Anti-sst2 antibodies and uses thereof

Abstract

The application discloses an antibody against sST2 and application thereof, and relates to the field of antibodies. The antibody against sST2 disclosed by the application comprises a heavy chain complementarity determining region and a light chain complementarity determining region, the antibody provides an important raw material source for detection of sST2, and has good affinity or activity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of antibodies, in particular to an anti-sST2 antibody and application thereof. BACKGROUND

[0002] Growth stimulating expression gene 2 protein (ST2) is also known as T1, IL1RL1 or Fit1 gene and is located on human chromosome 2q12, about 40KD, there are four subtypes of transcription products of ST2 gene, and two of them are the most important subtypes due to being regulated by different promoters: the first one is transmembrane ST2 (ST2L), which is composed of three extracellular immunoglobulin G domains, a transmembrane domain and an intracellular domain, and is a member of the interleukin-1 receptor family; the other one is soluble ST2 (sST2), which can freely flow in the blood due to lacking transmembrane and intracellular domains, so that it can be detected in serum.

[0003] Cardiovascular disease (CVD) is the leading cause of death worldwide, brain natriuretic peptide (BNP) and NT-proBNP are the most famous markers of heart failure (HF), and the troponin marker improves the diagnosis of acute and chronic coronary artery disease, however, a single biomarker can only reflect one-sided pathological mechanism of heart failure, and is also affected by renal function, age, BMI and other factors such as pulmonary hypertension, which reduces the accuracy of these biomarkers. With the increasing understanding of the role of sST2 and ST2L in the cardiovascular system, people began to consider the evaluation of plasma sST2 level as a new marker of cardiovascular events, especially related to heart failure and ischemic heart disease, and a clinical condition, and sST2 is not affected by renal function, age, body weight and other indices. sST2 is also considered as a possible biomarker for patients with acute heart failure and asthma. Studies have also confirmed the role of sST2 in predicting mortality, sST2 can be combined with other biomarkers as a prognostic biomarker, sST2 can also be used to monitor the pharmacological response of heart failure, and is related to the recommended treatment drugs for heart failure, which can provide a reference for the selection of treatment drugs and schemes in clinic. At present, the main methods for detecting sST2 in China are enzyme-linked immunosorbent assay (ELISA), magnetic microparticle chemiluminescence method, gold labeling method, etc.; different detection methods need specific monoclonal antibodies for sST2. Therefore, there is a strong demand in the art for antibodies that effectively bind to sST2 and detect it. SUMMARY

[0004] The present application provides an anti-sST2 antibody or antigen binding fragment thereof, which provides an important raw material source for the detection of sST2 and has good activity or affinity.

[0005] To achieve the above object, according to a first aspect of the present application, there is provided an anti-sST2 antibody or an antigen-binding fragment thereof, having three complementarity determining regions of any one of the heavy chain variable regions of the amino acid sequences of SEQ ID NOs: 21, 22, 23, 24 and three complementarity determining regions of any one of the light chain variable regions of the amino acid sequences of SEQ ID NOs: 29, 30.

[0006] To achieve the above object, according to a second aspect of the present application, there is provided an anti-sST2 antibody or an antigen-binding fragment thereof, comprising the following complementarity determining regions:

[0007] HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1;

[0008] HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 2;

[0009] HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 3 or 17;

[0010] LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 4 or 18;

[0011] LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 5; and

[0012] LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 6.

[0013] To achieve the above object, according to a third aspect of the present application, there is provided an anti-sST2 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and / or a light chain variable region, the amino acid sequence of the heavy chain variable region being as shown in any one of SEQ ID NOs: 21, 22, 23, 24; the amino acid sequence of the light chain variable region being as shown in any one of SEQ ID NOs: 29, 30.

[0014] To achieve the above object, according to a fourth aspect of the present application, there is provided an anti-sST2 antibody or an antigen-binding fragment thereof, comprising a heavy chain and / or a light chain, the amino acid sequence of the heavy chain being as shown in any one of SEQ ID NOs: 25, 26, 27, 28; the amino acid sequence of the light chain being as shown in any one of SEQ ID NOs: 31, 32.

[0015] To achieve the above object, according to a fifth aspect of the present application, there is provided an antibody conjugate comprising the anti-sST2 antibody or antigen-binding fragment thereof described above.

[0016] To achieve the above object, according to a sixth aspect of the present application, there is provided a reagent or kit comprising the anti-sST2 antibody or antigen-binding fragment thereof described above or the antibody conjugate described above.

[0017] To achieve the above object, according to a seventh aspect of the present application, there is provided a method of detecting sST2, comprising: a) contacting the anti-sST2 antibody or antigen-binding fragment thereof, the antibody conjugate, or the reagent or kit described above with sST2 in a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to occur to form an immunocomplex; and b) detecting the presence of the immunocomplex, the presence of the complex indicating the presence of the antigen in the test sample.

[0018] To achieve the above object, according to an eighth aspect of the present application, there is provided a nucleic acid encoding the anti-sST2 antibody or antigen-binding fragment thereof described above.

[0019] To achieve the above object, according to a ninth aspect of the present application, there is provided a vector comprising the nucleic acid described above.

[0020] To achieve the above object, according to a tenth aspect of the present application, there is provided a cell comprising the nucleic acid, the vector, or expressing the anti-sST2 antibody or antigen-binding fragment thereof described above.

[0021] To achieve the above object, according to an eleventh aspect of the present application, there is provided a method of producing the anti-sST2 antibody or antigen-binding fragment thereof described above, the method comprising culturing the cell described above.

[0022] To achieve the above object, according to a twelfth aspect of the present application, there is provided use of the anti-sST2 antibody or antigen-binding fragment thereof, the antibody conjugate, the reagent or kit described above in the manufacture of a product for detecting sST2. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced as follows. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be considered as a limitation on the scope, and for those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0024] Figure 1Results of reducing SDS-PAGE of Anti-sST2 3F9 Rmb1-5. DETAILED DESCRIPTION

[0025] In a first aspect, the embodiments of the present application provide an anti-sST2 antibody or antigen binding fragment thereof, which has the three complementarity determining regions of any one of the heavy chain variable regions of the amino acid sequence of SEQ ID NO: 21, 22, 23, 24 and the three complementarity determining regions of any one of the light chain variable regions of the amino acid sequence of SEQ ID NO: 29, 30.

[0026] It should be noted that the HCDR1, HCDR2 and HCDR3 are the amino acid sequences identical to the HCDR1, HCDR2 and HCDR3 of the same heavy chain variable region defined in the anti-sST2 antibody or antigen binding fragment thereof of the first aspect, and the LCDR1, LCDR2 and LCDR3 are the amino acid sequences identical to the LCDR1, LCDR2 and LCDR3 of the same light chain variable region defined in the anti-sST2 antibody or antigen binding fragment thereof of the first aspect.

[0027] For example, the HCDR1, HCDR2 and HCDR3 are the amino acid sequences identical to the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 21, and the LCDR1, LCDR2 and LCDR3 are the amino acid sequences identical to the LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO: 29.

[0028] In the present application, the term "antibody" is used in the broadest sense, and can include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, as long as they exhibit the desired biological activity.

[0029] In the present application, the term "complementarity determining region", "CDR" or "CDRs" refers to the highly variable regions of the heavy and light chains of immunoglobulins, which refer to the regions containing one or more, or even all, of the major amino acid residues that play a role in the binding of the antibody or antigen binding fragment to the antigen or epitope it recognizes.

[0030] In the present application, the heavy chain complementarity determining region is denoted as HCDR, which includes HCDR1, HCDR2 and HCDR3; and the light chain complementarity determining region is denoted as LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0031] Methods for defining CDRs are well known in the art and include the Kabat definition, the Chothia definition, the IMGT definition, the Contact definition, and the AbM definition. As used herein, the "Kabat definition" refers to the definition system set forth in Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" is set forth in Chothia et al., J Mol Biol 196:901-917 (1987). Still other CDR definition methods can not strictly follow one of the above schemes but will still overlap at least a portion of the CDR regions defined by Kabat, although they can be shortened or lengthened according to prediction or experimental results for particular residues or groups of residues. Exemplary defined CDRs are listed in Table 1 below, which vary slightly from definition to definition in the literature. One of skill in the art can routinely determine which residues comprise particular CDRs given the variable region amino acid sequence of a given antibody. It is noted that CDRs defined by other methods not listed in Table 1 are also within the scope of the present disclosure.

[0032] Table 1: CDR Definitions 1

[0033]

[0034]

[0035] 1 The numbering of all CDR definitions in Table 1 is according to the Kabat numbering system (see below), with "H + numbers" for amino acid positions on the heavy chain and "L + numbers" for amino acid positions on the light chain. One of ordinary skill in the art can unambiguously correlate this Kabat numbering system to any variable region sequence without depending on any experimental data other than the sequence itself. As used herein, the "Kabat numbering" refers to the numbering system set forth in Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0036] 2 "AbM" as used in Table 1 with a lower case "b" refers to CDRs defined by the "AbM" antibody modeling software of Oxford Molecular.

[0037] 3If H35A and H35B are both absent, then CDR-H1 ends at position 35; if only H35A is present, then CDR-H1 ends at position 35A; if H35A and H35B are both present, then CDR-H1 ends at position 35B.

[0038] 4 If H35A and H35B are both absent, then CDR-H1 ends at position 32; if only H35A is present, then CDR-H1 ends at position 33; if H35A and H35B are both present, then CDR-H1 ends at position 34.

[0039] 5 If H35A and H35B are both absent, then CDR-H1 ends at position 33; if only H35A is present, then CDR-H1 ends at position 34; if H35A and H35B are both present, then CDR-H1 ends at position 35.

[0040] According to embodiments of the application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of more than one of the systems.

[0041] In some alternative embodiments of the application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0042] In some alternative embodiments of the application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0043] In some alternative embodiments of the application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT system.

[0044] In some alternative embodiments of the application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0045] In some alternative embodiments of the application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Contact system.

[0046] In some alternative embodiments of the present application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat, Chothia, IMGT, AbM or Contact system combination.

[0047] According to embodiments of the present application, the Kabat, Chothia, AbM or IMGT system defined HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 amino acid sequence corresponding Kabat numbering position is as follows:

[0048]

[0049]

[0050] In a second aspect, embodiments of the present application provide an anti-sST2 antibody or antigen binding fragment thereof, comprising the following complementarity determining regions:

[0051] HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1;

[0052] HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 2;

[0053] HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 3 or 17;

[0054] LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4 or 18;

[0055] LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 5; and

[0056] LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 6.

[0057] According to embodiments of the present application, the HCDRs and LCDRs are defined by the Kabat system.

[0058] In the present application, the "framework region" or "FR" region includes heavy chain framework regions and light chain framework regions, which refers to the regions of the antibody heavy chain variable region and light chain variable region other than CDRs; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDRs, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDRs, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0059] In the present application, the heavy chain variable region is obtained by connecting the CDRs and FRs numbered as follows in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; and the light chain variable region is obtained by connecting the CDRs and FRs numbered as follows in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0060] In an alternative embodiment, the anti-sST2 antibody or antigen binding fragment thereof of the first aspect or the second aspect comprises a heavy chain variable region comprising HFR1, HFR2, HFR3, HFR4, and a light chain variable region comprising LFR1, LFR2, LFR3, and LFR4.

[0061] In an alternative embodiment, the HFR1 comprises / is an amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least 80% identity thereto;

[0062] the HFR2 comprises / is an amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto;

[0063] the HFR3 comprises / is an amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto;

[0064] the HFR4 comprises / is an amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto;

[0065] the LFR1 comprises / is an amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto;

[0066] the LFR2 comprises / is an amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto;

[0067] the LFR3 comprises / is an amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto; and

[0068] the LFR4 comprises / is an amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto.

[0069] It should be noted that in other embodiments, each of the framework region amino acid sequences of the anti-sST2 antibody or antigen-binding fragment thereof provided by the present application can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the corresponding framework region (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, or 14) described above.

[0070] In an alternative embodiment, the HFR2 comprises / has the sequence set forth in SEQ ID NO: 19.

[0071] In an alternative embodiment, the HFR3 comprises / has the sequence set forth in SEQ ID NO: 20.

[0072] In an alternative embodiment, the anti-sST2 antibody or antigen-binding fragment thereof binds sST2 with an affinity of KD<8.12x10 -9 M.

[0073] In an alternative embodiment, the anti-sST2 antibody or antigen-binding fragment thereof binds sST2 with an affinity of KD≤10 -7 M, KD≤10 - 8 M, KD≤10 -9 M, KD≤10 -10 M, KD≤10 -11 M, KD≤10 -12 M, KD≤10 -13 M.

[0074] In an alternative embodiment, the anti-sST2 antibody or antigen-binding fragment thereof binds sST2 with an affinity of KD≤2.22x10 -10 M.

[0075] There are many methods for determining the affinity (KD) of an antibody, which can be classified into thermodynamic detection methods, kinetic detection methods, and dynamic equilibrium detection methods according to the detection principle. Among them, the thermodynamic detection method is commonly known as isothermal titration calorimetry (ITC); the kinetic detection method is commonly known as surface plasmon resonance (SPR) and bio-layer interferometry (BLI); and the dynamic equilibrium detection method is commonly known as enzyme-linked immunosorbent assay (ELISA) and the like.

[0076] In a third aspect, an anti-sST2 antibody or antigen-binding fragment thereof is provided, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as set forth in any one of SEQ ID NOs: 21, 22, 23, 24, and the amino acid sequence of the light chain variable region is as set forth in any one of SEQ ID NOs: 29, 30.

[0077] In an optional embodiment, the combination of the heavy chain variable region and the light chain variable region of the anti-sST2 antibody or antigen-binding fragment thereof of the first aspect, the second aspect, or the third aspect is selected from any one of the following combinations:

[0078] Combination Heavy chain variable region Light chain variable region 1 SEQ ID NO: 21 SEQ ID NO: 29 2 SEQ ID NO: 23 SEQ ID NO: 29 3 SEQ ID NO: 22 SEQ ID NO: 29 4 SEQ ID NO: 24 SEQ ID NO: 29 5 SEQ ID NO: 21 SEQ ID NO: 30 .

[0079] In an optional embodiment, the anti-sST2 antibody or antigen-binding fragment thereof of the first aspect, the second aspect, or the third aspect further comprises a constant region.

[0080] In an optional embodiment, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0081] In an optional embodiment, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, IgD, or a combination of segments of multiple constant regions.

[0082] In an optional embodiment, the heavy chain constant region comprises CH1 of IgG, a hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

[0083] In an optional embodiment, the IgG is selected from IgG1, IgG2, IgG3, or IgG4.

[0084] In an optional embodiment, the light chain constant region is selected from a kappa type or a lambda type light chain constant region.

[0085] In an optional embodiment, the species origin of the constant region is bovine, equine, bovine, porcine, ovine, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, cock, or human.

[0086] In an optional embodiment, the species origin of the constant region is human.

[0087] In an optional embodiment, the sequence of the heavy chain constant region (CH) is as set forth in SEQ ID NO: 15, and the sequence of the light chain constant region (CL) is as set forth in SEQ ID NO: 16.

[0088] It is to be noted that in other embodiments, the constant region sequence can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the constant region (SEQ ID NO: 15 or 16) described above.

[0089] In alternative embodiments, the antigen-binding fragment is selected from any one of F(ab)2, F(ab’)2, Fab’, Fab, Fv and scFv of the antibody.

[0090] The antigen-binding fragment of the antibody described above generally has the same binding specificity as the antibody from which it is derived. It is readily understood by those skilled in the art that the antigen-binding fragment of the antibody described above can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by methods of splitting disulfide bonds by chemical reduction, based on the structural basis of the complete antibody disclosed in the present application. The antigen-binding fragment described above can be readily obtained by those skilled in the art based on the structural basis of the complete antibody disclosed in the present application.

[0091] The antigen-binding fragment of the antibody described above can also be obtained by recombinant genetic engineering techniques, which are also known to those skilled in the art, or by synthesis, for example, by an automatic peptide synthesizer, such as an automatic peptide synthesizer sold by Applied BioSystems and the like.

[0092] In a fourth aspect, the present application provides an anti-sST2 antibody or an antigen-binding fragment thereof, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO: 25, 26, 27, 28, and the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 31, 32.

[0093] In alternative embodiments, the antibody or antigen-binding fragment thereof of the first aspect, the second aspect, the third aspect or the fourth aspect described above comprises a heavy chain and a light chain in any one of the following combinations:

[0094] Combination Heavy chain Light chain 1 SEQ ID NO: 25 SEQ ID NO: 31 2 SEQ ID NO: 27 SEQ ID NO: 31 3 SEQ ID NO: 26 SEQ ID NO: 31 4 SEQ ID NO: 28 SEQ ID NO: 31 5 SEQ ID NO: 25 SEQ ID NO: 32 .

[0095] In a fifth aspect, the present application provides an antibody conjugate, comprising the anti-sST2 antibody or antigen-binding fragment thereof described above.

[0096] In alternative embodiments, the antibody conjugate described above further comprises biotin or a biotin derivative conjugated to the anti-sST2 antibody or antigen-binding fragment thereof.

[0097] In alternative embodiments, the antibody conjugate described above further comprises a label conjugated to the anti-sST2 antibody or antigen-binding fragment thereof.

[0098] In optional embodiments, the above-mentioned markers refer to a class of substances having properties such as luminescence, color development, radioactivity, etc. that can be directly observed by the naked eye or detected or probed by instruments, through which qualitative or quantitative detection of the corresponding target substances can be achieved.

[0099] In optional embodiments, the markers include but are not limited to fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0100] In actual use, the skilled person in the art can select appropriate markers according to the detection conditions or actual needs, and no matter what kind of marker is used, it falls within the protection scope of the present application.

[0101] In optional embodiments, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (such as but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorine (FAM), tetrafluorophorin (TET), etc. or their analogues), rhodamine dyes and their derivatives (such as but not limited to red rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogues), Cy series dyes and their derivatives (such as but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc. or their analogues), Alexa series dyes and their derivatives (such as but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogues), and protein dyes and their derivatives (such as but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0102] In optional embodiments, the enzymes include but are not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphogluconate dehydrogenase.

[0103] In optional embodiments, the radioisotope includes, but is not limited to, 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, and 18F.

[0104] In optional embodiments, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacyanin and its derivatives, ruthenium bispyridyl and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, lucigenin and its derivatives, and peroxyoxalate and its derivatives.

[0105] In optional embodiments, the nanoparticle-based label includes, but is not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0106] In optional embodiments, the colloids include, but are not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latex.

[0107] In optional embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0108] In optional embodiments, the colloidal metal is colloidal gold.

[0109] In optional embodiments, the above antibody conjugate further includes a solid support coupled to the anti-sST2 antibody or antigen-binding fragment thereof.

[0110] In optional embodiments, the solid support is selected from the group consisting of microspheres, plates, and membranes.

[0111] In optional embodiments, the solid support includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, micro-well plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0112] In a sixth aspect, the present application provides a reagent or kit, which includes the above-mentioned anti-sST2 antibody or antigen-binding fragment thereof or the above-mentioned antibody conjugate.

[0113] As described above, the anti-sST2 antibody or antigen-binding fragment thereof in some embodiments or examples of the present application can effectively bind to sST2, and thus the reagent or kit comprising the anti-sST2 antibody or antigen-binding fragment thereof can effectively detect sST2 qualitatively or quantitatively. The reagent or kit provided by the present application can be used in, for example, immunoblotting, immunoprecipitation, and other detection methods involving the specific binding of sST2 and its antibody. As described above, the anti-sST2 antibody or antigen-binding fragment thereof in some embodiments or examples of the present application has higher binding activity or affinity to sST2, and thus the reagent or kit comprising the anti-sST2 antibody or antigen-binding fragment thereof has higher detection sensitivity or specificity.

[0114] In a seventh aspect, the present application provides a method for detecting sST2, comprising: a) contacting the anti-sST2 antibody or antigen-binding fragment thereof, antibody conjugate, reagent or kit described above with sST2 in a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to occur to form an immunocomplex; and b) detecting the presence of the immunocomplex, the presence of the complex indicating the presence of the antigen in the test sample.

[0115] In an alternative embodiment, the immunocomplex further comprises a second antibody that binds to the anti-sST2 antibody or antigen-binding fragment thereof.

[0116] In an alternative embodiment, the immunocomplex further comprises a second antibody that binds to sST2.

[0117] In an eighth aspect, the present application provides a nucleic acid molecule encoding the anti-sST2 antibody or antigen-binding fragment thereof described above.

[0118] In a ninth aspect, the present application provides a vector comprising the nucleic acid molecule described above.

[0119] In a tenth aspect, the present application provides a cell comprising the vector described above.

[0120] In an eleventh aspect, the present application provides a method for producing the anti-sST2 antibody or antigen-binding fragment thereof, comprising culturing the cell described above.

[0121] In a twelfth aspect, the present application provides the use of the anti-sST2 antibody or antigen-binding fragment thereof, antibody conjugate, or reagent or kit described above in the manufacture of a product for detecting sST2.

[0122] On the basis of the amino acid sequences of the anti-sST2 antibodies or antigen-binding fragments thereof disclosed in the present application, it is easy for those skilled in the art to prepare the anti-sST2 antibodies or antigen-binding fragments thereof by using genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, by isolating and purifying the anti-sST2 antibodies or antigen-binding fragments thereof from the culture products of recombinant cells capable of recombinantly expressing the anti-sST2 antibodies or antigen-binding fragments thereof as described in any one of the above, which is easy for those skilled in the art to achieve. Therefore, no matter what technology is used to prepare the anti-sST2 antibodies or antigen-binding fragments thereof of the present application, they all belong to the protection scope of the present application.

[0123] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. If specific conditions are not indicated in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are used. If the manufacturers of the reagents or instruments are not indicated, the conventional products that can be purchased in the market are used.

[0124] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the preparations or unit doses herein, some methods and materials are now described. The techniques employed or contemplated herein are standard methodologies unless otherwise indicated. The materials, methods, and examples are illustrative only and not limiting.

[0125] Unless otherwise indicated, the practice of the present application will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (J. E. Coligan et al., eds., 1991), each of which is incorporated herein by reference in its entirety.

[0126] The features and advantages of the present application are further illustrated in the following examples.

[0127] Example 1. Preparation of Anti-sST2 3F9 Monoclonal Antibody

[0128] Restriction enzymes and Prime Star DNA polymerase used in this example were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TMRACE cDNA Amplification Kit was purchased from Takara. Plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen. Hybridoma cell line secreting Anti-sST2 3F9 monoclonal antibody was the existing hybridoma cell line, which was recovered for use.

[0129] 1.1 Anti-sST2 3F9 antibody gene preparation

[0130] The mRNA was extracted from the hybridoma cell line secreting Anti-sST2 3F9 monoclonal antibody, and the DNA product was obtained by RT-PCR method, and inserted into pMD-18T vector, transformed into DH5α competent cells, and after the colonies were grown, 4 colonies of positive clones of Heavy Chain and Light Chain genes were sent to the gene sequencing company for sequencing.

[0131] 1.2 Sequence analysis of Anti-sST2 3F9 antibody variable region gene

[0132] The gene sequence obtained by the above sequencing was placed in the KABAT antibody database for analysis, and the VNTI11.5 software was used for analysis to determine that the amplified genes by the heavy chain and light chain primer pairs were correct. Among them, the VL gene sequence in the Light Chain amplified gene fragment was 321 bp, and there was a 57 bp leader peptide sequence in front of it; the VH gene sequence in the Heavy Chain primer pair amplified gene fragment was 366 bp, belonging to the VH1 gene family, and there was a 57 bp leader peptide sequence in front of it. The heavy and light chain of the antibody sequence obtained above were humanized and constant region modified, and then the sequence correctness was determined by subsequent eukaryotic recombinant expression.

[0133] 1.3 Construction of recombinant antibody expression plasmid

[0134] pcDNA TM 3.4 vector is the constructed recombinant antibody eukaryotic expression vector, which is modified to introduce multiple cloning enzyme digestion sites, and is referred to as 3.4A expression vector in the following; according to the above antibody variable region gene sequencing results in pMD-18T, Anti-sST2 3F9 antibody VL and VH gene specific primers were designed, which were respectively labeled with restriction endonuclease digestion sites and protection bases at both ends, and 0.70Kb Light Chain gene fragment and 1.41Kb Heavy Chain gene fragment were amplified by PCR method.

[0135] The Heavy Chain and Light Chain gene fragments were double-digested with restriction endonucleases, and the 3.4A vector was also double-digested with restriction endonucleases. After purification and recovery of the fragments and vector, the Heavy Chain gene and Light Chain gene were ligated into the 3.4A expression vector to obtain recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0136] 2. Sample preparation of recombinant antibodies

[0137] HEK293 cells were revived early and passaged to a 200ml volume to achieve a cell density of 3–5 × 10⁻⁶ cells / mL. 6 Cell density reached the required antibody concentration and cell viability >95%; cells were washed by centrifugation, reconstituted with culture medium, and the cell density was adjusted to 2.9 × 10⁻⁶ cells / ml. 6 Cells were washed at a concentration of cells / ml and reconstituted with culture medium, which served as a cell dilution buffer. Plasmid DNA and transfection reagent dilution buffers were prepared separately using culture medium. The transfection reagent dilution buffer was added to the plasmid DNA dilution buffer, mixed well, and incubated at room temperature for 15 min. This mixture was then slowly added to the cell dilution buffer over 1 min, mixed well, and samples were taken for cell counting. Cell viability after transfection was recorded and observed. The cells were then incubated at 35°C with a rotation speed of 120 rpm and a CO2 concentration of 8%. After 13 days, the samples were centrifuged and collected. The supernatant was purified using a protein A affinity chromatography column. 6 μg of the purified antibody was subjected to reducing SDS-PAGE, as shown in the figure. The reducing SDS-PAGE showed two bands: one with a Mr of 50 kDa (heavy chain) and the other with a Mr of 28 kDa (light chain).

[0138] The resulting antibody was named Anti-sST2 3F9Rmb1. Mutations were performed on Anti-sST2 3F9Rmb1 to obtain a mutant antibody. The heavy chain (H) and light chain (L) sequences of the above antibody are shown in the table below:

[0139] Table 2 Antibody Sequences

[0140] Antibody name Heavy chain Light chain Anti-sST2 3F9Mb1 SEQ ID NO: 25 SEQ ID NO: 31 Anti-sST2 3F9Mb2 SEQ ID NO: 27 SEQ ID NO: 31 Anti-sST2 3F9Mb3 SEQ ID NO: 26 SEQ ID NO: 31 Anti-sST2 3F9Mb4 SEQ ID NO: 28 SEQ ID NO: 31 Anti-sST2 3F9Mb5 SEQ ID NO: 25 SEQ ID NO: 32

[0141] 1. Affinity Analysis

[0142] Purified antibodies were diluted in advance, and sST2 recombinant antigen (obtained from Phoenix Biotech) was gradient diluted; the antigen-antibody binding dissociation curve was tested on a Biacore 8K+ device using a CM5 chip pre-coupled with goat anti-mouse IgG, and the instrument automatically fitted to obtain the affinity constant, binding rate, and dissociation rate. (KD represents the equilibrium dissociation constant, i.e., the affinity constant; ka represents the binding rate; and kd represents the dissociation rate).

[0143] Table 3 Affinity data

[0144] Sample name KD (M) ka kd Control 8.12E-9 1.06E+05 8.61E-04 Anti-sST2 3F9Mb1 1.92E-10 6.85E+05 1.31E-04 Anti-sST2 3F9Mb2 2.08E-10 5.72E+05 1.19E-04 Anti-sST2 3F9Mb3 1.89E-10 5.59E+05 1.06E-04 Anti-sST2 3F9Mb4 2.06E-10 1.56E+05 3.22E-05 Anti-sST2 3F9Mb5 2.22E-10 6.01E+05 1.33E-04

[0145] 2. Activity identification

[0146] Coating solution (main component NaHCO3) diluted sST2 recombinant antigen to 3 ug / ml, 100 uL per well, 4°C overnight; the next day, wash solution (main components Na2HPO4+Nacl) was washed twice, and then dried; blocking solution (20% BSA+80% PBS) was added, 120 uL per well, 37°C, 1 h, and then dried; diluted purified antibodies and control antibodies were added, 100 uL per well, 37°C, 30 min; wash solution was washed 5 times, and then dried; goat anti-human IgG-HRP was added, 100 uL per well, 37°C, 30 min; wash solution was washed 5 times, and then dried; color developing solution A (50 uL per well) was added, color developing solution B (50 uL per well) was added, and then 10 min; stop solution was added, 50 uL per well; OD value was read on a microplate reader at 450 nm (reference 630 nm).

[0147] Note: A solution (main components citric acid+ sodium acetate+acetanilide+urea peroxide); B solution (main components citric acid+EDTA·2Na+TMB+concentrated HCL); stop solution (EDTA·2Na+concentrated H2SO4).

[0148] Table 4 Activity data

[0149] Concentration (ng / ml) 31.25 15.63 7.81 3.91 1.95 0.00 Control 1.521 0.912 0.502 0.289 0.151 0.059 Anti-sST2 3F9Mb1 2.023 1.594 1.011 0.572 0.463 0.054 Anti-sST2 3F9Mb2 2.031 1.527 1.012 0.572 0.426 0.055 Anti-sST2 3F9Mb3 2.008 1.558 1.073 0.568 0.443 0.053 Anti-sST2 3F9Mb4 2.014 1.512 1.076 0.599 0.452 0.055 Anti-sST2 3F9Mb5 2.079 1.523 1.031 0.556 0.428 0.058

[0150] 3. Stability evaluation

[0151] The above antibodies were placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days, and samples taken at 7 days, 14 days, and 21 days were observed for state, and the 21-day samples were subjected to activity detection. The results showed that under the three evaluation conditions, the antibodies were placed for 21 days, and no obvious protein state change was observed, and the activity did not show a downward trend with the increase of the evaluation temperature, indicating that the above antibodies were stable. The following table is the OD result of enzyme immunoactivity detection of antibody Anti-sST2 3F9Rmb1 evaluated for 21 days.

[0152] Table 5 Stability data

[0153] Sample concentration (ng / ml) 15.63 7.81 0 4°C, 21-day sample 1.512 1.031 0.022 -80°C, 21-day sample 1.553 1.022 0.021 37°C, 21-day sample 4°C, 21-day sample -80°C, 21-day sample 37°C, 21-day sample 4°C, 21-day sample -80°C, 21-day sample 37°C, 21-day sample 4°C, 21-day sample -80°C, 21-day sample 37°C, 21-day sample 4°C, 21-day sample -80°C, 21-day sample 37°C, 21-day sample 4°C, 21-day sample -80°C, 21-day sample 37°C, 21-day sample 4°C, 21-day sample -80°C, 21-day sample 37°C, 21-day sample 4°C, 21-day sample -80°C, 21-day sample 37°C, 21-day sample 4°C, 21-day sample -80°C, 21-day sample 37°C, 21-day sample 4°C, 21-day sample -80°C, 21-day sample 37°C, 21-day sample 4°C, 21-day sample -80°C, 21-day sample 37°C, 21-day sample 4°C, 21-day sample -80°C, 21-day sample 37°C, 21-day sample 4°C, 21-day sample -80°C, 21-day sample 37°C, 21-day sample 4°C, 21-day sample -80°C, 21-day sample 37°C, 21-day sample 4°C, 21-day sample -80°C, 21-day sample 37°C, 21-day sample 4°C, 21-day sample -80°C, 21-day sample 37°C, 21-day sample 4°C, 21-day sample -80°C, 21-day sample 37°C, 21-day sample 4°C, 21-day sample -80°C, 21-day sample 37 1.587 1.034 0.022

[0154] The preferred embodiments of the present application have been described above with the purpose of not limiting the present application but of providing practical examples for a person skilled in the art. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

[0155] Part of the amino acid sequences involved in the present application are shown in Table 6:

[0156]

[0157]

[0158]

Claims

1. An anti-sST2 antibody or its antigen-binding fragment, characterized in that, The anti-sST2 antibody or its antigen-binding fragment has three complementary determinant regions of the heavy chain variable region as shown in any one of SEQ ID NO: 21, 22, 24, and three complementary determinant regions of the light chain variable region as shown in any one of SEQ ID NO: 29, 30; or The anti-sST2 antibody or its antigen-binding fragment has three complementary determinant regions of the heavy chain variable region as shown in SEQ ID NO:23 and three complementary determinant regions of the light chain variable region as shown in SEQ ID NO:

29. The complementary determination region of the variable region is defined by any one of the systems Kabat, Chothia, IMGT, AbM, or Contact.

2. An anti-sST2 antibody or its antigen-binding fragment, characterized in that, The anti-sST2 antibody or its antigen-binding fragment includes the following complementarity-determining region: HCDR1, its amino acid sequence is shown in SEQ ID NO:

1. HCDR2, its amino acid sequence is shown in SEQ ID NO:

2. HCDR3, its amino acid sequence is shown in SEQ ID NO:

3. LCDR1, whose amino acid sequence is shown in SEQ ID NO:4 or 18. LCDR2, whose amino acid sequence is shown in SEQ ID NO:5, and LCDR3, whose amino acid sequence is shown in SEQ ID NO:6; or HCDR1, its amino acid sequence is shown in SEQ ID NO:

1. HCDR2, its amino acid sequence is shown in SEQ ID NO:

2. HCDR3, its amino acid sequence is shown in SEQ ID NO:

17. LCDR1, whose amino acid sequence is shown in SEQ ID NO:

4. LCDR2, whose amino acid sequence is shown in SEQ ID NO:5, and LCDR3, whose amino acid sequence is shown in SEQ ID NO:

6.

3. The anti-sST2 antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The anti-sST2 antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HFR1, HFR2, HFR3, and HFR4, and the light chain variable region includes LFR1, LFR2, LFR3, and LFR4.

4. The anti-sST2 antibody or its antigen-binding fragment according to claim 3, characterized in that, The HFR1 comprises SEQ ID NO:7 or an amino acid sequence having at least 80% identity with it; The HFR2 comprises SEQ ID NO:8 or an amino acid sequence having at least 80% identity with it; The HFR3 comprises SEQ ID NO:9 or an amino acid sequence having at least 80% identity with it; The HFR4 comprises SEQ ID NO:10 or an amino acid sequence having at least 80% identity with it; The LFR1 includes SEQ ID NO:11 or an amino acid sequence having at least 80% identity with it; The LFR2 comprises SEQ ID NO:12 or an amino acid sequence having at least 80% identity with it; The LFR3 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:13; and The LFR4 includes SEQ ID NO:14 or an amino acid sequence that is at least 80% identical to it.

5. The anti-sST2 antibody or its antigen-binding fragment according to any one of claims 1, 2, and 4, characterized in that, The anti-sST2 antibody or its antigen-binding fragment has a KD < 8.12 × 10⁻⁶. -9 M binds to sST2 with affinity.

6. An anti-sST2 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, characterized in that, The amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NO: 21, 22, 23, 24; the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO: 29, 30. The combination of the heavy chain variable region and the light chain variable region is selected from any of the following combinations: 。 7. The anti-sST2 antibody or its antigen-binding fragment according to any one of claims 1, 2, 4, and 6, characterized in that, The anti-sST2 antibody or its antigen-binding fragment further includes a constant region.

8. The anti-sST2 antibody or its antigen-binding fragment according to claim 7, characterized in that, The constant region includes the heavy chain constant region and / or the light chain constant region.

9. The anti-sST2 antibody or its antigen-binding fragment according to claim 8, characterized in that, The heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

10. The anti-sST2 antibody or its antigen-binding fragment according to claim 8, characterized in that, The heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

11. The anti-sST2 antibody or its antigen-binding fragment according to claim 7, characterized in that, The species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

12. The anti-sST2 antibody or its antigen-binding fragment according to claim 8, characterized in that, The heavy chain constant region sequence is as shown in SEQ ID NO:15 or has at least 80% identity with it.

13. The anti-sST2 antibody or its antigen-binding fragment according to claim 8, characterized in that, The light chain constant region sequence is as shown in SEQ ID NO:16 or has at least 80% identity with it.

14. The anti-sST2 antibody or its antigen-binding fragment according to any one of claims 1, 2, 4, and 6, characterized in that, The antigen-binding fragment is selected from any one of the antibody's F(ab')2, Fab', Fab, Fv, and scFv.

15. An anti-sST2 antibody or an antigen-binding fragment thereof, said antibody comprising a heavy chain and a light chain, characterized in that, The combination of heavy chain and light chain is selected from any of the following groups: 。 16. An antibody conjugate, characterized in that, The antibody-drug conjugate comprises the anti-sST2 antibody or its antigen-binding fragment as described in any one of claims 1 to 15 and biotin conjugated to the anti-sST2 antibody or its antigen-binding fragment.

17. An antibody conjugate, characterized in that, The antibody-drug conjugate comprises the anti-sST2 antibody or its antigen-binding fragment as described in any one of claims 1 to 15, and a label or solid-phase carrier conjugated to the anti-sST2 antibody or its antigen-binding fragment.

18. The antibody conjugate according to claim 17, characterized in that, The markers are selected from fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers.

19. A reagent or kit, characterized in that, The reagent or kit comprises the anti-sST2 antibody or its antigen-binding fragment as described in any one of claims 1 to 15, or the antibody conjugate as described in any one of claims 16 to 18.

20. Use of the anti-sST2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, the antibody conjugate according to any one of claims 16 to 18, or the reagent or kit according to claim 19 in the preparation of products for detecting sST2.

21. The use according to claim 20, characterized in that, include: a) Under conditions sufficient to induce an antibody / antigen binding reaction, the anti-sST2 antibody or its antigen-binding fragment according to any one of claims 1 to 15, the antibody conjugate according to any one of claims 16 to 18, or the reagent or kit according to claim 19 is brought into contact with sST2 in the sample to be tested to form an immune complex. and b) Detect the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample.

22. The use according to claim 21, characterized in that, The immune complex further includes a second antibody, which binds to the anti-sST2 antibody or its antigen-binding fragment.

23. The use according to claim 21, characterized in that, The immune complex also includes a second antibody that binds to sST2.

24. A nucleic acid, characterized in that, It encodes the anti-sST2 antibody or its antigen-binding fragment as described in any one of claims 1 to 15.

25. A carrier, characterized in that, It contains the nucleic acid as described in claim 24.

26. A cell characterized by, It contains the nucleic acid as described in claim 24 or the vector as described in claim 25.

27. A method for preparing the anti-sST2 antibody or its antigen-binding fragment according to any one of claims 1 to 15, characterized in that, It includes: Culture the cells as described in claim 26.

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