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

Biomarkers and kits for immune-related adverse reactions Technical Field

[0001] This application relates to the field of biomedical technology, and more specifically, to an immune-related adverse reaction biomarker and kit. Background Technology

[0002] Immune checkpoint inhibitors (ICIs) primarily target antibodies against cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1). ICIs enhance the immune killing of tumor cells and improve the efficacy of immunotherapy by blocking the activation of T cells by CTLA-4, PD-1, and their ligands. However, because ICIs rely on activating the body's own immune system to attack tumor cells, they disrupt the body's immune homeostasis, leading to the attack of normal cells and resulting in immune-related adverse events (irAEs).

[0003] Immune-related adverse events (irAEs) can affect organs throughout the body and can lead to death in severe cases. Current biomarkers for immune-related adverse events rely on immunohistochemical detection of tumor tissue sections, which is highly sample-dependent, invasive, and unsuitable for patients without access to tumor samples. Furthermore, immunohistochemical testing is time-consuming and susceptible to operator errors, hindering the development of standardized methods and systems. Therefore, effectively predicting immune-related adverse events is a pressing technical challenge.

[0004] Summary of the Invention

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

[0006] Firstly, an immune-related adverse reaction biomarker is provided, including CD183.

[0007] The immune-related adverse reactions mentioned are those caused by programmed death receptor 1 antibody / programmed death ligand antibody therapy.

[0008] Secondly, the application of CD183 in the preparation of kits for predicting immune-related adverse reactions is provided.

[0009] Thirdly, a kit is provided comprising: reagents for detecting the expression level of CD183, the kit being used to predict immune-related adverse reactions, the immune-related adverse reactions being immune-related adverse reactions induced by programmed death receptor 1 antibody / programmed death ligand antibody therapy.

[0010] In some possible implementations, the expression level of CD183 includes the expression level of CD183 in T cells.

[0011] In some possible implementations, the expression level of CD183 includes the expression level of CD183 in CD4+ T cell subsets, CD8+ T cell subsets, DNT subsets, gdT subsets, B cell subsets, NK cell subsets, monocytes subsets, and DC subsets.

[0012] In some possible implementations, the expression level of CD183 includes CD183 in the Treg subgroup, Th1 subgroup, Th1-exhuasted subgroup, Th2 subgroup, Th9 subgroup, Th17 subgroup, Th17-CD27+ subgroup, Tfh subgroup, etc. Expression levels in the 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.

[0013] In some possible implementations, the programmed death receptor 1 antibody includes at least one of the following antibodies: nivolumab, pembrolizumab, or cimetidine; and the programmed death ligand antibody includes at least one of the following antibodies: atezolizumab, avelumab, or duvalbumin.

[0014] In some possible implementations, the immune-related adverse reactions include: pneumonia, myocarditis, colitis, pancreatitis, hypothyroidism, hyperthyroidism, thyroiditis, hypopituitarism, type I diabetes, adrenal insufficiency, sarcoidosis, vitiligo, severe skin adverse reactions, thrombocytopenia, hepatitis, gastrointestinal toxicity, nervous system disorders, nephritis, uveitis, or localized pneumonia.

[0015] In some possible implementations, the programmed death receptor 1 antibody / programmed death ligand antibody therapy is used to treat: lung adenocarcinoma, skin melanoma, prostate cancer, bladder urothelial carcinoma, mesothelioma, invasive breast cancer, cervical squamous cell carcinoma, cervical adenocarcinoma, pancreatic cancer, ovarian serous cystadenocarcinoma, head and neck squamous cell carcinoma, gastric adenocarcinoma, thyroid cancer, bile duct carcinoma, adrenocortical carcinoma, rectal adenocarcinoma, colon cancer, hepatocellular carcinoma, low-grade glioma of the brain, glioblastoma multiforme, uveal melanoma, or uterine carcinosarcoma.

[0016] The immunologically related adverse reaction biomarkers and kits provided in the embodiments of this application can effectively predict immunologically related adverse reactions, thereby facilitating intervention and treatment of immunologically related adverse reactions. Attached Figure Description

[0017] Figure 1 is an expression diagram of each marker in monocytes and T cell subsets in Example 1 of this application.

[0018] Figures 2 and 3 are expression diagrams of CD183 in different immune cell groups and different T cell groups in Example 1 of this application.

[0019] Figures 4 and 5 show the ROC of CD183 predicting irAE in Embodiment 1 of this application.

[0020] Figure 6 shows the CD183 prediction result of irAE in Embodiment 2 of this application.

[0021] Figures 7 to 9 show the results of CD183 predicting irAE in Embodiment 3 of this application. Detailed Implementation

[0022] The technical solutions in the embodiments of this application will now be described with reference to the accompanying drawings. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. Unless otherwise specified, the reagents involved in the embodiments of this invention are all commercially available products and can be purchased through commercial channels.

[0023] Immune-related adverse events (irAEs) refer to adverse reactions against normal body tissues caused by the enhanced or reactivated immune system in patients receiving immunotherapy, especially immune checkpoint inhibitors (ICIs). These reactions occur because immunotherapy alters the body's tolerance to its own antigens, causing the immune system to attack not only tumor cells but also potentially normal body tissues, leading to various tissue and organ damage. Anti-programmed death 1 (PD-1) antibody / anti-programmed death ligand 1 (PD-L1) antibody therapy is a typical ICI that relies on activating the body's immune response to treat cancer and tumors.

[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 subsets. CXCR3 primarily 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 inflammatory responses and immune responses. When CXCR3 binds to its corresponding chemokines, it can guide these immune cells to migrate to sites of inflammation or viral infections, participating in antiviral and antitumor immunity. Under pathological conditions, CXCR3 and its chemokine system are closely related to the occurrence and development of various autoimmune diseases (such as multiple sclerosis and rheumatoid arthritis), allergic reactions, and certain types of tumors. Therefore, CXCR3 has become one of the important targets for immunomodulation and therapeutic research.

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

[0026] Regulatory T cell (Treg) subsets are a group of T cells with immunosuppressive functions, playing a crucial role in maintaining immune homeostasis and preventing autoimmune responses. These regulatory T cell subsets work together through various mechanisms to ensure the balance of the immune system, preventing autoimmune diseases and other immune-mediated pathological states caused by excessive immune responses.

[0027] Th1-exhuasted cell subsets refer to a state in which Th1 cells (T helper cell type 1), which originally possess potent antiviral and antitumor activity, gradually weaken or even lose their effector function after prolonged antigen stimulation under conditions of chronic infection, cancer, or other chronic diseases. Th1 cells are characterized by the secretion of pro-inflammatory cytokines such as IFN-γ (interferon-γ) and TNF-α (tumor necrosis factor-α), and typically participate in cell-mediated immune responses to combat intracellular pathogens and certain types of tumors. In chronic disease environments, such as chronic viral infections or the tumor microenvironment, persistent antigens drive continuous activation of Th1 cells, but they also suffer from strong inhibitory signals, such as overexpression of inhibitory receptors like PD-1 (programmed death receptor 1), Tim-3 (T cell immunoglobulin and mucin domain 3), and CTLA-4 (cytotoxic T lymphocyte-associated protein 4), leading to Th1 cell exhaustion. Exhausted Th1 cells exhibit reduced proliferation, decreased cytokine production, and diminished killing function, thus failing to effectively eliminate pathogens or tumor cells. Restoring the function of exhausted T cells, especially exhausted Th1 cells, has become an important research direction in immunotherapy. For example, the use of immune checkpoint inhibitors (such as PD-1 antibodies) can relieve inhibitory signals on the surface of T cells, which helps to reactivate exhausted Th1 cells and enhance their disease-fighting ability. This has been successfully applied in the clinical treatment of various cancers.

[0028] Follicular helper T cells (Tfh cells) are a subset of CD4+ T cells that play a crucial auxiliary role in humoral immune responses. Tfh cells are primarily found in secondary lymphoid organs such as the spleen and germinal centers of lymph nodes, playing a vital role in B cell activation, proliferation, class switching, and plasma cell formation. Tfh cells distinguish themselves from other CD4+ T cell subsets by highly expressing a series of specific markers, such as B-cell lymphoma-6 protein (Bcl-6), cell surface molecules CXCR5, PD-1 (Programmed Death-1), and ICOS (Inducible T-cell costimulator). Through direct contact with B cells, they secrete various cytokines such as IL-21, IL-4, and IFN-γ, guiding B cell development and antibody affinity maturation, ultimately leading to the production of high-titer and specific antibodies, achieving long-term immune protection. Dysregulation of Tfh cells may lead to the development of autoimmune diseases, such as systemic lupus erythematosus (SLE) and certain types of immunodeficiency diseases; conversely, regulating Tfh cell function can provide new strategies for vaccine design and the treatment of autoimmune diseases.

[0029] Unactivated T cells, also known as resting T cells or naive T cells, are a subset of cells that remain inactive in the body and have not yet encountered and responded to specific antigens. These cells are primarily located in the peripheral blood and secondary lymphoid organs (such as lymph nodes and the spleen) and patrol throughout the body via the circulatory system. Unactivated T cells are a reserve force in the immune system, waiting for the right opportunity to respond to initial or re-exposure to pathogens, thereby initiating an effective adaptive immune response.

[0030] Antigen-specific central memory T cells (TCMs) are a subset of memory T cells that form after the initial antigen stimulation and the acute immune response phase.

[0031] Exhausted precursor T cells (TPEX or precursors of exhausted T cells) are a subset of T cells that emerge in the context of chronic infection or cancer. They represent T cells that are transitioning to an exhausted state. Under prolonged antigen stimulation and inflammatory conditions, normally functioning T cells may first transform into exhausted precursor T cells during differentiation, and then further evolve into completely exhausted T cells (TEX cells). These cells lose their original effector functions and proliferative capacity.

[0032] Effector memory T cells (TEM) are a subset of memory T cells that form after the primary immune response and possess the ability to rapidly recognize and clear the same antigens. Effector memory T cells are memory T cells that have experienced antigen stimulation and possess immediate immune response capabilities, playing a crucial role in maintaining long-term immune protection.

[0033] T-cell effector cells (TEs) are a subset of T cells that, once activated, can directly perform immune functions. The activation and function of T-cell effectors demonstrate the high specificity and flexibility of adaptive immune responses, and they play a crucial role in fighting infection, eliminating tumors, and maintaining immune homeostasis.

[0034] T cells composed of g / d chains refer to the γδT cell (gdT) subset, a type of T cell distinct from conventional αβT cells. In the human immune system, most T cells express T cell receptors (TCRs) composed of α and β chains, but γδT cells express TCRs composed of both γ and δ chains. This type of T cell has unique functions and distribution in immune defense; they are found in the skin, mucous membranes, lymphatic organs, and blood, playing a particularly important role in mucosal immunity and early immune responses. The unique feature of γδT cells is that they can perform both innate and adaptive immune functions, and they can be activated without the typical major histocompatibility complex (MHC) antigen presentation. They can recognize a variety of non-peptide antigens, including phospholipids, carbohydrates, and other small molecule metabolites, and can also sense cellular stress signals, thus possessing unique advantages in anti-infection, anti-tumor, and immune surveillance.

[0035] Double-negative T cells (DNTs) are a subset of T cells that develop early in the thymus, and their membranes do not express both CD4 and CD8 co-stimulatory molecules. After migrating from the bone marrow to the thymus, T cells undergo a complex differentiation process. In this process, the initial stage of T cells is called double-negative T cells (CD4-CD8-), and a portion of these cells continue to differentiate into double-positive T cells (CD4+CD8+), which then further differentiate into mature single-positive T cells (either CD4+ helper cells or CD8+ cytotoxic T cells) through positive and negative selection. Double-negative T cells are relatively few in number under physiological conditions, but their numbers may be abnormally high under certain pathological conditions, such as autoimmune diseases, infections, or tumor immune responses. In these cases, changes in double-negative T cells may reflect immune system dysregulation or disease progression.

[0036] Double-positive T cells (DPT) represent an intermediate stage in T cell development within the thymus, characterized by the simultaneous expression of both CD4 and CD8 co-stimulatory molecules on their cell membranes. During the maturation of T cells from early progenitor cells in the thymus, double-negative T cells, which do not express CD4 or CD8, are initially formed. Through a series of complex differentiation steps, these double-negative T cells further develop into double-positive T cells that simultaneously express both CD4 and CD8. Double-positive T cells undergo positive and negative selection processes within the thymus. Positive selection refers to the binding of double-positive T cells to their own MHC molecules on thymic epithelial cells; if their TCRs (T cell receptors) can recognize and bind to the MHC-peptide complex, they are retained and continue differentiating. Negative selection, on the other hand, aims to eliminate T cells that react excessively to their own antigens to prevent subsequent autoimmune responses. Ultimately, double-positive T cells will further differentiate into helper T cells (Th cells) that express CD4 or cytotoxic T cells (CTL cells) that express CD8. These two types of cells play important roles in humoral immunity and cellular immunity, respectively.

[0037] Natural killer (NK) cells are a subset of cells that form an important part of the human innate immune system. They are lymphocytes that can recognize and effectively kill tumor cells, virus-infected cells, and certain parasite-infected cells without prior sensitization. NK cells originate from bone marrow lymphoid stem cells, and their development and maturation depend on the influence of the bone marrow and possibly the thymic microenvironment. NK cells are distributed in multiple tissues, including bone marrow, peripheral blood, spleen, liver, lungs, and lymph nodes. NK cells have various receptors on their surface, including activating receptors (such as NKG2D and natural killer cell activation receptor) and inhibitory receptors (such as KIR and NKR-P1C). These receptors interact with ligands on target cells, determining whether NK cells will kill the target cells. When the activation signal on NK cells exceeds the inhibitory signal, NK cells will kill the target cells. NK cells exert their cytotoxic effect primarily through two pathways: In addition, NK cells can also assist B cell-mediated immune responses through the ADCC (antibody-dependent cell-mediated cytotoxicity) mechanism, utilizing Fc receptors to bind to antibodies, thereby enhancing the killing effect on antibody-labeled target cells.

[0038] Monocytes are a subset of large white blood cells in the blood, originating from hematopoietic stem cells in the bone marrow and belonging to the Mononuclear Phagocyte System (MPS). In the bone marrow, pluripotent hematopoietic stem cells differentiate into myeloid progenitor cells, which further develop into primitive monocytes, immature monocytes, and finally mature and enter the peripheral blood. Monocytes account for approximately 3%-8% of the total white blood cell count in the blood of a normal adult.

[0039] Dendritic cells (DCs) are a crucial subset of immune cells, often considered the most potent professional antigen-presenting cells (APCs) in the body. They are named for the numerous dendrite-like pseudopodia-like projections they develop upon maturation, increasing their surface area in contact with the surrounding environment and facilitating the capture and uptake of antigens. Dendritic cells originate from hematopoietic stem cells and are widely distributed at interfaces with the external environment, such as in the skin, respiratory tract, gastrointestinal tract, and lymphatic tissues. Examples include Langerhans cells in the skin and DCs in the intestinal epithelium.

[0040] PD-1 / PD-L1 antibody therapy is a typical ICI (immune-mediated immunotherapy) that relies on activating the body's immune response to treat cancer and tumors. However, PD-1 / PD-L1 antibody therapy disrupts the body's immune homeostasis, and the resulting immune-related adverse reactions can affect organs throughout the body, potentially leading to death in severe cases. Therefore, the discovery of biomarkers for immune-related adverse reactions is of great significance.

[0041] In view of this, this application provides an immune-related adverse reaction biomarker and kit, which can effectively predict immune-related adverse reactions, thereby facilitating the intervention and treatment of immune-related adverse reactions.

[0042] In this embodiment of the application, the biomarker for immune-related adverse reactions includes CD183.

[0043] In this application embodiment, the kit for predicting immune-related adverse reactions includes: a reagent for detecting the expression level of CD183. The kit is used to predict immune-related adverse reactions, which are immune-related adverse reactions caused by programmed death receptor 1 antibody / programmed death ligand antibody therapy.

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

[0045] Optionally, the expression level of CD183 includes the expression level of CD183 in CD4+ T cell subsets, CD8+ T cell subsets, DNT subsets, gdT subsets, B cell subsets, NK cell subsets, monocytes subsets, and DC subsets.

[0046] Optionally, the expression level of CD183 includes the expression levels of CD183 in the Treg subgroup, Th1 subgroup, Th1-exhuasted subgroup, Th2 subgroup, Th9 subgroup, Th17 subgroup, Th17-CD27+ subgroup, and Tfh subgroup. Expression levels in the 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.

[0047] Optionally, the programmed death receptor 1 antibody includes at least one of the following antibodies: nivolumab, pembrolizumab, or cimetidine; the programmed death ligand antibody includes at least one of the following antibodies: atezolizumab, avelumab, or duvalumab. It should be understood that the specific types of programmed death receptor 1 antibodies or programmed death ligand antibodies are not limited in the embodiments of this application. That is, any currently available or future programmed death receptor 1 antibodies or programmed death ligand antibodies may be applicable.

[0048] Optionally, the immune-related adverse reactions include: pneumonia, myocarditis, colitis, pancreatitis, hypothyroidism, hyperthyroidism, thyroiditis, hypopituitarism, type I diabetes, adrenal insufficiency, sarcoidosis, vitiligo, severe skin adverse reactions, thrombocytopenia, hepatitis, gastrointestinal toxicity, neurological disorders, nephritis, uveitis, or localized pneumonia. It should be understood that the embodiments of this application do not limit the specific types of immune-related adverse reactions. That is, immune-related adverse reactions caused by programmed death receptor 1 antibody / programmed death ligand antibody treatment are all applicable.

[0049] Optionally, this programmed death receptor 1 antibody / programmed death ligand antibody therapy is used to treat: lung adenocarcinoma, skin melanoma, prostate cancer, bladder urothelial carcinoma, mesothelioma, invasive breast cancer, cervical squamous cell carcinoma, cervical adenocarcinoma, pancreatic cancer, ovarian serous cystadenocarcinoma, head and neck squamous cell carcinoma, gastric adenocarcinoma, thyroid cancer, bile duct carcinoma, adrenocortical carcinoma, rectal adenocarcinoma, colon cancer, hepatocellular carcinoma, low-grade glioma of the brain, glioblastoma multiforme, uveal melanoma, or uterine carcinosarcoma. It should be understood that the diseases treated with programmed death receptor 1 antibody / programmed death ligand antibody in this application are not limited. That is, any disease that can be treated with programmed death receptor 1 antibody / programmed death ligand antibody currently or in the future is applicable.

[0050] Example

[0051] This application embodiment uses mass cytometry (CyTOF) / flow cytometry to monitor the expression of different proteins in peripheral blood mononuclear cells (PBMCs) of cancer patients to distinguish whether or not patients experience immune-related adverse reactions (irAEs) after using PD-1 / PD-L1 antibodies, in order to determine biomarkers for irAEs.

[0052] In mass cytometry, cells labeled with metal-tagged antibodies enter a mass cytometer and are then individually passed through an inductively coupled plasma (ICP) mass spectrometer. The various metal tags in each cell are then quantitatively detected, allowing the determination of the content of each target protein in each cell.

[0053] In flow cytometry, dispersed cells suspended in a liquid generate electrical signals (representing fluorescence, scattered light, light absorption, or cell photoresistance) as they pass through the measurement zone one by one. This allows for rapid multi-parameter measurement and analysis of a large number of cells and the sorting of specific cell subpopulations from the entire population.

[0054] In the embodiments of this application, staining includes staining after whole blood PBMCs are extracted, cryopreserved, and thawed, staining PBMCs directly extracted from whole blood, or staining whole blood directly.

[0055] The cell staining procedure is as follows:

[0056] Day 1: Extracellular staining

[0057] Live and dead staining:

[0058] Prepare a 0.25 μM (1 mM) 194Pt (dead and live) staining solution using phosphate-buffered saline (PBS). Resuspend the cells in 100 μL of the 194Pt (dead and live) staining solution and stain on ice for 5 min (100 μL per sample).

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

[0060] Cell blocking:

[0061] Prepare a block mix by using 4 μL of blocking solution and 50 μL of FACS buffer for each sample.

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

[0063] Extracellular staining:

[0064] Prepare the antibody mix (Surface) by mixing 1.1 μL of each antibody and (55-1.1n) μL of FACS Buffer for each sample, where n is the number of antibodies.

[0065] Add 50 μL of Antibody mix (Surface) to each sample (together with the 50 μL of Block mix mentioned earlier, the total incubation volume is 100 μL).

[0066] Gently pipette to mix the cells and stain on ice for 30 minutes.

[0067] Add 1 mL of FACS Buffer to each sample, resuspend the cells, centrifuge at 400 g for 5 min at 4°C, and discard the supernatant.

[0068] overnight fixation + DNA staining:

[0069] Prepare a staining solution with a final concentration of 250 nM Ir (500 μM) using fixation and permeation buffer. Take 200 μL of each sample to resuspend the cells and incubate at room temperature for 1 hour or at 4°C overnight.

[0070] Day 2: Intracellular staining + machine operation

[0071] Preparation of freshly prepared reagents:

[0072] Preparation of 1x Perm Buffer: Dilute 10x Perm Buffer with ddH2O.

[0073] Foxp3 prefixation solution preparation: Dilute Fixation / Permeabilization Concentrate (concentrate) with Fixation / Permeabilization Diluent at a volume ratio of 3:1. Add 100 μL to each sample.

[0074] Pre-fixation:

[0075] 1. Add 1 ml of 1x Perm Buffer to each sample to wash the cells, centrifuge at 4°C for 800 g / 5 min, and discard the supernatant.

[0076] 2. Repeat step 1 once.

[0077] 3. Add 100 μL of Foxp3 prefixation solution to each sample, resuspend the cells, and incubate at room temperature for 30 minutes.

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

[0079] 5. Repeat step 4 once.

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

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

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

[0083] 9. Add 1 mL of ddH2O to each sample to resuspend the cells and transfer them to a 5 mL flow cytometer tube with a filter using a pipette. Then add 1 mL of ddH2O to a 1.5 mL centrifuge tube to wash the tube wall and transfer all the solution to a 5 mL flow cytometer tube with a filter.

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

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

[0086] Example 1

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

[0088] Cohort A contained 11 patients whose peripheral blood mononuclear cells were prepared and cryopreserved prior to treatment.

[0089] After treatment, patients were divided into an irAE group (with immune-related adverse reactions, 6 patients) and a non-irAE group (without immune-related adverse reactions, 5 patients) based on whether they had immune-related adverse reactions.

[0090] Cell resuscitation

[0091] The procedure for thawing frozen cells is as follows:

[0092] Preheat the water bath 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] Data analysis was performed on two groups: the irAE group (with immune-related adverse reactions) and the non-irAE group (without immune-related adverse reactions). Cluster analysis was conducted on monocytes and their T cell subsets to reveal the different expression patterns of each marker in the two groups, as shown in Figure 1. It was found that CD183 expression was significantly reduced in T cells of the irAE group.

[0108] Furthermore, the expression of CD183 in different immune cell populations and different T cell populations was analyzed, as shown in Figures 2 and 3.

[0109] As shown in Figure 2, the expression of CD183 was significantly decreased in the CD4+ T cell subset, CD8+ T cell subset, DNT subset, gdT subset, B cell subset, NK cell subset, monocytes subset, and DC subset in the irAE group.

[0110] Figure 3 shows that CD183 is present in the irAE group Treg subgroup, Th1 subgroup, Th1-exhuasted subgroup, Th2 subgroup, Th9 subgroup, Th17 subgroup, Th17-CD27+ subgroup, and Tfh subgroup. Expression was significantly decreased in the 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.

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

[0112] Furthermore, CD4+ T cells, CD8+ T cells, DC cells, NK cells, DNT cells, Th2 cells, gDT cells, monocytes, The expression levels of CD183 on the surface of 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 irAEs. The receiver operating characteristic (ROC) curves are shown in Figures 4 and 5, and the corresponding prediction results are shown in Table 3. It can be seen that CD183 has a good predictive ability for irAEs.

[0113] Table 3

[0114] Example 2

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

[0116] Cohort B included 16 patients, of whom 4 experienced irAEs after receiving PD-1 / PD-L1 antibody therapy and 12 had no irAEs after receiving PD-1 / PD-L1 antibody therapy.

[0117] For cohort B, the expression levels of CD183 on the surface of CD8+ T cells and gDT cells were detected using the aforementioned staining and detection methods, and irAE prediction was verified. The results are shown in Figure 6, and the corresponding prediction results are shown in Table 4. It can be seen from the results that the results verify the predictive ability of CD183 for irAE.

[0118] Table 4

[0119] Example 3

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

[0121] Cohort C included 2 patients, one of whom experienced an irAE after receiving PD-1 / PD-L1 antibody therapy, and the other of whom did not experience an irAE after receiving PD-1 / PD-L1 antibody therapy.

[0122] For cohort C, the expression levels of CD183 on the surface of CD4+ T cells, CD8+ T cells, DC cells, DNT cells, DPT cells, gdT cells, monocytes, and NK cells were detected using the aforementioned staining and detection methods. The results are shown in Figures 7-9. These results demonstrate that the expression levels of CD183 on the surface of CD4+ T cells, CD8+ T cells, DC cells, gdT cells, monocytes, and NK cells can predict irAE. This result also validates the predictive ability of CD183 for irAE.

[0123] The data above show that CD183 has a good predictive ability for irAE and can be used as a biomarker for predicting immune-related adverse reactions.

[0124] In addition, from the perspective of sample acquisition, this application can use only blood samples to determine immune-related adverse reactions caused by PD-1 / PD-L1 antibody therapy by staining CD45, CD3, and CD183, thereby enabling early prediction and active intervention, improving the patient's prognosis, and eliminating the previous reliance on the detection of the patient's tumor tissue.

[0125] Moreover, from the perspective of experimental cycle, this application can obtain test results within 3 hours of collecting blood samples, which greatly improves the testing efficiency and can quickly and accurately provide doctors with diagnostic and treatment assistance information in the first time.

[0126] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. The use of CD183 in the preparation of a kit for predicting immune-related adverse reactions, wherein the immune-related adverse reactions are immune-related adverse reactions caused by programmed death receptor 1 antibody / programmed death ligand antibody therapy.

2. A kit comprising: A reagent for detecting CD183 expression levels, the kit being used to predict immune-related adverse reactions, which are immune-related adverse reactions induced by programmed death receptor 1 antibody / programmed death ligand antibody therapy.

3. The kit according to claim 2, wherein the expression level of CD183 includes the expression level of CD183 in T cells.

4. The kit according to claim 2, wherein the expression level of CD183 includes the expression level of CD183 in CD4+ T cell subsets, CD8+ T cell subsets, DNT subsets, gdT subsets, B cell subsets, NK cell subsets, monocytes subsets, and DC subsets.

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 according to any one of claims 2 to 5, wherein the programmed death receptor 1 antibody comprises at least one of the following antibodies: nivolumab, pembrolizumab, or cimetidine; and the programmed death ligand antibody comprises at least one of the following antibodies: atezolizumab, avelumab, or duvalbumin.

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

8. The kit according to any one of claims 2 to 5, wherein the programmed death receptor 1 antibody / programmed death ligand antibody therapy is used to treat: lung adenocarcinoma, skin melanoma, prostate cancer, bladder urothelial carcinoma, mesothelioma, invasive breast cancer, cervical squamous cell carcinoma, cervical adenocarcinoma, pancreatic cancer, ovarian serous cystadenocarcinoma, head and neck squamous cell carcinoma, gastric adenocarcinoma, thyroid cancer, bile duct carcinoma, adrenocortical carcinoma, rectal adenocarcinoma, colon cancer, hepatocellular carcinoma, low-grade glioma of the brain, glioblastoma multiforme, uveal melanoma, or uterine carcinosarcoma.

Images