Immune related adverse event biomarker and kit

Abstract

An immune related adverse event marker and a kit. The immune related adverse event marker comprises CD183. The immune related adverse event marker can effectively predict immune related adverse events caused by programmed death receptor 1 antibody / programmed death-ligand antibody therapy, thereby facilitating intervention and treatment of immune related adverse events.

Description

Immune related adverse reaction biomarker and kit TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and more particularly, to an immune related adverse reaction biomarker and kit. BACKGROUND

[0002] Immune checkpoint inhibits (ICIs) mainly target antibodies of cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1). ICIs activate T cells by blocking CTLA-4, PD-1 and their ligands, promote immune killing of tumor cells, and improve the effect of immunotherapy. Since ICIs rely on activating the body's own immune system to attack tumor cells to achieve the purpose of treatment, the body's immune homeostasis will be broken, and some normal cells of the body will also be attacked by the immune system, thereby causing immune related adverse reactions (irAEs).

[0003] irAEs can involve all organs of the body, and can cause death in severe cases. Existing immune related adverse reaction biomarkers are based on immunohistochemical detection of tumor tissue sections of patients, have high dependence on samples, cause trauma to patients during sampling, and cannot be used to detect patients who cannot obtain tumor samples. At the same time, the immunohistochemical detection test has a long cycle and is interfered by personal factors of the operator, and cannot form a standardized detection method and system. Therefore, how to effectively predict immune related adverse reactions is a technical problem that needs to be solved.

[0004] SUMMARY

[0005] The present application provides an immune related adverse reaction biomarker and kit, which can effectively predict immune related adverse reactions.

[0006] In a first aspect, an immune related adverse reaction biomarker is provided, comprising: CD183.

[0007] The immune related adverse reaction is an immune related adverse reaction caused by programmed death receptor 1 antibody / programmed death ligand antibody treatment.

[0008] In a second aspect, the use of CD183 in the preparation of a kit for predicting immune related adverse reactions is provided.

[0009] In a third aspect, a kit is provided, the kit comprising: a reagent for detecting an expression level of CD183, the kit being used for predicting an immune-related adverse reaction, the immune-related adverse reaction being an immune-related adverse reaction caused by a programmed death 1 antibody / programmed death ligand antibody treatment.

[0010] In some possible implementation manners, the expression level of CD183 comprises an expression level of the CD183 in T cells.

[0011] In some possible implementation manners, the expression level of CD183 comprises an expression level of the CD183 in a CD4+ T cell subpopulation, a CD8+ T cell subpopulation, a DNT subpopulation, a gdT subpopulation, a B cell subpopulation, a NK cell subpopulation, a monocyte subpopulation, a DC subpopulation.

[0012] In some possible implementation manners, the expression level of CD183 comprises an expression level of the CD183 in a Treg subpopulation, a Th1 subpopulation, a Th1-exhausted subpopulation, a Th2 subpopulation, a Th9 subpopulation, a Th17 subpopulation, a Th17-CD27+ subpopulation, a Tfh subpopulation, a T subpopulation, a TEM-CD57+ subpopulation, a TEM-Tbet+ subpopulation, a TEM-CCR4+ subpopulation, a TE subpopulation, a T-CD161+ subpopulation, a gdT-CD57+ subpopulation, a gdT-CD57- subpopulation, a DNT subpopulation.

[0013] In some possible implementation manners, the programmed death 1 antibody comprises at least one of the following antibodies: Nivolumab, Pembrolizumab or Cemiplimab; and the programmed death ligand antibody comprises at least one of the following antibodies: Atezolizumab, Avelumab or Durvalumab.

[0014] In some possible implementation manners, the immune-related adverse reaction comprises: pneumonitis, myocarditis, colitis, pancreatitis, hypothyroidism, hyperthyroidism, thyroiditis, hypophysitis, type I diabetes, adrenal insufficiency, sarcoidosis, vitiligo, serious skin adverse reactions, thrombocytopenia, hepatitis, gastrointestinal toxicity, nervous system disorders, nephritis, uveitis or local pneumonitis.

[0015] In some possible implementation manners, the programmed death receptor 1 antibody / programmed death ligand antibody treatment is used for treating: lung adenocarcinoma, skin melanoma, prostate cancer, bladder urothelial carcinoma, mesothelioma, breast invasive carcinoma, cervical squamous carcinoma, cervical adenocarcinoma, pancreatic cancer, ovarian serous cystadenocarcinoma, head and neck squamous cell carcinoma, gastric adenocarcinoma, thyroid carcinoma, cholangiocarcinoma, adrenal cortical carcinoma, rectal adenocarcinoma, colon cancer, hepatocellular carcinoma, brain low-grade glioma, glioblastoma multiforme, uveal melanoma or uterine carcinosarcoma.

[0016] The immune-related adverse reaction marker and kit provided by the technical scheme of the embodiments of the present application can effectively predict immune-related adverse reactions, thereby facilitating intervention treatment of immune-related adverse reactions. BRIEF DESCRIPTION OF DRAWINGS

[0017] FIG. 1 is a diagram of expression of each marker in mononuclear cells and T cell subgroups in the embodiments of the present application.

[0018] FIGS. 2 and 3 are diagrams of expression of CD183 in different immune cell groups and different T cell groups in the embodiments of the present application.

[0019] FIGS. 4 and 5 are ROC of CD183 predicting irAE in the embodiments of the present application.

[0020] FIG. 6 is a result of CD183 predicting irAE in the embodiments of the present application.

[0021] FIGS. 7 to 9 are results of CD183 predicting irAE in the embodiments of the present application. DETAILED DESCRIPTION

[0022] The technical scheme in the embodiments of the present application will be described below with reference to the accompanying drawings. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application. Unless otherwise specified, the reagents involved in the embodiments of the present application are all commercially available and can be purchased through commercial channels.

[0023] Immune-related adverse effects (irAEs) refer to adverse reactions against normal self-tissues in patients receiving immunotherapy, especially immune checkpoint inhibitors (ICIs), due to the enhanced or reactivated immune system. These reactions are caused by the change of the body's tolerance to self-antigens by immunotherapy, so that the immune system not only attacks tumor cells, but also mistakenly attacks normal body tissues, leading to damage of various tissues and organs. Anti-programmed death 1 (PD-1) antibody / anti-programmed death ligand 1 (PD-L1) antibody therapy is a typical ICI, which relies on the activation of the body's immune response for cancer and tumor treatment.

[0024] Leukocyte differentiation antigen 183 (CD183), also known as CXCR3, is a CXC chemokine receptor widely distributed on the surface of various immune cells, including T helper cells (especially Th1 cells), natural killer cells (NK cells), B lymphocytes, and some monocyte / macrophage subpopulations. CXCR3 mainly interacts with its ligands CXCL9 (MIG), CXCL10 (IP-10), and CXCL11 (I-TAC), which are secreted by activated macrophages, endothelial cells, and other cell types during the inflammatory response and immune response. When CXCR3 binds to the corresponding chemokines, it can guide the above-mentioned immune cells to migrate to the inflammation site or viral infection area, participating in antiviral immunity and antitumor immunity. Under pathological conditions, CXCR3 and its chemokine system are closely related to various autoimmune diseases (such as multiple sclerosis, rheumatoid arthritis, etc.), allergic reactions, and the occurrence and development of some types of tumors, therefore, CXCR3 has become one of the important targets for immunomodulation and treatment research.

[0025] Leukocyte differentiation antigen 196 (CD196), also known as CCR6 (C-C chemokine receptor type 6), is a G protein-coupled receptor belonging to the chemokine receptor family. CCR6 is mainly expressed on Th17 cells, memory T cells, some γδ T cells, B lymphocytes, and other immune cells in T cell subsets, and it participates in regulating the migration of these immune cells to the inflammation site or specific tissues after binding to the specific chemokine CCL20 (also known as MIP-3α). CCR6 plays an important role in various immune responses and inflammatory diseases, for example, in autoimmune diseases (such as rheumatoid arthritis, multiple sclerosis), infectious diseases, and tumor metastasis processes, its expression and functional status can affect the progression of the disease.

[0026] Regulatory T cell (Treg) subsets are a subset of T cells with immunosuppressive function, which plays a key role in maintaining the body's immune homeostasis and preventing autoimmune reactions. Regulatory T cell subsets work together through different mechanisms to ensure the balance of the immune system, prevent autoimmune diseases caused by excessive immune responses, and other immune-mediated pathological states.

[0027] Exhausted Th1 (Th1-exhausted) cell subsets refer to a state in which Th1 cells (T helper cell type 1) with strong anti-viral and anti-tumor activity undergo long-term antigen stimulation and gradually lose their effector function under chronic infection, cancer or other chronic disease conditions. Th1 cells are characterized by the secretion of IFN-γ (interferon gamma), TNF-α (tumor necrosis factor alpha) and other pro-inflammatory cytokines, and are usually involved in cell-mediated immune responses against intracellular pathogens and some types of tumors. In the environment of chronic diseases such as chronic viral infection or tumor microenvironment, the persistent presence of antigens drives the continuous activation of Th1 cells, but at the same time they are subjected to strong inhibitory signals such as overexpression of inhibitory receptors such as PD-1 (programmed death receptor 1), Tim-3 (T cell immunoglobulin and mucin domain 3), CTLA-4 (cytotoxic T lymphocyte-associated protein 4), which leads to the entry of Th1 cells into an exhausted state. Exhausted Th1 cells exhibit reduced proliferation capacity, reduced cytokine production, and reduced killing function, and thus cannot effectively clear pathogens or tumor cells. The functional recovery of exhausted T cells, especially exhausted Th1 cells, has become an important research direction of immunotherapy. For example, the use of immune checkpoint inhibitors (such as PD-1 antibodies) can relieve the inhibitory signal on the surface of T cells, which helps to reactivate exhausted Th1 cells and enhance their anti-disease ability, and has been successfully applied to the treatment of various cancers in clinical practice.

[0028] Follicular Helper T Cells (Tfh cells) are a special subset of CD4+ T cells that play a critical role in humoral immune responses. Tfh cells are mainly found in the germinal centers of secondary lymphoid organs such as the spleen and lymph nodes, and play an important role in the activation, proliferation, class switching, and plasma cell formation of B cells. Tfh cells are distinguished from other CD4+ T cell subsets by the high expression of a series of specific markers, such as B-cell lymphoma-6 protein (Bcl-6), cell surface molecule CXCR5, PD-1 (Programmed Death-1), and ICOS (Inducible T-cell co-stimulator). They guide the development and antibody affinity maturation of B cells by directly contacting B cells and secreting various cytokines such as IL-21, IL-4, and IFN-γ, ultimately promoting the production of high-titer and specific antibodies to achieve long-term immune protection. The dysregulation of Tfh cells can lead to the occurrence of autoimmune diseases such as systemic lupus erythematosus (SLE) and certain types of immunodeficiency diseases; conversely, by regulating the function of Tfh cells, new strategies can be provided for vaccine design and treatment of autoimmune diseases.

[0029] Naive T cells, also known as resting T cells or initial T cells, are in a non-activated state in the body, and have not yet encountered and reacted to specific antigens. These cells are mainly located in peripheral blood and secondary lymphoid organs (such as lymph nodes and spleen), and patrol the entire body through the circulatory system. Naive T cells are a reserve force in the immune system, waiting for the right opportunity to respond to primary or secondary exposure to pathogens, thereby initiating an effective adaptive immune response.

[0030] Antigen-specific Central Memory T Cells (TCM) are a subset of memory T cells that form after experiencing primary antigen stimulation and passing through the acute immune response phase.

[0031] TPEX or precursors of exhausted T cells are a subset of T cells that appear in the context of chronic infection or cancer, and represent a T cell that is transitioning to an exhausted state. Under long-term antigen stimulation and inflammatory environments, normal T cells may first transform into TPEX during differentiation, and then further evolve into fully exhausted T cells (TEX cells), which lose their original effector function and proliferation capacity.

[0032] Effector Memory T Cells (TEM) are a subset of memory T cells that form after the initial immune response and have the ability to rapidly recognize and eliminate the same antigen. Effector Memory T Cells are memory T cells that have undergone antigen stimulation and possess the ability for immediate immune response, which is important for maintaining long-term immune protection of the body.

[0033] T Effector Cells (TE) are a subset of T cells that are capable of directly executing immune functions after activation. The activation and function of T Effector Cells embody the high specificity and flexibility of adaptive immune response, and they play a crucial role in resisting infection, eliminating tumors, and maintaining immune balance.

[0034] T cells composed of γ and δ chains refer to the Gamma delta T cells (gdT) subset, which is a type of T cell subset different from conventional αβ T cells. In the human immune system, most T cells have T cell receptors (TCR) composed of α and β chains, but γδ T cells express TCR composed of γ and δ chains. This type of T cell has unique functions and distribution in immune defense, and they are distributed in the skin, mucosal tissue, lymphoid organs, and blood, especially playing an important role in mucosal immunity and early immune response. The unique feature of γδ T cells is that they can perform both innate immune functions and adaptive immune functions, and they can be activated without the presentation of antigens by typical major histocompatibility complex (MHC) molecules. They can recognize a variety of non-peptide antigens, including phospholipids, sugars, and other small molecule metabolites, and can also sense cell stress signals, thus having unique advantages in anti-infection, anti-tumor, and immune surveillance.

[0035] Double Negative T Cells (DNT) subpopulation is a type of T cells at an early stage of development in the thymus, which does not express both CD4 and CD8 co-stimulatory molecules on the cell membrane surface. After T cells migrate from the bone marrow to the thymus, they begin to undergo a complex differentiation process. In this process, the initial stage of T cells is called double negative T cells (CD4-CD8-), and then a part of the cells continue to differentiate into double positive T cells (CD4+CD8+), and then further differentiate into mature single positive T cells (either CD4+ T helper cells or CD8+ cytotoxic T cells) through positive selection and negative selection. Double negative T cells are relatively small in number in physiological conditions, but their number may abnormally increase in certain pathological conditions, such as autoimmune diseases, infections or tumor immune responses. In these cases, the changes in double negative T cells may reflect the state of immune system disorder or disease progression.

[0036] Double Positive T Cells (DPT) subpopulation is an intermediate stage of T cell development in the thymus, characterized by the expression of both CD4 and CD8 co-stimulatory molecules on the cell membrane. In the process of T cell maturation from early progenitor cells in the thymus, double negative T cells that do not express CD4 and CD8 are first formed, and then through a series of complex differentiation steps, double negative T cells further develop into double positive T cells that express both CD4 and CD8. Double positive T cells undergo positive selection and negative selection in the thymus. Positive selection refers to the binding of double positive T cells to self-MHC molecules on thymic epithelial cells, and if their TCR (T cell receptor) can recognize and bind to MHC-peptide complexes, they will be retained to continue differentiation; negative selection is to eliminate T cells that are too strong in response to self-antigens to prevent subsequent initiation of autoimmune reactions. Ultimately, double positive T cells will further differentiate into CD4-expressing helper T cells (Th cells) or CD8-expressing cytotoxic T cells (CTL cells), which play important roles in humoral immunity and cellular immunity, respectively.

[0037] Natural Killer Cells (NK cells) are an important component of the human innate immune system, and are a type of lymphocyte that can recognize and effectively kill tumor cells, virus-infected cells, and some parasitic-infected cells without prior sensitization. NK cells are derived from bone marrow lymphoid stem cells, and their development and maturation depend on the influence of the bone marrow and possibly the thymus microenvironment. NK cells are distributed in multiple tissues, including bone marrow, peripheral blood, spleen, liver, lung, and lymph nodes. NK cells have a variety of receptors on their surface, including activating receptors (such as NKG2D, Natural Killer Cell Activating Receptors) and inhibitory receptors (such as KIR, NKR-P1C, etc.), which interact with ligands on target cells to determine whether NK cells will kill target cells. When the activating signal on the NK cell exceeds the inhibitory signal, the NK cell will kill the target cell. The killing effect of NK cells is mainly achieved through two pathways: In addition, NK cells can also assist B cell-mediated immune response through ADCC (antibody-dependent cell-mediated cytotoxicity) mechanism, using Fc receptors to bind with antibodies, thereby enhancing the killing effect on target cells marked with antibodies.

[0038] Monocytes are a type of large white blood cell in the blood, derived from hematopoietic stem cells in the bone marrow, and belong to the Mononuclear Phagocyte System (MPS). In the bone marrow, multipotent hematopoietic stem cells undergo differentiation into myeloid progenitor cells, which further develop into primitive monocytes, immature monocytes, and finally mature into peripheral blood. Monocytes account for about 3-8% of the total number of white blood cells in normal adult blood.

[0039] Dendritic Cells (DCs) are a type of extremely important immune cells, and are known as the strongest professional antigen-presenting cells (APCs) in the body. This cell is named because it will extend many dendrite-like pseudopod-like protrusions when it matures, increasing the surface area of the cell in contact with the surrounding environment, which helps to capture and uptake antigens. Dendritic cells are derived from hematopoietic stem cells and widely distributed in the interface parts that contact the outside world, such as skin, respiratory tract, gastrointestinal tract, and lymphoid tissue, such as Langerhans cells in the skin and DCs in the intestinal epithelium.

[0040] PD-1 / PD-L1 antibody treatment is a typical ICI, which relies on activating the body's immune response to treat cancer and tumor. However, PD-1 / PD-L1 antibody treatment breaks the body's immune homeostasis, and the immune-related adverse reactions caused thereby can involve the whole body organs, and even cause death in severe cases. Therefore, the discovery of immune-related adverse reaction biomarkers is of great significance.

[0041] Therefore, the present application provides an immune-related adverse reaction marker and kit, which can effectively predict immune-related adverse reactions, thereby facilitating the intervention treatment of immune-related adverse reactions.

[0042] In the embodiments of the present application, the immune-related adverse reaction marker comprises CD183.

[0043] In the embodiments of the present application, the kit for predicting immune-related adverse reactions comprises a reagent for detecting the expression level of CD183, and the kit is used for predicting immune-related adverse reactions caused by programmed death receptor 1 antibody / programmed death ligand antibody treatment.

[0044] Optionally, the expression level of CD183 comprises the expression level of CD183 in T cells.

[0045] Optionally, the expression level of CD183 comprises the expression level of CD183 in CD4+ T cell subpopulation, CD8+ T cell subpopulation, DNT subpopulation, gdT subpopulation, B cell subpopulation, NK cell subpopulation, monocyte subpopulation, DC subpopulation.

[0046] Optionally, the expression level of CD183 comprises the expression level of CD183 in Treg subpopulation, Th1 subpopulation, Th1-exhuasted subpopulation, Th2 subpopulation, Th9 subpopulation, Th17 subpopulation, Th17-CD27+ subpopulation, Tfh subpopulation, T subpopulation, TEM-CD57+ subpopulation, TEM-Tbet+ subpopulation, TEM-CCR4+ subpopulation, TE subpopulation, T-CD161+ subpopulation, gdT-CD57+ subpopulation, gdT-CD57- subpopulation, DNT subpopulation.

[0047] Optionally, the programmed death receptor 1 antibody comprises at least one of nivolumab, pembrolizumab or cemiplimab; and the programmed death ligand antibody comprises at least one of atezolizumab, avelumab or durvalumab. It should be understood that the embodiments of the present application do not limit the specific kinds of programmed death receptor 1 antibody or programmed death ligand antibody. That is, the embodiments of the present application can be applied to any possible programmed death receptor 1 antibody or programmed death ligand antibody at present or in the future.

[0048] Optionally, the immune-related adverse reaction comprises pneumonia, myocarditis, colitis, pancreatitis, hypothyroidism, hyperthyroidism, thyroiditis, hypophysitis, type I diabetes, adrenal insufficiency, sarcoidosis, vitiligo, severe skin adverse reactions, thrombocytopenia, hepatitis, gastrointestinal toxicity, nervous system disorders, nephritis, uveitis or local pneumonia. It should be understood that the embodiments of the present application do not limit the specific kinds of immune-related adverse reaction. That is, the embodiments of the present application can be applied to any immune-related adverse reaction caused by programmed death receptor 1 antibody / programmed death ligand antibody treatment.

[0049] Optionally, the programmed death receptor 1 antibody / programmed death ligand antibody treatment is used for treating lung adenocarcinoma, skin melanoma, prostate cancer, bladder urothelial carcinoma, mesothelioma, breast invasive carcinoma, cervical squamous cell carcinoma, cervical adenocarcinoma, pancreatic cancer, ovarian serous cystadenocarcinoma, head and neck squamous cell carcinoma, gastric adenocarcinoma, thyroid cancer, cholangiocarcinoma, adrenal cortex carcinoma, rectal adenocarcinoma, colon cancer, hepatocellular carcinoma, brain low-grade glioma, glioblastoma multiforme, uveal melanoma or uterine carcinosarcoma. It should be understood that the embodiments of the present application do not limit the diseases treated by programmed death receptor 1 antibody / programmed death ligand antibody. That is, the embodiments of the present application can be applied to any disease that can be treated by programmed death receptor 1 antibody / programmed death ligand antibody at present or in the future.

[0050] Embodiments

[0051] The embodiments of the present application distinguish patients who have or do not have immune-related adverse reactions after using PD-1 / PD-L1 antibody by monitoring the expression of different proteins in peripheral blood mononuclear cells (PBMC) of tumor patients through mass cytometry (CyTOF) / flow cytometry, so as to determine the biomarkers of irAE.

[0052] In mass cytometry, the cells labeled by metal-tagged antibodies are introduced into a mass cytometer, and then pass through an inductively coupled plasma (ICP) mass spectrometry detection device one by one, and then the various metal tags in each cell are quantitatively detected, and then the content of each target protein in each cell is known.

[0053] In flow cytometry, dispersed cells suspended in a liquid pass through a measuring zone one by one to generate an electrical signal (representing fluorescence, scattered light, light absorption or cell light impedance, respectively), so that a large number of cells can be quickly measured and analyzed in multiple parameters, and specific cell subgroups can be sorted from the entire population.

[0054] In the embodiments of the present application, the staining includes staining after thawing and recovering of PBMC extracted from whole blood, direct staining of PBMC extracted from whole blood, or direct staining of whole blood.

[0055] The cell staining process is as follows:

[0056] Day 1: extracellular staining

[0057] Dead-alive staining:

[0058] Prepare a final concentration of 0.25 μM 194Pt (1 mM) dead-alive staining solution with phosphate buffered saline (PBS), take 100 μL 194Pt dead-alive staining solution to resuspend the cells, and stain on ice for 5 min (100 μL for each sample).

[0059] Add 1 mL FACS Buffer (flow cytometry buffer) to each sample to resuspend the cells, centrifuge at 400g / 5min at 4°C, and discard the supernatant.

[0060] Cell blocking:

[0061] Prepare a blocking mixture (Block mix), wherein, for each sample, 4 μL of blocking solution (Blocking Solution) and 50 μL of FACS Buffer are obtained.

[0062] Add 50 μL of Block mix to each sample to resuspend the cells, and block on ice for 20 min.

[0063] Extracellular staining:

[0064] Antibody mix (Surface) was prepared, where for each sample, 1.1 μL of each antibody, FACS Buffer (55-1.1 n) μL, n is the number of antibodies, resulting in Antibody mix (Surface).

[0065] To each sample, 50 μL of Antibody mix (Surface) was added (plus the previous 50 μL of Block mix, total incubation volume was 100 μL).

[0066] The cells were gently mixed and stained for 30 min on ice.

[0067] To each sample, 1 mL of FACS Buffer was added, the cells were resuspended, and centrifuged at 400 g / 5 min at 4°C, and the supernatant was discarded.

[0068] Fixation overnight + DNA staining:

[0069] A final concentration of 250 nM Ir (500 μM) staining solution was prepared using Fix and Perm Buffer, and 200 μL of resuspended cells were taken for each sample, and incubated for 1 hour at room temperature or overnight at 4°C.

[0070] Next day: Intracellular staining + machine

[0071] Reagent preparation:

[0072] 1x Perm Buffer preparation: 10x Perm Buffer was diluted with ddH2O.

[0073] Foxp3 pre-fixation solution preparation: Fixation / Permeabilization Concentrate was diluted with Fixation / Permeabilization Diluent at a volume ratio of 3:1. 100 μL was added to each sample.

[0074] Pre-fixation:

[0075] 1. To each sample, 1 ml of 1x Perm Buffer was added to wash the cells, and centrifuged at 800 g / 5 min at 4°C, and the supernatant was discarded.

[0076] 2. Step 1 was repeated once.

[0077] 3. To each sample, 100 μL of Foxp3 pre-fixation solution was added to resuspend the cells, and incubated for 30 minutes at room temperature.

[0078] 4. Add 1 ml 1x Perm Buffer to each sample, centrifuge at 800g / 5min at 4°C, discard the supernatant.

[0079] 5. Repeat step 4 once.

[0080] 6. Prepare Antibody mix (Intracelluar) by adding 1.1 μL of each antibody and 105-1.1n μL of 1x Perm Buffer to each sample, where n is the number of antibodies, to obtain Antibody mix (Intracelluar).

[0081] 7. Add 100 μL Antibody mix (Intracelluar) to each sample to resuspend the cells, and incubate on ice for 30 min.

[0082] 8. Add 1 mL 1x FACS Buffer to each sample to wash the cells, centrifuge at 800g / 5min at 4°C, discard the supernatant.

[0083] 9. Add 1 mL ddH2O to each sample to resuspend the cells and transfer to a 5 mL flow tube with a filter screen using a pipette, and add 1 mL ddH2O to a 1.5 mL centrifuge tube to wash the tube wall and transfer to the 5 mL flow tube with a filter screen.

[0084] 10. Centrifuge at 800g / 5min at 4°C, discard the supernatant.

[0085] 11. Add 1 mL ddH2O to each sample to resuspend the cells, and take 10 μL for counting.

[0086] Example 1

[0087] To screen biomarkers of irAE, patient cohort A was collected.

[0088] Cohort A contained 11 patients, and peripheral blood mononuclear cells of the 11 patients were prepared and frozen before the patients were treated.

[0089] After the patients were treated, the patients were divided into an irAE group (6 patients with immune-related adverse reactions) and a non-irAE group (5 patients without immune-related adverse reactions) according to whether the patients had immune-related adverse reactions.

[0090] Cell recovery

[0091] The frozen cells were recovered, and the process was as follows:

[0092] A water bath was preheated to 37°C;

[0093] Preheat the complete culture medium to 37°C in a water bath. Add 100 μL of DNase to 50 mL of complete culture medium. The working concentration of DNase is 25 μg / mL. Invert the culture medium to mix well and set aside.

[0094] Prepare 15mL centrifuge tubes, label them, and add 5mL of preheated complete culture medium;

[0095] Based on the pre-located cell cryopreservation location, remove the cell cryopreservation tubes from the liquid nitrogen tank. This process should be completed within 2 minutes. Immediately after removal, place the cryopreservation tubes in a 37°C water bath to thaw for 2-3 minutes, shaking them continuously. Do not immerse the entire tubes in water. Remove the tubes after they are completely thawed.

[0096] First, add 1 mL of complete culture medium to 1 mL of thawed cell suspension, and then transfer it to a 15 mL centrifuge tube containing 5 mL of complete culture medium.

[0097] Rinse the cryovial with 1 mL of complete culture medium and add the rinsing solution to a 15 mL centrifuge tube.

[0098] Centrifuge at 800 rpm for 8 minutes at room temperature;

[0099] Discard the supernatant and resuspend the cells in 2 mL of complete culture medium;

[0100] Centrifuge at 300g for 5 minutes at 4℃, and carefully discard the supernatant.

[0101] Resuspend the cells in 1 mL of FACS Buffer;

[0102] Blood cell resuscitation was performed using an automated cell counter to determine cell concentration and viability. The results are shown in Table 1.

[0103] Table 1

[0104] Using the aforementioned staining procedure, after cell resuscitation, the cells were incubated with an antibody mixture to allow the antibody, which has a stable heavy metal isotope conjugated to the polymer chain through chelating groups, to fully bind with the cell surface antigen, forming an antigen-antibody complex, i.e., a peripheral blood mononuclear cell suspension labeled with metal antibodies. The incubation antibody and the metal isotope it chelates are shown in Table 2.

[0105] Table 2

[0106] Subsequently, the peripheral blood mononuclear cell suspension labeled with metal antibodies was detected using a PLT-MC601 mass spectrometry flow cytometry system to enrich heavy metal reporter ions, which were then separated according to their mass-to-charge ratio in a time-of-flight mass spectrometer. The ion counts were then converted into electrical signals to form data.

[0107] The data analysis was performed on two groups of data, i.e., irAE group (with immune-related adverse reactions) and non-irAE group (without immune-related adverse reactions), and the cluster analysis was performed on monocytes and T cell subgroups. The different expression of each marker in the two groups was analyzed and is shown in FIG. 1. It can be found from the figure that the expression of CD183 in T cells in the irAE group is significantly reduced.

[0108] Further, for CD183, the expression of CD183 in different immune cell groups and different T cell groups was analyzed, and FIG. 2-FIG. 3 were obtained.

[0109] It can be found from FIG. 2 that the expression of CD183 in CD4+ T cell subgroup, CD8+ T cell subgroup, DNT subgroup, gdT subgroup, B cell subgroup, NK cell subgroup, monocytes subgroup, and DC subgroup in the irAE group is significantly reduced.

[0110] It can be found from FIG. 3 that the expression of CD183 in Treg subgroup, Th1 subgroup, Th1-exhausted subgroup, Th2 subgroup, Th9 subgroup, Th17 subgroup, Th17-CD27+ subgroup, Tfh subgroup, T subgroup, TEM-CD57+ subgroup, TEM-Tbet+ subgroup, TEM-CCR4+ subgroup, TE subgroup, T-CD161+ subgroup, gdT-CD57+ subgroup, gdT-CD57- subgroup, and DNT subgroup in the irAE group is significantly reduced.

[0111] Therefore, CD183 can be used as a biomarker for predicting immune-related adverse reactions.

[0112] Further, the expression levels of CD183 on the surface of CD4+ T cells, CD8+ T cells, DC cells, NK cells, DNT cells, Th2 cells, gDT cells, monocytes cells, T cells, TCM cells, TE cells, TEM-CCR4+ cells, TEM-CD57+ cells, TEM-Tbet+ cells, Tfh cells, Th1 cells, Th17 cells, Th17-CD27+ cells, Th1-exhausted cells, and Th9 cells were used to predict the receiver operating characteristic curve (ROC) of irAE, as shown in FIG. 4-FIG. 5, and the corresponding prediction is shown in Table 3. It can be seen from the table that CD183 has better prediction ability for irAE.

[0113] Table 3

[0114] Example 2

[0115] To verify the prediction ability of CD183, a patient cohort B was collected:

[0116] Cohort B contained 16 patients, of which 4 patients had irAE after receiving PD-1 / PD-L1 antibody treatment, and 12 patients had no irAE after receiving PD-1 / PD-L1 antibody treatment.

[0117] For cohort B, the expression levels of CD183 on the surfaces of CD8+ T cells and gdT cells were detected respectively by using the aforementioned staining and detection methods, and the irAE prediction verification was performed, and the results are shown in FIG. 6, and the corresponding prediction is shown in Table 4, from which it can be seen that the results verify the prediction ability of CD183 for irAE.

[0118] Table 4

[0119] Example 3

[0120] To verify the prediction ability of CD183, a patient cohort C was collected:

[0121] Cohort C contained 2 patients, of which 1 patient had irAE after receiving PD-1 / PD-L1 antibody treatment, and 1 patient had no irAE after receiving PD-1 / PD-L1 antibody treatment.

[0122] For cohort C, the expression levels of CD183 on the surfaces of CD4+ T cells, CD8+ T cells, DC cells, DNT cells, DPT cells, gdT cells, monocytes cells and NK cells were detected by using the aforementioned staining and detection methods, and the results are shown in FIGS. 7-9. From which it can be seen that the expression levels of CD183 on the surfaces of CD4+ T cells, CD8+ T cells, DC cells, gdT cells, monocytes cells and NK cells can predict irAE. The results also verify the prediction ability of CD183 for irAE.

[0123] From the above data, it can be seen that CD183 has good prediction ability for irAE, and can be used as a biomarker for predicting immune-related adverse reactions.

[0124] In addition, from the sample acquisition point of view, the present application can only use blood samples, and by staining CD45, CD3 and CD183, the immune-related adverse reactions caused by PD-1 / PD-L1 antibody treatment can be judged, so that early prediction and active intervention can be performed, and the prognosis of the patient can be improved, and the dependence on tumor tissue detection of the patient in the past can be eliminated.

[0125] Moreover, from the experimental cycle angle, the application can obtain the detection result within 3 hours of collecting the blood sample, greatly improves the detection efficiency, and can quickly and accurately provide the doctor with diagnosis and treatment auxiliary information in the first time.

[0126] The above merely provides a specific implementation of the application, but the protection scope of the application is not limited thereto, and any person skilled in the art can easily think of various equivalent modifications or replacements within the technical range disclosed by the application, and these modifications or replacements shall be encompassed within the protection scope of the application. Therefore, the protection scope of the application shall be subject to the protection scope of the claims.

Claims

1. Use of CD183 in the manufacture of a kit for predicting immune-related adverse reactions caused by a programmed death receptor 1 antibody / programmed death ligand antibody treatment.

2. A kit comprising: a reagent for detecting the expression level of CD183, the kit being used for predicting immune-related adverse reactions caused by a programmed death receptor 1 antibody / programmed death ligand antibody treatment.

3. The kit of claim 2, the expression level of CD183 comprising the expression level of CD183 in T cells.

4. The kit of claim 2, the expression level of CD183 comprising the expression level of CD183 in CD4+ T cell subpopulation, CD8+ T cell subpopulation, DNT subpopulation, gdT subpopulation, B cell subpopulation, NK cell subpopulation, monocytes subpopulation, DC subpopulation.

5. The kit of claim 2, the expression level of CD183 comprises an expression level in a Treg subset, a Thl subset, a Thl-exhuasted subset, a Th2 subset, a Th9 subset, a Thl7 subset, a Thl7-CD27+ subset, a Tfh subset, a T subset, a TEM-CD57+ subset, a TEM-Tbet+ subset, a TEM-CCR4+ subset, a TE subset, a T-CD161+ subset, a gdT-CD57+ subset, a gdT-CD57- subset, a DNT subset.

6. The kit of any one of claims 2 to 5, the programmed death receptor 1 antibody comprising at least one of the following antibodies: nivolumab, pembrolizumab or cemiplimab; the programmed death ligand antibody comprising at least one of the following antibodies: atezolizumab, avelumab or durvalumab.

7. The kit of any one of claims 2 to 5, the immune-related adverse reactions comprising: pneumonitis, myocarditis, colitis, pancreatitis, hypothyroidism, hyperthyroidism, thyroiditis, hypophysitis, type I diabetes mellitus, adrenal insufficiency, sarcoidosis, vitiligo, severe skin adverse reactions, thrombocytopenia, hepatitis, gastrointestinal toxicity, nervous system disorders, nephritis, uveitis or pneumonitis.

8. The kit of any one of claims 2 to 5, the programmed death receptor 1 antibody / programmed death ligand antibody treatment being used for treating: lung adenocarcinoma, cutaneous melanoma, prostate cancer, urothelial carcinoma of the bladder, mesothelioma, breast invasive carcinoma, cervical squamous carcinoma, cervical adenocarcinoma, pancreatic carcinoma, ovarian serous cystadenocarcinoma, head and neck squamous cell carcinoma, gastric adenocarcinoma, thyroid carcinoma, cholangiocarcinoma, adrenocortical carcinoma, rectal adenocarcinoma, colon carcinoma, hepatocellular carcinoma, brain low grade glioma, glioblastoma multiforme, uveal melanoma or uterine carcinosarcoma.

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