Methods and compositions for quantifying il-33

Abstract

Methods and compositions for detecting and quantifying cytokines are provided. The disclosed assays have reduced assay interference relative to commercially available assays and / or control assays. The interference can be cytokine-dependent, non-cytokine-dependent, or both. One embodiment provides an IL-33 immunoassay that can reduce assay interference caused by endogenous soluble IL-33 binding molecules present in the sample. Exemplary soluble IL-33 binding molecules include, but are not limited to, anti-IL-33 antibodies, soluble ST2 receptors, and serum components. In some embodiments, a blocking agent is added to the sample to reduce, inhibit, or block reformation of IL-33 complexes in the sample after acid dissociation of the IL-33 complexes in the sample. In one embodiment, the blocking agent and the detection reagent do not compete for binding to IL-33.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201980025050.6, filed on April 10, 2019, entitled "Method and Composition for Quantifying IL-33". The original application was a national phase application with international application No. PCT / US2019 / 026699, which claimed priority to U.S. Provisional Patent Application No. 62 / 655,887, filed on April 11, 2018.

[0002] Cross-reference to related applications

[0003] This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 655,887, filed April 11, 2018, which is incorporated herein by reference in its entirety. Technical Field

[0004] All aspects of the present invention relate to immunoassays for detecting and quantifying cytokines, including but not limited to interleukin-33 (IL-33). Background Technology

[0005] Interleukin-33 (IL-33) is a cytokine that is upregulated in response to damage signals or aggression, such as cigarette smoke. It is released by barrier tissues upon necrosis and propagates the immune response to the presence of damaged tissue. This "alarmin" signal amplifies the inflammatory responses of both innate and adaptive immunity. Published data suggest that commercial kits for detecting soluble IL-33 produce unreliable results, likely due to interference from endogenous binding partners, such as the soluble ST2 receptor (Nygaard, U. et al., SpringerPlus, 5:33 (2016); Ketelaar, ME, Clin Exp Allergy, 46(6):884-7 (2016); Rivière, E. et al., Ann Rheum Dis, 75(3):633-5 (2016)).

[0006] The performance of ligand binding assays in complex matrices, such as serum, can be affected by specific endogenous components that interfere with the assay (Zhong, ZD et al., AAPS J, 19:1564 (2017)). Interference in immunoassays can lead to laboratory misinterpretation of patient results and physicians administering incorrect treatments (Tate, J. et al., ClinBiochem Rev. 2004, 25(2):105–120).

[0007] Others have attempted to develop better immunoassays. Doucet, J. et al. reported an improved IL-13 assay that includes an acid dissociation step (Doucet, J. et al., Disease Markers, 35(5):465-474(2013)). The immunoassay described by Doucet is IL-13 specific and does not detect IL-33. Furthermore, Doucet's immunoassay does not require sample neutralization prior to detection and does not use blocking agents to prevent the reformation of cytokine complexes during the assay.

[0008] Therefore, new assays and methods are needed for the detection of IL-33. Summary of the Invention

[0009] Methods and compositions for detecting and quantifying cytokines are provided. The disclosed assays have reduced assay interference compared to commercially available assays and / or control assays. This interference may be cytokine-dependent, cytokine-independent, or both. One embodiment provides an IL-33 immunoassay that reduces assay interference caused by endogenous soluble IL-33 binding molecules present in the sample. Exemplary soluble IL-33 binding molecules include, but are not limited to, anti-IL-33 antibodies, soluble ST2 receptors, and serum components. In some embodiments, a blocking agent is added to the sample to reduce, inhibit, or block the reformation of IL-33 complexes in the sample after acid dissociation of the IL-33 complexes in the sample. In one embodiment, the blocking agent and the detection reagent do not compete for binding to IL-33.

[0010] Another embodiment provides a method for determining the concentration of total IL-33 in a human serum sample using an electrochemiluminescence immunoassay. This assay includes acid pretreatment of the serum sample to dissociate soluble ligand:drug complexes present in the sample and improve the detection of IL-33 in the presence of the drug, thereby providing a quantitative measurement of total IL-33 levels. In this implementation, the procedure employs a streptavidin-coated plate (containing a biotinylated anti-human IL-33 antibody as a trapping reagent, and using recombinant IL-33 as a standard). The standard, control, and sample are diluted in acetic acid and neutralized with a Tris-based solution containing a detection reagent (e.g., a ruthenium-labeled anti-human IL-33 antibody). Antibodies binding to the anti-IL-33 antibody and anti-ST2 receptor antibodies are also added to the Tris solution to minimize interference from drugs (i.e., antibodies binding to IL-33) or endogenous soluble ST2 receptors. The neutralized standard, control, and sample are then added to the plate. The IL-33 trapped on the plate is measured using a plate reader based on the chemiluminescent signal generated by the ruthenium tag when a voltage is applied to the plate. The resulting electrochemiluminescent signal (i.e., count) is proportional to the amount of IL-33 present in the serum sample.

[0011] In one implementation, the method has a sensitivity of 12.5 pg / mL and ST2 tolerance of >50 ng / mL. Attached Figure Description

[0012] Figure 1 This is a schematic diagram of an exemplary measurement method according to one embodiment of the present invention. --Ruthenium tag, ■-Biotin, -Streptavidin

[0013] Figure 2A is a line graph of recovery rate versus rhST2 concentration (ng / ml). (■) C-AD acid treatment; (●) C-AD acid-free; (□) CAD acid treatment; (○) CAD acid-free. Figure 2B is a bar graph showing the detection of endogenous IL-33 in serum from three individuals (individual 1, individual 2, and individual 3) under specified conditions: CAD acid-free, C-AD acid-free, CAD acid, and C-AD acid.

[0014] Figure 3 The bar chart shows the endogenous IL-33 specificity in the serum of individuals 1 and 3 using a modified assay with the addition of rST2 and anti-IL-33 monoclonal antibody.

[0015] Figure 4 The graph shows the IL-33 concentration (pg / mL) of individuals 1 and 3 after multiple measurements, demonstrating the accuracy of the assay.

[0016] Figure 5 This is a line graph showing the tolerance of the method for detecting IL-33 in the presence of anti-IL-33 mAb, which was determined under specified conditions: C-AD acid treatment and CAD acid treatment.

[0017] Figure 6 This is a graph showing the serum endogenous IL-33 (pg / mL) levels in healthy individuals and individuals with asthma, COPD, and atopic dermatitis. Twenty-five individuals from four different populations were purchased from a commercial supplier, and endogenous IL-33 was screened using an acid-treated C-AD assay.

[0018] Figure 7A and Figure 7B This is a graph showing human IL-33 (pg / mL) from treated mice and control mice. Detailed Implementation

[0019] I. Definition

[0020] It should be understood that this disclosure is not limited to the compositions and methods described herein, nor the experimental conditions described herein, as they may vary. It should also be understood that the terminology used herein is for the purpose of describing certain embodiments only and is not intended to be limiting, as the scope of this disclosure will be limited only by the appended claims.

[0021] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although any compositions, methods, and materials similar or equivalent to those described herein may be used in the practice or testing of this invention. All publications mentioned are incorporated herein by reference in their entirety.

[0022] As used herein, the term "binding molecule" is intended to refer to a molecule that specifically interacts with and binds to a particular target. Targets may comprise biomolecules or small (chemical) molecules. Target molecules may define an antigen or an antigenic moiety. Examples of binding molecules include, but are not limited to, antibodies (including monoclonal antibodies, bispecific antibodies, and antibody fragments), fusion proteins, and other antigen-binding molecules known to those skilled in the art.

[0023] As used herein, the term "antibody" is an example of a binding molecule and refers to an immunoglobulin, which typically comprises four polypeptide chains (two heavy chains (H) and two light chains (L)) linked together by disulfide bonds. Each heavy chain contains a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2, and CH3. Each light chain contains one light chain variable region (LCVR or VL) and one light chain constant region. The light chain constant region contains one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0024] As used herein, the term “antigen-binding portion” (or simply “antibody portion” or “antibody fragment”) of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (such as hIL-33). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments covered in the term “antigen-binding portion” of an antibody include (i) Fab fragments, which are monovalent fragments containing VL, VH, CL1, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments containing two F(ab)' fragments linked by disulfide bonds in the hinge region; (iii) Fc fragments containing VH and CH1 domains; (iv) Fv fragments containing VL and VH domains of a single arm of the antibody; (v) dAb fragments (Ward, ES et al., Nature 241:544-546 (1989)) containing a VH domain; and (vi) CDR fragments. Furthermore, although the two domains (VL and VH) of the Fv fragment are encoded by independent genes, they can be linked by synthetic linkers using recombination methods, allowing them to be prepared as a single continuous chain, where the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird, RE et al., Science 242:423-426 (1988); and Huston, JS et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). This type of single-chain antibody is also intended to be covered within the term "antigen-binding portion" of antibody. Other forms of single-chain antibodies, such as biantibodies, are also included (see, for example, Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)).

[0025] "CDRs," or complementarity-determining regions, are highly variable regions scattered within a more conservative area called "frame regions" (FRs). FRs can be identical to phylogenetic sequences or can be naturally or artificially modified.

[0026] The term "epitope" is an antigenic determinant that interacts with a specific antigen-binding site called a complementary site in the variable region of an antibody molecule. A single antigen may have more than one epitope. Epitopes can be conformational epitopes or linear epitopes. Conformational epitopes are generated from amino acids from different segments of a linear polypeptide chain that are spatially juxtaposed. Linear epitopes are generated from adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may include a sugar, phosphoryl, or sulfonyl moiety on the antigen.

[0027] The term "assay interference" refers to an endogenous component in an assay that blocks or inhibits the detection of an analyte (such as IL-33 or anti-IL-33 antibody). Altering the measurable concentration of an analyte or altering the substance binding to an antibody can potentially lead to immunoassay interference. Assay interference can be analyte-dependent or non-analyte-dependent. Non-analyte-dependent interference refers to common interferences such as hemolysis, lipemia, and the effects of anticoagulants and sample storage, and is independent of analyte concentration. Analyte-dependent interference in immunoassays refers to the interaction between components in the sample and one or more reagent antibodies. These include chemically different but structurally similar compounds that can cross-react with antibodies. Naturally occurring interfering endogenous substances, multireactive antibodies or autoantibodies (heterophiles), human anti-animal antibodies, or antidrug antibodies (ADAs), and other unrecognized binding proteins specific to the individual can interfere with the reaction between the analyte and the reagent antibody in an immunoassay. Interference may be caused by soluble binding targets of the analyte, endogenous ligands of the analyte (including but not limited to soluble receptors, soluble ligands, and exfoliated receptors of the analyte), or serum factors such as rheumatoid factor and biotin. Interference may also be caused by antibodies against the analyte.

[0028] The term "ST2" refers to the receptor for IL-33, also known as interleukin-1 receptor-like 1 (IL-1RL1). ST2 can be membrane-bound or soluble.

[0029] The term "immunoassay" refers to a detection assay that incorporates a binding moiety of an analyte, either directly or indirectly, that is immune-specifically bound. Typically, the binding moiety is an antibody. Antibodies can be labeled with detectable tags.

[0030] The term "IL-33 complex" refers to the non-covalent association of IL-33 with a component of blood, plasma, or serum. Typically, an IL-33 complex contains IL-33 non-covalently bound to proteins such as protein drug products, anti-drug antibodies, IL-33 ligands, or combinations thereof.

[0031] II. IL-33 Detection and Quantitative Determination Methods and Their Application

[0032] Assays and methods for detecting and quantifying IL-33 are provided. The disclosed assays and methods can be used to detect and quantify IL-33 in samples. In one embodiment, the sample is obtained from a subject who has been treated with or is currently being treated with IL-33, or who has used an IL-33 antagonist.

[0033] Compared to commercially available assays and / or control assays, the disclosed assay has reduced assay interference. This interference may be IL-33-dependent, IL-33-independent, or both. In one embodiment, the method reduces or inhibits assay interference caused by endogenous soluble IL-33-binding molecules present in the sample. Exemplary soluble IL-33-binding molecules include, but are not limited to, anti-IL-33 antibodies, soluble ST2, and serum components. In some embodiments, a blocking agent is added to the sample to reduce, inhibit, or block the reformation of IL-33 complexes in the sample upon acid denaturation. In one embodiment, the blocking agent and the detection reagent do not compete for IL-33 binding.

[0034] In one implementation, the method has a sensitivity of 12.5 pg / mL and ST2 tolerance of >50 ng / mL.

[0035] A. Interleukin-33

[0036] In one embodiment, IL-33, or “human interleukin-33” or “human IL-33”, refers to the full-length, untreated IL-33 of 270 amino acids (see, for example, U.S. Patent Application No. O95760 and Publication No. 20180155436, both of which are incorporated herein by reference in their entirety), or a biologically active fragment thereof, as well as any form of IL-33 produced by intracellular processing. The term also covers naturally occurring variants of IL-33, such as splice variants (see, for example, Hong et al., (2011), J. Biol. Chem. 286(22):20078-20086), or allelic variants or any other isoform of IL-33, such as the oxidized or reduced form of IL-33 as described in WO2016 / 156440. IL-33 activity can be neutralized, inhibited, blocked, eliminated, weakened, reduced, or interfered with by antibodies or their antigen-binding fragments or by IL-33 trap proteins, including but not limited to the inhibition of IL-33 receptor-mediated signal transduction or the inhibition of IL-33-mediated inflammation.

[0037] B. Methods for detecting IL-33

[0038] One embodiment provides a method for detecting IL-33 in a sample by acidifying the sample to a pH sufficient to dissociate the IL-33 complex in the sample and neutralizing the acidified sample with a buffered alkaline solution. A trapping reagent and a detection reagent are also added to the sample to detect IL-33. In some embodiments, a blocking agent is added to the sample to inhibit, reduce, or block the binding of components in the sample to IL-33.

[0039] Another embodiment provides a method for reducing assay interference by acidifying the sample to a pH sufficient to dissociate IL-33 from the IL-33 complex in the sample, followed by neutralizing the acidified sample with a buffered alkaline solution containing a detection reagent. Optionally, an inhibitor that inhibits the reformation of the IL-33 complex in the sample may be added to the sample in a separate step, or the inhibitor may be present in the buffered alkaline solution. The method includes adding a trapping reagent to the sample and detecting the detection reagent, wherein the amount of the detection reagent detected is related to the amount of IL-33 in the sample. The trapping agent may be added as a separate step, or it may be present in the acidification step or in the buffered alkaline solution. Typically, the IL-33 complex present in the sample contains IL-33 that is non-covalently associated with soluble ST2.

[0040] Another embodiment provides a method for quantifying interleukin-33 in a serum sample by acidifying the serum sample to a pH sufficient to dissociate the IL-33 complex in the sample, followed by neutralizing the acidified sample with a buffered alkaline solution containing (a) an anti-human IL-33 antibody labeled with a detectable tag, and optionally (b) an anti-human ST2 antibody. The method includes adding the sample to a streptavidin-coated solid support containing the biotinylated anti-human IL-33 antibody and detecting the detectable tag on the streptavidin-coated solid support, wherein the amount of the detected detectable tag is correlated with the amount of IL-33 in the sample.

[0041] Another embodiment provides a method for determining the concentration of total IL-33 in a human serum sample using an electrochemiluminescence immunoassay. This assay includes acid pretreatment of the serum sample to dissociate soluble ligand-drug complexes present in the sample and improve the detection of IL-33 in the presence of a drug, thereby providing a quantitative measurement of total IL-33 levels. The ligand-drug complex includes, but is not limited to, IL-33 non-covalently bound to an anti-IL-33 antibody. Exemplary anti-IL-33 antibodies are disclosed in U.S. Patent Nos. 10,000,564 and 9,453,072, which are incorporated herein by reference in their entirety. In this implementation, the procedure employs a streptavidin-coated plate with a biotinylated anti-human IL-33 antibody as the trapping reagent and recombinant IL-33 as a standard. The standard, control, and sample are diluted in acetic acid and neutralized with a Tris-based solution containing a detection reagent (e.g., a ruthenium-labeled anti-human IL-33 antibody). Antibodies binding to the anti-IL-33 antibody and anti-ST2 receptor antibodies are also added to the Tris solution to minimize interference from drugs or endogenous soluble ST2 receptors. The neutralized standard, control, and sample are then added to the plate. The IL-33 trapped on the plate is measured using a plate reader based on the chemiluminescent signal generated by the ruthenium tag when a voltage is applied to the plate. The resulting electrochemiluminescent signal (i.e., count) is proportional to the amount of IL-33 present in the serum sample.

[0042] In one implementation, the amount of label detected in the sample is compared to a reference standard calibrated with a known concentration of IL-33 and the corresponding amount of detected label for that concentration. The amount of IL-33 in the sample can be determined by comparing the amount of label detected in the sample to the reference standard and matching the amount of detected label to the concentration shown on the reference standard for that amount of detected label.

[0043] 1. Biological samples

[0044] The samples used in the disclosed methods are typically biological samples, such as biofluids containing the analyte to be detected (e.g., IL-33 or a complex thereof). Biofluids include, but are not limited to, blood, plasma, serum, and saliva. In a preferred embodiment, the biological sample is a human biological sample.

[0045] In some implementations, samples are obtained from subjects who have or are suspected of having an inflammatory condition, disease, or disorder. Representative inflammatory conditions, diseases, and disorders include, but are not limited to, Crohn's disease, colitis, ulcerative colitis, atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, eosinophilic esophagitis, nasal polyps, or combinations thereof.

[0046] The analytes to be detected are typically components of the serum of the subject, preferably a human subject. Representative analytes to be detected and / or quantified in the sample include, but are not limited to, cytokines, protein pharmaceutical products, and their metabolites or fragments.

[0047] Representative cytokines include interleukins. Interleukins include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL -19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, and IL-36. In one embodiment, the analyte is IL-33.

[0048] Representative protein drug products include, but are not limited to, recombinant proteins, antibodies, and fusion proteins. Antibodies can be multireactive antibodies or autoantibodies (heterophiles), human anti-animal antibodies, or anti-drug antibodies. In one embodiment, the protein drug product is an anti-IL-33 antibody.

[0049] 2. Ligands of IL-33

[0050] IL-33 ligands are molecules that non-covalently bind to IL-33, including but not limited to endogenous components of the sample such as antibodies or soluble binding targets of the analyte, and endogenous ligands of IL-33, including but not limited to soluble receptors of IL-33, soluble ligands of IL-33, and exfoliated receptors of the analyte. In one embodiment, the ligand of IL-33 is a soluble ST2 receptor or a soluble co-receptor IL-1 receptor accessory protein (IL-1RAcP).

[0051] ST2 (also known as IL1RL1, DER4, T1, and FIT-1) is a member of the Toll-like / IL-1 receptor superfamily. Members of this superfamily are defined by a common intracellular domain, the Toll / interleukin-1 receptor (TIR) ​​domain. This approximately 160-amino acid domain consists of a five-stranded β-sheet located centrally at the cytoplasmic terminus of the protein, surrounded by five α-helices. The Toll-like / IL-1 receptor superfamily can be divided into three subfamilies based on their extracellular domains: the IL-1 receptor-like subfamily, the Toll receptor subfamily, and the family composed of their adaptor proteins.

[0052] 3. Acid denaturation of the IL-33 complex

[0053] The sample in the disclosed method may be acidified for 1 to 60 minutes. In one embodiment, the sample is acidified for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 minutes.

[0054] Acids that can be used in the acidification step include, but are not limited to, acetic acid, hydrochloric acid, and sulfuric acid.

[0055] The pH sufficient to dissociate the IL-33 complex can be from 3.0 to 5.0. In one embodiment, the pH is 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0056] In one embodiment, a denaturing agent may be added to the sample to dissociate the IL-33 complex, including but not limited to urea.

[0057] 4. Blockers

[0058] The blocking agent used in the disclosed method is a reagent that reduces or prevents the reformation of the IL-33 complex in the sample after dissociating the IL-33 complex during the acidification step. When a sample is obtained from a subject, such as a human subject, the sample typically contains the IL-33 complex to be detected or quantified. The blocking agent can be an antibody that specifically binds to components of the IL-33 complex other than IL-33. In one embodiment, the blocking agent is an anti-ST2 antibody.

[0059] In one implementation, the blocker is an antibody that binds to an IL-33 drug product, such as an antibody that binds to an anti-IL-33 antibody.

[0060] 5. Test reagents

[0061] The detection reagents used in the disclosed methods can be IL-33 antagonists or inhibitors, such as anti-IL-33 antibodies, preferably anti-human IL-33 antibodies or IL-33 trap proteins. The antibodies can be monoclonal, polyclonal, or humanized.

[0062] In some embodiments, the detection reagent is labeled with a detectable tag. Detectable tags are known in the art and include, but are not limited to, rare transition metal particles, fluorophores, chromophores, quantum dots, noble metal nanoparticles, radioactive components, enzymes, biotin / avidin tags, and chemiluminescent tags. In one embodiment, the detectable tag is ruthenium.

[0063] a. Anti-IL-33 antagonists

[0064] In some embodiments, the IL-33 antagonist or inhibitor that can be used in the disclosed assays and methods is an anti-IL-33 antibody or an antigen-binding fragment of an antibody that specifically binds to human IL-33. In one embodiment, the anti-IL-33 antibody described herein for use in the disclosed methods and assays is disclosed in U.S. Patent Nos. 10,000,564 and 9,453,072, which are incorporated herein by reference in their entirety.

[0065] The anti-IL-33 antibodies used in the disclosed assays and methods specifically bind to IL-33. The term "specifically binds" refers to the formation of a relatively stable complex between the antibody or its antigen-binding fragment and the antigen under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis and surface plasmon resonance. For example, antibodies that "specifically bind" IL-33 as used herein include those that bind to IL-33 or its biologically active moiety at KD values ​​of less than about 1000 nM, less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM, as measured in surface plasmon resonance assays. However, isolated antibodies that specifically bind to human IL-33 may exhibit cross-reactivity with other antigens, such as IL-33 molecules from other (non-human) species.

[0066] In some embodiments, the IL-33 antagonist is an anti-IL-33 antibody or its antigen-binding fragment (containing a heavy chain variable region (HCVR), a light chain variable region (LCVR), and / or a complementarity-determining region (CDR)) comprising any of the amino acid sequences of anti-IL-33 antibodies as shown in U.S. Patent Nos. 10,000,564 and 9,453,072. In some embodiments, the IL-33 antagonist is an anti-IL-33 antibody having the binding characteristics of the reference antibodies described in U.S. Patent Nos. 10,000,564 and 9,453,072.

[0067] Other anti-IL-33 antibodies and their antigen-binding fragments that can be used in the methods described herein are disclosed in EP1725261 and U.S. Patent Nos. 9,970,944, 8,187,596, WO2011031600, WO2015099175 and 9,758,578, WO2015106080, and U.S. Patent Nos. 10,059,764 (ANB020), US2016 / 0168242, WO2016 / 077381 and 10,093,730, WO2016 / 077366 and US2018 / 0171405, or WO2016 / 156440 and US2018 / 0207265, each of which is incorporated herein by reference in its entirety.

[0068] b. IL-33 trap protein

[0069] In some embodiments, the IL-33 antagonist or inhibitor used in the disclosed methods and assays may be a receptor-based IL-33 trap protein. An exemplary IL-33 trap protein includes at least one IL-33 binding domain containing the IL-33 binding moiety of an IL-33 receptor protein (e.g., ST2). In some embodiments, the IL-33 trap protein further includes an extracellular portion of an IL-33 co-receptor (e.g., an IL-1 receptor helper protein or IL-1RAcP). The IL-33 trap protein may also contain at least one polymerizing component that serves to interconnect the various components of the trap protein.

[0070] In one embodiment, the IL-33 trap protein described herein for the disclosed methods and assays is disclosed in U.S. Patent Nos. 9,637,535 and WO2014 / 152195, each of which is incorporated herein by reference in its entirety.

[0071] Generally, IL-33 trap proteins include a first IL-33-binding domain (D1) attached to a polymerizing domain (M). In some embodiments, the IL-33 antagonist includes a second IL-33-binding domain (D2) attached to D1 and / or M. According to some embodiments, D1 includes the IL-33-binding portion of the ST2 protein. The IL-33-binding portion of the ST2 protein may include or consist of all or part of the extracellular domains of the ST2 protein. In some embodiments, the ST2 protein is the human ST2 protein. As used herein, “human ST2 protein” means the ST2 protein as shown in amino acids 1-556 of accession number NP_057316.3, which is incorporated herein by reference in its entirety. According to some embodiments, D2 includes the extracellular portion of the IL-1RAcP protein.

[0072] The components of the IL-33 trap protein can be arranged relative to each other in various ways to create a functional antagonist molecule capable of binding IL-33. For example, D1 and / or D2 can be attached to the N-terminus of M. In other embodiments, D1 and / or D2 are attached to the C-terminus of M. In other embodiments, D1 is attached to the N-terminus of D2 and D2 is attached to the N-terminus of M, resulting in a tandem fusion of the antagonist molecule represented by the formula D1-D2-M from the N-terminus to the C-terminus.

[0073] In some embodiments, the IL-33 antagonist has two polymerized domains, M1 and M2, wherein M1 and M2 are identical to each other. For example, M1 may be an Fc domain having a specific amino acid sequence, and M2 may be an Fc domain having the same amino acid sequence as M1.

[0074] c. Other IL-33 antagonists

[0075] Other reagents that can act as IL-33 antagonists and can be used in the disclosed methods and assays include immunoadhesins, peptibody, soluble ST2, or derivatives thereof; anti-IL-33 receptor antibodies (e.g., anti-ST2 antibodies, such as AMG-282 (Amgen) or STLM15 (Janssen)); or WO2012 / 113813 and U.S. Patent Nos. 9,309,319, WO2013 / 173761 and 9,982,054, WO2013 / 165894 and U.S. Patent Nos. Any of the anti-ST2 antibodies described in Patent No. 9,951,137, U.S. Patent No. 8,444,987, or U.S. Patent No. 7,452,980, the entire contents of each of these patents are incorporated herein by reference. Other IL-33 antagonists used in the disclosed methods and assays include ST2-Fc proteins, such as those described in WO2013 / 173761 and U.S. Patent No. 9,982,054, or WO2013 / 165894 and U.S. Patent No. 9,951,137, the entire contents of each of these patents are incorporated herein by reference.

[0076] 6. Trapping reagent

[0077] In one embodiment, the trapping reagent used in the disclosed methods and assays is an IL-33 antagonist as described above, such as an antibody, preferably an anti-IL-33 antibody, and more preferably an anti-human IL-33 antibody. Exemplary anti-IL-33 antibodies are disclosed in U.S. Patent No. 10,000,564, which is incorporated herein by reference in its entirety. Other anti-IL-33 antibodies that may be used are described above. In one embodiment, the trapping reagent is biotinylated.

[0078] In some implementations, the capture reagent is immobilized to or attached to a solid support. The solid support may be a microplate, such as a streptavidin-coated microplate.

[0079] In some implementations, the trapping agent is the aforementioned IL-33 antagonist.

[0080] C. Companion Diagnoses

[0081] In one embodiment, the disclosed methods and assays for detecting and quantifying IL-33 are used as companion diagnostic methods in conjunction with treatments designed to reduce or increase circulating IL-33 levels in subjects receiving treatment. The treatment may include administration of an IL-33 antagonist or administration of IL-33. Exemplary IL-33 antagonists are those antagonists described above, and include, but are not limited to, anti-IL-33 antibodies, anti-IL-33 fusion proteins, sST decoy receptors, or combinations thereof.

[0082] One implementation provides a method for monitoring IL-33-related disease in subjects receiving treatment including administration of an IL-33 antagonist as described above or administration of IL-33. Representative diseases, disorders, or pathologies treated with IL-33 inhibitors or IL-33 include, but are not limited to, asthma, eosinophilic or non-eosinophilic asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD), inflammatory bowel disease, Crohn's disease, colitis, ulcerative colitis, multiple sclerosis, arthritis, allergic rhinitis, atopic dermatitis, eosinophilic esophagitis, psoriasis, nasal polyps, Alzheimer's disease, atherosclerosis, myocardial infarction, ischemic stroke, and fibrotic conditions including, but not limited to, Dupuytren's disease, adhesive capsulitis, periarticular fibrosis, keloids or hypertrophic scars, endometriosis, abdominal adhesions, perineural fibrosis, Ledderhose disease, Peyronie's disease, peritendinous adhesions, and periarticular fibrosis.

[0083] The methods and assays described herein can be used to assess, detect, or quantify circulating IL-33 levels in a subject before and after treatment with one or more pharmaceutical compositions containing one or more IL-33 antagonists as described above (e.g., particularly drugs relating to the mechanism of action involving IL-33), with anti-inflammatory therapy, or by immunoadsorption therapy, or wherein IL-33 is assessed after such treatment and the concentration or amount of IL-33 is compared with a predetermined level or with a level measured in the subject before treatment. The observation of an unfavorable IL-33 concentration or amount after treatment confirms that the subject will not benefit from further or continued treatment, while the observation of a favorable IL-33 concentration or amount after treatment confirms that the subject will benefit from further or continued treatment.

[0084] In one embodiment, the adverse IL-33 concentration detected by the disclosed methods and assays in samples from subjects treated with an IL-33 inhibitor is the same as or higher than the IL-33 level measured in the subjects prior to treatment. In one embodiment, the favorable IL-33 concentration detected by the disclosed methods and assays in samples from subjects treated with an IL-33 inhibitor is the IL-33 level lower than the IL-33 level measured in samples from the subjects prior to treatment.

[0085] In another embodiment, the adverse IL-33 concentration detected using the disclosed methods and assays in samples from subjects receiving treatment with IL-33 incorporation is a level lower than the IL-33 level measured in samples from the subjects prior to treatment. In another embodiment, the favorable IL-33 concentration detected using the disclosed methods and assays in samples from subjects receiving treatment with IL-33 incorporation is an IL-33 level higher than the IL-33 level measured in samples from the subjects prior to treatment.

[0086] Confirming circulating IL-33 levels can help manage clinical research and provide improved patient care.

[0087] III. Reagent Kit

[0088] A kit is also provided for determining the presence, amount, or concentration of IL-33 (or a fragment thereof) in a test sample. The kit includes at least one component for determining IL-33 (or a fragment thereof) in the test sample and instructions for determining IL-33 (or a fragment thereof) in the test sample.

[0089] In one embodiment, the kit includes the detection reagent as described above, the trapping reagent as described above, a solid support, other reagents, and written instructions for performing the assay. The detection reagent and the trapping reagent may be the same or different IL-33 antagonists described above, optionally immobilized on a solid phase, such as monoclonal antibodies (or fragments, variants, or fragments of variants thereof), fusion proteins, IL-33 trapping proteins, or aptamers.

[0090] Assay reagents are typically labeled with detectable tags such as chemiluminescent tags. Assay reagents may incorporate detectable tags as described herein, such as fluorophores, radioactive components, enzymes, biotin / avidin tags, chromophores, chemiluminescent tags, etc., or the kit may include reagents labeled with detectable tags for use. Antibodies, calibrators, and / or controls may be provided in separate containers or may be pre-dispensed into suitable assay formats, such as microtiter plates. In one embodiment, the IL-33 assay reagent is labeled with ruthenium.

[0091] In some embodiments, the kit contains a biotin-labeled trapping reagent and a streptavidin-coated solid support, such as a microtiter plate or electrochemiluminescence platform. In some embodiments, the trapping reagent and the microtiter plate or electrochemiluminescence platform are provided in separate containers. In other embodiments, the kit contains a microtiter plate or electrochemiluminescence platform plate coated with the trapping reagent. The kit also contains acid solutions and buffer solutions for sample processing as described above.

[0092] The kit may include calibrators or controls, such as isolated or purified IL-33. The kit may include at least one container (e.g., tube, microtiter plate, strip, or electrochemiluminescence platform) for performing the assay, and / or buffers, such as assay buffer or wash buffer (either of which may be provided as a concentrated solution), substrate solution for a detectable tag (e.g., enzyme tag), or stop solution. Preferably, the kit includes all components necessary for performing the assay, i.e., reagents, standards, buffers, diluents, etc. Instructions may be in paper or computer-readable form.

[0093] Optionally, the kit includes quality control components (e.g., a sensitivity panel, calibrators, and positive controls). The preparation of quality control reagents is well known in the art and is described in inserts for various immunodiagnostic products. The sensitivity panel members are optionally used to establish the performance characteristics of the assay and are also optionally useful indicators of the integrity of the reagents in the immunoassay kit and the standardization of the assay.

[0094] The kit may optionally include other reagents required for diagnostic assays or to aid in quality control evaluation, such as buffers, salts, enzymes, enzyme cofactors, enzyme substrates, assay reagents, etc. The kit may also include other components, such as buffers and solutions (e.g., pretreatment reagents) for separating and / or processing test samples. The kit may additionally include one or more other controls. One or more components of the kit may be lyophilized; in this case, the kit may also contain reagents suitable for reconstitution of the lyophilized components.

[0095] If necessary, the various components of the kit may optionally be placed in suitable containers, such as microtiter plates. The kit may also include containers for containing or storing samples (e.g., containers or tubes for urine samples). Where appropriate, the kit may optionally also contain reaction vessels, mixing vessels, and components that facilitate the preparation of reagents or test samples. The kit may also include one or more instruments for assisting in obtaining test samples, such as syringes, pipettes, tweezers, measuring spoons, etc.

[0096] Example

[0097] Example 1: Determination Method

[0098] Figure 1 This is a schematic diagram of an exemplary assay for detecting IL-33. This method for quantifying IL-33 is a sandwich immunoassay performed on an electrochemiluminescence platform. The procedure uses streptavidin-coated microplates and a biotinylated anti-IL-33 antibody as a trapping reagent. The method includes acid pretreatment of a serum sample to dissociate soluble ligands present in the sample: binding chaperone complexes and improved detection of IL-33 in the presence of binding chaperones (e.g., anti-IL-33 therapeutic agents and / or soluble ST2), thereby providing a quantitative measurement of total IL-33 levels. The acidified sample is then neutralized in an alkaline solution containing a labeled anti-IL-33 detection antibody, in one embodiment of which may competitively bind IL-33 to soluble ST2 receptors and drugs.

[0099] Exemplary acids that can be used include, but are not limited to, acetic acid, sulfuric acid, and hydrochloric acid. Samples can be neutralized using a buffered alkaline solution (e.g., Tris solution).

[0100] Exemplary IL-33 antibodies that may be used are described in U.S. Patent No. 10,000,564, which is incorporated herein by reference in its entirety. Other anti-IL-33 antibodies that may be used in the disclosed methods include, but are not limited to, those described in U.S. Patent No. 9,758,578 and U.S. Patent Application Publication 2018 / 0037644.

[0101] In one embodiment, standards, controls, and samples are diluted in acetic acid and neutralized with a Tris-based solution containing a detection reagent (e.g., a ruthenium-labeled anti-human IL-33 antibody). Anti-IL-33 and anti-ST2 antibodies are also added to the Tris solution to minimize interference from drugs or endogenous binding partners. The neutralized standards, controls, and samples are then added to the plate. IL-33 trapped on the plate is measured using a plate reader based on the chemiluminescent signal generated by the ruthenium tag when a voltage is applied to the plate. The resulting electrochemiluminescent signal (i.e., count) is proportional to the amount of IL-33 present in the serum sample.

[0102] Example II: Minimizing Endogenous Binding Partner Interference

[0103] Materials and methods

[0104] Recombinant human ST2 tolerance was tested using four different assay conditions (described below), where for all conditions the first (capture) step was to add biotinylated anti-IL-33mAb to a streptavidin plate.

[0105] For the stepwise assay (Catch-Analyte-Detect, CAD), the sample (containing the analyte, i.e., IL-33) is first diluted in an acidic solution and incubated. After washing the plate, the acidified sample is further diluted in an alkaline neutralization buffer and then added to the assay plate. After sample incubation, the assay plate is washed and labeled detection mAb solution is added. After detection reagent incubation, the plate is washed, buffer is added, and the plate is read. The assay can also be performed without the acidification step, in which the sample is diluted in the assay buffer and then added to the assay plate.

[0106] For assays performed with simultaneous addition of analyte and detector (C-AD), the sample is first diluted in an acidic solution and incubated. After plate washing, the acidified sample is further diluted in an alkaline neutralization buffer containing the labeled detector mAb and then added to the assay plate. After sample incubation, the plate is washed, buffer is added, and the plate is read. The assay can also be performed without the acidification step, in which the sample is first diluted in assay buffer, followed by dilution in assay buffer containing the labeled detector mAb, and then the sample is added to the assay plate.

[0107] Recombinant IL-33 at 36 pg / mL was tested under four different assay conditions as described above in the presence of increasing concentrations of recombinant soluble ST2, as shown in Figure 2A.

[0108] result

[0109] Acid treatment is necessary for detecting recombinant human IL-33 in the presence of recombinant ST2. Adding an acid treatment step to the CAD assay improves ST2 tolerance to approximately 12 ng / mL, and further improves it to >50 ng / mL using the acid-treated C-AD assay (Figure 2A). Acid treatment is also necessary for improving the detection of endogenous IL-33 in human serum. The C-AD assay improves the detection of endogenous IL-33 compared to the CAD assay due to reduced assay interference from endogenous binding chaperones (Figure 2B).

[0110] Example III: Precision and Specificity: Endogenous IL-33

[0111] Materials and methods

[0112] The specificity for IL-33 was confirmed by analyzing serum samples from two different individuals in an acid-treated C-AD assay as described in Example II, wherein the assay is performed with or without the addition of excess recombinant human ST2 or 200 μg / mL of anti-IL-33 antibody to an alkaline neutralized solution containing the detection reagent.

[0113] Two individuals were tested using the acid-treated C-AD assay as described in Example II. The data represent four independent replicate assays performed on five separate dates.

[0114] result

[0115] This assay is specific for endogenous IL-33 because the addition of a binding chaperone (such as recombinant ST2 or anti-IL-33 mAb) to the neutralization solution inhibits the detection of IL-33. Figure 3 It is a bar chart showing the specificity of endogenous IL-33.

[0116] The assay demonstrated acceptable accuracy, with percentage coefficients of variation (%CV) of 7.1 and 12.3 for individuals 1 and 3, respectively. Figure 4 This is a graph showing the accuracy of this assay in quantitative human serum endogenous IL-33.

[0117] Example IV: Detection of IL-33 (anti-IL-33mAb tolerance) in the presence of anti-IL-33mAb

[0118] Materials and methods

[0119] Figure 5 This is a graph showing the tolerance of the method for detecting IL-33 in the presence of anti-IL-33 mAb. Recombinant IL-33 at 90 pg / mL was tested in the acid-treated C-AD method described in Example II in the presence of escalating concentrations of anti-IL-33 mAb. Figure 5 As shown in the figure. The assay was improved by adding excess antidrug mAb and antiST2 mAb to the neutralization solution.

[0120] result

[0121] Acid treatment is important for detecting human IL-33 in the presence of anti-IL-33 mAb. Adding the assay reagent to the neutralization solution, as well as adding anti-drug mAb and anti-ST2 mAb, can improve the tolerance of anti-IL-33 mAb in this assay to >500 μg / mL.

[0122] Example V: Endogenous IL-33 levels in normal and diseased individuals

[0123] Materials and methods

[0124] Twenty-five individuals from four different groups were purchased from commercial suppliers and endogenous IL-33 was screened using the assay method described in Example II, namely the acid-treated C-AD assay.

[0125] result

[0126] Figure 6 This is a graph showing the endogenous IL-33 (pg / mL) in the serum of healthy individuals and individuals with asthma, COPD, and atopic dermatitis. Results for all tested individuals showed IL-33 concentrations below 31 pg / mL.

[0127] Example VI: Humanized Mouse Data

[0128] Materials and methods

[0129] Humanized IL-33 mice were treated with house dust mites (HDM) for 15 weeks to serve as a model of human respiratory disease. An exemplary humanized IL-33 mouse is disclosed in U.S. Patent Publication No. 20170311580, which is incorporated herein by reference in its entirety. Samples were tested in a modified form of the C-AD assay with acid treatment as described in Example IV.

[0130] result

[0131] Figure 7A and 7B Human IL-33 (pg / mL) from treated and control mice is shown. At the end of treatment, saline control, HDM-treated control, and isotype control showed similar human IL-33 levels. Mice administered with anti-IL-33 monoclonal antibodies showed significantly elevated serum IL-33 levels.

[0132] A total human IL-33 assay has been developed with a sensitivity of 12.5 pg / mL in pure serum. Overall, the data indicate that the acid treatment step is crucial for removing the endogenous binding partner of IL-33. Adding the detection antibody to the neutralization solution improves recombinant / endogenous ST2 tolerance. This assay has demonstrated specificity for endogenous human IL-33. HDM mice treated with anti-IL-33 antibodies showed elevated human IL-33 levels due to the binding target acquiring the drug's half-life, while HDM controls and isotype controls appeared to remain at baseline.

[0133] Although the invention has been described in the foregoing specification with respect to certain embodiments thereof, and many details have been set forth for illustrative purposes, it will be apparent to those skilled in the art that the invention is open to additional embodiments, and that some of the details described herein may be modified considerably without departing from the basic principles of the invention.

[0134] All references cited herein are incorporated herein by reference in their entirety. The invention may be embodied in other specific forms without departing from its spirit or essential nature, and therefore the scope of the invention should be indicated by reference to the appended claims rather than the foregoing description.

Claims

1. A method for reducing interference in IL-33 assays, wherein the method comprises the following steps: The sample was acidified to a pH level sufficient to cause the IL-33 complex in the sample to dissociate and release IL-33, wherein, The sample is acidified for 5 to 60 minutes, and the pH is adjusted to 3 to 5. The acidified sample was then neutralized with a buffer alkaline solution containing a detection reagent labeled with a detectable tag and a blocking agent that inhibits the formation of the IL-33 complex, wherein the detection reagent and the blocking agent do not compete for binding to IL-33, and wherein the detection reagent contains an anti-IL-33 antibody conjugated to the detectable tag. A trapping reagent is added to the sample, wherein the trapping reagent is added to the sample in a separate step independent of acidification or neutralization of the sample, and wherein the trapping reagent is an anti-human IL-33 antibody; and The detection reagent is tested, wherein the amount of the detection reagent detected is related to the amount of IL-33 in the sample.

2. The method of claim 1, wherein the sample comprises blood, serum or plasma.

3. The method of claim 2, wherein the sample is from a subject who has been given the IL-33 drug product.

4. The method of claim 3, wherein the IL-33 pharmaceutical product comprises an anti-IL-33 antibody.

5. The method according to any one of claims 1-4, wherein the IL-33 complex comprises IL-33 non-covalently bound to the protein.

6. The method of claim 5, wherein the protein is an endogenous serum protein.

7. The method of claim 5, wherein the protein is ST2 or its IL-33 binding fragment.

8. The method of claim 5, wherein the protein is an anti-IL-33 antibody or an IL-33-binding fragment thereof.

9. The method of claim 1, wherein the detection reagent comprises a detectable agent selected from rare transition metal particles, chromophores, quantum dots, and noble metal nanoparticles.

10. The method of claim 1, wherein the detection reagent comprises a fluorophore.

11. The method of claim 1, wherein the detection reagent is labeled with ruthenium.

12. The method of claim 1, wherein the trapping agent is conjugated to a solid support.

13. The method of claim 1, wherein the trapping agent is biotinylated.

14. The method of claim 1, wherein the sample is acidified with acetic acid.

15. The method of claim 1, wherein the buffered alkaline solution contains a blocking agent, and the blocking agent is an antibody.

16. The method of claim 1, wherein the buffered alkaline solution contains a blocking agent, and the blocking agent is an anti-ST2 antibody.

17. The method of claim 1, wherein the method has an IL-33 sensitivity of 12.5 pg / mL.

18. The method of claim 1, wherein the method has ST2 tolerance of >50 ng / mL.

19. The method of claim 1, wherein the sample is obtained from a subject diagnosed with or suspected of having an inflammatory disease or disorder.

20. The method of claim 1, wherein the sample is obtained from a subject diagnosed with or suspected of having asthma, chronic obstructive pulmonary disease, or atopic dermatitis.

21. The method of claim 12, wherein the solid support is an electrochemiluminescence platform.

22. The method of claim 1, wherein the amount of IL-33 is determined by relating the amount of the detected test reagent to a predetermined reference standard.

23. A method for quantifying interleukin-33 in a serum sample, comprising the following steps: The serum sample was acidified to a pH sufficient to dissociate the IL-33 complex in the sample, wherein, The sample is acidified for 5 to 60 minutes, and the pH is adjusted to 3 to 5. The acidified sample is neutralized with a buffer alkaline solution comprising (a) an anti-human IL-33 antibody labeled with a detectable tag and (b) an anti-human ST2 antibody, wherein the anti-human IL-33 antibody and the anti-human ST2 antibody do not compete for binding to IL-33; The sample is added to an avidin-coated solid support containing a biotinylated anti-human IL-33 antibody in a separate step, independent of acidification or neutralization of the sample. as well as The detectable tag on the avidin-coated solid support is detected, wherein the amount of the detectable tag detected is correlated with the amount of IL-33 in the sample.

24. The method of claim 23, wherein the solid support is a streptavidin-coated electrochemiluminescence platform.

25. The method of claim 23 or 24, wherein the detectable tag is ruthenium.

26. The method of claim 23, wherein the amount of the anti-human ST2 antibody present is sufficient to reduce ST2 binding to IL-33 in the sample.

27. The method of claim 23, wherein the method has an IL-33 sensitivity of 12.5 pg / mL and ST2 tolerance of >50 ng / mL.

28. The method of claim 1 or 23, wherein acidifying the sample comprises adding an additional denaturant to the sample.

29. The method of claim 28, wherein the denaturing agent is urea.

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