Application of IL-1R2 in identifying immune state of object

Abstract

The invention provides a method for identifying the immune state of a subject, and the use of an agent for detecting the level of IL-1R2 in a sample from the subject in the preparation of a product for identifying the immune state of the subject. The invention also provides an antibody binding to human IL-1R2 and a use thereof, and a hybridoma cell generating the antibody binding to human IL-1R2 and a use thereof.

Description

Technical Field

[0001] The present application relates to the field of biological detection and antibody preparation. Specifically, the present application provides a method for identifying the immune status of a subject, use of a reagent for detecting the level of IL-1R2 in a sample from a subject in the preparation of a product for identifying the immune status of the subject, antibodies that bind to human IL-1R2 and uses thereof, and hybridoma cells that produce antibodies that bind to human IL-1R2 and uses thereof. Background Art

[0002] Systemic inflammatory response syndrome caused by infection can lead to sepsis. Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Sepsis has high morbidity and mortality, and is still the leading cause of death from nosocomial infections. A recent statistical report emphasized that there are nearly 50 million cases of sepsis each year worldwide, and the age of onset covers all age groups. In 2017, deaths from sepsis accounted for nearly 20% of all deaths worldwide. In addition, patients who have had sepsis in the past have a higher risk of re-hospitalization and death. Sepsis seriously threatens human health and also causes a huge socioeconomic burden.

[0003] Immune dysfunction is closely related to the occurrence and development of sepsis, and patients may show excessive inflammatory response and immunosuppression at the same time. The former will cause early tissue damage and organ dysfunction, while the latter, when severe and persistent, will further induce a variety of fatal complications, thereby significantly increasing the mortality of patients with mid-to-late sepsis. In recent years, with the continuous improvement of organ support technology, the mortality rate in the proinflammatory response stage has decreased, but the mortality rate in the immunosuppression stage remains high. Therefore, the focus of research on immune dysfunction in sepsis has shifted from excessive inflammation to immunosuppression. However, for a long time, people have lacked understanding of the exact mechanism of immune dysfunction caused by severe damage and its role in sepsis, and there is a lack of effective immune monitoring and conditioning measures in clinical practice. Dynamic and accurate assessment of immune status is a prerequisite for timely identification of immune dysfunction in patients with sepsis and determination of the timing of immunomodulatory treatment. However, due to insufficient understanding of the precise molecular mechanism and cellular basis of sepsis-induced immunosuppression, there is a lack of clinical monitoring indicators and evaluation systems that can effectively reflect the immune status of patients with sepsis.

[0004] In the latest "Expert Consensus on the Diagnosis and Treatment of Sepsis Immunosuppression", experts pointed out that lymphocyte counts and monocyte human leukocyte antigen DR (mHLA-DR) are currently widely used to evaluate changes in immune function in patients with sepsis, but there are still limitations. Although lymphocyte counts are easy to obtain, their specificity is relatively low due to many confounding factors. At the same time, mHLA-DR testing requires flow cytometry, complex equipment, high testing costs, and the definition of warning thresholds has not yet been determined. In recent years, researchers have applied multi-omics methods to explore immune monitoring, aiming to discover new sepsis-related cell subsets and biomarkers, but their translational significance and value need to be further verified through large-scale clinical trials.

[0005] Therefore, the development of new methods and products for identifying the immune status of patients with sepsis or even systemic inflammatory response syndrome has important biological and medical significance. Summary of the invention

[0006] In a first aspect, the present application provides a method for identifying the immune status of a subject, comprising:

[0007] The level of interleukin 1 receptor 2 (IL-1R2) in a sample from the subject is determined.

[0008] In one or more embodiments of the present application, determining the level of IL-1R2 in the sample from the subject comprises contacting the sample with a reagent that detects the level of IL-1R2.

[0009] In one or more embodiments of the present application, determining the level of IL-1R2 in the sample from the subject comprises contacting the sample with a first antibody and a second antibody,

[0010] wherein the first antibody comprises a heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:3 and HCDR3 as shown in SEQ ID NO:4, and a light chain variable region comprising LCDR1 as shown in SEQ ID NO:5, LCDR2 as shown in SEQ ID NO:6 and LCDR3 as shown in SEQ ID NO:7; and / or

[0011] The second antibody comprises a heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:8, HCDR2 as shown in SEQ ID NO:9, and HCDR3 as shown in SEQ ID NO:10, and a light chain variable region comprising LCDR1 as shown in SEQ ID NO:11, LCDR2 as shown in SEQ ID NO:12, and LCDR3 as shown in SEQ ID NO:13;

[0012] The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

[0013] In one or more embodiments of the present application, the contacting is simultaneous or sequential contacting.

[0014] In one or more embodiments of the present application, the level of IL-1R2 in a sample is detected by an immunological method.

[0015] In one or more embodiments of the present application, the level of IL-1R2 is compared to a cut-off value of IL-1R2 level for identifying the immune status of the subject.

[0016] In a second aspect, the present application provides use of a reagent for detecting the level of IL-1R2 in a sample from a subject in the preparation of a product for identifying the immune status of the subject.

[0017] In one or more embodiments of the present application, the immune state is an immunosuppressive state.

[0018] In one or more embodiments of the present application, the subject is a mammal.

[0019] In one or more embodiments of the present application, the subject suffers from systemic inflammatory response syndrome, cancer, dengue fever, or colitis.

[0020] In one or more embodiments of the present application, the agent is a protein.

[0021] In one or more embodiments of the present application, the product for identifying the immune status of the subject is selected from the group consisting of: a kit, a test strip, a test card, and a microfluidic detection device.

[0022] In one or more embodiments of the present application, the IL-1R2 is soluble IL-1R2 or membrane-bound protein IL-1R2.

[0023] In one or more embodiments of the present application, the antibody comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein

[0024] The amino acid sequence of the HCDR1 is shown in SEQ ID NO:2, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:3, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:4, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:5, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:7; or

[0025] The amino acid sequence of the HCDR1 is shown in SEQ ID NO:8, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:9, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:10, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:11, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:12, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:13;

[0026] The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

[0027] In one or more embodiments of the present application, the product for identifying the immune status of the subject comprises a first antibody and / or a second antibody;

[0028] wherein the first antibody comprises a heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:3, and HCDR3 as shown in SEQ ID NO:4, and a light chain variable region comprising LCDR1 as shown in SEQ ID NO:5, LCDR2 as shown in SEQ ID NO:6, and LCDR3 as shown in SEQ ID NO:7; and / or

[0029] The second antibody comprises a heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:8, HCDR2 as shown in SEQ ID NO:9, and HCDR3 as shown in SEQ ID NO:10, and a light chain variable region comprising LCDR1 as shown in SEQ ID NO:11, LCDR2 as shown in SEQ ID NO:12, and LCDR3 as shown in SEQ ID NO:13;

[0030] The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

[0031] In one or more embodiments of the present application, the cut-off value of the IL-1R2 level used to identify the immune status of the subject is any value within the range of 19-29 ng / mL.

[0032] In one or more embodiments of the present application, the immune state of the subject whose IL-1R2 level is greater than the cut-off value is identified as an immunosuppressive state. In one or more embodiments of the present application, the immune state of the subject whose IL-1R2 level is less than the cut-off value is identified as a non-immunosuppressive state.

[0033] In one or more embodiments of the present application, the immune status of the subject (e.g., immunosuppressive status) also needs to be identified in combination with at least one of the following clinical indicators: absolute lymphocyte count (ALC), monocyte HLA-DR positivity rate, disease history, medication history, age, body mass index, and albumin level.

[0034] In a third aspect, the present application provides an antibody that binds to human IL-1R2, comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein

[0035] The amino acid sequence of the HCDR1 is shown in SEQ ID NO:2, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:3, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:4, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:5, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:7; or

[0036] The amino acid sequence of the HCDR1 is shown in SEQ ID NO:8, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:9, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:10, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:11, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:12, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:13;

[0037] The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

[0038] In one or more embodiments of the present application, the amino acid sequence of the heavy chain variable region of the antibody has at least 90% identity with SEQ ID NO: 14 or 15; and / or

[0039] The amino acid sequence of the light chain variable region of the antibody is at least 90% identical to SEQ ID NO: 16 or 17.

[0040] In one or more embodiments of the present application, the antibody is a whole antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a single-chain Fv fragment (scFv), a Fd fragment, a single domain antibody or a dAb fragment; and / or

[0041] The antibody is a monoclonal antibody; and / or

[0042] The antibody comprises a heavy chain constant region selected from the IgG1 subtype, IgG2 subtype or IgG4 subtype; and / or

[0043] The antibody comprises a light chain constant region selected from the κ subtype or the λ subtype; and / or

[0044] The antibody binds to human IL-1R2 having an amino acid sequence as shown in SEQ ID NO:1.

[0045] In one or more embodiments of the present application, the antibody is produced by a hybridoma cell line having a deposit number of CCTCC NO: C2024249 or CCTCC NO: C2024250.

[0046] In a fourth aspect, the present application provides a hybridoma cell that produces an antibody that binds to human IL-1R2, which is deposited with CCTCC NO: C2024249 or CCTCC NO: C2024250.

[0047] In a fifth aspect, the present application provides a product for detecting the level of human IL-1R2 in a sample, comprising a first antibody and / or a second antibody;

[0048] wherein the first antibody comprises a heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:3, and HCDR3 as shown in SEQ ID NO:4, and a light chain variable region comprising LCDR1 as shown in SEQ ID NO:5, LCDR2 as shown in SEQ ID NO:6, and LCDR3 as shown in SEQ ID NO:7; and / or

[0049] The second antibody comprises a heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:8, HCDR2 as shown in SEQ ID NO:9, and HCDR3 as shown in SEQ ID NO:10, and a light chain variable region comprising LCDR1 as shown in SEQ ID NO:11, LCDR2 as shown in SEQ ID NO:12, and LCDR3 as shown in SEQ ID NO:13;

[0050] The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

[0051] In a sixth aspect, the present application provides a method for detecting the level of human IL-1R2 in a sample, the method comprising contacting a first antibody and a second antibody with the sample; optionally, the contacting is simultaneous or sequential;

[0052] wherein the first antibody comprises a heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:3, and HCDR3 as shown in SEQ ID NO:4, and a light chain variable region comprising LCDR1 as shown in SEQ ID NO:5, LCDR2 as shown in SEQ ID NO:6, and LCDR3 as shown in SEQ ID NO:7; and / or

[0053] The second antibody comprises a heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:8, HCDR2 as shown in SEQ ID NO:9, and HCDR3 as shown in SEQ ID NO:10, and a light chain variable region comprising LCDR1 as shown in SEQ ID NO:11, LCDR2 as shown in SEQ ID NO:12, and LCDR3 as shown in SEQ ID NO:13;

[0054] The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

[0055] In one or more embodiments of the present application, the level of human IL-1R2 in a sample is detected by an immunological method.

[0056] In a seventh aspect, the present application provides use of the antibody described in the third aspect or the hybridoma cell described in the fourth aspect in preparing a product for detecting the level of human IL-1R2 in a sample.

[0057] In one or more embodiments of the present application, the product for detecting the level of human IL-1R2 in a sample is selected from the group consisting of: a kit, a test strip, a test card, and a microfluidic detection device.

[0058] In one or more embodiments of the present application, the human IL-1R2 is human soluble IL-1R2 or human membrane-bound protein IL-1R2.

[0059] In one or more embodiments of the present application, the first antibody or the second antibody is labeled with a detectable marker, and the detectable marker is selected from: enzymes, fluorescent molecules, radioactive isotopes, chemiluminescent molecules, latex particles, gold particles, detectable ligands and any combination thereof.

[0060] In one or more embodiments of the present application, the first antibody or the second antibody is linked to a solid support.

[0061] In one or more embodiments of the present application, the sample is selected from the group consisting of: plasma, blood, urine, serum, lymph, gastric juice, bile, saliva, sweat, cerebrospinal fluid, and any combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 The results of correct expression and purity identification of human soluble IL-1R2 recombinant protein are shown. A shows the results of using His tag antibody Western Blot to verify the correct expression of soluble IL-1R2 in cell culture supernatant; B shows the results of using SDS-PAGE to verify the purity of human soluble IL-1R2 recombinant protein.

[0063] Figure 2 The SPR detection of the binding and dissociation curves of monoclonal antibody 2E10 with different concentrations and IL-1R2 protein is shown.

[0064] Figure 3 The SPR detection of the binding and dissociation curves of monoclonal antibody 3D10 with different concentrations and IL-1R2 protein is shown.

[0065] Figure 4 Shown are the results of ROC curve analysis using the monoclonal antibodies 2E10 and 3D10 paired to detect human plasma IL-1R2 levels to identify immunosuppression.

[0066] Figure 5 Shown are the results of plasma IL-1R2 levels in apparently healthy subjects, non-immunosuppressed septic patients, and immunosuppressed septic patients using the paired monoclonal antibodies 2E10 and 3D10. Collection Instructions

[0067] 1. Cell name: Hybridoma cell line ZIL1R-2E10

[0068] Collection institution: China Center for Type Culture Collection

[0069] Abbreviation of depository institution: CCTCC

[0070] Address: Wuhan University, Wuhan, China (No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province)

[0071] Storage date: August 22, 2024

[0072] CCTCC registration number: CCTCC NO: C2024249

[0073] 2. Cell name: Hybridoma cell line ZIL1R-3D10

[0074] Collection institution: China Center for Type Culture Collection

[0075] Abbreviation of depository institution: CCTCC

[0076] Address: Wuhan University, Wuhan, China (No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province)

[0077] Storage date: August 22, 2024

[0078] CCTCC registration number: CCTCC NO: C2024250 Sequence Description

[0079] SEQ ID NO: 1 shows the amino acid sequence of positions 1 to 343 of soluble IL-1R2 with Uniprot number P27930-1.

[0080] SEQ ID NOs: 2-4 respectively show the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the anti-soluble human IL-1R2 monoclonal antibody 2E10.

[0081] SEQ ID NOs: 5-7 respectively show the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the anti-soluble human IL-1R2 monoclonal antibody 2E10.

[0082] SEQ ID NOs: 8-10 respectively show the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the anti-soluble human IL-1R2 monoclonal antibody 3D10.

[0083] SEQ ID NOs: 11-13 respectively show the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the anti-soluble human IL-1R2 monoclonal antibody 3D10.

[0084] SEQ ID NO: 14 shows the amino acid sequence of the heavy chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 2E10.

[0085] SEQ ID NO: 15 shows the amino acid sequence of the heavy chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 3D10.

[0086] SEQ ID NO: 16 shows the amino acid sequence of the light chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 2E10.

[0087] SEQ ID NO: 17 shows the amino acid sequence of the light chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 3D10.

[0088] SEQ ID NO: 18 shows the nucleotide sequence encoding the heavy chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 2E10.

[0089] SEQ ID NO: 19 shows the amino acid sequence of FR1 of the heavy chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 2E10.

[0090] SEQ ID NO: 20 shows the amino acid sequence of FR2 of the heavy chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 2E10.

[0091] SEQ ID NO: 21 shows the amino acid sequence of FR3 of the heavy chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 2E10.

[0092] SEQ ID NO: 22 shows the amino acid sequence of FR4 of the heavy chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 2E10.

[0093] SEQ ID NO: 23 shows the nucleotide sequence encoding the light chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 2E10.

[0094] SEQ ID NO: 24 shows the amino acid sequence encoding FR1 of the light chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 2E10.

[0095] SEQ ID NO: 25 shows the amino acid sequence encoding FR2 of the light chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 2E10.

[0096] SEQ ID NO: 26 shows the amino acid sequence encoding FR3 of the light chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 2E10.

[0097] SEQ ID NO: 27 shows the amino acid sequence encoding FR4 of the light chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 2E10.

[0098] SEQ ID NO: 28 shows the nucleotide sequence encoding the heavy chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 3D10.

[0099] SEQ ID NO: 29 shows the amino acid sequence of FR1 of the heavy chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 3D10.

[0100] SEQ ID NO: 30 shows the amino acid sequence of FR2 of the heavy chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 3D10.

[0101] SEQ ID NO: 31 shows the amino acid sequence of FR3 of the heavy chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 3D10.

[0102] SEQ ID NO: 32 shows the amino acid sequence of FR4 of the heavy chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 3D10.

[0103] SEQ ID NO: 33 shows the nucleotide sequence encoding the light chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 3D10.

[0104] SEQ ID NO: 34 shows the amino acid sequence encoding FR1 of the light chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 3D10.

[0105] SEQ ID NO: 35 shows the amino acid sequence encoding FR2 of the light chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 3D10.

[0106] SEQ ID NO: 36 shows the amino acid sequence encoding FR3 of the light chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 3D10.

[0107] SEQ ID NO: 37 shows the amino acid sequence encoding FR4 of the light chain variable region of the anti-soluble human IL-1R2 monoclonal antibody 3D10. DETAILED DESCRIPTION

[0108] Interleukin-1 (IL-1) is a cytokine with potent proinflammatory and immune response-amplifying effects, mainly produced by macrophages during defense responses. IL-1 is one of several proinflammatory cytokines produced during infection, sepsis, and systemic inflammatory response syndrome (SIRS), used to initiate host inflammatory responses and integrate nonspecific immunity. IL-1 is an important mediator involved in the pathogenesis of SIRS. The dominant cytokines in the early stages of SIRS are IL-1 and TNF-α, which induce the release of other proinflammatory cytokines (IL-6, IL-8, IFN-γ) and trigger the coagulation cascade. IL-1 can also induce the production of TNF-α.

[0109] Interleukin-1 receptor type 2 (IL-1R2) belongs to the IL-1 receptor family. IL-1R1, IL-1R2 and IL-1RAP are important components of the IL-1 receptor family. The IL-1R2 gene is located on chromosome 2, band 2q12. Like other members of the IL-1 receptor family, it consists of an extracellular part containing three glycosylated immunoglobulin (Ig)-like domains and has 28% amino acid homology with the extracellular part of IL-1R1. However, compared with other members of the family, it cannot initiate downstream signal transduction because of its shorter cytoplasmic structure and lack of Toll / IL-1 receptor (TIR) ​​domain. The IL-1R2 protein exists in two forms: cell membrane-bound and soluble (sIL-1R2).

[0110] Under the action of IL-1RAP, IL-1 forms an IL-1 / IL-1R1 / IL-1RAP complex with IL-1R1, activates the downstream signal transduction pathway of IL-1, and exerts pro-inflammatory biological functions. The membrane-bound protein IL-1R2 competitively captures IL-1 on the cell surface, while sIL-1R2 binds to IL-1 in the extracellular microenvironment, preventing IL-1 from binding to IL-1R1 on the cell surface and preventing the initiation of cell activation. It is one of the main mechanisms for inhibiting IL-1 activity and is defined as a decoy receptor.

[0111] IL-1R2 has a high affinity for IL-1β and a low affinity for interleukin 1 receptor antagonist (IL-1Ra), so it can play its role in inhibiting IL-1 activity and is only partially interfered by IL-1Ra. Compared with the membrane-bound protein IL-1R2, sIL-1R2 has different ligand binding abilities. The binding ability of sIL-1R2 to IL-1β is comparable to that of membrane receptors, but its ability to bind to IL-1α is better than that of membrane receptors. More importantly, sIL-1R2 cannot bind to IL-1Ra, which indicates that sIL-1R2 is a better IL-1 inhibitor than the membrane-bound protein IL-1R2 because its action is not interfered by IL-1Ra. In addition, sIL-1R2 can also bind to the IL-1β precursor Pro-IL-1β, preventing Pro-IL-1β from being cleaved and processed by proteases in cells to be converted into mature IL-1β, thereby playing a negative regulatory role in the inflammatory response. In summary, soluble IL-1R2 inhibits the pro-inflammatory effect of IL-1 and thus suppresses the immune response.

[0112] The inventors of the present application found that in critically ill patients with systemic inflammatory response syndromes such as sepsis, the plasma level of soluble IL-1R2 was significantly increased, and the plasma IL-1R2 level was positively correlated with the severity of the disease. Increased IL-1R2 reflects the disorder of IL-1, indicating the transition of SIRS to compensatory anti-inflammatory response syndrome (CARS). In addition, stimulation of anti-inflammatory factors such as IL-4, IL-10, and glucocorticoids can upregulate IL-1R2. IL-1R2 mainly plays a negative regulatory role in the inflammatory response of different cell types. In summary, increased soluble IL-1R2 indicates that the patient is in a state of relative immunosuppression, which can be used as one of the auxiliary detection indicators of the immunosuppressive state of patients with sepsis and even systemic inflammatory response syndrome.

[0113] In addition, in order to measure the level of human IL-1R2 (especially human soluble IL-1R2), the inventors of the present application also obtained new antibodies that bind to human IL-1R2 (especially human soluble IL-1R2) through antibody engineering technology.

[0114] In various aspects of the present application, methods for identifying the immune status of a subject are provided, use of a reagent for detecting the level of IL-1R2 in a sample from a subject in the preparation of a product for identifying the immune status of the subject, antibodies that bind to human IL-1R2 and their uses, hybridoma cells that produce antibodies that bind to human IL-1R2 and their uses, products for detecting the level of human IL-1R2 in a sample, and methods for detecting the level of human IL-1R2 in a sample.

[0115] The practice of the present application will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, and immunology within the skill of the art.

[0116] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0117] Unless otherwise specified, all experimental reagents were commercially available.

[0118] definition

[0119] As used herein, the term "immune status" refers to the functional status and responsiveness of an individual's immune system at a particular time. It reflects the immune system's ability to respond to antigens (such as pathogens, vaccines or other foreign substances) and overall health.

[0120] As used herein, the term "immunosuppressed state" refers to a state in which the immune system of an individual is weakened or suppressed, resulting in a reduced ability to respond to antigens (such as pathogens, tumor cells, or vaccines). Such a state may make an individual more susceptible to infection, the development of certain diseases, or less able to effectively fight off disease.

[0121] As used herein, the term "relative immunosuppression" refers to a state in which the immune system of an individual is partially weakened or suppressed under certain specific conditions or relative to healthy standards, but such suppression is not absolute or complete. This state may make an individual more susceptible to infection or other immune-related problems in certain situations (such as long-term hospitalization), but still maintain a certain degree of immune response capacity in other situations.

[0122] As used herein, the term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains, i.e., two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH). The heavy chain constant region comprises three domains, i.e., CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated as VL) and a light chain constant region (abbreviated as CL). The light chain constant region comprises one domain (CL1). The VH region and the VL region can be further subdivided into hypervariable regions called complementary determining regions (CDRs), interspersed with conserved regions called framework regions (FRs). In some embodiments, from N-terminus to C-terminus, both the light chain and heavy chain variable regions comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0123] Antibodies are immunoglobulin molecules that specifically bind to targets via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, polypeptides, and the like. "Antibodies" as used herein include not only complete (i.e., full-length) antibodies, but also antigen-binding fragments thereof, variants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, double antibodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configurations of immunoglobulin molecules comprising antigen recognition sites of desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0124] Full-length antibodies can be of any class, such as IgD, IgE, IgG, IgA or IgM (or subclasses thereof), but antibodies need not belong to any particular class. Immunoglobulins can be assigned to different classes based on the antibody amino acid sequence of the heavy chain constant region. In general, there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional structures of the different classes of immunoglobulins are well known.

[0125] As used herein, the term "antigen binding fragment" of an antibody refers to a portion or segment of a complete antibody molecule that is responsible for binding to an antigen. An antigen binding fragment may include a heavy chain variable region (VH), a light chain variable region (VL), or both. Antigen binding fragments of an antibody may be prepared from a complete antibody molecule using any suitable standard technique, including proteolytic digestion or recombinant genetic engineering techniques, etc. Non-limiting examples of antigen binding fragments include: Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, single-chain Fv fragments (scFv), Fd fragments, single domain antibodies, dAb fragments, and the minimum recognition unit (e.g., isolated CDR) composed of amino acid residues that mimic the hypervariable region of an antibody. Antigen binding fragments may also include other engineered molecules, such as double antibodies, three antibodies, four antibodies, and mini antibodies, etc. For example, the Fd fragment mentioned herein refers to an antibody fragment consisting of VH and CH1 regions; the Fv fragment consists of the VL and VH regions in a single arm of an antibody; and the dAb fragment (Ward et al., Nature 1989; 341:544-546) consists of the VH region.

[0126] It is well known to those skilled in the art that the complementarity determining region (CDR, usually CDR1, CDR2 and CDR3) is the region in the variable region that has the greatest impact on the affinity and specificity of the antibody. The CDR amino acid sequence of VH or VL is defined by common definitions, such as the IMGT numbering scheme, the Chothia numbering scheme and the Kabat numbering scheme. See, for example, Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991) 7; Al-Lazikani, et al., J. Mol. Biol. 273: 927-948 (1997); and Martin, et al., Proc. Natl. Acad. Sci. USA 86: 9268-9272 (1989). For the variable region amino acid sequence of a given antibody, the CDR amino acid sequences in the VH and VL amino acid sequences can be determined according to the IMGT numbering scheme definition, the Chothia numbering scheme definition, or the Kabat numbering scheme definition.

[0127] For a given antibody variable region amino acid sequence, the CDR amino acid sequence in the variable region amino acid sequence can be analyzed in a variety of ways, for example, it can be determined using the online software Abysis (http: / / www.abysis.org / ).

[0128] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic epitope, such as the ability of an antibody to bind to a specific antigen with an affinity at least two times greater than its affinity for a non-specific antigen. However, it should be understood that an antibody can specifically bind to two or more antigens related to its sequence.

[0129] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may occur in minor individuals.

[0130] As used herein, the term "murinized antibody" refers to any antibody in which all constant region sequences are mouse sequences. Such antibodies can be produced by hybridomas.

[0131] As used herein, the term "monoclonal antibody reactivity" refers to the ability of a monoclonal antibody to bind to an antigen under appropriate reaction conditions.

[0132] As used herein, the term "cell line" refers to a single cell culture obtained from a primary culture or cell line by screening or limiting dilution methods.

[0133] As used herein, the term "homology" is defined as the percentage of identical residues in amino acid or nucleotide sequence variants after sequence alignment and introduction of gaps, if necessary, to achieve the maximum percentage of homology. Methods and computer programs for alignment are well known in the art. "At least 90% homology" as described herein refers to any value of homology from 90% to 100%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0134] As used herein, the term "cut-off value" is a critical value used to determine whether a test result is abnormal. In medical tests, the cut-off value is used to determine whether the test result is higher or lower than the clinical or analytical decision point. If the test result is lower than the cut-off value, the test result is negative, and if it is higher than the cut-off value, the test result is positive. In some embodiments of the present application, when the human plasma IL-1R2 level is higher than the cut-off value, it indicates that the patient is in an immunosuppressive state (e.g., a relative immunosuppressive state); when the human plasma IL-1R2 level is lower than the cut-off value, it indicates that the patient is in a non-immunosuppressive state.

[0135] In a first aspect, the present application provides a method for identifying the immune status of a subject, comprising:

[0136] The level of interleukin 1 receptor 2 (IL-1R2) in a sample from the subject is determined.

[0137] In one or more embodiments of the present application, determining the level of IL-1R2 in the sample from the subject comprises contacting the sample with a reagent that detects the level of IL-1R2.

[0138] In one or more embodiments of the present application, determining the level of IL-1R2 in the sample from the subject comprises contacting the sample with a first antibody and a second antibody,

[0139] wherein the first antibody comprises a heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:3 and HCDR3 as shown in SEQ ID NO:4, and a light chain variable region comprising LCDR1 as shown in SEQ ID NO:5, LCDR2 as shown in SEQ ID NO:6 and LCDR3 as shown in SEQ ID NO:7; and / or

[0140] The second antibody comprises a heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:8, HCDR2 as shown in SEQ ID NO:9, and HCDR3 as shown in SEQ ID NO:10, and a light chain variable region comprising LCDR1 as shown in SEQ ID NO:11, LCDR2 as shown in SEQ ID NO:12, and LCDR3 as shown in SEQ ID NO:13;

[0141] The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

[0142] In one or more embodiments of the present application, the method for identifying the immune status of a subject is a method for assisting in identifying the immune status (eg, immunosuppressive status) of a subject.

[0143] In one or more embodiments of the present application, the level of IL-1R2 is compared to a cut-off value of IL-1R2 level for identifying the immune status of the subject.

[0144] In a second aspect, the present application provides use of a reagent for detecting the level of IL-1R2 in a sample from a subject in the preparation of a product for identifying the immune status of the subject.

[0145] In one or more embodiments of the present application, the immune state is an immunosuppressive state. In one or more embodiments of the present application, the immune state is a relatively immunosuppressive state.

[0146] In one or more embodiments of the present application, the subject is a mammal, such as a human.

[0147] In one or more embodiments of the present application, the subject suffers from systemic inflammatory response syndrome, cancer, dengue fever or colitis. In one or more embodiments of the present application, the systemic inflammatory response syndrome is sepsis or acute respiratory distress syndrome. In one or more embodiments of the present application, the cancer is gastric cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, lung cancer or leukemia.

[0148] In one or more embodiments of the present application, the agent is a protein, such as an antibody. In one or more embodiments of the present application, the antibody can be any antibody capable of binding to IL-1R2, such as a commercially available antibody that binds to IL-1R2.

[0149] In one or more embodiments of the present application, the IL-1R2 is soluble IL-1R2 or membrane-bound protein IL-1R2. In one or more embodiments of the present application, the IL-1R2 is soluble IL-1R2, such as human soluble IL-1R2.

[0150] In one or more embodiments of the present application, the antibody comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein

[0151] The amino acid sequence of the HCDR1 is shown in SEQ ID NO:2, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:3, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:4, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:5, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:7; or

[0152] The amino acid sequence of the HCDR1 is shown in SEQ ID NO:8, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:9, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:10, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:11, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:12, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:13;

[0153] The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

[0154] In one or more embodiments of the present application, the product for identifying the immune status of the subject is selected from: a kit, a test strip (eg, a colloidal gold test strip), a test card, and a microfluidic detection device.

[0155] In one or more embodiments of the present application, the product for identifying the immune status of the subject comprises a first antibody and / or a second antibody;

[0156] wherein the first antibody comprises a heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:3, and HCDR3 as shown in SEQ ID NO:4, and a light chain variable region comprising LCDR1 as shown in SEQ ID NO:5, LCDR2 as shown in SEQ ID NO:6, and LCDR3 as shown in SEQ ID NO:7; and / or

[0157] The second antibody comprises a heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:8, HCDR2 as shown in SEQ ID NO:9, and HCDR3 as shown in SEQ ID NO:10, and a light chain variable region comprising LCDR1 as shown in SEQ ID NO:11, LCDR2 as shown in SEQ ID NO:12, and LCDR3 as shown in SEQ ID NO:13;

[0158] The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

[0159] In one or more embodiments of the present application, the product for identifying the immune status of the subject may also include other reagents for detection, such as substrates, reference standards, diluents, washing solutions, and the like.

[0160] In one or more embodiments of the present application, the product for identifying the immune status of the subject may further include instructions for use of the product.

[0161] In one or more embodiments of the present application, the product for identifying the immune status of the subject may further include a container for mixing the sample with the antibody. Such a container may be suitable for use in a detection instrument capable of detecting a signal generated by the detection monoclonal antibody.

[0162] In one or more embodiments of the present application, the product for identifying the immune status of the subject is used to assist in identifying the immune status of the subject, such as the immunosuppressive status.

[0163] In one or more embodiments of the present application, the cut-off value of the IL-1R2 level used to identify the immune status of the subject is 19-29. ng / mL, for example, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23, 23.1, 23.2, 23.3, 23.4, 23.5, 23.6, 23.7, 23.8 8, 23.9, 24, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, 25, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, 25.9, 26, 26.1, 26.2, 26.3, 26. 4, 26.5, 26.6, 26.7, 26.8, 26.9, 27, 27.1, 27.2, 27.3, 27.4, 27.5, 27.6, 27.7, 27.8, 27.9, 28, 28.1, 28.2, 28.3, 28.4, 28.5, 28.6, 28.7, 28.8, 28.9 or 29 ng / mL. In one or more embodiments of the present application, the cut-off value of the IL-1R2 level for identifying the immune status of the subject is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 ng / mL. In one or more specific embodiments of the present application, the cut-off value of the IL-1R2 level for identifying the immune status of the subject is 24 ng / mL.

[0164] In one or more specific embodiments of the present application, the immune state of the subject whose IL-1R2 level is greater than the cut-off value is identified as an immunosuppressive state. In one or more embodiments of the present application, the immune state of the subject whose IL-1R2 level is less than the cut-off value is identified as a non-immunosuppressive state.

[0165] In one or more embodiments of the present application, the immune status of the subject (e.g., immunosuppressive status) also needs to be identified in combination with at least one of the following clinical indicators: absolute number of lymphocytes, monocyte HLA-DR positivity rate, disease history, medication history, age, body mass index, and albumin level.

[0166] In one or more embodiments of the present application, the immune status (e.g., immunosuppressive status) of the subject also needs to be identified in combination with the absolute number of lymphocytes and the monocyte HLA-DR positive rate. In one or more embodiments of the present application, the absolute number of lymphocytes in the blood routine report for three consecutive days is less than 1.0×10 9 The immune status of the patient with a peripheral blood mononuclear cell HLA-DR positive rate of less than 60% is identified as an immunosuppressive state, such as a relative immunosuppressive state. In one or more embodiments of the present application, whether the patient is in an immunosuppressive state requires a doctor to comprehensively identify the patient in combination with clinical indicators (such as absolute lymphocyte count and / or peripheral blood mononuclear cell HLA-DR positive rate and other factors).

[0167] In a third aspect, the present application provides an antibody that binds to human IL-1R2, comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein

[0168] The amino acid sequence of the HCDR1 is shown in SEQ ID NO:2, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:3, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:4, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:5, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:7; or

[0169] The amino acid sequence of the HCDR1 is shown in SEQ ID NO:8, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:9, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:10, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:11, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:12, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:13;

[0170] The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

[0171] In one or more embodiments of the present application, the amino acid sequence of the heavy chain variable region of the antibody has at least 90% identity with SEQ ID NO: 14 or 15, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity.

[0172] In one or more embodiments of the present application, the amino acid sequence of the light chain variable region of the antibody has at least 90% identity with SEQ ID NO: 16 or 17, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity.

[0173] In one or more embodiments of the present application, the amino acid sequence of the heavy chain variable region of the antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 14 or 15; and the amino acid sequence of the light chain variable region of the antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 16 or 17.

[0174] In one or more embodiments of the present application, the amino acid sequence of the heavy chain variable region of the antibody differs from the amino acid sequence shown in SEQ ID NO: 14 or 15 by about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions and / or additions.

[0175] In one or more embodiments of the present application, the amino acid sequence of the light chain variable region of the antibody differs from the amino acid sequence shown in SEQ ID NO: 16 or 17 by about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions and / or additions.

[0176] In one or more embodiments of the present application, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 14 or 15 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still maintaining similar function of the heavy chain variable region of the antibody.

[0177] In one or more embodiments of the present application, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 14 or 15, and the resulting amino acid sequence still maintains the function of the heavy chain variable region of the similar antibody.

[0178] In one or more embodiments of the present application, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 14 or 15, as long as the altered amino acid sequence substantially maintains the function of the heavy chain variable region of the similar antibody.

[0179] In one or more embodiments of the present application, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 16 or 17 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still maintaining similar function of the light chain variable region of the antibody.

[0180] In one or more embodiments of the present application, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 16 or 17, and the resulting amino acid sequence still maintains the function of the light chain variable region of the similar antibody.

[0181] In one or more embodiments of the present application, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 16 or 17, as long as the altered amino acid sequence substantially maintains the function of the light chain variable region of the similar antibody.

[0182] In one or more embodiments of the present application, the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO: 14 or 15.

[0183] In one or more embodiments of the present application, the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO: 16 or 17.

[0184] In one or more specific embodiments of the present application, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 16; or

[0185] The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 17.

[0186] In one or more embodiments of the present application, the antibody is a whole antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a single-chain Fv fragment (scFv), a Fd fragment, a single domain antibody or a dAb fragment.

[0187] In one or more embodiments of the present application, the antibody is a monoclonal antibody.

[0188] In one or more specific embodiments of the present application, the antibody is a murinized monoclonal antibody.

[0189] In one or more embodiments of the present application, the antibody comprises a heavy chain constant region selected from the IgG1 subtype, IgG2 subtype or IgG4 subtype.

[0190] In one or more embodiments of the present application, the antibody comprises a heavy chain constant region of the IgG1 subtype or the IgG2 subtype.

[0191] In one or more specific embodiments of the present application, when the antibody is the anti-soluble IL-1R2 monoclonal antibody 2E10, it comprises a heavy chain constant region of the IgG1 subtype.

[0192] In some specific embodiments of the first aspect, when the antibody is the anti-soluble IL-1R2 monoclonal antibody 3D10, it comprises a heavy chain constant region of the IgG2 subtype (eg, IgG2α subtype).

[0193] In one or more embodiments of the present application, the antibody comprises a light chain constant region selected from the κ subtype or the λ subtype.

[0194] In one or more embodiments of the present application, the antibody binds to human IL-1R2 having an amino acid sequence as shown in SEQ ID NO:1.

[0195] In one or more embodiments of the present application, the antibody is produced by a hybridoma cell line having a deposit number of CCTCC NO: C2024249 or CCTCC NO: C2024250.

[0196] In one or more embodiments of the present application, the anti-human soluble IL-1R2 monoclonal antibody 2E10 is produced by a hybridoma cell line with a deposit number of CCTCC NO: C2024249.

[0197] In one or more embodiments of the present application, the anti-human soluble IL-1R2 monoclonal antibody 3D10 is produced by a hybridoma cell line with a deposit number of CCTCC NO: C2024250.

[0198] In one or more embodiments of the present application, the antibody or the product comprising the antibody can be used for point of care testing (POCT).

[0199] In one or more embodiments of the present application, the antibody has the advantages of good specificity, high titer and high sensitivity.

[0200] In one or more embodiments of the present application, the antibody can effectively detect the level of IL-1R2 in the patient's body fluids (such as plasma, blood or urine) to determine whether the patient is in an immunosuppressive state, thereby guiding the doctor to determine the severity of the patient's condition, take effective treatment measures in a timely manner, and reduce the mortality rate, which has important clinical significance.

[0201] In a fourth aspect, the present application provides a hybridoma cell that produces an antibody that binds to human IL-1R2, which is deposited with CCTCC NO: C2024249 or CCTCC NO: C2024250.

[0202] In one or more embodiments of the present application, after the animal is inoculated with human IL-1R2 protein (e.g., human soluble IL-1R2 protein) antigen, antibodies and / or cells producing antibodies can be obtained in the animal. Immortalized cell lines producing antibodies can be prepared by cells isolated from immunized animals. After immunization, the animal is killed, lymph nodes and / or spleen B cells are taken for immortalization treatment, treated with carcinogenic compounds and mutagenic compounds, and fused with immortalized cells (e.g., myeloma cells, such as mouse myeloma cells SP2 / 0) to remove the activity of tumor suppressor genes. If myeloma cells are used for fusion, the myeloma cells preferably do not secrete immunoglobulin polypeptides (non-secreting cell lines). Immortalized cells are screened using human IL-1R2 protein (e.g., human soluble IL-1R2 protein) or cells expressing human IL-1R2 protein (e.g., human soluble IL-1R2 protein). In some embodiments, the initial screening is performed using enzyme-linked immunosorbent assay (ELISA). Cells such as hybridomas that produce antibodies against human IL-1R2 protein (e.g., human soluble IL-1R2 protein) are selected for cloning, and further screened for desired characteristics, including good growth, high antibody production, and desired antibody characteristics, etc. Methods for screening, cloning, and amplifying hybridomas are well known to those of ordinary skill in the art.

[0203] In one or more embodiments of the present application, the immunized animal is a non-human animal, wherein the splenic B cells are fused with a myeloma cell line from the same species as the non-human animal.

[0204] In one or more embodiments of the present application, the immunized animal is a BALB / c mouse.

[0205] In one or more embodiments of the present application, the hybridoma cell line is a mouse hybridoma cell line, such as a BALB / c mouse hybridoma cell line.

[0206] In a fifth aspect, the present application provides a product for detecting the level of human IL-1R2 in a sample, comprising a first antibody and / or a second antibody;

[0207] wherein the first antibody comprises a heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:3, and HCDR3 as shown in SEQ ID NO:4, and a light chain variable region comprising LCDR1 as shown in SEQ ID NO:5, LCDR2 as shown in SEQ ID NO:6, and LCDR3 as shown in SEQ ID NO:7; and / or

[0208] The second antibody comprises a heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:8, HCDR2 as shown in SEQ ID NO:9, and HCDR3 as shown in SEQ ID NO:10, and a light chain variable region comprising LCDR1 as shown in SEQ ID NO:11, LCDR2 as shown in SEQ ID NO:12, and LCDR3 as shown in SEQ ID NO:13;

[0209] The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

[0210] In a sixth aspect, the present application provides a method for detecting the level of human IL-1R2 in a sample, the method comprising contacting a first antibody and a second antibody with the sample;

[0211] wherein the first antibody comprises a heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:3 and HCDR3 as shown in SEQ ID NO:4, and a light chain variable region comprising LCDR1 as shown in SEQ ID NO:5, LCDR2 as shown in SEQ ID NO:6 and LCDR3 as shown in SEQ ID NO:7; and / or

[0212] The second antibody comprises a heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:8, HCDR2 as shown in SEQ ID NO:9, and HCDR3 as shown in SEQ ID NO:10, and a light chain variable region comprising LCDR1 as shown in SEQ ID NO:11, LCDR2 as shown in SEQ ID NO:12, and LCDR3 as shown in SEQ ID NO:13;

[0213] The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

[0214] In a seventh aspect, the present application provides use of the antibody described in the first aspect or the hybridoma cell described in the second aspect in preparing a product for detecting the level of human IL-1R2 in a sample.

[0215] In one or more embodiments of the present application, the human IL-1R2 level is an important indicator for monitoring the immune status (eg, immunosuppressive status) of the subject.

[0216] In one or more embodiments of the present application, the product for detecting the level of human IL-1R2 in a sample is selected from the group consisting of: a kit, a test strip (eg, a colloidal gold test strip), a test card, and a microfluidic detection device.

[0217] In one or more embodiments of the present application, the product for detecting the level of human IL-1R2 in a sample may further include other reagents for detection, such as substrates, reference standards, diluents, washing solutions, and the like.

[0218] In one or more embodiments of the present application, the product for detecting the level of human IL-1R2 in a sample may further include instructions for use of the product.

[0219] In one or more embodiments of the present application, the product for detecting the level of human IL-1R2 in a sample may further include a container for mixing the sample with the antibody. Such a container may be suitable for use in a detection instrument capable of detecting the signal generated by the detection monoclonal antibody.

[0220] In one or more embodiments of the present application, the contacting is simultaneous or sequential contacting.

[0221] In one or more embodiments of the present application, the level of IL-1R2, such as the level of human IL-1R2, in a sample is detected by an immunological method.

[0222] In one or more embodiments of the present application, the immunological method is selected from the group consisting of: enzyme-linked immunosorbent assay, fluorescent immunoquantitative analysis, chemiluminescent immunoassay, immunochromatography, immunoturbidimetric assay, immunoprecipitation, and any combination thereof.

[0223] According to the present application, antibodies that bind to IL-1R2 can be used to detect the level of IL-1R2 by immunological methods. For example, a "sandwich" assay can be used to detect and quantify IL-1R2. In this method, generally, an antibody is fixed on a solid surface to bind and capture IL-1R2, and the antibody is thus referred to as a capture antibody in this article. Another antibody is detectably labeled, for example, by a fluorescent group, an enzyme or a colored particle, and its binding to the IL-1R2-capture antibody complex indicates that IL-1R2 has been captured, and the intensity of the signal is proportional to the concentration of IL-1R2 in the sample. Therefore, another antibody is also referred to as a detection antibody or a labeled antibody in this article. Such assay methods can be referred to as a two-site immunoassay, a "sandwich" method or (when the antibody is an adhesive) a "sandwich immunoassay". As known in the art, the capture antibody and the detection antibody can be contacted with the test sample simultaneously or successively. Sequential method, sometimes referred to as a "forward" method, can be completed by incubating the capture antibody with the sample and adding the labeled detection antibody at a predetermined time thereafter. Alternatively, a labeled detection antibody may first be incubated with the sample, and then the sample may be contacted with the capture antibody (sometimes referred to as a "reverse" approach). Such assays may be performed in a number of specific formats known to those skilled in the art, including through the use of different high-throughput clinical laboratory analyzers or using point of care or home testing devices.

[0224] The most commonly used enzyme immunoassay is "enzyme-linked immunosorbent assay (ELISA)". ELISA is a technique that uses a labeled (e.g., enzyme-linked) form of an antibody to detect and measure the concentration of an antigen. There are different ELISA formats that are well known to those skilled in the art. Standard techniques for ELISA known in the art are described in "Methods in Immunodiagnosis", 2nd edition, Rose and Bigazzi, eds. John Wiley & Sons, 1980; Campbell, et al., "Methods and Immunology", W.A. Benjamin, Inc., 1964 and Oelleric, M. (1984, J. Clin. Chem. Clin. Biochem .22:895-904. In a "sandwich ELISA", an antibody (e.g., an anti-human IL-1R2 antibody) is attached to a solid phase (i.e., a microtiter plate) and contacted with a biological sample containing an antigen (e.g., human IL-1R2). The solid phase is then washed to remove unbound antigen. A labeled antibody (e.g., an enzyme-linked antibody) is then bound to the bound antigen, thereby forming an antibody-antigen-antibody sandwich. Examples of enzymes that can be linked to the antibody are horseradish peroxidase, alkaline phosphatase, luciferase, urease, and beta-galactosidase. The enzyme-linked antibody reacts with a substrate to produce a color reaction product that can be measured. The measurement can be used, for example, to deduce the concentration of human IL-1R2 present in the sample by comparing the measured value with a human IL-1R2 standard curve.

[0225] Quantitative fluorescence immunoassay is based on the principle of antigen-antibody reaction. First, the known antigen or antibody is labeled with fluorescein to make a fluorescent marker, and then the fluorescent antibody (or antigen) is used as a molecular probe to examine the corresponding antigen (or antibody) in cells or tissues. The antigen-antibody complex formed in cells or tissues contains fluorescein. When the specimen is observed under a fluorescence microscope, the fluorescein emits bright fluorescence under the irradiation of the excitation light, and the cells or tissues where the fluorescence is located can be seen, thereby determining the nature and location of the antigen or antibody, and measuring the content using quantitative technology.

[0226] Chemiluminescence immunoassay (CLIA) is a technique that combines highly sensitive chemiluminescence assay technology with highly specific immune reactions. It is used for the detection and analysis of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins and drugs. It is the latest immunoassay technology developed after radioimmunoassay, enzyme immunoassay, fluorescence immunoassay and time-resolved fluorescence immunoassay. Chemiluminescence immunoassay uses chemiluminescent agents to directly label antigens or antibodies for immunoassay. Commonly used chemiluminescent substances for labeling include acridinium ester compounds (Acridinium Ester, AE), which are effective luminescent markers. They emit light by starting the action of luminescent agents. The strong direct luminescence is completed within one second, which is a fast flashing luminescence. Acridinium ester is used as a marker for immunoassay. Its chemical reaction is simple, fast and does not require a catalyst. The competition method is used to detect small molecule antigens, and the sandwich method is used to detect large molecule antibodies. There is less nonspecific binding and low background. The binding with large molecules will not reduce the amount of light generated, thereby increasing sensitivity.

[0227] The principle of immunochromatography is to fix specific antibodies to a certain zone of the nitrocellulose membrane. When one end of the dry nitrocellulose membrane is immersed in the sample, the sample will move forward along the membrane due to capillary action. When it moves to the area where the antibody is fixed, the corresponding antigen in the sample will specifically bind to the antibody. If immunocolloidal gold or immunoenzyme staining is used, the area can show a certain color, thereby achieving specific immunodiagnosis.

[0228] Immunoturbidimetric assay is a dynamic determination method for antigen-antibody binding. Its basic principle is: when antigen and antibody react in a special dilution system and the ratio is appropriate (generally the antibody is in excess), the soluble immune complex formed will precipitate from the liquid phase under the action of the aggregation promoter in the dilution system to form particles, causing turbidity in the reaction solution. When the antibody concentration is fixed, the amount of immune complex formed increases with the increase of the amount of antigen in the test sample, and the turbidity of the reaction solution also increases accordingly. By measuring the turbidity of the reaction solution and comparing it with a series of standards, the content of the antigen in the test sample can be calculated.

[0229] Immunoprecipitation is a method of purifying and enriching target proteins using antibody-specific reactions. After the antibody binds to the corresponding protein in the sample, it is incubated with protein A / G or secondary antibody-coupled agarose or agarose beads, and the bead-protein A / G or secondary antibody-antibody-target protein complex is obtained by centrifugation. After washing, the precipitate is resuspended in electrophoresis loading buffer and boiled. Under the action of high temperature and reducing agent, the antigen and antibody are dissociated, and the supernatant is collected by centrifugation. The supernatant includes antibodies, target proteins and a small amount of miscellaneous proteins.

[0230] In one or more embodiments of the present application, the first antibody or the second antibody is labeled with a detectable marker. In one or more embodiments of the present application, the detectable marker can be selected from any marker generally known in the art. In one or more embodiments of the present application, the detectable marker is a marker that allows more accurate quantification. In one or more embodiments of the present application, the example of the detectable marker includes but is not limited to: enzyme, fluorescent molecule, radioisotope, chemiluminescent molecule, latex particle, gold particle, detectable ligand and any combination thereof.

[0231] In one or more embodiments of the present application, the detectable label is an enzyme or a fluorescent molecule. Methods for attaching the detectable label to an antibody are well known in the art and include covalent and non-covalent linkages.

[0232] In one or more embodiments of the present application, the detection antibody is detectably labeled by connecting the antibody to an enzyme, so that the enzyme will react with the substrate when in contact with its substrate, and this reaction can be detected, for example, by spectrophotometric measurement, fluorometry or visual inspection. Enzymes that can be used to detectably label the antibodies of the present application include, but are not limited to: horseradish peroxidase, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, alkaline phosphatase, luciferase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. In one or more embodiments of the present application, the enzyme is horseradish peroxidase.

[0233] In one or more embodiments of the present application, antibodies can be labeled with fluorescent molecules. When the fluorescently labeled antibody is exposed to light of an appropriate wavelength, its presence can be detected by the emitted fluorescence. Among them, the most commonly used fluorescent molecules can be selected from: Cy3 and Cy5 (water-soluble fluorescent dyes of the cyanine dye family-"Cy" dyes), fluorescein isothiocyanate, rhodamine, phycocyanin, allophycocyanin, o-phthalaldehyde and fluorescamine.

[0234] In one or more embodiments of the present application, detection can also be achieved using radiolabeled antibodies, which can then be detected using radioimmunoassay. Radioisotopes can be detected using, for example, a gamma counter or a scintillation counter or using autoradiography, for example 3 H. 131 I. 35 S. 14 C and 125 I.

[0235] In one or more embodiments of the present application, the antibody can also be detectably labeled by coupling the antibody to a chemiluminescent molecule, and then the presence of the chemiluminescent antibody is determined by detecting the presence of luminescence generated during the chemical reaction. The chemiluminescent molecule can be selected from: luminol, luciferin, isoaminophthalhydrazide, imidazole, acridinium salt and oxalate.

[0236] In one or more embodiments of the present application, the first antibody or the second antibody is linked to a solid support.

[0237] In one or more embodiments of the present application, the solid phase carrier can be a plastic product (such as a polystyrene plate), microparticles (such as magnetized microparticles) or a membrane carrier (such as a nitrocellulose membrane, a glass cellulose membrane or a nylon membrane).

[0238] In one or more embodiments of the present application, one of the first antibody and the second antibody is used as a capture antibody and is immobilized on a solid support to capture IL-1R2. The other of the first antibody and the second antibody is used as a detection antibody and is linked to a detectable marker.

[0239] In one or more embodiments of the present application, the sample is selected from: plasma, blood, urine, serum, lymph, gastric juice, bile, saliva, sweat, cerebrospinal fluid and any combination thereof. In one or more embodiments of the present application, the sample can be plasma, blood or urine.

[0240] In one or more embodiments of the present application, the human IL-1R2 is human soluble IL-1R2 or human membrane-bound protein IL-1R2.

[0241] In one or more specific embodiments of the present application, the human IL-1R2 is human soluble IL-1R2.

[0242] In other aspects, the application also provides nucleic acid molecules encoding antibodies that bind to human IL-1R2 as described in the application, vectors comprising the nucleic acid molecules, and host cells comprising the nucleic acid molecules or the vectors. In some embodiments, the nucleic acid molecules are operably linked to a regulatory nucleotide sequence that can be recognized by a host cell transformed with the vector.

[0243] It should be understood that the above detailed description is only for the purpose of enabling those skilled in the art to more clearly understand the content of the present application, and is not intended to be limiting in any aspect. Those skilled in the art can make various modifications and changes to the embodiments. Example

[0244] Example 1: Eukaryotic expression and purification of human soluble IL-1R2 recombinant protein

[0245] The codon-optimized gene of artificially synthesized soluble IL-1R2 (Uniprot: P27930-1, amino acid sequence from position 1 to position 343 as shown in SEQ ID NO: 1) was introduced with 6×His-Tag at the C-terminus, inserted into the pTT5 expression vector through the EcoRI / HindIII restriction site, and transferred into 293 cells for eukaryotic expression. The cell culture supernatant was collected and the protein was purified to finally obtain 5 mg of soluble IL-1R2 protein. Figure 1 As shown in Figure A, the results of immunoblotting with His tag antibody in the 293 cell culture supernatant and cell pellet showed that the 293 cell culture supernatant contained the target protein IL-1R2 (as indicated by the red arrow). Figure 1 As shown in B, SDS-PAGE electrophoresis of the purified protein confirmed that the purity and size of the IL-1R2 protein were consistent with expectations (as indicated by the red arrow).

[0246] Example 2: Preparation of anti-human IL-1R2 monoclonal antibodies and screening of antibody pairs

[0247] 2.1 Immunization of mice and cell fusion

[0248] Five 6-week-old female BALB / c mice were subcutaneously injected with soluble IL-1R2 protein emulsified with Freund's complete adjuvant at multiple points for three immunizations. After three immunizations, blood was collected from the tail vein and serum was collected for ELISA to detect serum titer. The mouse with the highest serum titer was intraperitoneally injected with IL-1R2 protein for shock immunization. The spleen of the mouse was taken, spleen cells were collected, and cell fusion was performed with mouse myeloma cells SP2 / 0, and plated in 96-well plates for culture.

[0249] 2.2 Hybridoma screening, stable cell line establishment and antibody subtype identification

[0250] Perform ELISA test on the culture supernatant of fusion cells, screen out positive wells, re-test, and subclone the re-test positive wells by limiting dilution method. Perform ELISA test after subcloning, select positive wells and continue to subclone by limiting dilution method. After multiple subcloning until the ELISA test positive rate is 100%, select clones with high absorbance, select wells with vigorous growth for expansion culture, and freeze for seed preservation. Perform ELISA test on the culture supernatant of stable cell lines to identify antibody subtypes.

[0251] 2.3 Preparation of monoclonal antibodies

[0252] The hybridoma cells were injected into the mouse peritoneum, and the ascites was collected and centrifuged. The supernatant was filtered through a 0.22 μm filter membrane, and then the antibody was purified through a protein A chromatography column (HiScreen Fibro™ PrismA, cytiva). The solution was replaced and concentrated with an ultrafiltration tube, and finally replaced with PBS and sterile filtered through a 0.22 μm filter membrane to obtain the monoclonal antibody. The antibody was gradiently diluted with PBS buffer, and the antibody titer was detected by ELISA. As shown in Table 1, the titers of the two antibodies screened were both greater than 1:128000.

[0253] Table 1. OD of monoclonal antibody titer detected by ELISA 450 value

[0254]

[0255] 2.4 Antibody pairing verification

[0256] The antibodies 2E10 and 3D10 obtained in Section 2.3, as well as the commercially available antibodies ALS11331 (rabbit anti-human IL-1R2 monoclonal antibody, purchased from Abgent, with the catalog number A-ALS11331) and R020 (rabbit anti-human IL-1R2 monoclonal antibody, purchased from Beijing Sino Biological Technology Co., Ltd., with the catalog number Cat: 10111-R020) were used as capture antibodies and labeling antibodies for pairing verification. The capture antibody was diluted to 1 μg / mL and added to the ELISA plate, coated with the antibody overnight at 4°C, then washed, blocked, and incubated with the antigen IL-1R2. The diluted detection antibody conjugated to horseradish peroxidase (HRP) was added, incubated, washed, color developed, and read. The pairing results are shown in Table 2. The sensitivity of the paired antibodies 2E10 (subtype IgG1) and 3D10 (subtype IgG2α) was the highest, reaching 10 pg / mL.

[0257] Table 2. OD of anti-soluble IL-1R2 monoclonal antibody pairs detected by ELISA 450 value

[0258]

[0259] Example 3: Affinity Identification of Anti-Human IL-1R2 Monoclonal Antibodies 2E10 and 3D10

[0260] The Biacore 8k+ surface plasmon resonance system (cytiva) was used to perform SPR experiments to detect the affinity of 2E10 and 3D10 antibodies to soluble human IL-1R2 protein. The eukaryotic expressed soluble human recombinant IL-1R2 protein was diluted to 10 μg / mL with sodium acetate at pH 5.5 and coupled to a CM5 chip (cytiva). 2E10 and 3D10 antibodies were diluted to 1.5625 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM and 50 nM with PBS + 0.025% P20, respectively, and the analytes were flowed over the chip surface from low to high concentrations, with a binding time of 120 s, a dissociation time of 300 s, a flow rate of 30 μL / min, and a glycine (pH 1.5) regeneration of 30 s at a flow rate of 30 μL / min. Fitting model (1:1 binding), analysis method using multi-cycle kinetics, according to different concentrations of antibody binding, dissociation response curve fitting, calculate the binding rate constant k a , the dissociation rate constant k d and the affinity constant K D The binding and dissociation curves of 2E10 antibody and IL-1R2 protein are shown in Figure 2 The binding and dissociation curves of 3D10 antibody and IL-1R2 protein are shown in Figure 3 According to the k obtained by fitting the binding and dissociation curves a , k d , K D See Table 3 for the affinity K values ​​of 2E10 and IL-1R2. D The value is 1.19×10^-10, and the affinity K of 3D10 to IL-1R2 is D The value is 1.35×10^-11. The affinity of antibody 2E10 to IL-1R2 is comparable to or better than the affinity of antibody ALS11331 or R020 to IL-1R2. The affinity of antibody 3D10 to IL-1R2 is comparable to or better than the affinity of antibody ALS11331 or R020 to IL-1R2.

[0261] Table 3. Affinity constants of monoclonal antibodies 2E10 and 3D10 for human IL-1R2

[0262]

[0263] Example 4: Increased plasma IL-R2 in sepsis patients reflects the patient's immunosuppressive state

[0264] 4.1 Identification of immunosuppression in patients with sepsis

[0265] According to the expert consensus on the diagnosis and treatment of sepsis immunosuppression (Shock and Sepsis Committee of the Chinese Society of Research Hospitals, Critical Care Medicine Committee of the Chinese People's Liberation Army, Critical Immunity Research Collaborative Group, etc., Expert consensus on the diagnosis and treatment of sepsis immunosuppression [J], Chinese Journal of Critical Care Medicine, 2020, 32(11):9. and Pei, Fei et al., "Expert consensus on the monitoring and treatment of sepsis-induced immunosuppression." Military Medical Research vol. 9,1 74. 26 Dec. 2022), the absolute number of lymphocytes in the routine blood test report for three consecutive days is less than 1.0×10 9 The criteria for identifying immunosuppression in patients were as follows: 1.1% of the patients had leukemia / mL, peripheral blood mononuclear cell HLA-DR positive rate less than 60%, age greater than 65 years, BMI less than 18.5, decreased albumin, and taking steroid drugs. The clinical test results, medical history, and medication information of patients with sepsis were collected to identify the immune status of the patients (whether they were immunosuppressed or not).

[0266] 4.2 Detection of HLA-DR positive rate in peripheral blood mononuclear cells of patients with sepsis

[0267] Collect EDTA anticoagulated blood from patients with sepsis, mix well, take 100 μL of blood, add 5 μL of PE-Anti-HLA-DR antibody (Biolegend), 5 μL of PerCP-CY5.5 Anti-CD14 antibody (Biolegend), mix well, and incubate in the dark for 30 min at 4°C. Add 900 μL of 1× BD FACS lysate (10× solution diluted with deionized water), mix well, and incubate in the dark for 15 min. Turn on the flow cytometer, adjust the voltage and compensation of each channel, and circle CD14 in the CD14-SSC graph. + For monocytes, collect 2,000 to 4,000 monocytes and circle the proportion of the PE-HLA-DR positive population.

[0268] 4.3 Detection of plasma IL-1R2 levels in patients with sepsis

[0269] EDTA anticoagulated blood was collected from patients and centrifuged at 4000 rpm for 15 min at 4°C. The supernatant was collected and the plasma IL-1R2 level was detected by ELISA using a kit for detecting IL-1R2 level prepared with anti-human IL-1R2 monoclonal antibodies 2E10 and 3D10.

[0270] 4.4 Comparative analysis of immune status

[0271] The immune status of the collected sepsis patients was determined based on multiple clinical indicators, as shown in Table 4 (absolute lymphocyte count ALC, monocyte HLA-DR positive rate, disease history, medication history, age, BMI, albumin level). A total of 47 sepsis patients were considered immunosuppressed, 20 sepsis patients were considered non-immunosuppressed, and 44 apparently healthy people (non-immunosuppressed) were added as controls (as shown in Table 5). ROC curves were drawn for the populations considered immunosuppressed and non-immunosuppressed, as shown in Table 4. Figure 4 As shown in the figure, the area under the ROC curve AUC = 0.9302, P < 0.0001, and AUC above 0.9, which has a high accuracy. According to the ROC curve, the cut-off value of IL-1R2 was calculated to be 24 ng / mL. Figure 5 The distribution of plasma IL-1R2 levels in three groups of people is shown. Among the 47 patients who were diagnosed as immunosuppressed according to clinical indicators, 39 had elevated IL-1R2 levels exceeding 24 ng / mL, with a sensitivity of 82.98%; among the 64 people who were clinically diagnosed as non-immunosuppressed, 58 had IL-1R2 levels below 24 ng / mL, with a specificity of 90.63%. Therefore, when the anti-human IL-1R2 monoclonal antibodies 2E10 and 3D10 are paired, the sensitivity of identifying the immunosuppressive state of patients is 82.98% and the specificity is 90.63% when the measured plasma IL-1R2 is greater than 24 ng / mL, which can reflect the immunosuppressive state of patients.

[0272] Table 4. Determination of immunosuppression status in patients with sepsis

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281] Table 5. Plasma IL-1R2 levels in apparently healthy subjects (non-immunosuppressed)

[0282]

[0283]

[0284] It is understood that although the present application describes the inventions involved in the above specific forms, these inventions are not limited to the specific contents described in these specific forms. It is obvious to those skilled in the art that, without departing from the spirit of the invention described in the present application, various equivalent changes may be made to the technical features included in the inventions involved therein, and these changes should all fall within the scope of the invention. Sequence information

[0285] The sequencing results of the antibody variable region sequences of this application were provided by Global Gene.

[0286] SEQ ID NO:1

[0287] MLRLYVLVMGVSAFTLQPAAHTGAARSCRFRGRHYKREFRLEGEPVALRCPQVPYWLWASVSPRINLTWHKNDSARTVPGEEETRMWAQDGALWLLPALQEDSGTYVCTTRNASYCDKMSIELRVFENTDAFLPFISYPQILTLSTSGVLVCPDLSEFTRDKTDVKIQWYK DSLLLDKDNEKFLSVRGTTHLLVHDVALEDAGYYRCVLTFAHEGQQYNITRSIELRIKKKKEETIPVIISPLKTISASLGSRLTIPCKVFLGTGTPLTTMLWWTANDTHIESAYPGGRVTEGPRQEYSENNENYIEVPLIFDPVTREDLHMDFKCVVHNTLSFQTLRTTVKE

[0288] SEQ ID NO:2

[0289] GYTFTSYA

[0290] SEQ ID NO:3

[0291] IYPSRDYT

[0292] SEQ ID NO:4

[0293] ARDGGFFGGHAMDY

[0294] SEQ ID NO:5

[0295] QNAV

[0296] SEQ ID NO:6

[0297] RAS

[0298] SEQ ID NO:7

[0299] QQGQSYPLT

[0300] SEQ ID NO:8

[0301] GYTFSSYW

[0302] SEQ ID NO:9

[0303] ILPGSGRN

[0304] SEQ ID NO:10

[0305] GRSGLY

[0306] SEQ ID NO:11

[0307] QSLLYSDGKTY

[0308] SEQ ID NO:12

[0309] LVS

[0310] SEQ ID NO:13

[0311] WQGTHSPFT

[0312] SEQ ID NO:14

[0313] QVQLQQSGAALARPGASVKMSCKASGYTFTSYAMHWVKQRPGRGLEWIGYIYPSRDYTNYNQRFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARDGGFFGGHAMDYWGQGTSVTVSS

[0314] SEQ ID NO:15

[0315] QVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVKQRPGHGLEWIGEILPGSGRNAYNEKFKGKATFTADSSSNTVYMQLSSLTSEDSAVYYCGRSGLYWGQGTLVTVSA

[0316] SEQ ID NO:16

[0317] DIQMNQSPSSLSASLGDTITITCHASQNINVWLSWYQQKPGNIPKLLIYRASNLHTGVPSRFSGSGSGTGFTFTISSLQPEDIATYYCQQGQSYPLTFGAGTKLELK

[0318] SEQ ID NO:17

[0319] DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSDGKTYLNWLLQRPGQSPKRLIYLVSNLDSGVPDRFTGSGSGTDFTLKIRRVEAEDLGVYYCWQGTHSPFTFGSGTKLEIK

[0320] SEQ ID NO:18

[0321] CAGGTCCAGCTGCAGCAGTCTGGGGCTGCACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACTAGCTACGCGATGCACTGGGTGAAACAGAGGCCTGGACGGGGTCTGGAATGGATTGGATACATTTATCCTAGTCGTGATTATACTAATTACAATCAGAGGTTCAAAGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGACGGAGGTTTCTTCGGGGGACATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

[0322] SEQ ID NO:19

[0323] QVQLQQSGAALARPGASVKMSCKAS

[0324] SEQ ID NO:20

[0325] MHWVKQRPGRGLEWIGY

[0326] SEQ ID NO:21

[0327] NYNQRFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYC

[0328] SEQ ID NO:22

[0329] WGQGTSVTVSS

[0330] SEQ ID NO:23

[0331] GACATCCAGATGAACCAGTCTCCATCCAGTCTGTCTGCATCCCTTGGAGACACAATTACCATCACTTGCCATGCCAGTCAGAACATTAATGTGTGGCTAAGCTGGTACCAGCAGAAACCAGGAAATATTCCTAAACTATTGATCTATAGGGCTTCCAACTTGCACACAGGCGTCCCATCAAGGTTTAGTGGCAGTGGATCTGGAACAGGTTTCACATTCACCATCAGCAGCCTGCAGCCTGAAGACATTGCCACTTACTACTGTCAACAGGGTCAAAGTTATCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0332] SEQ ID NO:24

[0333] DIQMNQSPSSLSASLGDTITITCHAS

[0334] SEQ ID NO:25

[0335] LSWYQQKPGNIPKLLIY

[0336] SEQ ID NO:26

[0337] NLHTGVPSRFSGSGSGTGFTFTISSLQPEDIATYYC

[0338] SEQ ID NO:27

[0339] FGAGTKLELK

[0340] SEQ ID NO:28

[0341] CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTACTGGCTACACATTCAGTAGCTACTGGATAGAGTGGGTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGTGGTCGTAATGCCTACAATGAGAAGTTCAAGGGCAAGGCCACGTTCACTGCAGATTCATCCTCCAACACAGTCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGGAAGATCTGGCCTCTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0342] SEQ ID NO:29

[0343] QVQLQQSGAELMKPGASVKISCKAT

[0344] SEQ ID NO:30

[0345] IEWVKQRPGHGLEWIGE

[0346] SEQ ID NO:31

[0347] AYNEKFKGKATFTADSSSNTVYMQLSSLTSEDSAVYYC

[0348] SEQ ID NO:32

[0349] WGQGTLVTVSA

[0350] SEQ ID NO:33

[0351] GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTATATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAATCTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGGAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTCTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA

[0352] SEQ ID NO:34

[0353] DVVMTQTPLTLSVTIGQPASISCKSS

[0354] SEQ ID NO:35

[0355] LNWLLQRPGQSPKRLIY

[0356] SEQ ID NO:36

[0357] NLDSGVPDRFTGSGSGTDFTLKIRRVEAEDLGVYYC

[0358] SEQ ID NO:37

[0359] FGSGTKLEIK

Claims

1. A method for identifying the immune status of a subject, comprising: determining the level of interleukin 1 receptor 2 (IL-1R2) in a sample from the subject; Preferably, the immune state is an immunosuppressive state; more preferably, the immune state is a relatively immunosuppressive state; and / or Preferably, the subject is a mammal; more preferably, the subject is a human; and / or Preferably, the subject suffers from systemic inflammatory response syndrome, cancer, dengue fever or colitis; more preferably, the systemic inflammatory response syndrome is sepsis or acute respiratory distress syndrome; and / or, the cancer is gastric cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, lung cancer or leukemia; and / or Preferably, the sample is selected from the group consisting of: plasma, blood, urine, serum, lymph, gastric juice, bile, saliva, sweat, cerebrospinal fluid and any combination thereof; more preferably, the sample is plasma, blood or urine; Optionally, the IL-1R2 is soluble IL-1R2 or membrane-bound protein IL-1R2.

2. The method of claim 1, wherein said determining the level of IL-1R2 in a sample from said subject comprises contacting said sample with a reagent that detects the level of IL-1R2; Preferably, the agent is a protein; more preferably, the agent is an antibody.

3. The method of claim 1 or 2, wherein said determining the level of IL-1R2 in a sample from said subject comprises contacting said sample with a first antibody and a second antibody, optionally, said contacting being simultaneous or sequential; in, The first antibody comprises a heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:3 and HCDR3 as shown in SEQ ID NO:4, and a light chain variable region comprising LCDR1 as shown in SEQ ID NO:5, LCDR2 as shown in SEQ ID NO:6 and LCDR3 as shown in SEQ ID NO:7; and / or The second antibody comprises a heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:8, HCDR2 as shown in SEQ ID NO:9, and HCDR3 as shown in SEQ ID NO:10, and a light chain variable region comprising LCDR1 as shown in SEQ ID NO:11, LCDR2 as shown in SEQ ID NO:12, and LCDR3 as shown in SEQ ID NO:13; Among them, the amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme; Preferably, the first antibody or the second antibody is labeled with a detectable marker, and the detectable marker is selected from: an enzyme, a fluorescent molecule, a radioisotope, a chemiluminescent molecule, a latex particle, a gold particle, a detectable ligand and any combination thereof; and / or Preferably, the first antibody or the second antibody is linked to a solid support; and / or Detecting the level of IL-1R2 in the sample by an immunological method; preferably, the immunological method is selected from the group consisting of: enzyme-linked immunosorbent assay, fluorescent immunoquantitative assay, chemiluminescent immunoassay, immunochromatography, immunoturbidimetric assay, immunoprecipitation and any combination thereof; more preferably, enzyme-linked immunosorbent assay; and / or Preferably, the level of IL-1R2 is compared with a cut-off value for the level of IL-1R2 used to identify the immune status of the subject; preferably, the cut-off value for the level of IL-1R2 used to identify the immune status of the subject is any value in the range of 19-29 ng / mL; more preferably, the cut-off value for the level of IL-1R2 used to identify the immune status of the subject is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 ng / mL; most preferably, the cut-off value for the level of IL-1R2 used to identify the immune status of the subject is 24 ng / mL; and / or Preferably, the immune state of the subject whose IL-1R2 level is greater than the cut-off value is identified as an immunosuppressive state; and / or, the immune state of the subject whose IL-1R2 level is less than the cut-off value is identified as a non-immunosuppressive state; Optionally, the immune status of the subject is further required to be identified in combination with at least one of the following clinical indicators: absolute number of lymphocytes, monocyte HLA-DR positivity rate, disease history, medication history, age, body mass index and albumin level.

4. Use of a reagent for detecting the level of IL-1R2 in a sample from a subject in the preparation of a product for identifying the immune status of the subject; Preferably, the immune state is an immunosuppressive state; more preferably, the immune state is a relatively immunosuppressive state; and / or Preferably, the subject is a mammal; more preferably, the subject is a human; and / or Preferably, the subject suffers from systemic inflammatory response syndrome, cancer, dengue fever or colitis; more preferably, the systemic inflammatory response syndrome is sepsis or acute respiratory distress syndrome; and / or, the cancer is gastric cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, lung cancer or leukemia; and / or Preferably, the sample is selected from the group consisting of: plasma, blood, urine, serum, lymph, gastric juice, bile, saliva, sweat, cerebrospinal fluid and any combination thereof; more preferably, the sample is plasma, blood or urine; and / or Preferably, the agent is a protein; more preferably, the agent is an antibody; and / or Preferably, the product for identifying the immune status of the subject is selected from the group consisting of: a test kit, a test strip, a test card, and a microfluidic detection device; Optionally, the IL-1R2 is soluble IL-1R2 or membrane-bound protein IL-1R2.

5. The use according to claim 4, wherein the antibody comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein The amino acid sequence of the HCDR1 is shown in SEQ ID NO:2, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:3, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:4, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:5, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:7; or The amino acid sequence of the HCDR1 is shown in SEQ ID NO:8, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:9, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:10, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:11, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:12, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:13; in, The amino acid sequences of the HCDRs and LCDRs are defined according to the IMGT numbering scheme.

6. The use according to claim 4 or 5, wherein the product for identifying the immune status of the subject comprises a first antibody and / or a second antibody; in, The first antibody comprises a heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:3, and HCDR3 as shown in SEQ ID NO:4, and a light chain variable region comprising LCDR1 as shown in SEQ ID NO:5, LCDR2 as shown in SEQ ID NO:6, and LCDR3 as shown in SEQ ID NO:7; and / or The second antibody comprises a heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:8, HCDR2 as shown in SEQ ID NO:9, and HCDR3 as shown in SEQ ID NO:10, and a light chain variable region comprising LCDR1 as shown in SEQ ID NO:11, LCDR2 as shown in SEQ ID NO:12, and LCDR3 as shown in SEQ ID NO:13; Among them, the amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme; Preferably, the first antibody or the second antibody is labeled with a detectable marker, and the detectable marker is selected from: an enzyme, a fluorescent molecule, a radioisotope, a chemiluminescent molecule, a latex particle, a gold particle, a detectable ligand and any combination thereof; and / or Preferably, the first antibody or the second antibody is linked to a solid support; and / or Preferably, the cut-off value of the IL-1R2 level for identifying the immune status of the subject is any value in the range of 19-29 ng / mL; more preferably, the cut-off value of the IL-1R2 level for identifying the immune status of the subject is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 ng / mL; most preferably, the cut-off value of the IL-1R2 level for identifying the immune status of the subject is 24 ng / mL; and / or Preferably, the immune state of the subject whose IL-1R2 level is greater than the cut-off value is identified as an immunosuppressive state; and / or, the immune state of the subject whose IL-1R2 level is less than the cut-off value is identified as a non-immunosuppressive state; Optionally, the immune status (eg, immunosuppressive status) of the subject needs to be identified in combination with at least one of the following clinical indicators: absolute lymphocyte count, monocyte HLA-DR positivity rate, disease history, medication history, age, body mass index, and albumin level.

7. An antibody that binds to human IL-1R2, comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein The amino acid sequence of the HCDR1 is shown in SEQ ID NO:2, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:3, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:4, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:5, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:7; or The amino acid sequence of the HCDR1 is shown in SEQ ID NO:8, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:9, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:10, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:11, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:12, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:13; in, The amino acid sequences of HCDRs and LCDRs are defined according to the IMGT numbering scheme; Preferably, the amino acid sequence of the heavy chain variable region of the antibody is at least 90% identical to SEQ ID NO: 14 or 15; and / or The amino acid sequence of the light chain variable region of the antibody is at least 90% identical to SEQ ID NO: 16 or 17; More preferably, the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO: 14 or 15; and / or The amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 16 or 17; Most preferably, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 16; or The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 17; Optionally, the human IL-1R2 is human soluble IL-1R2 or human membrane-bound protein IL-1R2.

8. The antibody of claim 7, wherein The antibody is a whole antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a single-chain Fv fragment (scFv), a Fd fragment, a single domain antibody or a dAb fragment; and / or The antibody is a monoclonal antibody, preferably a murinized monoclonal antibody; and / or The antibody comprises a heavy chain constant region selected from IgG1 subtype, IgG2 subtype or IgG4 subtype; preferably, the antibody comprises a heavy chain constant region of IgG1 subtype or IgG2 subtype; and / or The antibody comprises a light chain constant region selected from the κ subtype or the λ subtype; and / or The antibody binds to human IL-1R2 whose amino acid sequence is shown in SEQ ID NO: 1; and / or The antibody is produced by a hybridoma cell line with a deposit number of CCTCC NO: C2024249 or CCTCC NO: C2024250.

9. A hybridoma cell that produces an antibody that binds to human IL-1R2, which is deposited with CCTCC NO: C2024249 or CCTCC NO: C2024250; Optionally, the human IL-1R2 is human soluble IL-1R2 or human membrane-bound protein IL-1R2.

10. Use of the antibody according to claim 7 or 8, or the hybridoma cell according to claim 9, in the preparation of a product for detecting the level of human IL-1R2 in a sample; Preferably, the product for detecting the level of human IL-1R2 in a sample is selected from the group consisting of: a test kit, a test strip, a test card and a microfluidic detection device; and / or Preferably, the sample is selected from the group consisting of: plasma, blood, urine, serum, lymph, gastric juice, bile, saliva, sweat, cerebrospinal fluid and any combination thereof; more preferably, the sample is plasma, blood or urine; Optionally, the human IL-1R2 is human soluble IL-1R2 or human membrane-bound protein IL-1R2.

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