TIM-3+T cells (%)

[0080] 3. Data processing

[0081] 3.1. Substitute the lymphocyte subset expression measured in step 2 into the following calculation formula: model prediction value = -4.8 + 0.07*A + 0.23*B + 0.52*C - 0.02*D - 0.33*E + 0.53*F, where A, B, C, D, E, and F represent the proportion of CD3+T cells, the ratio of CD4+T cells to CD8+T cells, the proportion of CD3+CD8+PD-1+T cells, the proportion of CD3+γδT+Vδ2+PD-1+T cells, the proportion of CD3+CD8+TIM-3+T cells, and the proportion of CD3+γδT+Vδ2+TIM-3+T cells, respectively.

[0082] 3.2, according to Figure 2 The combined prediction model of immune indicators shown in Part B of the disclosure (see paragraph 0051 and attached Figure 2 ), the cutoff value of the model parameter with the best prediction effect is 0.450.

[0083] 3.3. Compare the model prediction value obtained in step 3.1 with the cutoff value 0.450 in step 3.2. If it is greater than or equal to this value, it is judged as "response, R", and if it is less than this value, it is judged as "no response, NR". The results are shown in Table 4.

[0084] Table 4

[0085]

[0086]

[0087] * Cutoff value = 0.450. Values ​​greater than or equal to this value are judged as “response, R”, and values ​​less than this value are judged as “no response, NR”.

[0088] 4. Verify the results

[0089] The judgment data in Table 4 are formed into Table 5. It can be seen from Table 5 that there are 7 cases with the evaluation model judgment result of R and the clinical judgment result of R, 1 case with the evaluation model judgment result of R and the clinical judgment result of NR, 3 cases with the evaluation model judgment result of NR and the clinical judgment result of R, and 8 cases with the evaluation model judgment result of NR and the clinical judgment result of NR; there are 8 cases with the evaluation model judgment result of R, 11 cases with the evaluation model judgment result of NR, 10 cases with the clinical judgment result of R, and 9 cases with the clinical judgment result of NR.

[0090] Therefore, the positive predictive value (PPV) refers to the proportion of people who are judged as "responsive" by the evaluation model and who actually benefit from PD-1 monoclonal antibody treatment. It is an indicator to measure the ability of early screening products to avoid "misdiagnosis". The PPV of this evaluation model is: (cases judged as R by both the evaluation model and the clinic) / (cases judged as R by the evaluation model)*100%=7 / 8*100%=87.50%; the negative predictive value (NPV) refers to the proportion of people who are judged as "unresponsive" by the evaluation model and who actually do not benefit from PD-1 monoclonal antibody treatment. It is an indicator to measure the early screening product's ability to "miss detection" and exclude negative results. The NPV of this evaluation model is: (cases judged as NR by both the evaluation model and the clinic) / (cases judged as NR by the evaluation model)*100%=8 / 11*100%=72.73%.

[0091] Sensitivity refers to the proportion of patients evaluated as "responsive" by the model among those who are clinically judged to respond well to PD-1 monoclonal antibody treatment, which reflects the product's ability to predict the therapeutic effect of PD-1 monoclonal antibody. The sensitivity of the evaluation model is: (cases judged as R by both the evaluation model and the clinic) / (cases judged as R by the clinic)*100%=7 / 10*100%=70.00%; specificity refers to the proportion of patients evaluated as "unresponsive" by the model among those who are clinically judged to respond well to PD-1 monoclonal antibody treatment. The higher the specificity, the fewer false positives the product detection produces. The specificity of the evaluation model is: (cases judged as NR by both the evaluation model and the clinic) / (cases judged as NR by the clinic)*100%=8 / 9*100%=88.89%.

[0092] Table 5

[0093]

[0094] Positive predictive value (PPV) = 87.50%

[0095] Negative predictive value (NPV) = 72.73%

[0096] Sensitivity = 70.00%

[0097] Specificity = 88.89%

[0098] The above is only a specific implementation of the present application. Those skilled in the art can clearly understand that for the convenience and simplicity of description, the specific working processes of the systems, modules and units described above can refer to the corresponding processes in the aforementioned method embodiments, and will not be repeated here. It should be understood that the protection scope of the present application is not limited to this. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed in this application, and these modifications or replacements should be included in the protection scope of this application.

Claims

1. Use of a peripheral blood lymphocyte subset in the preparation of a detection kit for evaluating the prognosis of anti-PD-1 treatment in patients with advanced non-small cell lung cancer, characterized in that: The kit is used to measure the proportion of lymphocyte subpopulations in the peripheral blood of patients with advanced non-small cell lung cancer who have received anti-PD-1 treatment, so as to evaluate the prognosis of patients with advanced non-small cell lung cancer after anti-PD-1 treatment; The lymphocyte subsets consist of CD3+T cells, CD4+T cells, CD8+T cells, CD3+CD8+PD-1+T cells, CD3+CD8+TIM-3+T cells, CD3+γδT+Vδ2+PD-1+T cells and CD3+γδT+Vδ2+TIM-3+T cells; The prognosis of patients with advanced non-small cell lung cancer after anti-PD-1 treatment was evaluated by the proportion of CD3+T cells, the ratio of CD4+T cells to CD8+T cells, the proportion of CD3+CD8+PD-1+T cells, the proportion of CD3+CD8+TIM-3+T cells, the proportion of CD3+γδT+Vδ2+PD-1+T cells and the proportion of CD3+γδT+Vδ2+TIM-3+T cells.

2. The use according to claim 1, characterized in that: When patients with advanced non-small cell lung cancer receive anti-PD-1 treatment and achieve clinical benefit, the proportion of CD3+T cells in their baseline peripheral blood is higher than 49.15%.

3. The use according to claim 1, characterized in that: When patients with advanced non-small cell lung cancer achieve clinical benefit after anti-PD-1 treatment, the ratio of CD4+T cells to CD8+T cells in their baseline peripheral blood is higher than 1.

06.

4. The use according to claim 1, characterized in that: When patients with advanced non-small cell lung cancer achieve clinical benefit after anti-PD-1 treatment, the proportion of CD3+CD8+PD-1+T cells in their baseline peripheral blood is higher than 0.43%.

5. The use according to claim 1, characterized in that: When patients with advanced non-small cell lung cancer achieve clinical benefit after anti-PD-1 treatment, the proportion of CD3+CD8+TIM-3+T cells in their baseline peripheral blood is higher than 3.41%.

6. The use according to claim 1, characterized in that: When patients with advanced non-small cell lung cancer achieve clinical benefit after anti-PD-1 treatment, the proportion of CD3+γδT+Vδ2+PD-1+T cells in their baseline peripheral blood is higher than 0.81%.

7. The use according to claim 1, characterized in that: When patients with advanced non-small cell lung cancer receive anti-PD-1 treatment and achieve clinical benefit, the proportion of CD3+γδT+Vδ2+TIM-3+T cells in their baseline peripheral blood is higher than 1.26%.

8. The use according to claim 1, characterized in that: The kit comprises fluorescently labeled monoclonal antibodies, which consist of anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-TCRγδ antibody, anti-TCRVδ2 antibody, anti-PD-1 antibody and anti-TIM-3 antibody.

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