Use of il-1r2 in identifying immune status in a subject

Abstract

The present application provides methods of identifying the immune status of a subject, and use of a reagent for detecting IL-1R2 levels in a sample from a subject in the manufacture of a product for identifying the immune status of the subject. The present application also provides antibodies that bind human IL-1R2 and uses thereof, and hybridoma cells that produce antibodies that bind human IL-1R2 and uses thereof.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological detection and antibody preparation, and in particular, the present application provides a method for identifying the immune status of a subject, use of a reagent for detecting IL-1R2 level in a sample from a subject in the preparation of a product for identifying the immune status of the subject, an antibody binding to human IL-1R2 and use thereof, and a hybridoma cell producing an antibody binding to human IL-1R2 and use thereof. BACKGROUND

[0002] Systemic inflammatory response syndrome triggered 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 currently the leading cause of death in hospital and outside of hospital. A recent statistical report highlights that there are nearly 50 million sepsis patients worldwide each year, and the age of onset covers all ages. The number of deaths from sepsis accounted for nearly 20% of all deaths worldwide in 2017. In addition, patients who have had sepsis before have a higher risk of readmission and death. Sepsis is a serious threat to human health and also causes a huge social and economic burden.

[0003] Immune dysfunction is closely related to the occurrence and development of sepsis, and patients may show both excessive inflammatory response and immune suppression. The former can trigger early tissue damage and organ dysfunction, while the latter, when severe and persistent, can further induce a variety of fatal complications, thereby significantly increasing the mortality of patients in the middle and late stages of sepsis. In recent years, with the continuous improvement of organ support technology, the mortality rate in the pro-inflammatory response stage has decreased, but the mortality rate in the immune suppression stage remains high, so the research focus of immune dysfunction in sepsis has shifted from excessive inflammation to immune suppression. However, for a long time, people have had insufficient understanding of the exact mechanism of immune dysfunction caused by severe injury 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 sepsis patients and determination of the timing of immune regulation therapy. However, due to insufficient understanding of the precise molecular mechanisms and cellular basis of sepsis-induced immune suppression, there is a lack of clinical monitoring indicators and evaluation systems that can effectively reflect the immune status of sepsis patients.

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

[0005] Therefore, it is of important biological and medical significance to develop new methods and products for identifying the immune status of patients with sepsis or even systemic inflammatory response syndrome. SUMMARY

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

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

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

[0009] In one or more embodiments of the present application, the determination of 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 of the amino acid sequence as shown in SEQ ID NO: 2, HCDR2 of the amino acid sequence as shown in SEQ ID NO: 3 and HCDR3 of the amino acid sequence as shown in SEQ ID NO: 4, and a light chain variable region comprising LCDR1 of the amino acid sequence as shown in SEQ ID NO: 5, LCDR2 of the amino acid sequence as shown in SEQ ID NO: 6 and LCDR3 of the amino acid sequence as shown in SEQ ID NO: 7; and / or

[0011] the second antibody comprises a heavy chain variable region comprising a HCDR1 of an amino acid sequence as set forth in SEQ ID NO: 8, a HCDR2 of an amino acid sequence as set forth in SEQ ID NO: 9, and a HCDR3 of an amino acid sequence as set forth in SEQ ID NO: 10, and a light chain variable region comprising a LCDR1 of an amino acid sequence as set forth in SEQ ID NO: 11, a LCDR2 of an amino acid sequence as set forth in SEQ ID NO: 12, and a LCDR3 of an amino acid sequence as set forth in SEQ ID NO: 13;

[0012] wherein the amino acid sequences of the HCDRs and LCDRs 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 the sample is detected by an immunological method.

[0015] In one or more embodiments of the present application, the level of IL-1R2 is compared with 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 a level of IL-1R2 in a sample from a subject in the manufacture of a product for identifying the immune status of the subject.

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

[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 has systemic inflammatory response syndrome, cancer, dengue fever or colitis.

[0020] In one or more embodiments of the present application, the reagent 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 a soluble IL-1R2 or a membrane-bound protein IL-1R2.

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

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

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

[0026] wherein the amino acid sequences of the HCDRs and the LCDRs are defined according to the IMGT numbering scheme.

[0027] In one or more embodiments of the 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 a HCDR1 having an amino acid sequence set forth in SEQ ID NO: 2, a HCDR2 having an amino acid sequence set forth in SEQ ID NO: 3, and a HCDR3 having an amino acid sequence set forth in SEQ ID NO: 4, and a light chain variable region comprising a LCDR1 having an amino acid sequence set forth in SEQ ID NO: 5, a LCDR2 having an amino acid sequence set forth in SEQ ID NO: 6, and a LCDR3 having an amino acid sequence set forth in SEQ ID NO: 7; and / or

[0029] the second antibody comprises a heavy chain variable region comprising a HCDR1 of an amino acid sequence as set forth in SEQ ID NO: 8, a HCDR2 of an amino acid sequence as set forth in SEQ ID NO: 9, and a HCDR3 of an amino acid sequence as set forth in SEQ ID NO: 10, and a light chain variable region comprising a LCDR1 of an amino acid sequence as set forth in SEQ ID NO: 11, a LCDR2 of an amino acid sequence as set forth in SEQ ID NO: 12, and a LCDR3 of an amino acid sequence as set forth in SEQ ID NO: 13;

[0030] wherein the amino acid sequences of the HCDRs and the LCDRs 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 for identifying the immune status of the subject is any value in the range of 19-29 ng / mL.

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

[0033] In one or more embodiments of the present application, the immune status (e.g. immunosuppressed status) of the subject is further 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 binding to human IL-1R2, comprising a heavy chain variable region comprising a HCDR1, a HCDR2, and a HCDR3, and a light chain variable region comprising a LCDR1, a LCDR2, and a LCDR3, wherein

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

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

[0037] wherein the amino acid sequences of the HCDRs and the LCDRs 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 to SEQ ID NO: 14 or 15; and / or

[0039] the amino acid sequence of the light chain variable region of the antibody has at least 90% identity 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 group consisting of an IgG1 subtype, an IgG2 subtype, or an IgG4 subtype; and / or

[0043] the antibody comprises a light chain constant region selected from the group consisting of a kappa subtype or a lambda subtype; and / or

[0044] the antibody binds to human IL-1R2 having the amino acid sequence set forth in SEQ ID NO: 1.

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

[0046] In a fourth aspect, the present application provides a hybridoma cell producing an antibody that binds to human IL-1R2, which is deposited under the accession number 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, which comprises a first antibody and / or a second antibody;

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

[0049] the second antibody comprises a heavy chain variable region comprising a HCDR1 of an amino acid sequence as set forth in SEQ ID NO: 8, a HCDR2 of an amino acid sequence as set forth in SEQ ID NO: 9, and a HCDR3 of an amino acid sequence as set forth in SEQ ID NO: 10, and a light chain variable region comprising a LCDR1 of an amino acid sequence as set forth in SEQ ID NO: 11, a LCDR2 of an amino acid sequence as set forth in SEQ ID NO: 12, and a LCDR3 of an amino acid sequence as set forth in SEQ ID NO: 13;

[0050] wherein the amino acid sequences of the HCDRs and LCDRs are defined according to the IMGT numbering scheme.

[0051] In a sixth aspect, the present application provides a method for detecting a 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 contacting;

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

[0053] the second antibody comprises a heavy chain variable region comprising a HCDR1 of an amino acid sequence as set forth in SEQ ID NO: 8, a HCDR2 of an amino acid sequence as set forth in SEQ ID NO: 9, and a HCDR3 of an amino acid sequence as set forth in SEQ ID NO: 10, and a light chain variable region comprising a LCDR1 of an amino acid sequence as set forth in SEQ ID NO: 11, a LCDR2 of an amino acid sequence as set forth in SEQ ID NO: 12, and a LCDR3 of an amino acid sequence as set forth in SEQ ID NO: 13;

[0054] wherein the amino acid sequences of HCDRs and LCDRs are defined according to IMGT numbering scheme.

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

[0056] In a seventh aspect, the present application provides use of the antibody of the third aspect, or the hybridoma cell of the fourth aspect, in the manufacture of 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 label selected from the group consisting of an enzyme, a fluorescent molecule, a radioisotope, a chemiluminescent molecule, a latex particle, a gold particle, a detectable ligand, and any combination thereof.

[0060] In one or more embodiments of the present application, the first antibody or the second antibody is attached 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, spinal fluid, and any combination thereof. BRIEF DESCRIPTION OF DRAWINGS

[0062] Figure 1 The results of correct expression and purity identification of human soluble IL-1R2 recombinant protein are shown. Among them, 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 binding and dissociation curves of SPR detection of different concentrations of monoclonal antibody 2E10 and IL-1R2 protein are shown.

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

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

[0066] Figure 5 Results of plasma IL-1R2 levels in healthy subjects, non-immunosuppressed sepsis patients and immunosuppressed sepsis patients detected using the pair of monoclonal antibodies 2E10 and 3D10.

[0067] DEPOSIT DESCRIPTION

[0068] 1. Cell name: Hybridoma cell line ZIL1R-2E10

[0069] Depository: China Center for Type Culture Collection

[0070] Abbreviation of the depository: CCTCC

[0071] Address: Wuhan University, Wuhan, China (Wuchang District, Wuhan City, Hubei Province, China, 299 Baoyi Road)

[0072] Date of deposit: August 22, 2024

[0073] Accession number: CCTCC NO: C2024249

[0074] 2. Cell name: Hybridoma cell line ZIL1R-3D10

[0075] Depository: China Center for Type Culture Collection

[0076] Abbreviation of the depository: CCTCC

[0077] Address: Wuhan University, Wuhan, China (Wuchang District, Wuhan City, Hubei Province, China, 299 Baoyi Road)

[0078] Date of deposit: August 22, 2024

[0079] Accession number: CCTCC NO: C2024250

[0080] SEQUENCE DESCRIPTION

[0081] SEQ ID NO: 1 shows the amino acid sequence of soluble IL-1R2 from position 1 to 343 of Uniprot No. P27930-1.

[0082] SEQ ID NO: 2-4 show the amino acid sequences of HCDR1, HCDR2 and HCDR3 of monoclonal antibody 2E10 against soluble human IL-1R2, respectively.

[0083] SEQ ID NOs: 5-7 show the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively, of monoclonal antibody 2E10 against soluble human IL-1R2.

[0084] SEQ ID NOs: 8-10 show the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively, of monoclonal antibody 3D10 against soluble human IL-1R2.

[0085] SEQ ID NOs: 11-13 show the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively, of monoclonal antibody 3D10 against soluble human IL-1R2.

[0086] SEQ ID NO: 14 shows the amino acid sequence of the heavy chain variable region of monoclonal antibody 2E10 against soluble human IL-1R2.

[0087] SEQ ID NO: 15 shows the amino acid sequence of the heavy chain variable region of monoclonal antibody 3D10 against soluble human IL-1R2.

[0088] SEQ ID NO: 16 shows the amino acid sequence of the light chain variable region of monoclonal antibody 2E10 against soluble human IL-1R2.

[0089] SEQ ID NO: 17 shows the amino acid sequence of the light chain variable region of monoclonal antibody 3D10 against soluble human IL-1R2.

[0090] SEQ ID NO: 18 shows the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 2E10 against soluble human IL-1R2.

[0091] SEQ ID NO: 19 shows the amino acid sequence of FR1 of the heavy chain variable region of monoclonal antibody 2E10 against soluble human IL-1R2.

[0092] SEQ ID NO: 20 shows the amino acid sequence of FR2 of the heavy chain variable region of monoclonal antibody 2E10 against soluble human IL-1R2.

[0093] SEQ ID NO: 21 shows the amino acid sequence of FR3 of the heavy chain variable region of monoclonal antibody 2E10 against soluble human IL-1R2.

[0094] SEQ ID NO: 22 shows the amino acid sequence of FR4 of the heavy chain variable region of monoclonal antibody 2E10 against soluble human IL-1R2.

[0095] SEQ ID NO: 23 shows a nucleotide sequence encoding the variable region of the light chain of monoclonal antibody 2E10 against soluble human IL-1R2.

[0096] SEQ ID NO: 24 shows an amino acid sequence of FR1 of the variable region of the light chain of monoclonal antibody 2E10 against soluble human IL-1R2.

[0097] SEQ ID NO: 25 shows an amino acid sequence of FR2 of the variable region of the light chain of monoclonal antibody 2E10 against soluble human IL-1R2.

[0098] SEQ ID NO: 26 shows an amino acid sequence of FR3 of the variable region of the light chain of monoclonal antibody 2E10 against soluble human IL-1R2.

[0099] SEQ ID NO: 27 shows an amino acid sequence of FR4 of the variable region of the light chain of monoclonal antibody 2E10 against soluble human IL-1R2.

[0100] SEQ ID NO: 28 shows a nucleotide sequence encoding the variable region of the heavy chain of monoclonal antibody 3D10 against soluble human IL-1R2.

[0101] SEQ ID NO: 29 shows an amino acid sequence of FR1 of the variable region of the heavy chain of monoclonal antibody 3D10 against soluble human IL-1R2.

[0102] SEQ ID NO: 30 shows an amino acid sequence of FR2 of the variable region of the heavy chain of monoclonal antibody 3D10 against soluble human IL-1R2.

[0103] SEQ ID NO: 31 shows an amino acid sequence of FR3 of the variable region of the heavy chain of monoclonal antibody 3D10 against soluble human IL-1R2.

[0104] SEQ ID NO: 32 shows an amino acid sequence of FR4 of the variable region of the heavy chain of monoclonal antibody 3D10 against soluble human IL-1R2.

[0105] SEQ ID NO: 33 shows a nucleotide sequence encoding the variable region of the light chain of monoclonal antibody 3D10 against soluble human IL-1R2.

[0106] SEQ ID NO: 34 shows an amino acid sequence of FR1 of the variable region of the light chain of monoclonal antibody 3D10 against soluble human IL-1R2.

[0107] SEQ ID NO: 35 shows an amino acid sequence of FR2 of the variable region of the light chain of monoclonal antibody 3D10 against soluble human IL-1R2.

[0108] SEQ ID NO: 36 shows an amino acid sequence encoding FR3 of the light chain variable region of monoclonal antibody 3D10 against soluble human IL-1R2.

[0109] SEQ ID NO: 37 shows an amino acid sequence encoding FR4 of the light chain variable region of monoclonal antibody 3D10 against soluble human IL-1R2. DETAILED DESCRIPTION

[0110] Interleukin-1 (IL-1) is a cytokine with potent pro-inflammatory and amplification of immune responses, mainly produced by macrophages during the defense response. IL-1 is one of several pro-inflammatory cytokines produced during infection, sepsis and systemic inflammatory response syndrome (SIRS), used to initiate the host inflammatory response and integrate the non-specific immunity. IL-1 is an important mediator involved in the pathogenesis of SIRS. The dominant cytokines in the early stage of SIRS are IL-1 and TNF-a, which induce the release of other pro-inflammatory cytokines (IL-6, IL-8, IFN-γ) and trigger the coagulation cascade. IL-1 can also induce the production of TNF-a.

[0111] 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. IL-1R2 gene is located on chromosome 2q12, like other members of the IL-1 receptor family, it is composed of an extracellular part containing three glycosylated immunoglobulin (Ig) like domains, and has 28% amino acid homology with the extracellular part of IL-1R1. But compared with other members of the family, it cannot initiate downstream signal transduction due to its short cytoplasmic structure and lack of Toll / IL-1 receptor (TIR) domain. IL-1R2 protein exists in two forms: cell membrane bound and soluble (sIL-1R2).

[0112] IL-1 forms an IL-1 / IL-1R1 / IL-1RAP complex with IL-1R1 under the action of IL-1RAP, activates the downstream signal transduction pathway of IL-1, and plays a pro-inflammatory biological function. Membrane-bound protein IL-1R2 competitively captures IL-1 on the cell surface, while sIL-1R2 binds IL-1 in the extracellular microenvironment, both preventing IL-1 from binding to IL-1R1 on the cell surface and preventing the initiation of cell activation, which is one of the main mechanisms to inhibit IL-1 activity, and is defined as a decoy receptor.

[0113] IL-1R2 has a high affinity for IL-1β and a low affinity for interleukin 1 receptor antagonist (IL-1Ra), and thus can play a role in inhibiting the activity of IL-1 and is only partially interfered by IL-1Ra. Compared with the membrane-bound protein IL-1R2, sIL-1R2 has different ligand binding capacity. The binding capacity of sIL-1R2 to IL-1β is comparable to that of the membrane receptor, but the binding capacity to IL-1α is superior to that of the membrane receptor. 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 IL-1β precursor Pro-IL-1β, preventing Pro-IL-1β from being processed into mature IL-1β by protease in cells, thereby playing a negative regulatory role in inflammatory response. In summary, soluble IL-1R2 can inhibit immune response by inhibiting the pro-inflammatory effect of IL-1.

[0114] The inventors of the present application found that the plasma level of soluble IL-1R2 is significantly increased in critically ill patients with sepsis and other systemic inflammatory response syndromes, and the plasma IL-1R2 level is positively correlated with the severity of the disease. The increase of IL-1R2 reflects the imbalance of IL-1, indicating the transition of SIRS to compensatory anti-inflammatory response syndrome (CARS). In addition, anti-inflammatory factors such as IL-4, IL-10, glucocorticoids, etc. can up-regulate IL-1R2. IL-1R2 mainly plays a negative regulatory role in the inflammatory response of different cell types. In summary, the increase of soluble IL-1R2 indicates that the patient is in a relatively immunosuppressed state, and can be used as one of the auxiliary detection indicators of the immunosuppressed state of patients with sepsis and even systemic inflammatory response syndrome.

[0115] In addition, in order to realize the determination of the level of human IL-1R2 (especially human soluble IL-1R2), the inventors of the present application also obtained new antibodies binding to human IL-1R2 (especially human soluble IL-1R2) through antibody engineering technology.

[0116] In various aspects of the present application, methods for identifying the immune status of a subject, uses of reagents for detecting the level of IL-1R2 in a sample from a subject in the preparation of products for identifying the immune status of the subject, antibodies binding to human IL-1R2 and uses thereof, hybridoma cells producing antibodies binding to human IL-1R2 and uses thereof, 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 are provided.

[0117] The practice of the present application will employ, unless otherwise indicated, conventional molecular biology, microbiology, cell biology, biochemistry, and immunology techniques.

[0118] The terms used in this application have the meanings as commonly understood by a person of ordinary skill in the art, unless otherwise indicated.

[0119] Unless otherwise indicated, all experimental reagents are commercially available.

[0120] Definitions

[0121] As used herein, the term "immune status" refers to the functional condition and responsiveness of an individual's immune system at a particular time. It reflects the responsiveness of the immune system to antigens (such as pathogens, vaccines, or other foreign substances) and overall health status.

[0122] As used herein, the term "immunosuppressed state" refers to the weakening or suppression of an individual's immune system function, resulting in reduced responsiveness to antigens (such as pathogens, tumor cells, or vaccines). Such a state can make the individual more susceptible to infection, development of certain diseases, or difficulty in effectively fighting off disease.

[0123] As used herein, the term "relatively immunosuppressed state" refers to a partial weakening or suppression of an individual's immune system function under certain specific conditions or relative to a healthy standard, but this suppression is not absolute or complete. This state can make the individual more susceptible to infection or other immune-related issues in specific situations (such as long-term hospitalization), but still maintains some degree of immune responsiveness in other situations.

[0124] As used herein, the term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) and two light (L) chains interconnected by disulfide bonds, as well as 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, 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 and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). In some embodiments, both light and heavy chain variable regions comprise, from N-terminus to C-terminus, FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0125] An antibody specifically binds to a target 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. As used herein, "antibody" includes not only intact (i.e., full-length) antibodies, but also antigen binding fragments thereof, variants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0126] Full-length antibodies can be of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), although the antibody need not be of any particular class. An immunoglobulin can be assigned to a class based on the amino acid sequence of its constant regions. In general, immunoglobulins belong to one of five major classes: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy chain regions that correspond to the different classes of immunoglobulins are designated a, d, e, g, and m, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0127] As used herein, the term "antigen binding fragment" of an antibody refers to a portion or segment of an intact antibody molecule responsible for binding an antigen. An antigen binding fragment can comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. An antigen binding fragment of an antibody can be prepared using any suitable standard techniques, including proteolytic digestion or recombinant genetic engineering techniques. 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 minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated CDR). Antigen binding fragments can also include other engineered molecules, such as diabodies, triabodies, tetrabodies, and minibodies, etc. For example, an Fd fragment as described herein refers to an antibody fragment consisting of a VH and CH1 domain; an Fv fragment consists of a VL and VH domain of a single arm of an antibody; a dAb fragment (Ward et al., Nature 1989; 341 :544-546) consists of a VH domain.

[0128] It is well known to those skilled in the art that the complementarity determining regions (CDRs, typically CDR1, CDR2, and CDR3) are the regions of the variable region that have the greatest impact on the affinity and specificity of an antibody. The CDR amino acid sequences of a VH or VL are defined by common definitions, such as the IMGT numbering scheme definition, the Chothia numbering scheme definition, and the Kabat numbering scheme definition. See, e.g., 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 a given variable region amino acid sequence of an 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.

[0129] For a given variable region amino acid sequence of an antibody, the CDR amino acid sequences in the variable region amino acid sequence can be analyzed in a number of ways, such as can be determined using the online software Abysis (http: / / www.abysis.org / ).

[0130] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an epitope of an antigen, e.g., the ability of an antibody to bind to a specific antigen with an affinity that is at least two-fold greater than its affinity for a non-specific antigen. It will be appreciated, however, that an antibody can be able to specifically bind to two or more antigens that are related in sequence.

[0131] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts.

[0132] As used herein, the term "murinized antibody" refers to any antibody in which all of the constant region sequences are of mouse sequence. Such antibodies can be produced by a hybridoma.

[0133] As used herein, the term "reactivity of a monoclonal antibody" refers to the ability of a monoclonal antibody to bind to an antigen under suitable reaction conditions.

[0134] As used herein, the term "cell line" refers to a single cell culture obtained from a primary culture or cell line by either selection or limiting dilution methods.

[0135] As used herein, the term "homology" is defined as the percentage of residues in an amino acid or nucleic acid sequence variant that are identical after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent 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%, for example 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0136] As used herein, the term "cut-off value" is a threshold value used to determine whether a test result is abnormal. In medical testing, the cut-off value is a value used to determine whether a test result is above or below a clinical or analytical decision point. If the test result is below the cut-off value, the test result is negative, and if the test result is above the cut-off value, the test result is positive. In some embodiments of the present application, when the level of human plasma IL-1R2 is higher than the cut-off value, it indicates that the patient is in an immunosuppressed state (e.g., a relative immunosuppressed state); when the level of human plasma IL-1R2 is lower than the cut-off value, it indicates that the patient is in a non-immunosuppressed state.

[0137] In a first aspect, the present application provides a method of identifying an immune status of a subject, comprising:

[0138] determining a level of interleukin 1 receptor 2 (IL-1R2) in a sample from the subject.

[0139] In one or more embodiments of the present application, the determining a level of IL-1R2 in a sample from the subject comprises contacting the sample with a reagent that detects the level of IL-1R2.

[0140] In one or more embodiments of the present application, the determining a level of IL-1R2 in a sample from the subject comprises contacting the sample with a first antibody and a second antibody,

[0141] wherein the first antibody comprises a heavy chain variable region comprising a HCDR1 of an amino acid sequence as set forth in SEQ ID NO: 2, a HCDR2 of an amino acid sequence as set forth in SEQ ID NO: 3, and a HCDR3 of an amino acid sequence as set forth in SEQ ID NO: 4, and a light chain variable region comprising a LCDR1 of an amino acid sequence as set forth in SEQ ID NO: 5, a LCDR2 of an amino acid sequence as set forth in SEQ ID NO: 6, and a LCDR3 of an amino acid sequence as set forth in SEQ ID NO: 7; and / or

[0142] the second antibody comprises a heavy chain variable region comprising a HCDR1 of an amino acid sequence as set forth in SEQ ID NO: 8, a HCDR2 of an amino acid sequence as set forth in SEQ ID NO: 9, and a HCDR3 of an amino acid sequence as set forth in SEQ ID NO: 10, and a light chain variable region comprising a LCDR1 of an amino acid sequence as set forth in SEQ ID NO: 11, a LCDR2 of an amino acid sequence as set forth in SEQ ID NO: 12, and a LCDR3 of an amino acid sequence as set forth in SEQ ID NO: 13;

[0143] wherein the amino acid sequences of HCDR and LCDR are defined according to IMGT numbering scheme.

[0144] 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 (e.g. immune suppression status) of a subject.

[0145] In one or more embodiments of the present application, the level of IL-1R2 is compared with a cut-off value of IL-1R2 level for identifying the immune status of the subject.

[0146] 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 manufacture of a product for identifying the immune status of the subject.

[0147] In one or more embodiments of the present application, the immune status is immune suppression status. In one or more embodiments of the present application, the immune status is relative immune suppression status.

[0148] In one or more embodiments of the present application, the subject is a mammal, e.g. a human.

[0149] In one or more embodiments of the application, the subject has systemic inflammatory response syndrome, cancer, dengue fever, or colitis. In one or more embodiments of the application, the systemic inflammatory response syndrome is sepsis or acute respiratory distress syndrome. In one or more embodiments of the application, the cancer is gastric cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, lung cancer, or leukemia.

[0150] In one or more embodiments of the application, the agent is a protein, e.g., an antibody. In one or more embodiments of the application, the antibody can be any antibody capable of binding to IL-1R2, e.g., a commercially available antibody that binds to IL-1R2.

[0151] In one or more embodiments of the application, the IL-1R2 is a soluble IL-1R2 or a membrane-bound protein IL-1R2. In one or more embodiments of the application, the IL-1R2 is a soluble IL-1R2, e.g., a human soluble IL-1R2.

[0152] In one or more embodiments of the 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

[0153] the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 3, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 4, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 5, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 6, and the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 7; or

[0154] the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 8, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 9, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 11, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 12, and the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 13;

[0155] wherein the amino acid sequences of the HCDRs and the LCDRs are defined according to the IMGT numbering scheme.

[0156] 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 (e.g., a colloidal gold detection test strip), a detection card, and a microfluidic detection device.

[0157] 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.

[0158] In one or more embodiments of the present application, the first antibody comprises a heavy chain variable region comprising a HCDR1 of an amino acid sequence as set forth in SEQ ID NO: 2, a HCDR2 of an amino acid sequence as set forth in SEQ ID NO: 3, and a HCDR3 of an amino acid sequence as set forth in SEQ ID NO: 4, and a light chain variable region comprising a LCDR1 of an amino acid sequence as set forth in SEQ ID NO: 5, a LCDR2 of an amino acid sequence as set forth in SEQ ID NO: 6, and a LCDR3 of an amino acid sequence as set forth in SEQ ID NO: 7; and / or

[0159] the second antibody comprises a heavy chain variable region comprising a HCDR1 of an amino acid sequence as set forth in SEQ ID NO: 8, a HCDR2 of an amino acid sequence as set forth in SEQ ID NO: 9, and a HCDR3 of an amino acid sequence as set forth in SEQ ID NO: 10, and a light chain variable region comprising a LCDR1 of an amino acid sequence as set forth in SEQ ID NO: 11, a LCDR2 of an amino acid sequence as set forth in SEQ ID NO: 12, and a LCDR3 of an amino acid sequence as set forth in SEQ ID NO: 13.

[0160] In one or more embodiments of the present application, the amino acid sequences of the HCDRs and the LCDRs are defined according to the IMGT numbering scheme.

[0161] In one or more embodiments of the present application, the product for identifying the immune status of the subject can further comprise other reagents for detection, such as a substrate, a reference standard, a diluent, a washing solution, and the like.

[0162] In one or more embodiments of the present application, the product for identifying the immune status of the subject can further comprise a product instruction manual.

[0163] In one or more embodiments of the present application, the product for identifying the immune status of the subject can further comprise a container for mixing the sample with the antibody. Such a container can be suitable for a detection instrument capable of detecting a signal generated by the detection monoclonal antibody.

[0164] 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 an immunosuppressed state.

[0165] In one or more embodiments of the application, the cut-off value for the IL-1R2 level used to identify the immune status of the subject is any value in the range of 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, 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 application, the cut-off value for the IL-1R2 level used to identify 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 application, the cut-off value for the IL-1R2 level used to identify the immune status of the subject is 24 ng / mL.

[0166] In one or more specific embodiments of the application, the immune status of the subject is identified as immune-suppressed if the IL-1R2 level is greater than the cut-off value. In one or more embodiments of the application, the immune status of the subject is identified as non-immune-suppressed if the IL-1R2 level is less than the cut-off value.

[0167] In one or more embodiments of the application, the immune status of the subject (e.g., immune-suppressed status) is further 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.

[0168] In one or more embodiments of the present application, the immune status (e.g., immune suppression status) of the subject also needs to be identified in combination with the absolute number of lymphocytes and the HLA-DR positive rate of mononuclear cells. In one or more embodiments of the present application, the immune status of the patient whose absolute number of lymphocytes in the blood routine report for three consecutive days is less than 1.0 x 10 9 9 / L; and / or the HLA-DR positive rate of peripheral blood mononuclear cells is less than 60% is identified as an immune suppression status, e.g., a relative immune suppression status. In one or more embodiments of the present application, whether the patient is in an immune suppression status needs to be identified by the doctor in combination with clinical indicators (e.g., factors such as the absolute number of lymphocytes and / or the HLA-DR positive rate of peripheral blood mononuclear cells, etc.).

[0169] In a third aspect, the present application provides an antibody binding 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

[0170] the amino acid sequence of the HCDR1 is shown as SEQ ID NO: 2, the amino acid sequence of the HCDR2 is shown as SEQ ID NO: 3, the amino acid sequence of the HCDR3 is shown as SEQ ID NO: 4, the amino acid sequence of the LCDR1 is shown as SEQ ID NO: 5, the amino acid sequence of the LCDR2 is shown as SEQ ID NO: 6, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO: 7; or

[0171] the amino acid sequence of the HCDR1 is shown as SEQ ID NO: 8, the amino acid sequence of the HCDR2 is shown as SEQ ID NO: 9, the amino acid sequence of the HCDR3 is shown as SEQ ID NO: 10, the amino acid sequence of the LCDR1 is shown as SEQ ID NO: 11, the amino acid sequence of the LCDR2 is shown as SEQ ID NO: 12, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO: 13;

[0172] wherein the amino acid sequences of the HCDRs and the LCDRs are defined according to the IMGT numbering scheme.

[0173] 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, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, to SEQ ID NO: 14 or 15.

[0174] In one or more embodiments of the application, the amino acid sequence of the heavy chain variable region of the antibody has at least 90% identity to SEQ ID NO: 14 or 15, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity.

[0175] In one or more embodiments of the application, the amino acid sequence of the heavy chain variable region of the antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to the amino acid sequence set forth in SEQ ID NO: 14 or 15; and the amino acid sequence of the light chain variable region of the antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to the amino acid sequence set forth in SEQ ID NO: 16 or 17.

[0176] In one or more embodiments of the application, the amino acid sequence of the heavy chain variable region of the antibody differs from the amino acid sequence set forth in SEQ ID NO: 14 or 15 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, deletions, and / or additions of amino acids.

[0177] In one or more embodiments of the application, the amino acid sequence of the light chain variable region of the antibody differs from the amino acid sequence set forth in SEQ ID NO: 16 or 17 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, deletions, and / or additions of amino acids.

[0178] In one or more embodiments of the application, the C-terminal or N-terminal region of the amino acid sequence set forth 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 retaining similar functionality of the heavy chain variable region of the antibody.

[0179] In one or more embodiments of the application, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids can also be added to the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 14 or 15, and the resulting amino acid sequence still retains similar functionality of the heavy 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 or deleted from a region other than the C-terminus or N-terminus of the amino acid sequence set forth in SEQ ID NO: 14 or 15, as long as the altered amino acid sequence substantially retains similar function of the heavy chain variable region of the antibody.

[0181] In one or more embodiments of the present application, the C-terminal or N-terminal region of the amino acid sequence set forth 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 retaining similar function of the light chain variable region of the antibody.

[0182] 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 set forth in SEQ ID NO: 16 or 17, and the resulting amino acid sequence still retains similar function of the light chain variable region of the antibody.

[0183] 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 or deleted from a region other than the C-terminus or N-terminus of the amino acid sequence set forth in SEQ ID NO: 16 or 17, as long as the altered amino acid sequence substantially retains similar function of the light chain variable region of the antibody.

[0184] In one or more embodiments of the present application, the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 14 or 15.

[0185] In one or more embodiments of the present application, the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 16 or 17.

[0186] In one or more specific embodiments of the present application, the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 16; or

[0187] the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 17.

[0188] In one or more embodiments of the 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.

[0189] In one or more embodiments of the application, the antibody is a monoclonal antibody.

[0190] In one or more embodiments of the application, the antibody is a murinized monoclonal antibody.

[0191] In one or more embodiments of the application, the antibody comprises a heavy chain constant region selected from the IgGl subtype, the IgG2 subtype, or the IgG4 subtype.

[0192] In one or more embodiments of the application, the antibody comprises a heavy chain constant region of the IgGl subtype or the IgG2 subtype.

[0193] In one or more embodiments of the application, when the antibody is the anti-soluble IL-1R2 monoclonal antibody 2E10, it comprises a heavy chain constant region of the IgGl subtype.

[0194] In some 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 (e.g., the IgG2a subtype).

[0195] In one or more embodiments of the application, the antibody comprises a light chain constant region selected from the kappa subtype or the lambda subtype.

[0196] In one or more embodiments of the application, the antibody binds to human IL-1R2 having the amino acid sequence set forth in SEQ ID NO: 1.

[0197] In one or more embodiments of the application, the antibody is produced by the hybridoma cell strain having the accession number CCTCC NO: C2024249 or CCTCC NO: C2024250.

[0198] In one or more embodiments of the application, the anti-soluble human IL-1R2 monoclonal antibody 2E10 is produced by the hybridoma cell strain having the accession number CCTCC NO: C2024249.

[0199] In one or more embodiments of the application, the anti-soluble human IL-1R2 monoclonal antibody 3D10 is produced by the hybridoma cell strain having the accession number CCTCC NO: C2024250.

[0200] In one or more embodiments of the present application, the antibody or product comprising the antibody can be used for point of care testing (POCT).

[0201] In one or more embodiments of the present application, the antibody has the advantages of good specificity, high titer and high sensitivity.

[0202] In one or more embodiments of the present application, the antibody can be used to detect the level of IL-1R2 in the body fluid (e.g. plasma, blood or urine) of a patient, so as to determine whether the patient is in an immunosuppressed state, thereby guiding the doctor to determine the severity of the patient's condition and take effective treatment measures in time to reduce mortality, which has important clinical significance.

[0203] In a fourth aspect, the present application provides a hybridoma cell producing an antibody binding to human IL-1R2, which is deposited under the accession number CCTCC NO: C2024249 or CCTCC NO: C2024250.

[0204] In one or more embodiments of the present application, the antibody and / or antibody-producing cells can be obtained from the animal in vivo after the animal is inoculated with the human IL-1R2 protein (e.g. human soluble IL-1R2 protein) antigen. An immortalized cell line producing the antibody can be prepared from the cells isolated from the immunized animal. After immunization, the animal is killed, and the lymph node and / or spleen B cells are taken for immortalization treatment, treated with carcinogenic compounds and inducible compounds, fused with immortalized cells (e.g. myeloma cells such as mouse myeloma cells SP2 / 0), and the activity of tumor suppressor genes is removed. If myeloma cells are used for fusion, the myeloma cells preferably do not secrete immunoglobulin polypeptides (non-secretory cell lines). The 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 primary screening is performed using enzyme-linked immunoassay (ELISA). The cells producing antibodies against human IL-1R2 protein (e.g. human soluble IL-1R2 protein) such as hybridomas are selected for cloning and further screening for desired properties, including good growth, high antibody production and desired antibody properties. The screening, cloning and amplification of hybridomas are well known to those skilled in the art.

[0205] In one or more embodiments of the present application, the immunized animal is a non-human animal, wherein the spleen B cells are fused with a myeloma cell line from the same species as the non-human animal.

[0206] In one or more embodiments of the present application, the immunized animal is a BALB / c mouse.

[0207] In one or more embodiments of the application, the hybridoma cell line is a mouse hybridoma cell line, for example a BALB / c mouse hybridoma cell line.

[0208] 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;

[0209] wherein the first antibody comprises a heavy chain variable region comprising a HCDR1 of an amino acid sequence as set forth in SEQ ID NO: 2, a HCDR2 of an amino acid sequence as set forth in SEQ ID NO: 3, and a HCDR3 of an amino acid sequence as set forth in SEQ ID NO: 4, and a light chain variable region comprising a LCDR1 of an amino acid sequence as set forth in SEQ ID NO: 5, a LCDR2 of an amino acid sequence as set forth in SEQ ID NO: 6, and a LCDR3 of an amino acid sequence as set forth in SEQ ID NO: 7; and / or

[0210] the second antibody comprises a heavy chain variable region comprising a HCDR1 of an amino acid sequence as set forth in SEQ ID NO: 8, a HCDR2 of an amino acid sequence as set forth in SEQ ID NO: 9, and a HCDR3 of an amino acid sequence as set forth in SEQ ID NO: 10, and a light chain variable region comprising a LCDR1 of an amino acid sequence as set forth in SEQ ID NO: 11, a LCDR2 of an amino acid sequence as set forth in SEQ ID NO: 12, and a LCDR3 of an amino acid sequence as set forth in SEQ ID NO: 13;

[0211] wherein the amino acid sequences of the HCDRs and the LCDRs are defined according to the IMGT numbering scheme.

[0212] In a sixth aspect, the present application provides a method for detecting the level of human IL-1R2 in a sample, comprising contacting a first antibody and a second antibody with the sample;

[0213] wherein the first antibody comprises a heavy chain variable region comprising a HCDR1 of an amino acid sequence as set forth in SEQ ID NO: 2, a HCDR2 of an amino acid sequence as set forth in SEQ ID NO: 3, and a HCDR3 of an amino acid sequence as set forth in SEQ ID NO: 4, and a light chain variable region comprising a LCDR1 of an amino acid sequence as set forth in SEQ ID NO: 5, a LCDR2 of an amino acid sequence as set forth in SEQ ID NO: 6, and a LCDR3 of an amino acid sequence as set forth in SEQ ID NO: 7; and / or

[0214] the second antibody comprises a heavy chain variable region comprising a HCDR1 of an amino acid sequence as set forth in SEQ ID NO: 8, a HCDR2 of an amino acid sequence as set forth in SEQ ID NO: 9, and a HCDR3 of an amino acid sequence as set forth in SEQ ID NO: 10, and a light chain variable region comprising a LCDR1 of an amino acid sequence as set forth in SEQ ID NO: 11, a LCDR2 of an amino acid sequence as set forth in SEQ ID NO: 12, and a LCDR3 of an amino acid sequence as set forth in SEQ ID NO: 13;

[0215] wherein the amino acid sequences of the HCDRs and LCDRs are defined according to the IMGT numbering scheme.

[0216] In a seventh aspect, the present application provides use of the antibody of the first aspect, or the hybridoma cell of the second aspect, in the manufacture of a product for detecting a level of human IL-1R2 in a sample.

[0217] In one or more embodiments of the present application, the level of human IL-1R2 is an important indicator for monitoring the immune status (e.g. immunosuppressive status) of the subject.

[0218] In one or more embodiments of the present application, the product for detecting a level of human IL-1R2 in a sample is selected from the group consisting of: a kit, a test strip (e.g. a colloidal gold detection test strip), a detection card, and a microfluidic detection device.

[0219] In one or more embodiments of the present application, the product for detecting a level of human IL-1R2 in a sample can further comprise other reagents for detection, such as a substrate, a reference standard, a diluent, a washing solution, and the like.

[0220] In one or more embodiments of the present application, the product for detecting a level of human IL-1R2 in a sample can further comprise instructions for use of the product.

[0221] In one or more embodiments of the present application, the product for detecting a level of human IL-1R2 in a sample can further comprise a container for mixing the sample with the antibody. Such a container can be suitable for a detection instrument capable of detecting a signal generated by the detection monoclonal antibody.

[0222] In one or more embodiments of the present application, the contacting is simultaneous or sequential contacting.

[0223] In one or more embodiments of the present application, the level of IL-1R2, e.g. human IL-1R2, in a sample is detected by an immunological method.

[0224] In one or more embodiments of the application, the immunological method is selected from the group consisting of: enzyme-linked immunosorbent assay, fluorescence immunoassay, chemiluminescent immunoassay, immunochromatography, immunoturbidimetry, immunoprecipitation, and any combination thereof.

[0225] According to the present application, the level of IL-1R2 can be detected by immunological methods using an antibody that binds to IL-1R2. For example, a "sandwich" assay can be used to detect and quantify IL-1R2. In this method, typically, one antibody is immobilized on a solid surface so as to bind to and capture IL-1R2, which antibody is thus referred to herein as a capture antibody. Another antibody is detectably labeled, e.g., with a fluorescent group, an enzyme, or a colored particle, whose binding to the IL-1R2-capture antibody complex indicates that IL-1R2 has been captured, the intensity of the signal being proportional to the concentration of IL-1R2 in the sample. The other antibody is thus also referred to herein as a detection antibody or a labeled antibody. Such an assay method can be referred to as a two-site immunoassay, a "sandwich" method, or a "sandwich immunoassay" (when the antibody is an adhesive). As is known in the art, the capture antibody and the detection antibody can be contacted with the test sample simultaneously or sequentially. The sequential method, sometimes referred to as the "forward" method, can be accomplished by incubating the capture antibody with the sample and thereafter adding the labeled detection antibody over a predetermined period of time. Alternatively, the labeled detection antibody can be incubated with the sample first, and then the sample can be contacted with the capture antibody (sometimes referred to as the "backward" method). Such assays can be performed in many specific formats known to those skilled in the art, including by use of different high-throughput clinical laboratory analyzers or with point of care or home-use test devices.

[0226] The most commonly used enzyme immunoassay is the "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 concentrations of an antigen. Different ELISA formats are known to those skilled in the art. Standard techniques for ELISA known in the art are described in "Methods in Immunodiagnosis," 2nd Ed., 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 attached to an antibody are horseradish peroxidase, alkaline phosphatase, luciferase, urease, and beta-galactosidase. The enzyme-linked antibody is reacted with a substrate to produce a chromogenic reaction product that can be measured. This measurement can be used to derive the concentration of human IL-1R2 present in the sample, for example, by comparing the measured value to a standard curve of human IL-1R2.

[0227] Quantitative fluorescence immunoassay is based on the principle of antigen-antibody reaction. A known antigen or antibody is labeled with fluorescein to make a fluorescent marker, and this 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 the cells or tissues contains fluorescein, and the specimen is observed using a fluorescence microscope. The fluorescein emits bright fluorescence under excitation light, and the cells or tissues where the fluorescence is present can be seen, thereby determining the nature, localization, and content of the antigen or antibody using quantitative techniques.

[0228] Chemiluminescence immunoassay (CLIA) is a combination of chemiluminescence determination technology with high sensitivity and high specificity of immune response, used for detection and analysis of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins and drugs, etc. It is a new immunological determination technology developed after radioimmunoassay, enzyme immunoassay, fluorescence immunoassay and time-resolved fluorescence immunoassay. Chemiluminescence immunoassay directly labels antigens or antibodies with chemiluminescent agents for immune analysis. The chemiluminescent substances commonly used for labeling include acridinium ester compounds (AE), which are effective luminescent markers. They emit light by starting the luminescent reagent, and the strong direct luminescence is completed in one second, which is a rapid flash luminescence. As a marker for immune analysis, acridinium ester has simple chemical reaction, rapidness, no catalyst, less non-specific binding, low background; and the binding with macromolecules does not reduce the amount of light generated, thereby increasing the sensitivity.

[0229] The principle of immunochromatography is to fix specific antibodies on a certain zone of a nitrocellulose membrane. When one end of the dry nitrocellulose membrane is immersed in a sample, the sample will move forward along the membrane due to capillary action. When it reaches the region where the antibodies are fixed, the corresponding antigens in the sample will specifically bind to the antibodies. If the region is stained with immunocolloidal gold or immunoenzyme, it will show a certain color, thereby realizing specific immunodiagnosis.

[0230] Immunoturbidimetric assay is a dynamic determination method of antigen-antibody combination. Its basic principle is that when antigen and antibody react in a special dilution system and the proportion is appropriate (generally, the antibody is in excess), the soluble immune complex formed under the action of the promoter in the dilution system precipitates from the liquid phase to form microparticles, resulting in turbidity of 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 sample, and the turbidity of the reaction solution also increases. By comparing the turbidity of the reaction solution with a series of standard samples, the content of antigen in the sample can be calculated.

[0231] Immunoprecipitation is a method for purifying and enriching a protein of interest by using the specific reaction of an antibody. 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. The bead-protein A / G or secondary antibody-antibody-protein of interest 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 dissociate. The supernatant is collected by centrifugation, which includes the antibody, the protein of interest and a small amount of impurities.

[0232] 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 commonly 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, examples of the detectable marker include, but are not limited to, enzymes, fluorescent molecules, radioisotopes, chemiluminescent molecules, latex particles, gold particles, detectable ligands, and any combination thereof.

[0233] In one or more embodiments of the present application, the detectable marker is an enzyme or a fluorescent molecule. Methods for attaching the detectable marker to the antibody are well known in the art and include covalent and non-covalent linkage.

[0234] In one or more embodiments of the present application, the detection antibody is detectably labeled by linking the antibody to an enzyme, such that the enzyme will react with a substrate when contacted therewith, and such reaction can be detected, for example, by spectrophotometry, fluorometry, or visually. Enzymes useful for detectably labeling 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, triose phosphate isomerase, alkaline phosphatase, luciferase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholine esterase. In one or more embodiments of the present application, the enzyme is horseradish peroxidase.

[0235] In one or more embodiments of the present application, the antibody can be labeled with a fluorescent molecule. When a fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence can be detected by the emitted fluorescence. Among the most commonly used fluorescent molecules can be selected from the group consisting of Cy3 and Cy5 (water-soluble fluorescent dyes of the cyanine dye family - "Cy" dyes), fluorescein isothiocyanate, rhodamine, phycobiliproteins, allophycocyanin, o-phenylenediamine, and fluorescamine.

[0236] In one or more embodiments of the application, the detection can also be achieved using a radiolabeled antibody, which can then be detected by using a radioimmunoassay. The radioactive isotope can be detected using, for example, a gamma counter or a scintillation counter or by autoradiography. 3 H、 131 I、 35 S、 14 C and 125 I.

[0237] In one or more embodiments of the application, the antibody can also be detectably labeled by coupling it to a chemiluminescent molecule, and the presence of the chemiluminescent antibody is then determined by detecting the presence of luminescence during the chemical reaction. The chemiluminescent molecule can be selected from the group consisting of luminol, luciferin, isatoic hydrazide, imidazole, acridinium salt, and oxalate ester.

[0238] In one or more embodiments of the application, the first antibody or the second antibody is attached to a solid support.

[0239] In one or more embodiments of the application, the solid support can be a plastic article (e.g., a polystyrene plate), a microparticle (e.g., a magnetized microparticle), or a membrane support (e.g., a nitrocellulose membrane, a glass cellulose membrane, or a nylon membrane).

[0240] In one or more embodiments of the application, one of the first antibody and the second antibody is used as a capture antibody and is immobilized on a solid support for capturing IL-1R2. The other of the first antibody and the second antibody is used as a detection antibody and is attached to a detectable label.

[0241] In one or more embodiments of the application, the sample is selected from the group consisting of plasma, blood, urine, serum, lymph, gastric juice, bile, saliva, sweat, spinal fluid, and any combination thereof. In one or more embodiments of the application, the sample can be plasma, blood, or urine.

[0242] In one or more embodiments of the application, the human IL-1R2 is human soluble IL-1R2 or human membrane-bound protein IL-1R2.

[0243] In one or more specific embodiments of the application, the human IL-1R2 is human soluble IL-1R2.

[0244] In other aspects, the present application also provides nucleic acid molecules encoding the antibodies binding to human IL-1R2 described in the present 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 regulatory nucleotide sequences, which can be recognized by the host cells transformed with the vectors.

[0245] It should be understood that the foregoing detailed description is only intended to acquaint others about the content of the present application and is not intended to limit the application in any way. Those skilled in the art can make various modifications and changes to the described embodiments. EMBODIMENT

[0246] Example 1: Eukaryotic expression and purification of human soluble IL-1R2 recombinant protein

[0247] The codon-optimized gene of the artificially synthesized soluble IL-1R2 (Uniprot: P27930-1, the amino acid sequence from 1st to 343rd is shown in SEQ ID NO: 1) was inserted into the pTT5 expression vector through EcoRI / HindIII enzyme cutting sites, and was transferred into 293 cells for eukaryotic expression. The cell culture supernatant was collected, and the protein was purified, and finally 5 mg of soluble IL-1R2 protein was obtained. As shown in FIG. 1A, the results of His-tag antibody immunoblotting in the 293 cell culture supernatant and cell precipitate showed that the 293 cell culture supernatant contained the target protein IL-1R2 (as shown by the red arrow). As shown in FIG. 1B, the SDS-PAGE electrophoresis of the purified protein verified that the IL-1R2 protein had the expected purity and size (as shown by the red arrow). Figure 1 Figure 1

[0248] Example 2: Preparation of anti-human IL-1R2 monoclonal antibody and antibody screening

[0249] 2.1 Immunization of mice and cell fusion

[0250] Five 6-week-old female BALB / c mice were taken, and were subcutaneously injected with soluble IL-1R2 protein emulsified with Freund's complete adjuvant at multiple points, for three times of immunization. After the three times of immunization, blood was collected from the tail vein, and the serum was collected for ELISA detection of serum titer. The mouse with the highest serum titer was taken, and was intraperitoneally injected with IL-1R2 protein for impact immunization. The spleen of the mouse was taken, and the spleen cells were collected and fused with mouse myeloma cells SP2 / 0, which were plated in 96-well plates for culture.

[0251] 2.2 Hybridoma screening, establishment of stable cell strain, and antibody subtype identification

[0252] ​​The culture supernatant of the fusion cells was subjected to ELISA detection, and positive wells were screened, rechecked, and the positive wells after rechecking were subjected to subcloning by limiting dilution method. After subcloning, ELISA detection was performed, and positive wells were selected for further subcloning by limiting dilution method. After multiple subcloning to an ELISA detection positive rate of 100%, clones with high absorbance were selected, and wells with vigorous growth were selected for expansion culture and cryopreservation. The culture supernatant of the stable cell strain was subjected to ELISA detection to identify the antibody subtype.

[0253] 2.3 Preparation of monoclonal antibody

[0254] The hybridoma cells were injected intraperitoneally into mice, and the ascites was collected and centrifuged. The supernatant was filtered through a 0.22 μm filter membrane, and then the antibody was purified by a protein A chromatography column (HiScreen Fibro™ PrismA, cytiva). The antibody was concentrated by replacing the liquid with an ultrafiltration tube, and finally replaced into PBS. The monoclonal antibody was obtained by sterile filtration through a 0.22 μm filter membrane. The antibody was gradiently diluted with PBS buffer, and subjected to ELISA detection of antibody titer. As shown in Table 1, the titer of the two selected antibodies was greater than 1:128000.

[0255] Table 1. OD of ELISA detection of monoclonal antibody titer 450 Value

[0256]

[0257] 2.4 Antibody pairing verification

[0258] The antibodies 2E10 and 3D10 obtained in Section 2.3, and the commercially available antibodies ALS11331 (rabbit anti-human IL-1R2 monoclonal antibody, purchased from Abgent Company, product number A-ALS11331) and R020 (rabbit anti-human IL-1R2 monoclonal antibody, purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., product number Cat: 10111-R020) were used as capture antibodies and labeling antibodies, respectively, for pairing verification. Among them, the capture antibody was diluted to 1 μg / mL and added to the enzyme-labeled plate, and the antibody was coated at 4°C overnight. Then the plate was washed, blocked, and incubated with the antigen IL-1R2. The diluted detection antibody coupled with horseradish peroxidase (HRP) was added, incubated, washed, developed, and read. The pairing results are shown in Table 2, in which the two paired antibodies 2E10 (subtype IgG1) and 3D10 (subtype IgG2a) have the highest sensitivity, which can reach 10 pg / mL.

[0259] Table 2. OD of ELISA detection of monoclonal antibody pairing against soluble IL-1R2 450 Value

[0260]

[0261] Example 3: Affinity identification of anti-human IL-1R2 monoclonal antibodies 2E10 and 3D10

[0262] SPR experiments were performed using a Biacore 8k+ surface plasmon resonance system (cytiva) to detect the affinity of 2E10 and 3D10 antibodies to soluble human IL-1R2 protein. 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 concentrations of 1.5625 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM and 50 nM with PBS+0.025% P20, respectively, and flowed over the chip surface from low to high concentration as analyte, with a binding time of 120 s, a dissociation time of 300 s, a flow rate of 30 μL / min, and glycine (pH 1.5) regeneration for 30 s at a flow rate of 30 μL / min. The model (1:1 binding) was fitted, the analysis method was multi-cycle kinetics, and the binding and dissociation response value curves of different concentrations of antibodies were fitted to calculate the binding rate constant k a , the dissociation rate constant k d and the affinity constant K D value. The binding and dissociation curves of 2E10 antibody to IL-1R2 protein are shown in Figure 2 , and the binding and dissociation curves of 3D10 antibody to IL-1R2 protein are shown in Figure 3 . The k a , k d , K D values obtained by fitting the binding and dissociation curves are shown in Table 3, the affinity K D value of 2E10 to IL-1R2 is 1.19 x 10^-10, and the affinity K D value of 3D10 to IL-1R2 is 1.35 x 10^-11. The affinity of antibody 2E10 to IL-1R2 is comparable or better than that of antibody ALS11331 or R020 to IL-1R2. The affinity of antibody 3D10 to IL-1R2 is comparable or better than that of antibody ALS11331 or R020 to IL-1R2.

[0263] Table 3. Affinity constants of monoclonal antibodies 2E10 and 3D10 to human IL-1R2

[0264]

[0265] Example 4: Elevated IL-R2 in sepsis patients reflects immune suppression status of patients

[0266] 4.1 Identification of immune suppression state of sepsis patients

[0267] According to the Expert Consensus on the Monitoring and Treatment of Sepsis-Induced Immunosuppression (Chinese Research Hospital Association Shock and Sepsis Professional Committee, Chinese People's Liberation Army Critical Care Medicine Professional Committee, Severe Immune Research Collaboration Group, et al. Expert consensus on the monitoring and treatment of sepsis-induced immunosuppression [J]. Chinese 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, 174. 26 Dec. 2022), the absolute number of lymphocytes in the blood routine report for three consecutive days is less than 1.0 × 10 9 6 / L, peripheral blood mononuclear cell HLA-DR positive rate is less than 60%, age is greater than 65 years old, BMI is less than 18.5, albumin is decreased, and steroid drugs are taken, etc. as the standard for identifying immune suppression of patients. Collect the clinical test results, disease history and medication information of sepsis patients, and identify the immune state of patients (whether immune suppression).

[0268] 4.2 Detection of peripheral blood mononuclear cell HLA-DR positive rate of sepsis patients

[0269] Collect EDTA anticoagulated blood of sepsis patients, mix well, take 100 μL blood, add 5 μL PE-Anti-HLA-DR antibody (Biolegend), 5 μL PerCP-CY5.5 Anti-CD14 antibody (Biolegend), mix well, incubate for 30 min in the dark. Add 900 μL 1 × BD FACS lysis solution (10 × solution diluted with deionized water), mix well, incubate in the dark for 15 min. Turn on the flow cytometer, adjust the voltage and compensation of each channel, circle CD14 + mononuclear cells in the CD14-SSC diagram, collect 2000 to 4000 mononuclear cells, and circle the proportion of PE-HLA-DR positive groups.

[0270] 4.3 Detection of plasma IL-1R2 level of sepsis patients

[0271] Collect EDTA anticoagulated blood of patients, centrifuge at 4000 rpm for 15 min at 4℃, take the supernatant, use the IL-1R2 level detection kit prepared by anti-human IL-1R2 monoclonal antibodies 2E10 and 3D10, and detect the plasma IL-1R2 level by ELISA.

[0272] 4.4 Immune status comparison analysis

[0273] According to the clinical indicators to determine the immune status of the collected sepsis patients, such as Table 4 (lymphocyte absolute number ALC, mononuclear cell HLA-DR positive rate, disease history, medication history, age, BMI, albumin level), a total of 47 cases of sepsis patients were considered immune suppressed, 20 cases of sepsis patients were considered non-immunosuppressed, and 44 cases of apparently healthy people (non-immunosuppressed) were added as controls (as shown in Table 5). The ROC curve was drawn for the people considered immune suppressed and non-immunosuppressed, as shown in Figure 4 The area under the ROC curve AUC=0.9302, P<0.0001, AUC is above 0.9, and has a higher accuracy. According to the ROC curve, the cut-off value of IL-1R2 is 24 ng / mL. As shown in Figure 5 The plasma IL-1R2 level distribution of the three groups of people is shown in Table 5. Among the 47 patients determined to be immune suppressed according to clinical indicators, 39 patients had elevated IL-1R2 levels, exceeding 24 ng / mL, with a sensitivity of 82.98%; among the 64 people determined to be non-immunosuppressed by clinical indicators, 58 people had IL-1R2 levels below 24 ng / mL, with a specificity of 90.63%. Therefore, using the anti-human IL-1R2 monoclonal antibodies 2E10 and 3D10 pair to determine plasma IL-1R2 > 24 ng / mL, the sensitivity for identifying the immune suppression status of patients is 82.98%, and the specificity is 90.63%, which can reflect the immune suppression status of patients.

[0274] Table 4. Sepsis patient immune suppression status determination

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283] Table 5. Plasma IL-1R2 levels of apparently healthy people (non-immunosuppressed)

[0284]

[0285]

[0286] It can be clearly understood that although the application has been described above with reference to the specific forms of the application involved, the application is not limited to the specific content described in these specific forms. It is obvious to those skilled in the art that various equivalent changes can be made to the technical features involved in the application without departing from the spirit of the application described in the application, and these changes should all belong to the scope of the application.

[0287] Sequence information

[0288] The antibody variable region sequence sequencing results of the application are provided by Globel Gen.

[0289] SEQ ID NO: 1

[0290] MLRLYVLVMGVSAFTLQPAAHTGAARSCRFRGRHYKREFRLEGEPVALRCPQVPYWLWASVSPRINLTWHKNDSARTVPGEEETRMWAQDGALWLLPALQEDSGTYVCTTRNASYCDKMSIELRVFENTDAFLPFISYPQILTLSTSGVLVCPDLSEFTRDKTDVKIQWYKDSLLLDKDNEKFLSVRGTTHLLVHDVALEDAGYYRCVLTFAHEGQQYNITRSIELRIKKKKEETIPVIISPLKTISASLGSRLTIPCKVFLGTGTPLTTMLWWTANDTHIESAYPGGRVTEGPRQEYSENNENYIEVPLIFDPVTREDLHMDFKCVVHNTLSFQTLRTTVKE

[0291] SEQ ID NO: 2

[0292] GYTFTSYA

[0293] SEQ ID NO: 3

[0294] IYPSRDYT

[0295] SEQ ID NO: 4

[0296] ARDGGFFGGHAMDY

[0297] SEQ ID NO: 5

[0298] QNINVW

[0299] SEQ ID NO: 6

[0300] RAS

[0301] SEQ ID NO: 7

[0302] QQGQSYPLT

[0303] SEQ ID NO: 8

[0304] GYTFSSYW

[0305] SEQ ID NO: 9

[0306] ILPGSGRN

[0307] SEQ ID NO: 10

[0308] GRSGLY

[0309] SEQ ID NO: 11

[0310] QSLLYSDGKTY

[0311] SEQ ID NO: 12

[0312] LVS

[0313] SEQ ID NO: 13

[0314] WQGTHSPFT

[0315] SEQ ID NO: 14

[0316] QVQLQQSGAALARPGASVKMSCKASGYTFTSYAMHWVKQRPGRGLEWIGYIYPSRDYTNYNQRFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARDGGFFGGHAMDYWGQGTSVTVSS

[0317] SEQ ID NO: 15

[0318] QVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVKQRPGHGLEWIGEILPGSGRNAYNEKFKGKATFTADSSSNTVYMQLSSLTSEDSAVYYCGRSGLYWGQGTLVTVSA

[0319] SEQ ID NO: 16

[0320] DIQMNQSPSSLSASLGDTITITCHASQNINVWLSWYQQKPGNIPKLLIYRASNLHTGVPSRFSGSGSGTGFTFTISSLQPEDIATYYCQQGQSYPLTFGAGTKLELK

[0321] SEQ ID NO: 17

[0322] DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSDGKTYLNWLLQRPGQSPKRLIYLVSNLDSGVPDRFTGSGSGTDFTLKIRRVEAEDLGVYYCWQGTHSPFTFGSGTKLEIK

[0323] SEQ ID NO: 18

[0324] CAGGTCCAGCTGCAGCAGTCTGGGGCTGCACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACTAGCTACGCGATGCACTGGGTGAAACAGAGGCCTGGACGGGGTCTGGAATGGATTGGATACATTTATCCTAGTCGTGATTATACTAATTACAATCAGAGGTTCAAAGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGACGGAGGTTTCTTCGGGGGACATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

[0325] SEQ ID NO: 19

[0326] QVQLQQSGAALARPGASVKMSCKAS

[0327] SEQ ID NO: 20

[0328] MHWVKQRPGRGLEWIGY

[0329] SEQ ID NO: 21

[0330] NYNQRFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYC

[0331] SEQ ID NO: 22

[0332] WGQGTSVTVSS

[0333] SEQ ID NO: 23

[0334] GACATCCAGATGAACCAGTCTCCATCCAGTCTGTCTGCATCCCTTGGAGACACAATTACCATCACTTGCCATGCCAGTCAGAACATTAATGTGTGGCTAAGCTGGTACCAGCAGAAACCAGGAAATATTCCTAAACTATTGATCTATAGGGCTTCCAACTTGCACACAGGCGTCCCATCAAGGTTTAGTGGCAGTGGATCTGGAACAGGTTTCACATTCACCATCAGCAGCCTGCAGCCTGAAGACATTGCCACTTACTACTGTCAACAGGGTCAAAGTTATCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0335] SEQ ID NO: 24

[0336] DIQMNQSPSSLSASLGDTITITCHAS

[0337] SEQ ID NO: 25

[0338] LSWYQQKPGNIPKLLIY

[0339] SEQ ID NO: 26

[0340] NLHTGVPSRFSGSGSGTGFTFTISSLQPEDIATYYC

[0341] SEQ ID NO: 27

[0342] FGAGTKLELK

[0343] SEQ ID NO: 28

[0344] CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTACTGGCTACACATTCAGTAGCTACTGGATAGAGTGGGTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGTGGTCGTAATGCCTACAATGAGAAGTTCAAGGGCAAGGCCACGTTCACTGCAGATTCATCCTCCAACACAGTCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGGAAGATCTGGCCTCTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0345] SEQ ID NO: 29

[0346] QVQLQQSGAELMKPGASVKISCKAT

[0347] SEQ ID NO: 30

[0348] IEWVKQRPGHGLEWIGE

[0349] SEQ ID NO: 31

[0350] AYNEKFKGKATFTADSSSNTVYMQLSSLTSEDSAVYYC

[0351] SEQ ID NO: 32

[0352] WGQGTLVTVSA

[0353] SEQ ID NO: 33

[0354] GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTATATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAATCTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGGAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTCTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA

[0355] SEQ ID NO: 34

[0356] DVVMTQTPLTLSVTIGQPASISCKSS

[0357] SEQ ID NO: 35

[0358] LNWLLQRPGQSPKRLIY

[0359] SEQ ID NO: 36

[0360] NLDSGVPDRFTGSGSGTDFTLKIRRVEAEDLGVYYC

[0361] SEQ ID NO: 37

[0362] FGSGTKLEIK

Claims

1. Use of a reagent for detecting IL-1R2 level in a sample from a subject in the preparation of a product for identifying immune status of the subject; wherein, the subject has sepsis; and the reagent is an antibody binding to IL-1R2, and the antibody comprises a first antibody and a second antibody, wherein the first antibody comprises a heavy chain variable region comprising a HCDR1 of an amino acid sequence as set forth in SEQ ID NO: 2, a HCDR2 of an amino acid sequence as set forth in SEQ ID NO: 3, and a HCDR3 of an amino acid sequence as set forth in SEQ ID NO: 4, and a light chain variable region comprising a LCDR1 of an amino acid sequence as set forth in SEQ ID NO: 5, a LCDR2 of an amino acid sequence as set forth in SEQ ID NO: 6, and a LCDR3 of an amino acid sequence as set forth in SEQ ID NO: 7; and the second antibody comprises a heavy chain variable region comprising a HCDR1 of an amino acid sequence as set forth in SEQ ID NO: 8, a HCDR2 of an amino acid sequence as set forth in SEQ ID NO: 9, and a HCDR3 of an amino acid sequence as set forth in SEQ ID NO: 10, and a light chain variable region comprising a LCDR1 of an amino acid sequence as set forth in SEQ ID NO: 11, a LCDR2 of an amino acid sequence as set forth in SEQ ID NO: 12, and a LCDR3 of an amino acid sequence as set forth in SEQ ID NO: 13; wherein the amino acid sequences of the HCDRs and the LCDRs are defined according to the IMGT numbering scheme.

2. The use of claim 1, wherein the immune status is an immunosuppressed status.

3. The use of claim 2, wherein the immune status is a relatively immunosuppressed status.

4. The use of claim 1, wherein the subject is a mammal.

5. The use of claim 4, wherein the subject is a human.

6. The use of claim 1, wherein the sample is selected from the group consisting of plasma, blood, urine, serum, lymph, gastric juice, bile, saliva, sweat, spinal fluid, and any combination thereof.

7. The use of claim 6, wherein the sample is plasma, blood, or urine.

8. The use of claim 1, wherein the product for identifying 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.

9. The use of claim 1, wherein the IL-1R2 is a soluble IL-1R2 or a membrane-bound protein IL-1R2.

10. The use of claim 1, wherein the first antibody or the second antibody is labeled with a detectable label selected from the group consisting of an enzyme, a fluorescent molecule, a radioisotope, a chemiluminescent molecule, a latex particle, a gold particle, a detectable ligand, and any combination thereof.

11. The use of claim 1, wherein the first antibody or the second antibody is attached to a solid support.

12. The use of claim 1, wherein 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.

13. The use of claim 12, wherein 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.

14. The use of claim 13, wherein the cut-off value of the IL-1R2 level for identifying the immune status of the subject is 24 ng / mL.

15. The use of any one of claims 12-14, wherein the immune status of the subject is identified as an immunosuppressed status if the IL-1R2 level is greater than the cut-off value.

16. The use of any one of claims 12-14, wherein the immune status of the subject is identified as a non-immunosuppressed status if the IL-1R2 level is less than the cut-off value.

17. The use of claim 1, wherein the immune status of the subject is 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.

18. The use of claim 17, wherein the immune status of the subject is an immunosuppressed status.

19. 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 set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 3, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 4, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 5, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 6, and the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 7; or the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 8, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 9, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 11, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 12, and the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 13; the amino acid sequences of the HCDRs and the LCDRs are defined according to the IMGT numbering scheme.

20. The antibody of claim 19, wherein the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 14 or 15. ​ wherein, ​ ​ 21. The antibody of claim 19, wherein the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 16 or 17.

22. The antibody of claim 20 or 21, wherein the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 16; or the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:

17.

23. The antibody of claim 19, wherein the human IL-1R2 is human soluble IL-1R2 or human membrane-bound protein IL-1R2.

24. The antibody of claim 19, 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.

25. The antibody of claim 19, wherein the antibody is a monoclonal antibody.

26. The antibody of claim 25, wherein the antibody is a murinized monoclonal antibody.

27. The antibody of claim 19, wherein the antibody comprises a heavy chain constant region selected from the group consisting of an IgG1 subtype, an IgG2 subtype, or an IgG4 subtype.

28. The antibody of claim 27, wherein the antibody comprises a heavy chain constant region of an IgG1 subtype or an IgG2 subtype.

29. The antibody of claim 19, wherein the antibody comprises a light chain constant region selected from the group consisting of a kappa subtype or a lambda subtype.

30. The antibody of claim 19, wherein the antibody binds to human IL-1R2 having the amino acid sequence set forth in SEQ ID NO:

1.

31. The antibody of claim 19, wherein the antibody is produced by a hybridoma cell line having the accession number CCTCC NO: C2024249 or CCTCC NO: C2024250.

32. A hybridoma cell producing an antibody that binds to human IL-1R2, which is deposited under the accession number CCTCC NO: C2024249 or CCTCC NO: C2024250.

33. The hybridoma cell of claim 32, wherein the human IL-1R2 is human soluble IL-1R2 or human membrane-bound protein IL-1R2. ​ ​

Images