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

Abstract

The application discloses application of peripheral blood lymphocyte subgroups 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 determining the proportion of lymphocyte subgroups in peripheral blood of the patient with advanced non-small cell lung cancer after receiving the anti-PD-1 treatment, so as to evaluate the prognosis of the patient with advanced non-small cell lung cancer. According to the embodiment of the application, the anti-PD-1 treatment effect of the patient with advanced NSCLC can be known in time by detecting the change of peripheral blood lymphocyte subgroups of the patient with advanced NSCLC.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biomedicine, and particularly relates to application of peripheral blood lymphocyte subgroups in preparation of a detection kit for evaluating prognosis of anti-PD-1 treatment of a patient with advanced non-small cell lung cancer. BACKGROUND

[0002] Among malignant tumors, the morbidity and mortality of lung cancer in China are in the forefront, and non-small cell lung cancer (NSCLC) is the main type of lung cancer, accounting for about 85% of the total lung cancer. Since the onset of NSCLC is relatively insidious, it is often diagnosed in the advanced stage. In addition to maintenance therapy, chemotherapy and targeted therapy, immunotherapy, especially PD-1 / L1 inhibitor, is a standard treatment method for first-line and second-line advanced NSCLC. For the population with high PD-L1 expression, immunotherapy alone can bring significant clinical benefits, and the 5-year survival rate is as high as 32%, which has changed the treatment concept of advanced lung cancer. However, the clinical benefit of immunotherapy alone in the population with low or no PD-L1 expression is not significant, and patients with PD-L1≥50% only account for 29.8% of the total population of NSCLC.

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

[0004] The application provides application of peripheral blood lymphocyte subgroups in preparation of a detection kit for evaluating prognosis of anti-PD-1 treatment of a patient with advanced non-small cell lung cancer, which can know the efficacy of anti-PD-1 treatment of a patient with NSCLC in time by detecting changes in peripheral blood lymphocyte subgroups of the patient with advanced NSCLC.

[0005] The application provides application of peripheral blood lymphocyte subgroups in preparation of a detection kit for evaluating prognosis of anti-PD-1 treatment of a patient with advanced non-small cell lung cancer, which utilizes the kit to determine the proportion of lymphocyte subgroups in peripheral blood of a patient with advanced non-small cell lung cancer who receives anti-PD-1 treatment, so as to evaluate the prognosis of anti-PD-1 treatment of the patient with advanced non-small cell lung cancer; the lymphocyte subgroups 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 for the patient with advanced non-small cell lung cancer is evaluated by at least one of 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.

[0007] In any embodiment of the present application, when the 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 peripheral blood at baseline is higher than 49.15%.

[0008] In any embodiment of the present application, when the 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 peripheral blood at baseline is higher than 1.06.

[0009] In any embodiment of the present application, when the 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 peripheral blood at baseline is higher than 0.43%.

[0010] In any embodiment of the present application, when the patient with advanced non-small cell lung cancer obtains clinical benefit after receiving anti-PD-1 treatment, the proportion of CD3+CD8+TIM-3+T cells in the peripheral blood at baseline is higher than 3.41%.

[0011] In any embodiment of the present application, when the 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 peripheral blood at baseline is higher than 0.81%.

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

[0013] In any embodiment of the present application, the prognosis of anti-PD-1 treatment for the patient with advanced non-small cell lung cancer is evaluated by at least one of 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.

[0014] In any embodiment of the present application, the kit comprises a fluorescently labeled monoclonal antibody, the monoclonal antibody comprising 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.

[0015] In any embodiment of the present application, the monoclonal antibody consists 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.

[0016] The peripheral blood lymphocyte subpopulation of the embodiments of the present application is used in the preparation of a detection kit for evaluating the prognosis of anti-PD-1 treatment of patients with advanced non-small cell lung cancer. The kit can detect 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 in the peripheral blood of patients with advanced NSCLC, so that the immune status of the tumor and the host in the body of the NSCLC patient after anti-PD-1 treatment can be conveniently, non-invasively, and repeatedly known. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required to be used in the embodiments of the present application will be briefly introduced below. For those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

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

[0019] Figure 2 Prediction model of key immune indicators for PD-1 monoclonal antibody treatment effect of non-small cell lung cancer;

[0020] Figures 3-25 Flow cytometry gating strategy for lymphocytes; wherein, Figure 3 Gating lymphocytes in peripheral blood whole cell subpopulations; Figure 4 Gating CD3-positive cells in the lymphocyte population, i.e., CD3+T cells; Figure 5 Gating γδ-positive cells in the CD3+T cell population, i.e., γδT cells; Figure 6 Gating CD4-positive cells and CD8-positive cells in the CD3+T cell population, i.e., CD3+CD4+T cells and CD3+CD8+T cells; Figure 7In some embodiments, Vδ1 positive cells and Vδ2 positive cells are circled in the γδ T cell population, i.e. CD3+γδ+Vδ1+T cells and CD3+γδ+Vδ2+T cells, respectively; Figures 8-10 In some embodiments, three subpopulations are circled in the CD3+γδ+Vδ2+T cell population, i.e. CD3+γδ+Vδ2+PD-1+T cells, CD3+γδ+Vδ2+TIM-3+T cells and CD3+γδ+Vδ2+PD-1+TIM-3+T cells, respectively; Figures 11-13 In some embodiments, three subpopulations are circled in the CD3+γδ+Vδ1+T cell population, i.e. CD3+γδ+Vδ1+PD-1+T cells, CD3+γδ+Vδ1+TIM-3+T cells and CD3+γδ+Vδ1+PD-1+TIM-3+T cells, respectively; Figures 14-16 In some embodiments, three subpopulations are circled in the γδ T cell population, i.e. CD3+γδ+PD-1+T cells, CD3+γδ+TIM-3+T cells and CD3+γδ+PD-1+TIM-3+T cells, respectively; Figures 17-19 In some embodiments, three subpopulations are circled in the CD3+T cell population, i.e. CD3+PD-1+T cells, CD3+TIM-3+T cells and CD3+PD-1+TIM-3+T cells, respectively; Figures 20-22 In some embodiments, three subpopulations are circled in the CD3+CD8+T cell population, i.e. CD3+CD8+PD-1+T cells, CD3+CD8+TIM-3+T cells and CD3+CD8+PD-1+TIM-3+T cells, respectively; Figures 23-25 In some embodiments, three subpopulations are circled in the CD3+CD4+T cell population, i.e. CD3+CD4+PD-1+T cells, CD3+CD4+TIM-3+T cells and CD3+CD4+PD-1+TIM-3+T cells, respectively. DETAILED DESCRIPTION

[0021] In order to more clearly understand the above-mentioned purposes, features and advantages of the present application, the schemes 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, a large number of specific details are set forth in order to facilitate a thorough understanding of the present disclosure, but the present application can also be implemented in other ways different from those described herein; obviously, the embodiments in the specification are only some of the embodiments of the present application, not all the embodiments.

[0023] Hereinafter, the peripheral blood lymphocyte subgroups of the present application are specifically disclosed in the preparation of a test kit for evaluating the prognosis of anti-PD-1 treatment for patients with advanced non-small cell lung cancer, and the embodiments of the kit are described in detail with appropriate reference to the accompanying drawings. However, unnecessary detailed descriptions will be omitted. For example, detailed descriptions of matters well known in the art, repeated descriptions of substantially identical structures will be omitted. This is to avoid the following description from becoming unnecessarily lengthy and to facilitate understanding by those skilled in the art. In addition, the accompanying drawings and the following description are provided to enable those skilled in the art to fully understand the present application, and are not intended to limit the subject matter recited in the claims.

[0024] The ranges disclosed herein are defined by their lower and upper limits, given that the range is defined by selecting a lower limit and an upper limit, the selected lower and upper limits define the boundaries of the particular range. Ranges defined by the endpoints can include the endpoints, or can not include the endpoints, and can be freely combined in any manner with any other ranges, i.e., any lower limit can be combined with any upper limit to form a range. For example, if a range of 60-120 and a range of 80-110 are listed, it is understood that a range of 60-110 and a range of 80-120 are also contemplated. Furthermore, if a minimum range value of 1 and 2 is listed, and if a maximum range value of 3, 4, and 5 is listed, then the following ranges are all contemplated: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In the present application, unless otherwise stated, a numerical range "a-b" indicates a shorthand way of describing all of the real combinations between a and b, where a and b are both real numbers. For example, the numerical range "0-5" indicates that all of the real numbers between "0-5" have been listed herein, and "0-5" is just a shorthand way of describing these numerical combinations. In addition, when it is stated that a certain parameter is an integer ≥ 2, it is equivalent to disclose that the parameter is, for example, an integer 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0025] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form new technical solutions, and such technical solutions should be considered 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 can be combined with each other to form new technical solutions, and such technical solutions should be considered to be included in the disclosure of the present application.

[0027] If not otherwise specified, all steps of the present application can be carried out in sequence or randomly, preferably in sequence. For example, the method comprises steps (a) and (b) means that the method can comprise steps (a) and (b) in sequence, or steps (b) and (a) in sequence. For example, the method can further comprise step (c) means that step (c) can be added to the method in any order, for example, the method can comprise steps (a), (b) and (c), or steps (a), (c) and (b), or steps (c), (a) and (b), etc.

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

[0029] Clinical benefit refers to all target lesions disappear, no new lesions appear, and tumor markers are normal, at least for 4 weeks, or the sum of the maximum diameters of target lesions is reduced by ≥30%, at least for 4 weeks.

[0030] In the prior art, the prognosis of advanced NSCLC is mainly through 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, while peripheral blood markers, as a supplement to tissue detection, have the advantages of convenient sampling, non-invasive, repeated sampling, etc., and can cover information on tumor and host immune status.

[0031] PD-1 is a membrane receptor protein mainly present on the surface of immune cells such as activated T cells, B cells, NK cells, etc. PD-L1 is a ligand protein of PD-1, which is a first type of transmembrane protein with a size of 40 KDa. It mainly exists on the surface of tumor cells and some antigen-presenting cells. PD-L1 on the surface of tumor cells can bind to PD-1 on the surface of activated T lymphocytes, reduce the activity of T lymphocytes through the PD-1 / PD-L1 pathway, and inhibit the proliferation of lymphocytes, 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 a T lymphocyte immunoglobulin mucin 3, which is expressed in T cells, macrophages and dendritic cells, mainly playing the function of inhibitory receptor. TIM3 can play an immunosuppressive role in tumor microenvironment through the following pathways: inhibiting Th1 and Th17, inducing CD8+ T cell exhaustion, promoting Treg cells to become a highly immunosuppressive cell population, promoting the massive expansion of myeloid-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 various types of cancer, including sarcoma, cervical cancer and gastric cancer, myeloma, melanoma and lung cancer, and its expression is associated with poor prognosis.

[0033] Gamma delta T cells are major histocompatibility complex (MHC) non-restricted innate-like lymphocytes, bridging the innate and adaptive immune systems. According to the expression of the gamma chain and the delta chain of TCR, gamma delta T cells are divided into three subtypes: Vδ1 T cells, Vδ2 T cells and Vδ3 T cells. Among them, Vδ2 type mainly plays a role in killing tumor cells, and plays an anti-tumor role through the following mechanisms: direct killing of tumor cells through NK cell receptors; inducing apoptosis of tumor cells through apoptosis-related factor ligand (TRAIL / FASL); killing tumor cells through secreted Granzyme / Perforin; killing tumor cells through ADCC effect; as an antigen presenting cell, activating αβ T cells; promoting the maturation of DC cells; promoting B cells to produce antibodies and improving humoral immune response; activating NK cells through expressed 4-1BBL; gamma delta T cells can sense the early changes of cell transformation into tumor cells, which makes gamma delta T cells in the first line of cancer immune surveillance.

[0034] In a study of non-small cell lung cancer (NSCLC) patients 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 non-small cell lung cancer (NSCLC) patients treated with PD-L1 inhibitors, fewer CD8+ T cells in circulation were associated with durable clinical benefit (DCB) (accuracy = 70%). In non-small cell lung cancer patients treated with PD-1 / PD-L1 inhibitors, increased senescent T cells were associated with poor prognosis. We found that the peripheral blood immune cells of NSCLC patients changed to varying degrees after anti-PD-1 treatment, and the functions performed by different subgroups may not be the same. There is currently no effective laboratory standard for evaluating the clinical efficacy and state of NSCLC patients related to peripheral blood lymphocyte subgroups; the industry has not explored and researched gamma delta T cells in NSCLC patients. The present application is a new solution based on the existing technical problems.

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

[0036] Use of peripheral blood lymphocyte subsets in preparation of a detection kit for evaluating prognosis of anti-PD-1 treatment for patients with advanced non-small cell lung cancer, using the kit to determine the proportion of lymphocyte subsets in the peripheral blood of patients with advanced non-small cell lung cancer after receiving anti-PD-1 treatment, in order to evaluate the prognosis of anti-PD-1 treatment for 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 for patients with advanced non-small cell lung cancer is evaluated by at least one of the proportion of CD3+ T cells, the ratio of the proportion 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. As shown in Table 2, on day 42 after treatment with PD-1 mAb, the proportion of CD3+ T cells in the R group increased, the proportion of CD3+ CD8+ PD-1+ T cells decreased, the proportion of CD3+ CD8+ TIM-3+ T cells increased, and the proportion of CD3+ γδT+ Vδ2+ PD-1+ T cells decreased, compared with the baseline data. The proportion of CD3+ CD8+ PD-1+ T cells in the NR group decreased, and the results were statistically significant. Figure 1

[0038] In some embodiments, when the patient with advanced non-small cell lung cancer receives anti-PD-1 treatment and achieves clinical benefit, the proportion of CD3+ T cells in the peripheral blood at baseline is higher than 49.15%. The clinical benefit refers to the disappearance of all target lesions, the absence of new lesions, and the normality of tumor markers, which is maintained for at least 4 weeks, or the sum of the maximum diameters of target lesions is reduced by ≥30%, which is maintained for at least 4 weeks.

[0039] In some embodiments, when the patient with advanced non-small cell lung cancer receives anti-PD-1 treatment and achieves clinical benefit, the ratio of CD4+ T cells to CD8+ T cells in the peripheral blood at baseline is higher than 1.06. The clinical benefit refers to the disappearance of all target lesions, the absence of new lesions, and the normality of tumor markers, which is maintained for at least 4 weeks, or the sum of the maximum diameters of target lesions is reduced by ≥30%, which is maintained for at least 4 weeks.

[0040] ​In some embodiments, 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 peripheral blood at baseline is higher than 0.43%. The clinical benefit refers to disappearance of all target lesions, no new lesion, and normal tumor markers for at least 4 weeks, or a reduction of ≥30% in the sum of the largest diameters of target lesions for at least 4 weeks.

[0041] In some embodiments, when a patient with advanced non-small cell lung cancer obtains clinical benefit after receiving anti-PD-1 treatment, the proportion of CD3+CD8+TIM-3+T cells in the peripheral blood at baseline is higher than 3.41%. The clinical benefit refers to disappearance of all target lesions, no new lesion, and normal tumor markers for at least 4 weeks, or a reduction of ≥30% in the sum of the largest diameters of target lesions for at least 4 weeks.

[0042] In some embodiments, 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 peripheral blood is higher than 0.81%. The clinical benefit refers to disappearance of all target lesions, no new lesion, and normal tumor markers for at least 4 weeks, or a reduction of ≥30% in the sum of the largest diameters of target lesions for at least 4 weeks.

[0043] In some embodiments, 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+TIM-3+T cells in the peripheral blood is higher than 1.26%. The clinical benefit refers to disappearance of all target lesions, no new lesion, and normal tumor markers for at least 4 weeks, or a reduction of ≥30% in the sum of the largest diameters of target lesions for at least 4 weeks.

[0044] In some embodiments, the prognosis of anti-PD-1 treatment for a patient with advanced non-small cell lung cancer is 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. When 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%, the patient with advanced non-small cell lung cancer is determined to obtain clinical benefit after receiving anti-PD-1 treatment.

[0045] like Figure 2 As shown in the results, the accuracy of evaluating the efficacy of anti-PD-1 treatment in NSCLC patients by jointly evaluating the six indicators, including 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, was 81.7%.

[0046] In some embodiments, the kit includes a fluorescently labeled monoclonal antibody, wherein the monoclonal antibody includes 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 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.

[0048] In some embodiments, the kit further comprises a lysis solution and / or a PBS buffer solution. The lysis solution can 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 removing the antibody from the 4°C refrigerator, place it in a centrifuge and briefly centrifuge at 3000 rpm. In a 1.5 ml EP tube, refer to the corresponding antibody instructions in Table 1 and prepare the antibody working solution at a ratio of 1:10 (the total antibody ratio in the system is 1:100). Then, gently pipette to mix and place on ice until ready to use. A blank tube should be set up each time as a control.

[0052] Table 1

[0053]

[0054] 2. Sample Incubation: Pipette 100 μl of whole blood from a patient with advanced NSCLC receiving anti-PD-1 therapy into a labeled flow cytometer tube. Mix the prepared antibody working solution thoroughly, then add 10 μl to the whole blood (or transfer 10 μl of the mixed antibody to the flow cytometer tube and then add 100 μl of whole blood). Gently shake to mix thoroughly, and incubate at 4°C in the dark for 30 min. Patients with advanced NSCLC receiving anti-PD-1 therapy are divided into responder (R) and non-responder (NR) groups, as shown in Table 2. The responder (R) group includes complete response (PR) and partial response (CR). A complete response refers to the disappearance of all target lesions, the absence of new lesions, and normal tumor markers, maintained for at least 4 weeks. A partial response refers to a ≥30% reduction in the sum of the largest diameters of target lesions, maintained for at least 4 weeks. The non-responder (NR) group includes stable disease (PD) and progressive disease (SD). Stable disease refers to a reduction in the sum of the largest diameters of target lesions that does not reach a PR or an increase that does not reach a PD. Disease progression refers to an increase of ≥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 at 1000 rpm for 5 minutes. If the lysis is not complete, add another 200 μL of lysis buffer, mix, lyse at room temperature for 3 minutes, and centrifuge at 1000 rpm for 5 minutes.

[0059] Remove 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 to wash, centrifuge at 1500rpm for 5min, remove the supernatant, add 200ul 1×PBS to resuspend, mix well and test within 3 hours. The flow cytometer model is BD FACSlyric. The test results are as follows: Figures 3-25 shown.

[0061] 4. According to Figures 3-25The key immune indicators of patients in group R and group NR before and after PD-1 monoclonal antibody treatment, namely, the proportion of CD3+T cells, the ratio of CD4+T cells to CD8+T cells, the proportion of CD3+TIM-3+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+T cells, the proportion of CD3+γδT+Vδ2+PD-1+T cells, and the proportion of CD3+γδT+Vδ2+TIM-3+T cells, were analyzed using the paired T test method. The results are shown in the figure below. 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 the R group showed that the proportion of CD3+T cells increased, the proportion of CD3+CD8+PD-1+T cells decreased, the proportion of CD3+CD8+TIM-3+T cells increased, and the proportion of CD3+γδT+Vδ2+PD-1+T cells decreased. The proportion of CD3+CD8+PD-1+T cells of patients in the NR group 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 curve (ROC curve) and perform multivariate logistic regression analysis on the key immune indicators and the effect of PD-1 monoclonal antibody treatment and draw ROC curve. The results are as follows: Figure 2 shown.

[0063] Depend on Figure 2 As shown in Part A, when patients with advanced non-small cell lung cancer receive anti-PD-1 treatment and achieve 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 can be seen from part B in Table 2, the ROC model of multi-index combined prediction is superior to the single-index prediction model. Among them, the combination of CD3+T cells, the ratio of CD4+T cells to CD8+T cells, CD3+CD8+PD-1+T cells, CD3+CD8+TIM-3+T cells, CD3+γδT+Vδ2+PD-1+T cells, CD3+γδT+Vδ2+TIM-3+T cells for evaluating the accuracy of NSCLC patient anti-PD-1 treatment effect is the highest, reaching 81.7%, the sensitivity for judging the treatment effect is 95.8%, and the specificity is 60.0%. The combined prediction of prognosis by the six indexes has higher accuracy than the single-index prediction, and also higher than the combined prediction of prognosis by 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] The verification cohort included a total of 19 patients diagnosed with non-small cell lung cancer and treated with PD-1 monoclonal antibody. According to the clinical evaluation results, they were divided into response group (R group) and non-response group (NR group), and there was no statistical difference in the composition of 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 lymphocyte subset expression in peripheral blood, and the experimental method was the same as steps 1-4 in Example 1. The final test results are as follows:

[0072]

[0073]

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

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

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

[0077] D: CD3+ gd T+ V52+ PD-1+ T cells (%);

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

[0079] F: CD3+ gd T+ V52+ TIM-3+ T cells (%)

[0080] 3. Data processing

[0081] 3.1, the lymphocyte subpopulation expression measured in step 2 is substituted 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, A, B, C, D, E, F in the formula respectively 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+ gd T+ V52+ PD-1+ T cells, the proportion of CD3+ CD8+ TIM-3+ T cells, and the proportion of CD3+ gd T+ V52+ TIM-3+ T cells.

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

[0083] 3.3, the model prediction value obtained in step 3.1 is compared with the cutoff value 0.450 in step 3.2, and greater than or equal to the value is determined as "response, R", and less than the value is determined as "non-response, NR", and the results are shown in Table 4.

[0084] Table 4

[0085]

[0086]

[0087] Cutoff value = 0.450, greater than or equal to the value is determined as "response, R", and less than the value is determined as "non-response, NR".

[0088] 4. Verification results

[0089] The determination data in Table 4 forms Table 5, and from Table 5, there are 7 cases in which the evaluation model determination result is R and the clinical determination result is R, 1 case in which the evaluation model determination result is R and the clinical determination result is NR, 3 cases in which the evaluation model determination result is NR and the clinical determination result is R, and 8 cases in which the evaluation model determination result is NR and the clinical determination result is NR; there are 8 cases in which the evaluation model determination result is R, 11 cases in which the evaluation model determination result is NR, 10 cases in which the clinical determination result is R, and 9 cases in which the clinical determination result is NR.

[0090] Therefore, the positive predictive value (PPV) refers to the proportion of people who truly benefit from PD-1 mAb treatment among the population determined as “response” by the evaluation model, and is an index for measuring the ability of the early screening product to avoid “misdiagnosis”. The PPV of the evaluation model is: (cases determined as R by both the evaluation model and the clinic) / (cases determined as R by the evaluation model) * 100% = 7 / 8 * 100% = 87.50%; the negative predictive value (NPV) refers to the proportion of people who do not truly benefit from PD-1 mAb treatment among the population determined as “non-response” by the evaluation model, and is an index for measuring the “missed detection” ability and the ability to eliminate negative results of the early screening product. The NPV of the evaluation model is: (cases determined as NR by both the evaluation model and the clinic) / (cases determined as NR by the evaluation model) * 100% = 8 / 11 * 100% = 72.73%.

[0091] The sensitivity refers to the proportion of cases determined as “response” by the evaluation model among the population determined as “response” to PD-1 mAb treatment by the clinic, and reflects the ability of the product to predict the effect of PD-1 mAb treatment. The sensitivity of the evaluation model is: (cases determined as R by both the evaluation model and the clinic) / (cases determined as R by the clinic) * 100% = 7 / 10 * 100% = 70.00%; the specificity refers to the proportion of cases determined as “non-response” by the evaluation model among the population determined as “non-response” to PD-1 mAb treatment by the clinic, and the higher the specificity, the fewer false positives produced by the product. The specificity of the evaluation model is: (cases determined as NR by both the evaluation model and the clinic) / (cases determined 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 (Sensitivity) = 70.00%

[0097] Specificity (Specificity) = 88.89%

[0098] The above is only a specific implementation of the present application, and those skilled in the art can clearly understand that, for the convenience and brevity of description, the specific working process of the system, module and unit described above can refer to the corresponding process in the foregoing method embodiments, which will not be described here. It should be understood that the protection scope of the present application is not limited to this, and any person skilled in the art can easily think of various equivalent modifications or replacements within the technical range disclosed in the present application, and these modifications or replacements should be covered within the protection scope of the present 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 subsets 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 are composed 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 receive anti-PD-1 treatment and achieve clinical benefit, 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 receive anti-PD-1 treatment and achieve clinical benefit, 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 receive anti-PD-1 treatment and achieve clinical benefit, 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 receive anti-PD-1 treatment and achieve clinical benefit, 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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