CN114720356A

CN114720356A - Flow cytometry detection kit for lung cancer late stage immune characterization and detection method thereof

Info

Publication number: CN114720356A
Application number: CN202210268118.7A
Authority: CN
Inventors: 李太生; 罗玲; 刘晓笙
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-03-18
Filing date: 2022-03-18
Publication date: 2022-07-08
Anticipated expiration: 2042-03-18
Also published as: CN114720356B

Abstract

The invention relates to a flow cytometer detection kit for lung cancer late stage immune characterization, wherein a reagent A comprises a CD45 monoclonal antibody, a CD3 monoclonal antibody, a CD4 monoclonal antibody, a CD8 monoclonal antibody and a PD-1 monoclonal antibody which are marked by fluorescent microspheres dyed by dyes with different colors, the kit takes peripheral blood of a patient suffering from late stage lung cancer and subjected to an immune checkpoint inhibitor as a sample for detection, and the kit is used for detecting CD8 in a sample to be detected⁺PD‑1⁺/CD8⁺T cell%, CD4⁺PD‑1⁺/CD4⁺T cell%, CD8⁺HLA‑DR⁺/CD8⁺T cell%, each index can comprehensively characterize the patientThe level of immunity in the subject.

Description

Flow cytometry detection kit for lung cancer late stage immune characterization and detection method thereof

Technical Field

The invention belongs to the field of flow cytometer detection kits, and particularly relates to a flow cytometer detection kit for lung cancer late-stage peripheral blood immune characterization and a detection method thereof.

Background

With the progress of lung cancer treatment studies, the clinical success of immune checkpoint inhibitors (antagonists of CTLA-4, PD-1 and PD-L1) has provided a new therapeutic option. A variety of PD-1/PD-L1 Immune Checkpoint Inhibitors (ICIs) have been approved for use in the treatment of advanced lung cancer and have achieved significant efficacy. Furthermore, the expression of PD-1 changes in depleted T cells as tumor patients develop depleted T cells due to sustained stimulation by tumor antigens. PD-1 is induced by T cell receptor signaling, and binding of PD-1 to its ligand inhibits TCR/CD28 signaling and T cell activation. Blocking the PD-1 signaling pathway restores the anti-tumor response of depleted T cells.

T lymphocyte subpopulation is closely related to the immune function of the body, and when the immune function is disordered, the relative percentage content of the process cell subpopulation and the numerical detection in the aspect of absolute counting are abnormal. In the case of specific diseases such as lung cancer, the T lymphocyte subpopulation expressing PD-1 is of great importance in monitoring the development of the disease, patient prognosis, and clinical treatment guidance. By utilizing the peripheral blood immunology characteristic information of the late-stage lung cancer patient, the T cell immunity level of the patient can be predicted, and whether the patient is suitable for further immunotherapy is judged in an auxiliary way, so that the curative effect of the immunotherapy is improved to the maximum extent, and immune-related adverse reactions are reduced.

Therefore, a detection kit and a detection method thereof for characterizing immune cell subsets and immune checkpoint molecule expression conditions of peripheral blood of patients with advanced lung cancer are urgently needed.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention provides a flow cytometer detection kit for lung cancer late stage immune characterization and a using method thereof, which provide an auxiliary judgment basis for immune cell characterization change for the effectiveness and safety of a patient receiving immune checkpoint inhibitor treatment.

The invention provides the following technical scheme:

a flow cytometry detection kit for lung cancer advanced stage immune characterization comprises a reagent A,

the reagent A comprises a CD45 monoclonal antibody, a CD3 monoclonal antibody, a CD4 monoclonal antibody, a CD8 monoclonal antibody and a PD-1 monoclonal antibody which are marked by fluorescent microspheres dyed by dyes with different colors, wherein the concentration ratio of each antibody is 1: 1: 1: 1:1, all of which are 0.05-0.1 mg/mL.

Further, the detection kit also comprises a reagent B, wherein the reagent B is erythrocyte lysate.

The detection method of the detection kit comprises the following steps:

step one, taking human venous blood, separating to obtain white cell liquid, and adding the white cell liquid into the bottom of a flow type tube;

step two, adding the reagent A into a tube, wherein the volume ratio of the reagent A to the leukocyte liquid is 1:10, and incubating the reagent A and the leukocyte liquid together;

step three, adding a PBS solution for cleaning, performing vortex mixing, centrifuging at 4 ℃, discarding supernatant, adding the PBS solution again to form cell suspension, and detecting by using a flow cytometer;

step four, screening cells which are positive to the CD45 antibody and have SSC value in the range of 0-50K, and determining the cells as lymphocytes; screening CD3 antibody positive cells from lymphocytes to determine T cells; screening for CD4 antibody-positive cells from T cells, screening for PD-1 antibody-positive cells from CD4 antibody-positive cells, and calculating CD4⁺PD-1⁺/CD4⁺Percentage of T cells; screening for CD8 antibody-positive cells from T cells, screening for PD-1 antibody-positive cells from CD8 antibody-positive cells, and calculating CD8⁺PD-1⁺/CD8⁺Percentage of T cells.

The other detection method of the detection kit comprises the following steps:

step one, respectively absorbing EDTA-K2 anticoagulated whole blood to the bottom of a flow tube by using a reverse pipetting technology to avoid the blood from touching the upper part of the tube wall;

adding a reagent A into the tube, wherein the volume ratio of the reagent A to the whole blood is 1:10, and uniformly mixing by using a vortex instrument; incubating at room temperature in a dark place; adding a reagent B into the tube, wherein the volume ratio of the reagent B to the whole blood is 20: 1; uniformly mixing by using a vortex instrument; reaction in dark place;

step three, after uniformly mixing by using a vortex instrument, centrifuging at 4 ℃, discarding supernatant, adding PBS (phosphate buffer solution) for cleaning, centrifuging at 4 ℃ after uniformly mixing by using a vortex instrument, discarding supernatant, adding PBS solution again to form cell suspension, and detecting by using a flow cytometer;

step four, screening cells which are positive to the CD45 antibody and have SSC value in the range of 0-50K, and determining the cells as lymphocytes; screening the lymphocytes for CD3 antibody positive cells, and determining the cells as T cells; screening for CD4 antibody-positive cells from T cells, screening for PD-1 antibody-positive cells from CD4 antibody-positive cells, and calculating CD4⁺PD-1⁺/CD4⁺Percentage of T cells, screening for cells positive for CD8 antibody from T cells, screening for cells positive for PD-1 antibody from cells positive for CD8 antibody, calculating CD8⁺PD-1⁺/CD8⁺Percentage of T cells.

Further, CD8⁺PD-1⁺/CD8⁺Percentage of T cells<2% and CD4⁺PD-1⁺/CD4⁺Percentage of T cells<2%, the proportion used to characterize functional depletion of T cells is low.

the reagent A comprises a CD45 monoclonal antibody, a CD3 monoclonal antibody, a CD4 monoclonal antibody, a CD8 monoclonal antibody, a PD-1 monoclonal antibody and an HLA-DR monoclonal antibody which are marked by fluorescent microspheres dyed by dyes with different colors, wherein the concentration ratio of each antibody is 1: 1: 1: 1: 1:1, all of which are 0.05-1 mg/mL.

The detection method of the detection kit comprises the following steps:

step two, adding a reagent A into the tube, wherein the volume ratio of the reagent A to the whole blood is 1:10, and uniformly mixing by using a vortex instrument; incubating at room temperature in a dark place; adding a reagent B into the tube, wherein the volume ratio of the reagent B to the whole blood is 20: 1; uniformly mixing by using a vortex instrument; reaction in dark place;

step four, screening cells which are positive to the CD45 antibody and have SSC value in the range of 0-50K, and determining the cells as lymphocytes; screening CD3 antibody positive cells from lymphocytes to determine T cells; screening for CD4 antibody-positive cells from T cells, screening for PD-1 antibody-positive cells from CD4 antibody-positive cells, and calculating CD4⁺PD-1⁺/CD4⁺Percentage of T cells, screening for cells positive for CD8 antibody from T cells, screening for cells positive for PD-1 antibody from cells positive for CD8 antibody, calculating CD8⁺PD-1⁺/CD8⁺Percentage of T cells, HLA-DR antibody-positive cells were selected from CD8 antibody-positive cells, and CD8 was calculated⁺HLA-DR⁺/CD8⁺Percentage of T cells.

Further, CD8⁺PD-1⁺/CD8⁺Percentage of T cells<2％、CD4⁺PD-1⁺/CD4⁺Percentage of T cells<2% and CD8⁺HLA-DR⁺/CD8⁺Percentage of T cells<19.5%, the ratio of functional depletion to activation used to characterize T cells was low.

A flow cytometer detection kit for lung cancer advanced stage immune characterization comprises a reagent A

The reagent A comprises a CD45 monoclonal antibody, a CD3 monoclonal antibody, a CD4 monoclonal antibody, a CD8 monoclonal antibody, a PD-1 monoclonal antibody and a CD38 monoclonal antibody which are marked by fluorescent microspheres dyed by different color dyes. The concentration ratio of each antibody was 1: 1: 1: 1: 1:1, all of which are 0.05-1 mg/mL.

11. The detection kit according to claim 10, wherein the detection kit further comprises a reagent B, and the reagent B is a red blood cell lysate.

The detection method of the detection kit comprises the following steps:

step three, after uniformly mixing by using a vortex instrument, centrifuging at 4 ℃, removing supernatant, adding PBS (phosphate buffer solution) for cleaning, centrifuging at 4 ℃ after uniformly mixing by using a vortex instrument, removing supernatant, adding PBS again to form cell suspension, and detecting by using a flow cytometer;

step four, screening cells which are positive to the CD45 antibody and have SSC value in the range of 0-50K, and determining the cells as lymphocytes; screening CD3 antibody positive cells from lymphocytes to determine T cells; screening for CD4 antibody-positive cells from T cells, screening for PD-1 antibody-positive cells from CD4 antibody-positive cells, and calculating CD4⁺PD-1⁺/CD4⁺Percentage of T cells, screening for cells positive for CD8 antibody from T cells, screening for cells positive for PD-1 antibody from cells positive for CD8 antibody, calculating CD8⁺PD-1⁺/CD8⁺Percentage of T cells, CD38 antibody-positive cells were selected from CD8 antibody-positive cells, and CD8 was calculated⁺CD38⁺/CD8⁺Percentage of T cells.

Further, CD8⁺PD-1⁺/CD8⁺Percentage of T cells<2％、CD4⁺PD-1⁺/CD4⁺Percentage of T cells<2% and CD8⁺CD38⁺/CD8⁺Percentage of T cells<27%, the level of depletion used to characterize T cells is low in proportion to abnormal activation.

By adopting the technical scheme, the invention has the following beneficial effects:

(1) the flow cytometry kit for analyzing peripheral blood immune cells provided by the invention detects peripheral blood of a patient with advanced lung cancer after receiving an immune checkpoint inhibitor as a sample, and detects CD8 in the sample to be detected by using the kit⁺PD-1⁺/CD8⁺T cell%, CD4⁺PD-1⁺/CD4⁺T cell%, CD8⁺HLA-DR⁺/CD8⁺T cell%, each index can be comprehensively characterizedThe patient's immune level, thereby aiding in the determination of the therapeutic efficacy of the patient checkpoint inhibitor;

(2) detection of CD8 in sample to be detected by using kit⁺PD-1⁺/CD8⁺T cell%, CD4⁺PD-1⁺/CD4⁺T cell%, CD8⁺CD38⁺/CD8⁺T cell%, and each index comprehensively represents the immune level of a patient, so as to assist in judging whether immune-related adverse reactions occur after the treatment of the checkpoint inhibitor of the patient.

Drawings

FIG. 1 is a sample streaming results graph numbered 24;

FIG. 2 is a sample streaming results graph numbered 13;

FIG. 3 is a diagram of the kit detecting CD8 of samples Nos. 9-17 and 24-34⁺PD-1⁺/CD8⁺T cell ratio results;

FIG. 4 is a diagram of the kit detecting CD4 of samples Nos. 9-17 and 24-34⁺PD-1⁺/CD4⁺T cell ratio results;

FIG. 5 is a sample streaming results graph numbered 4;

FIG. 6 is a sample streaming results graph numbered 13;

FIG. 7 is a diagram of the kit detecting CD8 of samples Nos. 1-8 and 9-23⁺HLA-DR⁺/CD8⁺T cell ratio results;

FIG. 8 is a diagram of the kit detecting CD4 of samples Nos. 1-8 and 9-23⁺HLA-DR⁺/CD4⁺T cell ratio result chart;

FIG. 9 is a sample streaming results graph numbered 13;

FIG. 10 is a sample streaming results graph numbered 23;

FIG. 11 is a diagram showing the detection of sample CD8 of Nos. 9-17 and 18-23 by the kit⁺PD-1⁺/CD8⁺A cell proportion result chart;

FIG. 12 is a diagram of the kit detecting sample CD4 of Nos. 9-17 and 18-23⁺PD-1⁺/CD4⁺Cell ratio results chart.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the block diagrams and specific examples described herein are only illustrative of the invention and are not intended to limit the invention.

Example 1

The invention provides a flow cytometer detection kit for lung cancer late stage immune characterization, which comprises a reagent A: reagent A includes CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody and PD-1 monoclonal antibody labeled by fluorescent microspheres dyed with different color dyes. The concentration ratio of each antibody was 1: 1: 1: 1:1, all of which are 0.05-0.1 mg/mL.

The amino acid sequence of each antibody binding protein is detailed in table 1.

TABLE 1 amino acid sequence

The antibody type sequences and the dye detection signals of the flow cytometer are matched as follows: CD45 antibody (2D1/IgG1, V500-C channel), CD3 antibody (UCHT1/IgG1, PE-Cy7 channel), CD4 antibody (SK3/IgG1, AF700 channel), CD8 antibody (53-6.7/IgG2a, APC-Cy7 channel), PD-1 antibody (J43/IgG2, BV510 channel).

The diameter of the fluorescent microsphere is 7-10 μm, and the fluorescent microsphere can be excited by 488-640nm laser, and the emission wavelength range is 500-719 nm.

Preferably, the reagent B is erythrocyte lysate, and comprises the following components of ammonium chloride (NH4Cl), potassium bicarbonate (KHCO3) and ethylenediaminetetraacetic acid (EDTA), wherein the proportion of each component is 15.5 mM: 1 nM: 0.01 mM.

The invention provides a detection method of a detection kit, which comprises the following steps:

step two, adding the reagent A into a tube, wherein the volume ratio of the reagent A to the leukocyte liquid is 1:10, and incubating the reagent A and the leukocyte liquid together; the fluorescently labeled antibody in reagent a will specifically bind to the leukocyte surface antigen.

Step three, adding a PBS solution for cleaning, performing vortex mixing, centrifuging at 4 ℃, removing supernatant, adding the PBS solution again to form cell suspension, and detecting by an up-flow cytometer; during the flow cytometry detection process, the laser beam enables the labeled antibody bound on the cell surface to excite fluorescence with specific wavelength. Fluorescence signals are detected to determine information about cell size (as characterized by Forward Scatter (FSC) values), internal complexity (as characterized by Side Scatter (SSC) values), and corresponding antigen expression intensity and abundance.

Another detection method of the detection kit takes whole blood as an examination starting material and comprises the following steps:

step three, after uniformly mixing by using a vortex apparatus, centrifuging at 4 ℃, discarding supernatant, adding PBS (phosphate buffer solution) for cleaning, centrifuging at 4 ℃ after uniformly mixing by using a vortex apparatus, discarding supernatant, adding PBS again to form cell suspension, and detecting by using an up-flow cytometer;

step four, screening cells which are positive to the CD45 antibody and have SSC value in the range of 0-50K, and determining the cells as lymphocytes; from lymphScreening cells positive to the CD3 antibody from the cells, and determining the cells as T cells; screening for CD4 antibody-positive cells from T cells, screening for PD-1 antibody-positive cells from CD4 antibody-positive cells, and calculating CD4⁺PD-1⁺/CD4⁺Percentage of T cells, screening for cells positive for CD8 antibody from T cells, screening for cells positive for PD-1 antibody from cells positive for CD8 antibody, calculating CD8⁺PD-1⁺/CD8⁺Percentage of T cells.

A total of 23 patients with advanced lung cancer who received immune checkpoint inhibitor therapy were selected and had no infection before and after receiving immune therapy. At the same time, 11 patients with advanced lung cancer who did not receive immune checkpoint inhibitor treatment were selected as a control group.

The clinical experimental cases are shown in Table 2

TABLE 2

The Progression Free Survival (PFS) for the specific disease in these 34 patients is shown in Table 3. The method for judging the immune-related adverse reaction comprises the following steps: according to the evaluation Standard of terms of common adverse reactions (CTCAE 4.0) established by the national institute of health and cancer.

TABLE 3

The detection kit is adopted to detect the CD8 of the samples with the numbers of 9-17 and 24-34⁺PD-1⁺/CD8⁺Percentage of T cells, CD4⁺PD-1⁺/CD4⁺Percentage of T cells.

FIG. 1 is a graph of the flow results for advanced lung cancer patient No. 24 who did not receive an immune checkpoint inhibitor; FIG. 2 is numbered as13, the flow result chart of the patients who have better curative effect and do not have immune-related adverse reaction. The gate circle strategy is as follows: (a) lymphocyte (LYM) delineation using SSC and CD45 channels; (b) cells positive for CD3 antibody were selected as T cells (T cells) in LYM; (c) cells positive for CD4 were selected as CD4 in T cells⁺T cell (CD 4)⁺T cells), CD8 antibody positive cells as CD8⁺T cell (CD 8)⁺T cells), (d) at CD4⁺Cells positive for PD-1 antibody were selected as PD-1 in T cells⁺CD4⁺T cells, the upper right-hand corner of the population representing CD4⁺Percentage of T cells. (g) In CD8⁺Cells positive for PD-1 antibody were individually selected as PD-1 in T cells⁺CD8⁺T cells, the upper right-hand corner of the population representing CD8⁺Percentage of T cells.

CD8⁺PD-1⁺/CD8⁺T cell ratio results are shown in FIG. 3, and the patients (Nos. 9-17) who received the immune checkpoint inhibitor for treatment of advanced lung cancer without immune-related adverse reactions had good therapeutic effects, and their treated peripheral blood CD8⁺PD-1⁺/CD8⁺A significant reduction in T cell proportion compared to advanced lung cancer not treated with immune checkpoint inhibitors (0.1(0.1-0.6) vs 9.3(6.1-12.1), p ═ 0.002), statistical method: comparisons were made using independent sample t-tests.

Treating advanced lung cancer patients with good curative effect by using immune checkpoint inhibitor, and treating with immune checkpoint inhibitor after immune therapy to obtain CD8⁺+PD-1⁺/CD8⁺The proportion of T cells is significantly reduced compared to patients with advanced lung cancer who do not receive immunotherapy.

CD4⁺PD-1⁺/CD4⁺T cell ratio results are shown in FIG. 4, and the patients (Nos. 9-17) who received the immune checkpoint inhibitor and had good curative effect and no immune-related adverse reaction were treated with CD4 in the peripheral blood⁺PD-1⁺/CD4⁺A significant reduction in the proportion of T cells compared to advanced lung cancer not treated with an immune checkpoint inhibitor, (0.1(0.1-0.5) vs 15.7(13.8-17.7), p<0.05), statistical method: comparisons were made using independent sample t-tests.

Immune checkpoint inhibitorsThe preparation can be used for treating patients with good therapeutic effect, and the patients are treated with CD4 after receiving immune checkpoint inhibitor⁺PD-1⁺/CD4⁺The T cell ratio is obviously reduced compared with the advanced lung cancer which is not treated by the immune checkpoint inhibitor.

Thus, using the kit of this example, CD8⁺PD-1⁺/CD8⁺Percentage of T cells<2% and CD4⁺PD-1⁺/CD4⁺Percentage of T cells<2%, the proportion for characterizing the function exhaustion of the T cells is lower, or the auxiliary characterization is performed by the treatment of an immune checkpoint inhibitor, and the curative effect is better.

Example 2

The invention provides another flow cytometry detection kit for lung cancer advanced stage immune characterization, which comprises a reagent A, wherein the reagent A comprises a CD45 monoclonal antibody, a CD3 monoclonal antibody, a CD4 monoclonal antibody, a CD8 monoclonal antibody, a PD-1 monoclonal antibody and an HLA-DR monoclonal antibody which are marked by fluorescent microspheres dyed by dyes with different colors, and the amino acid sequences of binding proteins of the antibodies are detailed in a table 1. The antibody type sequences and dye detection signals are matched as follows: CD45 antibody (2D1/IgG1, V500-C channel), CD3 antibody (UCHT1/IgG1, PE-Cy7 channel), CD4 antibody (SK3/IgG1, AF700 channel), CD8 antibody (53-6.7/IgG2a, APC-Cy7 channel), PD-1 antibody (J43/IgG2, BV510 channel), HLA-DR antibody (L243/IgG1a, PerCp5.5 channel). The concentration ratio of each antibody was 1: 1: 1: 1: 1:1, all of which are 0.05-1 mg/mL.

The detection kit also comprises a reagent B, and the reagent B is erythrocyte lysate.

The detection method of the detection kit comprises the following steps:

step four, screening cells which are positive to the CD45 antibody and have SSC value in the range of 0-50K, and determining the cells as lymphocytes; screening the lymphocytes for CD3 antibody positive cells, and determining the cells as T cells; screening for CD4 antibody-positive cells from T cells, screening for PD-1 antibody-positive cells from CD4 antibody-positive cells, and calculating CD4⁺PD-1⁺/CD4⁺Percentage of T cells, screening for CD8 antibody-positive cells from T cells, screening for PD-1 antibody-positive cells from CD8 antibody-positive cells, calculating CD8⁺PD-1⁺/CD8⁺Percentage of T cells, HLA-DR antibody-positive cells were selected from CD8 antibody-positive cells, and CD8 was calculated⁺HLA-DR⁺/CD8⁺Percentage of T cells.

The CD8 of the samples with the numbers 1-8 and 9-23 is detected by adopting the detection kit⁺HLA-DR⁺/ CD8⁺Percentage of T cells.

The results of the peripheral blood immune cell subtypes were compared between patients (nos. 9-23) with advanced lung cancer who received good treatment with the immune checkpoint inhibitor and patients (nos. 1-8) with advanced lung cancer who received poor treatment, as shown in table 4.

TABLE 4

	Patients with poor immunotherapy efficacy	Patients with good curative effect of immunotherapy	P value
				CD8+％	26.8(20.4-33.2)	29.0(21.2-31.3)	0.900
CD8+HLA-DR+％	36.6±16.7	14.2±5.8	0.002*
				CD8+CD38+％	20.4±12.9	22.9±14.0	0.675
CD8+PD-1+％	0.6(0.23-0.88)	0.4(0.1-0.9)	0.925
				CD4+％	27.4(22.0-38.3)	33.5(27-40)	0.506
CD4+HLA-DR+％	27.7±18.6	11.5±7.7	0.008*
				CD4+CD38+％	8.8(5.2-16.72)	7.5(4.9-11.2)	0.636
CD4+PD-1+％	0.35(0.23-0.90)	0.1(0.1-1.2)	0.975

The flow result of the sample number 4 is shown in fig. 5, and the flow result of the sample number 13 is shown in fig. 6. The gate circle strategy is as follows: (a) trapping Lymphocytes (LYM) using SSC and CD45 channels; (b) selecting cells positive for CD3 antibody in LYM as T cells (T cells); (c) cells positive for CD4 were selected as CD4 in T cells⁺T cell (CD 4)⁺T cells), CD8 antibody positive cells as CD8⁺T cell (CD 8)⁺T cells); (d, e) in CD4⁺Cells positive for PD-1/HLA-DR antibody were selected as PD-1 in T cells⁺CD4⁺T cell/HLA-DR⁺CD4⁺T cells, the upper right-hand corner of the population representing CD4⁺Percentage of T cells; (g-h) in CD8⁺Cells positive for PD-1/HLA-DR antibody were selected as PD-1 in T cells⁺CD⁺T cell/HLA-DR⁺CD8⁺T cells, the upper right-hand corner of the population representing CD8⁺Percentage of T cells.

As shown in FIG. 7, the results showed that patients with advanced lung cancer who received immune checkpoint inhibitor therapy (Nos. 9-23) were found to have good efficacy, and that CD8 was present in the peripheral blood after treatment⁺HLA-DR⁺/CD8⁺The proportion of T cells is higher than that of peripheral blood CD8 of late-stage lung cancer patient with poor curative effect⁺HLA-DR⁺/CD8⁺The proportion of T cells is significantly low (14.2 + -5.8% vs 36.6 + -16.7%, p)<0.05). The statistical method comprises the following steps: comparisons were made using independent sample t-tests.

As shown in FIG. 8, the results showed that patients with advanced lung cancer who received immune checkpoint inhibitor therapy (Nos. 9-23) were found to have good therapeutic effect on peripheral blood CD4 after treatment⁺HLA-DR⁺/CD4⁺The proportion of T cells is higher than that of peripheral blood CD4 of late-stage lung cancer patient with poor curative effect⁺HLA-DR⁺/CD4⁺The proportion of T cells is significantly low (11.5 + -7.7% vs 27.7 + -18.6%, p)<0.05). The statistical method comprises the following steps: comparisons were made using independent sample t-tests.

By adopting a logistic regression analysis method to carry out multi-factor analysis on a patient group with better immune treatment curative effect and a patient group with poor immune treatment curative effect and advanced lung cancer (shown in table 5), the proportion of CD8+ HLA-DR + cells in peripheral blood after immune treatment is less than 19.5 percent, and the independent prediction factor is an independent prediction factor for assisting in representing better curative effect of continuously receiving immune checkpoint inhibitor treatment.

TABLE 5

	OR	95％CI	P value
				CD8+HLA-DR+％<19.5％	0.004	0.001-0.479	0.024*
Age (age)	1.135	0.850-1.517	0.391
				Sex	2.814	0.046-172.93	0.623
Presence or absence of mutation	0.054	0.001-5.560	0.217
				Whether or not it is non-small cell lung cancer	0.004	0015-86.159	0.960

Therefore, the detection kit of this example, CD8, was used⁺PD-1⁺/CD8⁺Percentage of T cells<2％、CD4⁺PD-1⁺/CD4⁺Percentage of T cells<2% and CD8⁺HLA-DR⁺/CD8⁺Percentage of T cells<19.5%, can be used for characterizing that the function of the T cells is exhausted and activated in a lower proportion, and related patients continue to receive the immune checkpoint inhibitor to have better curative effect.

Example 3

A flow cytometry detection kit for lung cancer advanced stage immune characterization comprises a reagent A. The reagent A comprises a CD45 monoclonal antibody, a CD3 monoclonal antibody, a CD4 monoclonal antibody, a CD8 monoclonal antibody, a PD-1 monoclonal antibody and a CD38 monoclonal antibody which are marked by fluorescent microspheres dyed by different color dyes. The amino acid sequence of each antibody binding protein is detailed in table 1. The antibody type sequences and dye detection signals are matched as follows: CD45 antibody (2D1/IgG1, V500-C channel), CD3 antibody (UCHT1/IgG1, PE-Cy7 channel), CD4 antibody (SK3/IgG1, AF700 channel), CD8 antibody (53-6.7/IgG2a, APC-Cy7 channel), PD-1 antibody (J43/IgG2, BV510 channel), CD38 antibody (HB7/IgG1, V450 channel). The concentration ratio of each antibody was 1: 1: 1: 1: 1:1, all of which are 0.05-1 mg/mL. The diameter of the fluorescent microsphere is 7-10 μm, and the fluorescent microsphere can be excited by 488-640nm laser, and the emission wavelength range is 500-719 nm.

The detection kit also comprises a reagent B, wherein the reagent B is erythrocyte lysate. The components comprise ammonium chloride (NH4Cl), potassium bicarbonate (KHCO3) and Ethylene Diamine Tetraacetic Acid (EDTA), and the proportion of each component is 15.5 mM: 1 nM: 0.01 mM.

The detection method of the detection kit comprises the following steps:

step four, screening cells which are positive to the CD45 antibody and have SSC value in the range of 0-50K, and determining the cells as lymphocytes; screening the lymphocytes for CD3 antibody positive cells, and determining the cells as T cells; screening for CD4 antibody-positive cells from T cells, screening for PD-1 antibody-positive cells from CD4 antibody-positive cells, and calculating CD4⁺PD-1⁺/CD4⁺Percentage of T cells, screening for cells positive for CD8 antibody from T cells, screening for cells positive for PD-1 antibody from cells positive for CD8 antibody, calculating CD8⁺PD-1⁺/CD8⁺Percentage of T cells, CD38 antibody-positive cells were selected from CD8 antibody-positive cells, and CD8 was calculated⁺CD38⁺/CD8⁺Percentage of T cells.

The sample CD8 with the numbers of 9-17 and 18-23 is detected by the detection kit⁺ CD38⁺/CD8⁺Percentage of T cells.

The flow result of the sample number 13 is shown in fig. 9, and the flow result of the sample number 23 is shown in fig. 10. The gate circle strategy is as follows: (a) trapping Lymphocytes (LYM) using SSC and CD45 channels; (b) selecting cells positive for CD3 antibody in LYM as T cells (T cells); (c) CD4 positive cells were selected as CD4+ T cells (CD4+ T cells) and CD8 antibody positive cells as CD8+ T cells (CD8+ T cells) in T cells; (d-f) circling PD-1/HLA-DR/CD38 antibody positive cells in CD4+ T cells as PD-1+ CD4+ T cells/HLA-DR + CD4+ T cells/CD 38+ CD4+ T cells, respectively, the upper right-hand value being the percentage of the population of cells in CD4+ T cells; (g-i) cells positive for PD-1/HLA-DR/CD38 antibody were selected as PD-1+ CD + T cells/HLA-DR + CD8+ T cells/CD 38+ CD8+ T cells in CD8+ T cells, respectively, with the top right value being the percentage of the population of cells to CD8+ T cells.

Peripheral blood CD8 after treatment in patients with advanced lung cancer who have had immune-related adverse effects (ir-AE) who received immune checkpoint inhibitors⁺PD-1⁺/CD8⁺The percentage of T cells decreased significantly compared to advanced lung cancer that did not receive treatment with immune checkpoint inhibitors [0.7(0.2-1.4) vs 9.3(6.1-12.1), p<0.05]And the statistical method comprises the following steps: the comparison was performed using independent sample t-test and the results are shown in figure 11.

Patients with advanced lung cancer with immune-related adverse events receiving CD4 after receiving immune checkpoint inhibitor⁺PD-1⁺/CD4⁺The percentage of T cells showed a significant decrease in advanced lung cancer that did not receive treatment with immune checkpoint inhibitors [0.6(0.1-1.3) vs 15.7(13.8-17.7), p<0.05]And the statistical method comprises the following steps: a comparison was made using the independent sample t-test, as shown in figure 12.

The results of the peripheral blood immune cell subtypes of patients with advanced lung cancer who received immune checkpoint inhibitor therapy and developed immune-related adverse reactions were compared with those of patients with advanced lung cancer who developed no immune-related adverse reactions after immunotherapy, as shown in table 6. It was found that in patients with advanced lung cancer who had developed immune-related side effects treated with immune checkpoint inhibitors, the proportion of peripheral blood CD8+ CD38+ cells was significantly higher after treatment than in patients with advanced lung cancer who did not develop ir-AE (37.9 (33.5-40.7) vs 13.8(7.3-19.2), p ═ 0.001).

TABLE 6

The group of immune-related adverse reactions not occurring with immunotherapy and the group of advanced lung cancer patients not occurring with ir-AE were subjected to multi-factor analysis using logistic regression analysis, as shown in Table 7. The proportion of peripheral blood CD8+ CD38+ cells > 27% after immunotherapy was found to be an independent predictor of the development of immune-related adverse reactions.

TABLE 7

	OR	95％CI	P value
				CD8+CD38+％>27％	0.025	0.001-0.496	0.016

Therefore, the detection kit of this example, CD8, was used⁺PD-1⁺/CD8⁺Percentage of T cells<2％、CD4⁺PD-1⁺/CD4⁺Percentage of T cells<2% and CD8⁺CD38⁺/CD8⁺Percentage of T cells<27% of the total amount of T cells depleted and abnormally activated, the relevant patients continuing to receive treatment with immune checkpoint inhibitorsThe possibility of the adverse reaction related to the generation of immunity is low.

The above embodiments only express the embodiments of the present invention, and the description is specific and detailed, but it should be understood that the invention is not limited to the claims, and it should be noted that, for those skilled in the art, many variations and modifications can be made without departing from the spirit of the invention, and these embodiments are all within the scope of the invention.

Sequence listing

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Asn Thr Thr Ile Thr Ala Asn Thr Ser Asp Ala Tyr Leu Asn Ala Ser

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Glu Thr Thr Thr Leu Ser Pro Ser Gly Ser Ala Val Ile Ser Thr Thr

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Thr Ile Ala Thr Thr Pro Ser Lys Pro Thr Cys Asp Glu Lys Tyr Ala

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Asn Ile Thr Val Asp Tyr Leu Tyr Asn Lys Glu Thr Lys Leu Phe Thr

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Ala Lys Leu Asn Val Asn Glu Asn Val Glu Cys Gly Asn Asn Thr Cys

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Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg Gly

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Gln Ile Lys Ile Leu Gly Asn Gln Gly Ser Phe Leu Thr Lys Gly Pro

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Ser Lys Leu Asn Asp Arg Ala Asp Ser Arg Arg Ser Leu Trp Asp Gln

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Gly Asn Phe Pro Leu Ile Ile Lys Asn Leu Lys Ile Glu Asp Ser Asp

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Thr Tyr Ile Cys Glu Val Glu Asp Gln Lys Glu Glu Val Gln Leu Leu

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Val Phe Gly Leu Thr Ala Asn Ser Asp Thr His Leu Leu Gln Gly Gln

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Ser Leu Thr Leu Thr Leu Glu Ser Pro Pro Gly Ser Ser Pro Ser Val

115 120 125

Gln Cys Arg Ser Pro Arg Gly Lys Asn Ile Gln Gly Gly Lys Thr Leu

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Ser Val Ser Gln Leu Glu Leu Gln Asp Ser Gly Thr Trp Thr Cys Thr

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Val Leu Gln Asn Gln Lys Lys Val Glu Phe Lys Ile Asp Ile Val Val

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Leu Ala Phe Gln Lys Ala Ser Ser Ile Val Tyr Lys Lys Glu Gly Glu

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Gln Val Glu Phe Ser Phe Pro Leu Ala Phe Thr Val Glu Lys Leu Thr

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Gly Ser Gly Glu Leu Trp Trp Gln Ala Glu Arg Ala Ser Ser Ser Lys

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Ser Trp Ile Thr Phe Asp Leu Lys Asn Lys Glu Val Ser Val Lys Arg

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Val Thr Gln Asp Pro Lys Leu Gln Met Gly Lys Lys Leu Pro Leu His

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Leu Thr Leu Pro Gln Ala Leu Pro Gln Tyr Ala Gly Ser Gly Asn Leu

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Thr Leu Ala Leu Glu Ala Lys Thr Gly Lys Leu His Gln Glu Val Asn

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Leu Val Val Met Arg Ala Thr Gln Leu Gln Lys Asn Leu Thr Cys Glu

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Val Trp Gly Pro Thr Ser Pro Lys Leu Met Leu Ser Leu Lys Leu Glu

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Asn Lys Glu Ala Lys Val Ser Lys Arg Glu Lys Ala Val Trp Val Leu

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Asn Pro Glu Ala Gly Met Trp Gln Cys Leu Leu Ser Asp Ser Gly Gln

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Val Leu Leu Glu Ser Asn Ile Lys Val Leu Pro Thr Trp Ser Thr Pro

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Val Gln Pro

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<212> PRT

<213> CD8 antibody (Artificial sequence)

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Leu Gln Gln Thr Pro Ala Tyr Ile Lys Val Gln Thr Asn Lys Met Val

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Met Leu Ser Cys Glu Ala Lys Ile Ser Leu Ser Asn Met Arg Ile Tyr

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Trp Leu Arg Gln Arg Gln Ala Pro Ser Ser Asp Ser His His Glu Phe

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Leu Ala Leu Trp Asp Ser Ala Lys Gly Thr Ile His Gly Glu Glu Val

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Glu Gln Glu Lys Ile Ala Val Phe Arg Asp Ala Ser Arg Phe Ile Leu

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Asn Leu Thr Ser Val Lys Pro Glu Asp Ser Gly Ile Tyr Phe Cys Met

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Ile Val Gly Ser Pro Glu Leu Thr Phe Gly Lys Gly Thr Gln Leu Ser

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Val Val Asp Phe Leu Pro Thr Thr Ala Gln Pro Thr Lys Lys Ser Thr

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Leu Lys Lys Arg Val Cys Arg Leu Pro Arg Pro Glu Thr Gln Lys Gly

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Pro Leu Cys Ser Pro

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Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser

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Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu Pro Asn Gly

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Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly

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Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys

100 105 110

Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val

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Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln

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Thr Leu Val

145

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<213> HLA-DR antibody (Artificial sequence)

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Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe

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His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe

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Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala

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Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr

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Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro

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Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe

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Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val

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Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu

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Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val

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Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys

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His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu

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<212> PRT

<213> CD38 antibody (Artificial sequence)

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Val Pro Arg Trp Arg Gln Gln Trp Ser Gly Pro Gly Thr Thr Lys Arg

1 5 10 15

Phe Pro Glu Thr Val Leu Ala Arg Cys Val Lys Tyr Thr Glu Ile His

20 25 30

Pro Glu Met Arg His Val Asp Cys Gln Ser Val Trp Asp Ala Phe Lys

35 40 45

Gly Ala Phe Ile Ser Lys His Pro Cys Asn Ile Thr Glu Glu Asp Tyr

50 55 60

Gln Pro Leu Met Lys Leu Gly Thr Gln Thr Val Pro Cys Asn Lys Ile

65 70 75 80

Leu Leu Trp Ser Arg Ile Lys Asp Leu Ala His Gln Phe Thr Gln Val

85 90 95

Gln Arg Asp Met Phe Thr Leu Glu Asp Thr Leu Leu Gly Tyr Leu Ala

100 105 110

Asp Asp Leu Thr Trp Cys Gly Glu Phe Asn Thr Ser Lys Ile Asn Tyr

115 120 125

Gln Ser Cys Pro Asp Trp Arg Lys Asp Cys Ser Asn Asn Pro Val Ser

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Val Phe Trp Lys Thr Val Ser Arg Arg Phe Ala Glu Ala Ala Cys Asp

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Val Val His Val Met Leu Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys

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Asn Ser Thr Phe Gly Ser Val Glu Val His Asn Leu Gln Pro Glu Lys

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Val Gln Thr Leu Glu Ala Trp Val Ile His Gly Gly Arg Glu Asp Ser

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Arg Asp Leu Cys Gln Asp Pro Thr Ile Lys Glu Leu Glu Ser Ile Ile

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Ser Lys Arg Asn Ile Gln Phe Ser Cys Lys Asn Ile Tyr Arg Pro Asp

225 230 235 240

Lys Phe Leu Gln Cys Val Lys Asn Pro Glu Asp Ser Ser Cys Thr Ser

245 250 255

Glu Ile

Claims

1. a flow cytometer detection kit of lung cancer late stage immune signature, comprising reagent A, is characterized in that:

The reagent A includes CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody and PD-1 monoclonal antibody labeled with fluorescent microspheres stained with different color dyes, and the concentration ratio of each antibody is 1: 1:1:1:1, both are 0.05-0.1 mg/mL.

2. The detection kit according to claim 1, wherein the detection kit further comprises a reagent B, and the reagent B is an erythrocyte lysate.

3. the detection method of the described detection kit of claim 1, is characterized in that, comprises the following steps:

Step 1. Take human venous blood, separate and obtain leukocyte liquid, and add it to the bottom of the flow tube;

Step 2, adding the reagent A into the tube, the volume ratio of the reagent A and the white blood cell liquid is 1:10, and the reagent A and the white blood cell liquid are incubated together;

Step 3. Add PBS solution for washing, vortex and mix well, centrifuge at 4°C, discard the supernatant, add PBS solution again to form a cell suspension, and use flow cytometer for detection;

Step 4. Screen the cells that are positive for CD45 antibody and whose SSC value is in the range of 0-50K, and determine as lymphocytes; select cells that are positive for CD3 antibody from lymphocytes, and determine as T cells; select cells that are positive for CD4 antibody from T cells , screen PD-1 antibody-positive cells from CD4 antibody-positive cells, and calculate the percentage of CD4 ⁺ PD-1 ⁺ /CD4 ⁺ T cells; screen CD8 antibody-positive cells from T cells, and select CD8 antibody-positive cells from CD8 antibody-positive cells Cells positive for PD-1 antibody were screened, and the percentage of CD8 ⁺ PD-1 ⁺ /CD8 ⁺ T cells was calculated.

4. the detection method of the described detection kit of claim 2, is characterized in that, comprises the following steps:

Step 1. Use the reverse pipetting technique to draw the EDTA-K2 anticoagulated whole blood to the bottom of the flow tube to prevent the blood from touching the upper part of the tube wall;

Step 2: Add reagent A to the tube, the volume ratio of reagent A to whole blood is 1:10, and mix with a vortexer; incubate at room temperature in the dark; add reagent B to the tube, and the volume ratio of reagent B to whole blood is 20:1; Mix by vortexer; avoid light reaction;

Step 3. After mixing with a vortexer, centrifuge at 4°C to discard the supernatant, add PBS solution to wash, vortex and mix, centrifuge at 4°C, discard the supernatant, add PBS solution again to form a cell suspension, and use flow cytometry instrument to detect;

Step 4. Screen the cells that are positive for CD45 antibody and whose SSC value is in the range of 0-50K, and determine as lymphocytes; select cells that are positive for CD3 antibody from lymphocytes, and determine as T cells; select cells that are positive for CD4 antibody from T cells , screen PD-1 antibody positive cells from CD4 antibody positive cells, calculate the percentage of CD4 ⁺ PD-1 ⁺ /CD4 ⁺ T cells, screen CD8 antibody positive cells from T cells, select CD8 antibody positive cells from CD8 antibody positive cells Cells positive for PD-1 antibody were screened, and the percentage of CD8 ⁺ PD-1 ⁺ /CD8 ⁺ T cells was calculated.

5. The detection kit according to claim 1 or 2, wherein the percentage of CD8 ⁺ PD-1 ⁺ /CD8 ⁺ T cells <2% and the percentage of CD4 ⁺ PD-1 ⁺ /CD4 ⁺ T cells < A low ratio of 2% was used to characterize the functional exhaustion of T cells.

6. A flow cytometer detection kit for late stage immune characterization of lung cancer, comprising reagent A, characterized in that:

Reagent A includes CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, PD-1 monoclonal antibody, HLA-DR monoclonal antibody, and each antibody The concentration ratio is 1:1:1:1:1:1, both of which are 0.05-1 mg/mL.

7. The detection kit according to claim 6, wherein the detection kit further comprises a reagent B, and the reagent B is an erythrocyte lysate.

8. the detection method of the detection kit of claim 7, is characterized in that, comprises the following steps:

Step 4. Screen the cells that are positive for CD45 antibody and whose SSC value is in the range of 0-50K, and determine as lymphocytes; select cells that are positive for CD3 antibody from lymphocytes, and determine as T cells; select cells that are positive for CD4 antibody from T cells , screen PD-1 antibody positive cells from CD4 antibody positive cells, calculate the percentage of CD4 ⁺ PD-1 ⁺ /CD4 ⁺ T cells, screen CD8 antibody positive cells from T cells, select CD8 antibody positive cells from CD8 antibody positive cells Screen PD-1 antibody-positive cells, calculate the percentage of CD8 ⁺ PD-1 ⁺ /CD8 ⁺ T cells, screen HLA-DR antibody-positive cells from CD8 antibody-positive cells, and calculate CD8 ⁺ HLA-DR ⁺ /CD8 ⁺ percentage of T cells.

9. The detection kit according to claim 6 or 7, wherein the percentage of CD8 ⁺ PD-1 ⁺ /CD8 ⁺ T cells <2%, the percentage of CD4 ⁺ PD-1 ⁺ /CD4 ⁺ T cells < 2% and the percentage of CD8 ⁺ HLA-DR ⁺ /CD8 ⁺ T cells <19.5%, which was used to characterize the lower ratio of functional depletion to activation of T cells.

10. A flow cytometry detection kit for late-stage lung cancer immune characterization, comprising reagent A, characterized in that:

The reagent A includes CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, PD-1 monoclonal antibody, and CD38 monoclonal antibody labeled with fluorescent microspheres stained with different color dyes. The concentration ratio of each antibody is 1:1:1:1:1:1, both of which are 0.05-1 mg/mL.

11. The detection kit according to claim 10, wherein the detection kit further comprises a reagent B, and the reagent B is an erythrocyte lysate.

12. The detection method of the detection kit of claim 11, characterized in that, comprising the following steps:

Step 4. Screen the cells that are positive for CD45 antibody and whose SSC value is in the range of 0-50K, and determine as lymphocytes; select cells that are positive for CD3 antibody from lymphocytes, and determine as T cells; select cells that are positive for CD4 antibody from T cells , screen PD-1 antibody positive cells from CD4 antibody positive cells, calculate the percentage of CD4 ⁺ PD-1 ⁺ /CD4 ⁺ T cells, screen CD8 antibody positive cells from T cells, select CD8 antibody positive cells from CD8 antibody positive cells Screen PD-1 antibody positive cells, calculate the percentage of CD8 ⁺ PD-1 ⁺ /CD8 ⁺ T cells, select CD38 antibody positive cells from CD8 antibody positive cells, and calculate the percentage of CD8 ⁺ CD38 ⁺ /CD8 ⁺ T cells .

13. The detection kit according to claim 10 or 11, wherein the percentage of CD8 ⁺ PD-1 ⁺ /CD8 ⁺ T cells <2%, the percentage of CD4 ⁺ PD-1 ⁺ /CD4 ⁺ T cells < 2% and the percentage of CD8 ⁺ CD38 ⁺ /CD8 ⁺ T cells <27%, used to characterize the low level of depletion and abnormal activation of T cells.