Assay for detecting IL-33

Abstract

The present disclosure relates to a method for detecting different forms of antigenicity of IL-33 in a biological sample, kits for performing such methods, binding molecules and reporter molecules for use in such methods.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 371,932, filed on August 19, 2022, and U.S. Provisional Patent Application No. 63 / 385,817, filed on December 2, 2022, under 35 U.S.C. §119(e), which are incorporated herein by reference in their entirety for all purposes.

[0003] This application incorporates by reference the sequence listing submitted in computer readable form (CRF) with this application, which is a text file named "IL33-208-WO-PCT Sequence Listing" created on August 17, 2023 and has a size of 46,433 bytes. Technical Field

[0004] The present disclosure relates to a method for detecting antigenically distinct forms of IL-33 in a biological sample, kits for performing such methods, binding molecules and reporter molecules for use in such methods. Background Art

[0005] Interleukin-33 (IL-33), also known as IL-1F11, is a member of the IL-1 family of cytokines. IL-33 is a 270 amino acid protein composed of two domains: a homeodomain and a cytokine (IL-1-like) domain. The homeodomain contains a nuclear localization signal (NLS). IL-33 is known to exist in different forms; a reduced form (redIL-33) and an oxidized form (oxIL-33). Previous studies have shown that the reduced form is rapidly oxidized under physiological conditions to form at least one disulfide bond in the oxidized form, and that the two forms may have different binding modes and effects.

[0006] The reduced form of IL-33 has previously been found to bind to ST2 and is in fact the only known ligand for the ST2 receptor expressed by Th2 cells and mast cells. Reduced IL-33 stimulates target cells by binding to ST2 and subsequently activating the NFkB and MAP kinase pathways, leading to the production of cytokines and chemokines such as IL-4, IL-5, and IL-13 to promote inflammation. Soluble ST2 (sST2) is thought to be a decoy receptor that blocks reduced IL-33 signaling.

[0007] Recently, it was discovered that the oxidized form of IL-33 also has physiological effects. It was found that oxidized IL-33 does not bind to ST2, but instead binds to the receptor for advanced glycation end products (RAGE) and signals through this alternative pathway.

[0008] As a result, there has been intense interest in IL-33 as a therapeutic target, primarily because the now-referred reduced form of IL-33 can stimulate ST2 and lead to a potent inflammatory effect, and now also because the oxidized form can stimulate RAGE, which is implicated in a wide range of diseases.

[0009] Given the different forms of IL-33 currently known to exist and the different physiological effects associated with each form, it would be beneficial to be able to detect such different forms in a biological sample.

[0010] However, until now, there is no reliable means to distinguish the different forms of IL-33. Some assays for detecting IL-33 have been developed. However, the readings from these assays are generally not able to distinguish between different forms, and therefore cannot provide information about biological pathways that may be inhibited or activated in a given biological sample for the user. For example, the user has no way of knowing whether the result indicates the activation of the ST2 pathway or the RAGE pathway. In addition, because these assays cannot distinguish between different forms, it is unclear which form is measured in any given assay, and the results generally lack consistency and reliability.

[0011] The inventors have developed a highly sensitive assay that can detect not only one IL-33 form in a biological sample, but each of the different forms of IL-33; i.e., reduced IL33, oxidized IL33, and bound IL33 / sST2. The assay is based on the development of novel binding molecules that can bind to each of the different forms of IL-33. The inventors believe that this assay will be useful for analyzing the antigenic different forms of IL-33 present in samples from patients in order to provide information for medical treatment.

[0012] Accordingly, one or more aspects of the present disclosure and further features thereof are defined hereinafter. Summary of the invention

[0013] According to a first aspect of the present disclosure, there is provided a method for detecting one or more antigenically different forms of IL-33 in a biological sample, comprising:

[0014] (a) contacting the sample with one or more binding molecules under conditions sufficient to form a complex,

[0015] Each of the binding molecules is capable of binding to an antigenically different form of IL-33 selected from:

[0016] Reduced IL-33, oxidized IL-33 and / or IL-33 / sST2; and

[0017] (b) detecting the level of one or more reduced IL-33, oxidized IL-33 and / or IL-33 / sST2 complex in the sample, wherein:

[0018] (i) The binding molecule that binds to reduced IL-33 comprises:

[0019] a. A variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 1, 2 and 3, respectively, and a variable light domain having VLCDRs 1-3 of SEQ ID NOs: 5, 6 and 7, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions;

[0020] b. Variable heavy domains having VHCDRs 1-3 of SEQ ID NOs: 25, 26 and 27, respectively, and VLCDRs of SEQ ID NOs: 29, 30 and 31, respectively.

[0021] 1-3, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions; or

[0022] c. A variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 35, 36 and 37, respectively, and a VLCDR having VHCDRs of SEQ ID NOs: 39, 40 and 41, respectively.

[0023] 1-3 variable light domain, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions;

[0024] (ii) a binding molecule that binds to oxidized IL-33 comprises a corresponding SEQ ID NO: 9,

[0025] 10 and 11 of the variable heavy domain of VHCDR 1-3 and having the corresponding SEQ ID NO:

[0026] The variable light domain of VLCDR 1-3 of 13, 14 and 15, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions; and

[0027] (iii) The binding molecule that binds to IL-33 / ST2 comprises a binding molecule having the corresponding SEQ ID NOs: 17, 18

[0028] and 19 of the variable heavy domain of VHCDR 1-3 and having corresponding SEQ ID NO: 21,

[0029] The variable light domain of VLCDR 1-3 of 22 and 23, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions.

[0030] In one example, the method of the first aspect further comprises the step of contacting the complexes with one or more reporter molecules capable of binding to one or more complexes. In such examples, the detecting step (b) comprises detecting the level of bound reporter molecules in the sample.

[0031] In one example, the reporter molecule is defined as follows.

[0032] In one example, step (a) comprises contacting the sample with one or more binding molecules capable of binding to reduced IL-33. Suitably, in such examples, the one or more binding molecules include those defined in (i). Suitably, in such examples, the one or more binding molecules consist of those defined in (i). Suitably, in such examples, step (b) comprises detecting the level of reduced IL-33-binding molecule complexes.

[0033] In one example, step (a) comprises contacting the sample with one or more binding molecules capable of binding to oxidized IL-33. Suitably, in such examples, the one or more binding molecules include those defined in (ii). Suitably, in such examples, the one or more binding molecules consist of those defined in (ii). Suitably, in such examples, step (b) comprises detecting the level of oxidized IL-33-binding molecule complexes.

[0034] In one example, step (a) comprises contacting the sample with one or more binding molecules capable of binding to IL-33 / sST2. Suitably, in such examples, the one or more binding molecules include those defined in (iii). Suitably, in such examples, the one or more binding molecules consist of those defined in (iii). Suitably, in such examples, step (b) comprises detecting the level of IL-33 / sST2-binding molecule complexes.

[0035] In one example, step (a) comprises contacting the sample with one or more binding molecules capable of binding to reduced IL-33 and IL-33 / sST2. Suitably, in such examples, at least one binding molecule is as defined in (i) or (iii). Suitably, in such examples, the binding molecules include those defined in (i) and (iii). Suitably, in such examples, the binding molecules consist of those defined in (i) and (iii). Suitably, in such examples, step (b) comprises detecting the levels of reduced IL-33-binding molecule complexes and IL-33 / sST2-binding molecule complexes.

[0036] In one example, step (a) comprises contacting the sample with one or more binding molecules capable of binding to oxidized IL-33 and IL-33 / sST2. Suitably, in such examples, at least one binding molecule is as defined in (ii) or (iii). Suitably, in such examples, the binding molecules include those defined in (ii) and (iii). Suitably, in such examples, the binding molecules consist of those defined in (ii) and (iii). Suitably, in such examples, step (b) comprises detecting the levels of oxidized IL-33-binding molecule complexes and IL-33 / sST2-binding molecule complexes.

[0037] In one example, step (a) comprises contacting the sample with one or more binding molecules capable of binding to oxidized IL-33 and reduced IL-33. Suitably, in such examples, at least one binding molecule is as defined in (i) or (ii). Suitably, in such examples, the binding molecules include those defined in (i) and (ii). Suitably, in such examples, the binding molecules consist of those defined in (i) and (ii). Suitably, in such examples, step (b) comprises detecting the levels of reduced IL-33-binding molecule complexes and oxidized IL-33-binding molecule complexes.

[0038] In one example, step (a) comprises contacting the sample with one or more binding molecules capable of binding to oxidized IL-33, reduced IL-33 and IL-33 / sST2. Suitably, in such examples, at least one binding molecule is as defined in (i), (ii) or (iii). Suitably, in such examples, the binding molecules include those defined in (i), (ii) and (iii). Suitably, in such examples, the binding molecules consist of those defined in (i), (ii) and (iii). Suitably, in such examples, step (b) comprises detecting the levels of reduced IL-33-binding molecule complexes, oxidized IL-33-binding molecule complexes and IL-33 / sST2-binding molecule complexes.

[0039] According to a second aspect of the present disclosure, there is provided a method for detecting antigenically different forms of IL-33 in a biological sample, comprising:

[0040] (a) contacting the sample with one or more binding molecules capable of binding to reduced IL-33, oxidized IL-33, and IL-33 / sST2 under conditions sufficient to form a complex; and

[0041] (b) detecting the levels of reduced IL-33, oxidized IL-33 and IL-33 / sST2 complex in the sample.

[0042] In one example, the binding molecule is as defined in the fourth, fifth and sixth aspects.

[0043] In one example, the method of the second aspect further comprises the step of contacting the complex with one or more reporter molecules capable of binding to the complex. In such examples, the detecting step (b) comprises detecting the level of bound reporter molecules in the sample.

[0044] In one example, the reporter molecule is defined as follows.

[0045] According to a third aspect of the present disclosure, there is provided an assay kit for detecting antigenic different forms of IL-33 in a biological sample, comprising:

[0046] (a) a binding molecule capable of binding to an antigenically different form of IL-33 selected from the group consisting of reduced IL-33, oxidized IL-33 and / or IL-33 / sST2 to form a complex;

[0047] (b) a reporter molecule capable of binding to a binding molecule complex selected from:

[0048] Reduced IL-33, oxidized IL-33 and / or IL-33 / sST2 binding molecule complex.

[0049] In one example, the binding molecule is as defined in the fourth, fifth and sixth aspects. In one example, the binding molecule may be considered a capture molecule or a probe.

[0050] In one example, the reporter molecule is as defined below. In another example, the reporter molecule may be those defined in the seventh aspect.

[0051] According to a fourth aspect of the present disclosure, there is provided a binding molecule that binds to reduced IL-33, comprising:

[0052] a. A variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 1, 2 and 3, respectively, and a variable light domain having VLCDRs 1-3 of SEQ ID NOs: 5, 6 and 7, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions;

[0053] b. a variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 25, 26 and 27, respectively, and a variable light domain having VLCDRs 1-3 of SEQ ID NOs: 29, 30 and 31, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions; or

[0054] c. a variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 35, 36 and 37, respectively, and a variable light domain having VLCDRs 1-3 of SEQ ID NOs: 39, 40 and 41, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions.

[0055] According to a fifth aspect of the present disclosure, there is provided a binding molecule that binds to oxidized IL-33, comprising a variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 9, 10 and 11, respectively, and a variable light domain having VLCDRs 1-3 of SEQ ID NOs: 13, 14 and 15, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions.

[0056] According to a sixth aspect of the present disclosure, a binding molecule that binds to IL-33 / sST2 is provided, comprising a variable heavy domain having VHCDR 1-3 of SEQ ID NOs: 17, 18 and 19, respectively, and a variable light domain having VLCDR 1-3 of SEQ ID NOs: 21, 22 and 23, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions.

[0057] In one example, any binding molecule defined above may be considered a capture molecule or probe. Suitably, each capture molecule or probe may be used in conjunction with a reporter molecule, suitably as defined below. Suitably, in a method for detecting antigenically distinct forms of IL-33 in a biological sample as defined in the second aspect.

[0058] In one example, the or each binding molecule may comprise a detectable label. In one example, the or each binding molecule is conjugated to a detectable label. Thus, suitably, in one example, the or each binding molecule herein may be used as a reporter molecule.

[0059] According to a seventh aspect of the present disclosure, there is provided a reporter molecule that binds to a reduced IL33-binding molecule complex, comprising: (a) a variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 1, 2 and 3, respectively, and a variable light domain having VLCDRs 1-3 of SEQ ID NOs: 5, 6 and 7, respectively, wherein one or more CDRs have 3 or less single amino acid substitutions, deletions or insertions, (b) a variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 25, 26 and 27, respectively, and a variable light domain having VLCDRs 1-3 of SEQ ID NOs: 29, 30 and 31, respectively, wherein one or more CDRs have 3 or less single amino acid substitutions, deletions or insertions, or (b) a variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 35, 36 and 37, respectively, and a variable light domain having VLCDRs 1-3 of SEQ ID NOs: 39, 40 and 41, respectively. In one embodiment, the or each reporter molecule may comprise a detectable label. In one embodiment, the or each reporter molecule is conjugated to a detectable label.

[0060] According to an alternative seventh aspect of the present disclosure, there is provided a reporter molecule that binds to an oxidized IL33-binding molecule complex, comprising a variable heavy domain having VHCDR 1-3 of SEQ ID NOs: 9, 10 and 11, respectively, and a variable light domain having VLCDR 1-3 of SEQ ID NOs: 13, 14 and 15, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions. In one example, the or each reporter molecule may comprise a detectable label. In one example, the or each reporter molecule is conjugated to a detectable label.

[0061] According to an alternative seventh aspect of the present disclosure, a reporter molecule is provided that binds to an IL-33 / sST2-binding molecule complex, comprising a variable heavy domain having VHCDR 1-3 of SEQ ID NOs: 17, 18, and 19, and a variable light domain having VLCDR 1-3 of SEQ ID NOs: 21, 22, and 23, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions, or insertions. In one example, the or each reporter molecule may comprise a detectable label. In one example, the or each reporter molecule is conjugated to a detectable label.

[0062] In one example of the fourth to seventh aspects, the binding molecule and the reporter molecule are antibodies or antigen-binding fragments thereof.

[0063] In one example, a pair of molecules can be provided, which includes a binding molecule selected from the fourth to sixth aspects and a reporter molecule optionally selected from one of the seventh aspects. Suitably, wherein the binding molecule is according to the fourth aspect and the reporter molecule is according to one of the seventh aspects. Suitably, wherein the binding molecule is according to the fifth aspect and the reporter molecule is according to one of the seventh aspects. Suitably, wherein the binding molecule is according to the sixth aspect and the reporter molecule is according to one of the seventh aspects. Alternatively, the binding molecule can be selected from the fourth to sixth aspects, and the reporter molecule can be selected from commercially available reporter molecules. Suitably, any commercially available reporter molecule capable of binding to reduced IL33-binding molecule complex, oxidized IL33-binding molecule complex or IL-33 / sST2-binding molecule complex can be used. Suitable such commercially available reporter molecules are described elsewhere herein. Still alternatively, the binding molecule can be selected from commercially available binding molecules, and the reporter molecule can be selected from one of the seventh aspects. Suitably, any commercially available binding molecule capable of binding to reduced IL33, oxidized IL33 or IL-33 / sST2 can be used. Suitable such commercially available binding molecules are described elsewhere herein.

[0064] Suitably, the skilled person may select an appropriate binding molecule and reporter molecule pair from those described in the fourth to seventh aspects and / or commercially available molecules to detect reduced IL-33, oxidized IL-33 or IL33 / sST2.

[0065] For example, in one example, a pair of molecules is provided, comprising: a binding molecule according to the sixth aspect that binds to IL-33 / sST2, comprising a variable heavy domain having VHCDR 1-3 of SEQ ID NOs: 17, 18 and 19, respectively, and a variable light domain having VLCDR 1-3 of SEQ ID NOs: 21, 22 and 23, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions; and a reporter molecule capable of binding to the IL-33 / sST2-binding molecule complex, which optionally comprises a detectable label. Suitably, for detecting reduced IL-33 / sST2 in a biological sample.

[0066] For example, in one example, a pair of molecules is provided, comprising: a binding molecule capable of binding to IL-33 / sST2; and a reporter molecule comprising a variable heavy domain having VHCDR 1-3 of SEQ ID NOs: 17, 18 and 19, respectively, and a variable light domain having VLCDR 1-3 of SEQ ID NOs: 21, 22 and 23, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions, optionally comprising a detectable label. Suitably, it is also used to detect reduced IL-33 / sST2 in a biological sample.

[0067] In one example, the molecule pair is used to detect reduced IL-33 in a biological sample. In one example, the molecule pair is used to detect oxidized IL-33 in a biological sample. In one example, the molecule pair is used to detect reduced IL-33 / sST2 in a biological sample.

[0068] Suitably, as described above, when used in a molecule pair with a reporter molecule, the binding molecule may be considered a capture molecule or probe.

[0069] In the eighth aspect of the present disclosure, a polynucleotide encoding the VH or VL domain of any binding molecule of the fourth, fifth or sixth aspect is provided, or a polynucleotide encoding the VH or VL domain of the reporter molecule of the seventh aspect is provided. In the ninth aspect of the present disclosure, a polynucleotide encoding any binding molecule of the fourth, fifth or sixth aspect is provided, or a polynucleotide encoding the reporter molecule of the seventh aspect is provided.

[0070] In a tenth aspect, a vector comprising one or more polynucleotides of the eighth or ninth aspect is provided.

[0071] In the eleventh aspect, a host cell comprising one or more polynucleotides of the eighth or ninth aspect or one or more vectors of the tenth aspect is provided. In one embodiment, the host cell may comprise: a first vector comprising a polynucleotide encoding the VH domain of the binding molecule of the fourth, fifth or sixth aspect; and a second vector comprising a polynucleotide encoding the corresponding VL domain of the binding molecule of the fourth, fifth or sixth aspect.

[0072] In the twelfth aspect, a method for producing the binding molecule of the fourth, fifth or sixth aspect is provided, comprising: culturing the host cell of the eleventh aspect comprising one or more polynucleotides encoding the binding molecule under conditions suitable for the host cell to express the binding molecule, and recovering the binding molecule.

[0073] In a thirteenth aspect, a binding molecule produced by the twelfth aspect is provided.

[0074] In a fourteenth aspect, a composition comprising a binding molecule of the fourth, fifth or sixth aspect is provided.

[0075] In a fifteenth aspect, there is provided the use of the binding molecule of the fourth, fifth or sixth aspect or the reporter molecule of the seventh aspect in an assay. In one embodiment, the assay is an immunoassay, such as an ELISA, ECLIA or CLIA. In one embodiment, the assay comprises a method for detecting one or more antigenically distinct forms of IL-33 in a biological sample. In one embodiment, the method is according to the first aspect.

[0076] Further features and examples of the aspects defined above are described in the following subsections with headings. Each subsection can be combined with any of the above-mentioned aspects in any compatible combination. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] The present disclosure will now be described with reference to the following drawings, in which:

[0078] Figure 1A : Shows MSD determination (top) and MSDS-PLEX of recombinant protein standards for reduced and oxidized IL-33 (Bottom). AB1070012 was used as capture antibody and AB1070019 was used as detection antibody.

[0079] Figure 1B : Shows MSD determination (top) and MSDS-PLEX of recombinant protein standards for reduced and oxidized IL-33 (Bottom). AB1070141 was used as capture antibody and AF3625 (R&D Systems) was used as detection antibody.

[0080] Figure 1C : Shows MSD assay (top) and MSD S-PLEX for recombinant protein standards of IL-33 / sST2 (Bottom). AB1070008 was used as capture antibody and MAB5232 (R&D Systems) was used as detection antibody.

[0081] Figure 2A : Shows examples of using the assay of the present disclosure to measure released endogenous forms of human IL-33 in healthy individuals and patient samples and in an in vitro human epithelial cell culture system. (A) Shows the MSD S-PLEX for detecting different IL-33 forms in commercial human serum samples (n=50) from healthy individuals (n=20), asthma (severity GINA 2-3, n=14) and COPD patients (severity GOLD 2-3, n=16). Example of extrapolation of the concentration of IL-33 forms in samples using a standard curve of the relevant purified recombinant protein.

[0082] Figure 2B An example of measuring reduced IL-33 (n=14), oxidized IL-33 (n=4), and IL-33 / sST2 (n=6) in nasal mucosal lining fluid using the MSD assay at baseline (top) and after intranasal grass pollen allergen challenge (30 minutes before challenge to 480 minutes after challenge) (bottom) is shown. The concentration of IL-33 forms in the samples was extrapolated using the relevant purified recombinant protein standard curve.

[0083] Figure 2C Shown using MSD S-PLEX Example of measuring reduced and oxidized IL-33 release into the culture supernatant from submerged healthy human bronchial epithelial cells (NHBE) (release time course 1 min - 1440 min). The concentration of IL-33 forms in the samples was extrapolated using the relevant purified recombinant protein standard curve.

[0084] Figure 2D An example of measuring reduced and oxidized IL-33 released from the human epithelial air-liquid interface (ALI) into the culture supernatant using the MSD assay is shown (n=7 donors, after 24h). The concentration of IL-33 forms in the samples was extrapolated using the relevant purified recombinant protein standard curve.

[0085] Figure 3 : Showing two ACCORD recruitment periods during the pandemic.

[0086] Figure 4 : IL33-sST2 in patients during the ACCORD Phase 2a study is shown using the assay of the present disclosure. The number of patients treated with SoC relative to the combination of SoC and tozorakimab who died or had respiratory failure on Day 29 of the study is shown as (a) overall, (b) grouped according to low IL-33 / sST2 baseline levels as measured by the assay, or (c) grouped according to high IL-33 / sST2 baseline levels as measured by the assay.

[0087] Figure 5 It was shown that both AB1070012 and AB1070069 performed equally well in the MSD assay format for the detection of reduced IL-33 when paired with AB1070019. DETAILED DESCRIPTION

[0088] definition

[0089] As used herein, "IL-33" protein refers to interleukin 33, in particular mammalian interleukin 33 protein, such as the human protein deposited under UniProt No. 095760. However, in light of the inventors' discoveries, it is apparent that the entity is not a single substance, but exists in reduced and oxidized forms. The terms "IL-33" and "IL-33 polypeptide" are used interchangeably. In some instances, IL-33 is full-length. In another instance, IL-33 is mature, truncated IL-33 (amino acids 112-270). Recent studies have shown that full-length IL-33 is active (Cayrol and Girard, Proc Natl Acad Sci USA 106(22):9021-6 (2009); Hayakawa et al., Biochem Biophys Res Commun. 387(1):218-22 (2009); Talabot-Ayer et al., J Biol Chem. 284(29):19420-6 (2009)). However, N-terminally processed or truncated IL-33, including but not limited to aa 72-270, 79-270, 95-270, 99-270, 107-270, 109-270, 111-270, 112-270 may have enhanced activity (Lefrancais 2012, 2014). In another example, IL-33 may include full-length IL-33, fragments thereof, or IL-33 mutant or variant polypeptides, wherein the fragment of IL-33 or IL-33 variant polypeptide retains some or all of the functional properties of active IL-33.

[0090] As used herein, "oxidized IL-33" or "oxIL-33" refers to a form of IL-33 that binds to RAGE and triggers RAGE-EGFR-mediated signaling. Oxidized IL-33 is a protein that is visible as a distinct band, for example, by Western blot analysis under non-reducing conditions, particularly having a mass that is 4 Da less than the corresponding reduced form. Specifically, it refers to a protein that has one or two disulfide bonds between cysteines independently selected from cysteine ​​208, 227, 232, and 259. In one example, oxidized IL-33 appears to not bind to ST2.

[0091] As used herein, "reduced IL-33" or "redIL-33" refers to a form of IL-33 that binds to ST2 and triggers ST2-mediated signaling. Specifically, cysteine ​​208, 227, 232, and 259 in the reduced form are not disulfide bonded. In one example, reduced IL-33 is shown to not bind to RAGE.

[0092] As used herein, "IL-33 / sST2" refers to the reduced IL-33 form bound to the soluble ST2 receptor, which form may also be referred to as a complex.

[0093] As used herein, "antigenic different forms of IL-33" refers to any form of IL-33 that can act as an antigen and be recognized by an antibody or its binding fragment, which in the context of the present disclosure generally refers to oxidized IL-33, reduced IL-33, and reduced IL-33 / sST2 complex.

[0094] The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute the sequence of a protein or peptide. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides and oligomers) and longer chains (which are commonly referred to in the art as proteins, which have many types). "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, or combinations thereof.

[0095] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, as well as sequences explicitly specified. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0096] The term "conservative sequence modification" refers to amino acid modifications that do not significantly affect or change the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies or antibody fragments of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0097] It should be noted that the term "a" or "an" entity refers to one or more (one or more) of the entity; for example, "anti-IL-33 antibody" should be understood to mean one or more anti-IL-33 antibodies. Therefore, the terms "a" (or "a"), "one or more (one or more)" and "at least one (kind)" are used interchangeably herein.

[0098] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject, particularly a mammalian subject, for whom diagnosis, prognosis or therapy is desired, unless the subject is defined as a "healthy subject". Mammalian subjects include humans; domestic animals; farm animals; such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc.

[0099] sample

[0100] The present disclosure relates to assaying a biological sample by measuring the levels of antigenically distinct forms of IL-33 present in the sample.

[0101] Suitably, the sample is a biological sample. Suitably, the sample is a body fluid sample. Alternatively, a suitable sample may include a tissue sample, such as a biopsy.

[0102] Suitable body fluid samples may include: a blood sample (e.g., a whole blood sample, a plasma sample, or a serum sample); or a mucosal lining fluid sample (e.g., mucosal lining fluid from an epithelium); or a lavage sample (e.g., a lung or bronchoalveolar lavage sample); or a supernatant sample (e.g., from a culture of epithelial cells).

[0103] Suitably, the supernatant sample may be an epithelial cell supernatant sample. Suitably, from a bronchial epithelial culture. Suitably, the supernatant may be from the cells or from the air-liquid interface of the culture.

[0104] Suitably, different antigenically distinct forms of IL-33 may be present in different body fluids. Thus, suitably, different antigenically distinct forms of IL-33 may be measured in different body fluid samples.

[0105] Suitably, oxidized IL-33 is measured in a mucosal lining fluid sample or a lavage sample.

[0106] Suitably, reduced IL-33 is measured in a mucosal lining fluid sample or a lavage sample.

[0107] Suitably, the IL-33 / sST2 complex is measured in a serum sample.

[0108] Suitably, the mucosal lining fluid samples or lavage samples contain biological cells, suitably these samples contain epithelial cells.

[0109] Suitably, the method of the present disclosure may further comprise the step of obtaining a biological sample. Suitably, the biological sample is obtained from a subject. Suitably, this step occurs before the contacting step (a). Alternatively, the sample may have been previously obtained.

[0110] Suitably, the subject may be a healthy subject or may suffer from a disease. Thus, suitably, the subject may be a patient. Thus, suitably, the method of the present disclosure may be performed on a biological sample obtained from a patient suffering from a disease. Thus, suitably, the method of the present disclosure may further comprise the step of obtaining a biological sample from a patient.

[0111] Suitably, the blood sample may be obtained by drawing blood from the subject.

[0112] Suitably, the mucosal lining fluid sample may be obtained by nasal swab from the subject.

[0113] Suitably, the lavage sample may be obtained by lavage of the subject's lungs.

[0114] Suitably, the supernatant sample may be obtained by culturing cells from a subject. Suitably, by culturing epithelial cells isolated from an epithelium of a subject. Suitably, isolated from respiratory epithelium of a subject.

[0115] Suitably, the sample represents the cytokine levels in the subject on which the method is being practiced. Suitably, the sample represents the interleukin levels in the subject on which the method is being practiced. Suitably, the sample represents the IL-33 levels in the subject on which the method is being practiced.

[0116] Suitably, the sample is a sample of mucosal lining fluid from the respiratory tract, suitably from the epithelium of the respiratory tract. Suitably, this may be isolated from the upper or lower respiratory tract, suitably from the epithelium of the upper or lower respiratory tract. Suitably, it is isolated from the upper respiratory tract, suitably from the epithelium of the upper respiratory tract. Suitably, the sample is nasal mucosal lining fluid, suitably nasal mucosal lining fluid isolated from the epithelium of the nasal passages.

[0117] Nasal mucosal lining fluid samples are particularly suitable for use in the methods of the present disclosure. The use of such samples is advantageous because they are easily available. In addition, the risk and discomfort of obtaining such samples are much less than in the case of samples such as lung tissue samples.

[0118] The sample may be processed to enrich for antigenically distinct forms of IL-33. Techniques suitable for such enrichment may be determined with reference to the nature of the sample. In general, examples of suitable techniques (such as techniques for isolating biomolecules such as cytokines from a sample) will be well known to those skilled in the art.

[0119] Subjects

[0120] The methods of the present disclosure may be practiced on a sample obtained from a subject.

[0121] Suitably, the subject may be a human. The subject may be receiving medical care, or may be requesting medical care. Suitably, the subject is male or female. Suitably, the subject is an adult or a child.

[0122] As explained above, the subject may be healthy or may be suffering from a disease. Suitably, the subject may be a patient.

[0123] Suitably, in the methods of the present disclosure, a suitable subject may be a subject suspected of having abnormal levels of antigenic different forms of IL-33. Suitably, a subject may be a subject believed to have a physiological condition or disease involving abnormal levels of antigenic different forms of IL-33, or a subject in which the prognosis or diagnosis of the condition or disease depends on the level of one or more antigenic different forms of IL-33. Suitably, abnormal levels of antigenic different forms of IL-33 may be a result or cause of a disease or condition.

[0124] Suitably, the subject may exhibit symptoms of such a condition or disease.

[0125] Alternatively, in the context of the methods of the present disclosure, a suitable subject may be a subject believed to be at risk of developing such a condition or disease. For example, such a subject may have had contact with an individual suffering from such a disease or condition, may suffer from a related condition, or may meet risk factors associated with the condition, such as smoking, old age, allergies, etc.

[0126] touch

[0127] The methods of the present disclosure include the step of contacting a biological sample with one or more binding molecules capable of specifically binding to one or more antigenically different forms of IL-33 selected from the group consisting of reduced IL-33, oxidized IL-33 and / or IL-33 / sST2 under conditions sufficient to form a complex.

[0128] Suitably, the methods of the present disclosure comprise the step of contacting the biological sample with one or more binding molecules capable of specifically binding to one or more antigenically different forms of IL-33 selected from the group consisting of reduced IL-33, oxidized IL-33 and / or IL-33 / sST2.

[0129] Suitably, "specific binding" means that each binding molecule is capable of binding to a specific form of IL-33.

[0130] Suitably, the methods of the present disclosure may further include the step of contacting the complex with one or more reporter molecules capable of binding to one or more complexes. In such examples, the detection step (b) includes detecting the level of the reporter molecules bound in the sample. In such examples, suitably, these methods also include the step of contacting the biological sample with one or more reporter molecules capable of binding to one or more complexes therein. Suitably, sufficient time is continued to allow the reporter molecules to bind to the one or more complexes.

[0131] Suitably, the method of the present disclosure may further comprise the step of contacting the complexes with one or more reporter molecules capable of specifically binding to the one or more complexes. Suitably, contacting the complexes with one or more reporter molecules may comprise contacting the biological sample with one or more reporter molecules.

[0132] Suitably, "specific binding" means that each reporter molecule is capable of binding to a specific form of the IL-33-binding molecule complex.

[0133] Suitable binding molecules and reporter molecules are defined elsewhere herein.

[0134] Optionally, the methods of the present disclosure may include a further step prior to contacting the sample with one or more binding molecules capable of specifically binding to one or more antigenically different forms of IL-33, the further step comprising immobilizing the antigenically different isoforms of IL-33 by contacting the biological sample with a solid surface under conditions suitable for the antigenically different isoforms of IL-33 to bind to the solid surface by adsorption or covalent bonding. Suitably, the solid surface may include a plate such as a microtiter plate or a multiwell plate, a well, a slide such as a glass slide, a paper such as a filter paper, a cuvette, etc. Suitably, the solid surface is contained in a container in which the method is to be performed. Thus, suitably, a solid surface comprising antigenically different isoforms of IL-33 is generated. Suitably, in such examples, the binding molecule is then contacted with the solid surface comprising antigenically different isoforms of IL-33 for a sufficient time to form a complex.

[0135] Suitably, the binding molecule is contacted with the biological sample for a sufficient time to form a complex. Suitably, the reporter molecule is contacted with the biological sample for a sufficient time to bind to the one or more complexes.

[0136] Suitably, this time may be referred to as the incubation time. Suitably, the incubation time will vary depending on the binding molecule used and the reporter molecule used and the biological sample.

[0137] Suitably, sufficient incubation time may be between 5 minutes and 180 minutes, suitably between 10 minutes and 150 minutes, suitably between 20 minutes and 120 minutes, suitably between 30 minutes and 100 minutes, suitably between 45 minutes and 90 minutes, suitably about 60 minutes.

[0138] Suitably, one or more binding molecules are contacted with the biological sample for about 120 minutes, 100 minutes, 80 minutes, 60 minutes, 40 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes or 5 minutes. Suitably, one or more binding molecules are contacted with the biological sample for about 9 minutes.

[0139] Suitably, one or more reporter molecules are contacted with the complex / biological sample for about 120 minutes, 100 minutes, 80 minutes, 60 minutes, 40 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes or 5 minutes. Suitably, one or more reporter molecules are contacted with the complex / biological sample for about 9 minutes.

[0140] Suitably, contacting the biological sample with the binding molecule or reporter molecule comprises bringing the binding molecule or reporter molecule and the biological sample together such that binding can occur.

[0141] Suitably, the biological sample may be contacted with the binding molecule or reporter molecule in any suitable manner using known assay techniques.

[0142] The sample may be contacted with one or more binding molecules by using a solid support or carrier. Suitably, the sample or binding molecules may be immobilized on a support or carrier.

[0143] Suitably, the support may then be washed. Suitably, the support is washed with a suitable buffer. Suitably, the method of the present disclosure may include one or more washing steps. Suitably, washing occurs after adding the one or more binding molecules and before adding the one or more reporter molecules. Suitably, after adding the one or more reporter molecules and before the determining step.

[0144] Suitably, at least one washing step occurs after step (a), and suitably before step (b).

[0145] Suitably, the support may be contacted with one or more reporter molecules after step (a). Thus, suitably, the step of contacting the complex with one or more reporter molecules capable of binding to one or more complexes may comprise contacting the complex immobilized on the support with one or more reporter molecules capable of binding to one or more complexes. Suitably, the support may then be washed a second time to remove unbound reporter molecules. Thus, suitably, a second washing step may occur after contacting the complex with one or more reporter molecules.

[0146] Optionally, the binding molecule or reporter molecule may comprise a detectable label, or they may be subsequently labeled. Suitable labels are discussed elsewhere herein. The amount of the binding label that may be bound to the solid support may then be detected by conventional means, suitably as described in step (b) of the disclosed method.

[0147] Suitably, in some embodiments where the assay is an immunoassay, the binding molecule may be biotinylated. Suitably, in some embodiments where the assay is an immunoassay, the reporter molecule may be labelled with a chemiluminescent compound.

[0148] "Solid support or carrier" is intended to mean any support capable of binding an antigen or antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, nitrocellulose, amylase, natural and modified cellulose, polyacrylamide and magnetite. For the purposes of this disclosure, the nature of the carrier may be soluble or insoluble to a certain extent. The support material may have almost any possible structural configuration, as long as the coupled molecule is capable of binding to the antigen or antibody. Thus, the support configuration may be spherical (as in beads) or cylindrical (as in the inner surface of a test tube or the outer surface of a rod). Alternatively, the surface may be flat, such as a sheet, a test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable supports for binding antibodies or antigens, or will be able to determine them by using routine experiments.

[0149] Detection

[0150] The methods of the present disclosure include the step of detecting the level of one or more reduced IL-33, oxidized IL-33 and / or IL-33 / sST2 complex in a biological sample.

[0151] Suitably, detecting the level of one or more reduced IL-33, oxidized IL-33 and / or IL-33 / sST2 complexes refers to detecting the level of reduced IL-33-binding molecule complexes and / or oxidized IL-33-binding molecule complexes and / or IL-33 / sST2-binding molecule complexes. Suitably, such complexes are formed in step (a) by one or more binding molecules that specifically bind to one or more of reduced IL-33, oxidized IL-33 and / or IL-33 / sST2 in the biological sample.

[0152] Suitably, the complex detected in step (b) corresponds to the binding molecule used in the method. Thus, suitably, if the method is performed using only oxidized IL-33 binding molecules, only oxidized IL-33 complexes are detected in step (b).

[0153] Suitably, detection of one or more reduced IL-33, oxidized IL-33 and / or IL-33 / sST2 complex in a sample may be performed by any suitable assay technique known in the art.

[0154] Suitably, as described above, detecting one or more reduced IL-33, oxidized IL-33 and / or IL-33 / sST2 complexes in a sample may comprise detecting the level of one or more bound reporter molecules in the sample. Suitably, the level of one or more reporter molecules bound to one or more reduced IL-33, oxidized IL-33 and / or IL-33 / sST2 complexes in a sample is detected.

[0155] Suitably, the reporter molecules are detected by detecting a detectable label associated with each reporter molecule. Suitably, the detectable label may be part of the reporter molecule or may be added separately, suitably after the reporter molecule has bound to one or more reduced IL-33, oxidized IL-33 and / or IL-33 / sST2 complexes in the sample.

[0156] Alternatively, detecting one or more reduced IL-33, oxidized IL-33 and / or IL-33 / sST2 complexes in a sample may include detecting the level of one or more bound binding molecules in the sample. In such examples, suitably, the binding molecules are directly detected by detecting a detectable label associated with each binding molecule. Suitably, in such examples, a reporter molecule is not required. Suitably, the detectable label may be a part of the binding molecule or may be added separately, suitably, after the binding molecule has been bound to one or more reduced IL-33, oxidized IL-33 and / or IL-33 / sST2 complexes in the sample.

[0157] Suitable detectable labels are discussed elsewhere herein.

[0158] Thus, suitably, the methods of the present disclosure may further comprise the step of adding a detectable label to the sample. Suitably, step (b) may comprise detecting the detectable label associated with one or more reporter molecules.

[0159] Alternatively, step (b) may comprise detecting a detectable label associated with the one or more binding molecules.

[0160] In one example, the reporter molecule comprises a detectable label conjugated thereto. In such examples, the reporter molecule is detected by detecting the conjugated detectable label.

[0161] In one example, the binding molecule comprises a detectable label conjugated thereto. In such examples, the binding molecule is detected by detecting the conjugated detectable label.

[0162] Suitably, the assay used is highly sensitive and thus the methods of the disclosure are highly sensitive. Suitably, the methods of the disclosure are capable of detecting antigenically distinct forms of IL-33 in a sample at a concentration of micrograms / ml or less, nanograms / ml or less, picograms / ml or less, femtograms / ml or less.

[0163] Suitably, the lower limit of detection is a concentration of micrograms / ml or less, nanograms / ml or less, picograms / ml or less, femtograms / ml or less. Suitably, the lower limit of detection is a concentration of femtograms / ml.

[0164] In one example, the method is capable of detecting antigenically distinct forms of IL-33 in a sample at a concentration of at least femtogram / ml.

[0165] Determination

[0166] Suitably, the methods of the present disclosure may comprise detecting one or more antigenically distinct forms of IL-33 in a biological sample in a single assay.

[0167] Alternatively, the methods of the present disclosure may comprise detecting one or more antigenically distinct forms of IL-33 in a biological sample in more than one assay. Thus, suitably, the methods of the present disclosure may further comprise more than one biological sample. Thus, suitably, the methods of the present disclosure may comprise more than one step within step (a) and more than one step within step (b).

[0168] Suitably, the methods of the present disclosure may comprise contacting one or more biological samples separately with one or more binding molecules capable of binding reduced IL-33, oxidized IL-33 and / or IL-33 / sST2.

[0169] Suitably, step (a) of the method of the present disclosure may comprise: (ai) contacting the first sample with a binding molecule capable of binding to oxidized IL-33; (aii) contacting the second sample with a binding molecule capable of binding to reduced IL-33; and / or (aiii) contacting the third sample with a binding molecule capable of binding to IL-33 / sST2. Suitably, the first, second and third samples may be the same sample or different samples.

[0170] Suitably, the methods of the present disclosure may further comprise separately detecting the level of one or more reduced IL-33, oxidized IL-33 and / or IL-33 / sST2 complex in one or more samples.

[0171] Suitably, step (b) of the method of the present disclosure may comprise: (bi) detecting the level of oxidized IL-33-binding molecule complex in a first sample; (bii) detecting the level of reduced IL-33-binding molecule complex in a second sample; and / or (biii) detecting the level of IL-33 / sST2-binding molecule complex in a third sample. Suitably, the first, second or third sample may be the same sample or different samples.

[0172] As described above, different antigenic variants of IL-33 may be present in the same or different samples, and thus, suitably, different antigenic variants of IL-33 may require different assays for different samples. Thus, suitably, these methods may include one or more assays.

[0173] Suitably, step (a) of the method of the present disclosure may comprise: (ai) contacting a mucosal lining fluid or lung lavage sample with an oxidized IL-33 binding molecule; (aii) contacting a mucosal lining fluid or lung lavage sample with a reduced IL-33 binding molecule; and / or (aiii) contacting a serum sample with an IL-33 / sST2 binding molecule.

[0174] Suitably, any of the antigenically different forms of IL-33 may be detected in any combination in the same sample or in different samples.

[0175] For example, suitably, the methods of the present disclosure may include: (bi) detecting the level of oxidized IL-33-binding molecule complex and the level of reduced IL-33-binding molecule complex in a first sample; and / or (bii) detecting the level of IL-33 / sST2-binding molecule complex in a second sample. For example, suitably, the methods of the present disclosure may include (bi) detecting the level of oxidized IL-33-binding molecule complex and the level of reduced IL-33-binding molecule complex in a mucosal lining fluid or lung lavage sample; and / or (bii) detecting the level of IL-33 / sST2-binding molecule complex in a serum sample.

[0176] Prior to step (a), the method of the present disclosure may further comprise the step of isolating one or more antigenically distinct forms of IL-33 from the biological sample. Suitably, the isolating step may comprise separation by fractionation of the sample. Suitable separation techniques are known in the art and may comprise: chromatography, centrifugation, affinity purification, filtration, etc. Suitably, the isolating step may comprise immobilizing one or more antigenically distinct forms of IL-33 to a solid surface, as explained elsewhere herein.

[0177] Examples of suitable samples are described elsewhere in this specification.It will be appreciated that the nature of the sample may then determine the nature of the assay techniques that may be used to practice the methods of the present disclosure.

[0178] The various forms of IL-33 are proteins. Therefore, suitably, the assay technique is a protein assay technique.

[0179] Protein levels can be determined by various assay techniques such as S-plex, ELISA, ECLIA, CLIA, radioimmunoassay, immunoprecipitation, Western blot, and mass spectrometry.

[0180] Suitably, the level of one or more antigenically distinct forms of IL-33 is determined by immunoassay.

[0181] Immunoassays generally require a capture reagent (such as an antibody) to capture the analyte of interest and optionally a probe reagent to detect the analyte of interest. Suitable immunoassay techniques are: ELISA (enzyme-linked immunosorbent assay), ECLIA (enzyme electrochemiluminescent immunoassay) such as the ELISA from Roche S-plex, CLIA (chemiluminescent immunoassay), Western blot, immunocytochemistry, immunoprecipitation, affinity chromatography, Bio-Layer Interferometry (Octet, ForteBio), and biochemical assays such as dissociation-enhanced lanthanide fluorescent immunoassay ( PerkinElmer), Förster resonance energy transfer (FRET) assays (e.g., homogeneous time-resolved fluorescence ( CisBiointernational) and radioimmunoassay / radioligand binding assays.

[0182] Western blot analysis generally includes preparation of protein samples, electrophoresis of protein samples in polyacrylamide gels (e.g., 8-20 SDS-PAGE, depending on the molecular weight of the antigen), transfer of protein samples from polyacrylamide gels to membranes such as nitrocellulose, PVDF or nylon, blocking of the membrane in a blocking solution (e.g., PBS with 3% BSA or skim milk), washing of the membrane in a wash buffer (e.g., PBS-Tween 20), blocking of the membrane with a primary antibody (antibody of interest) diluted in a blocking buffer, washing of the membrane in a wash buffer, blocking of the membrane with a secondary antibody (which recognizes the primary antibody, e.g., anti-human antibody) conjugated to an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase) or a radioactive molecule (e.g., 32P or 125I) diluted in a blocking buffer, washing of the membrane in a wash buffer, and detection of the presence of the antigen. Those skilled in the art will know the parameters that can be modified to increase the detected signal and reduce background noise. For further discussion of Western blotting protocols, see, e.g., Ausubel et al., eds. (1994) Current Protocols in Molecular Biology (John Wiley & Sons, Inc., NY) Vol. 1 10.8.1.

[0183] ELISA includes preparing an antigen, coating the wells of a 96-well microtiter plate with the antigen, adding an antibody conjugated to a detectable compound such as an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the wells and incubating for a period of time, and detecting the presence of the antigen. In ELISA, the antibody does not have to be conjugated to the detectable compound; instead, a second antibody (which recognizes the first antibody) conjugated to the detectable compound can be added to the well. Both the first antibody and the second antibody bind to the antigen and form a "sandwich". In addition, instead of coating the wells with the antigen, the antibody can be coated onto the wells. In this case, the second antibody conjugated to the detectable compound can be added after the antigen of interest is added to the coated wells. Those skilled in the art will know that the parameters that can be modified to increase the detected signal and other variations of ELISA known in the art. For further discussion of ELISA, see, for example, Ausubel et al., ed. (1994) Current Protocols in Molecular Biology (John Wiley & Sons, Inc., NY) Vol. 1 13.2.1.

[0184] Suitably, the level of one or more antigenic different forms of IL-33 is determined by immunoassay. Suitably, by using a modified ELISA known as the S-plex assay. The S-plex assay is available from Meso Scale Diagnostics LLC together with appropriate instructions for use.

[0185] Alternatively, the level of one or more antigenic different forms of IL-33 is determined by ECLIA. Suitably, the ECLIA assay includes contacting a sample containing an antigen with a first antibody specific for the antigen conjugated to biotin and incubating for a period of time to allow the first antibody to bind to the antigen in the sample. Then add a second antibody specific for the antigen but conjugated to a labeling molecule, typically a ruthenium compound, suitably ruthenium bipyridine. Both the first and second antibodies bind to the antigen and form a "sandwich". In some cases, the first and second antibodies may be added simultaneously. Then, simultaneously or separately, streptavidin-coated beads or microparticles are added to the sample, which bind to the biotin of the first antibody and form an antibody complex. Suitably, the beads or microparticles are magnetic. The sample is then passed over an electrode, and the antibody complex is bound to the electrode by the streptavidin beads or microparticles. Before adding a reactant and passing an electric current through the electrode to induce chemiluminescence of the labeling molecule, wash off the unbound antibody with a wash buffer such as ProCell. Suitably, the reactant may be an amine, such as tripropylamine or dibutylethanolamine. The level of chemiluminescence can be measured by a photomultiplier tube to indicate the amount of bound complex, and therefore the amount of antigen in the sample. An example of such an ECLIA immunoassay is the ELISA available from Roche Assay, which may suitably be performed on a Cobas e immunoassay analyser.

[0186] In one embodiment of the present disclosure, there is provided a method for detecting one or more antigenically distinct forms of IL-33 in a biological sample, comprising performing an ECLIA assay, suitably using one or more binding molecules and / or reporter molecules described herein.

[0187] Therefore, in one embodiment of the present disclosure, there is provided a method for detecting one or more antigenically different forms of IL-33 in a biological sample, comprising:

[0188] (a) contacting the sample with one or more binding molecules, each of which is capable of specifically binding to an antigenically different form of IL-33 selected from the group consisting of reduced IL-33, oxidized IL-33 and / or IL-33 / sST2, under conditions sufficient to form a complex, wherein each of the binding molecules comprises a tag;

[0189] (b) contacting the sample with one or more reporter molecules each capable of binding to the complex produced in (a) under conditions sufficient to form a sandwich complex, wherein each of the reporter molecules comprises a label;

[0190] (c) contacting the sample with one or more capture partners, each capable of binding to a tag contained in each binding molecule, under conditions sufficient to form a captured sandwich complex;

[0191] (d) Detection of the amount of captured sandwich complex using a label.

[0192] Suitably, the binding molecules and reporter molecules are as defined elsewhere herein.

[0193] Suitably, the label is biotin. Therefore, suitably, the capture partner is streptavidin. However, suitably, other known labels and capture partners can be used. Suitably, the capture molecule can be attached to a support, suitably attached to a solid support, suitably attached to a bead or microparticle. Therefore, suitably, step (c) comprises contacting the sample with one or more capture beads comprising streptavidin, each of which is capable of binding to the biotin label contained in each binding molecule. Therefore, suitably, step (c) produces beads comprising one or more sandwich complexes.

[0194] Suitably, the beads are formed of a magnetic material, suitably a magnetic metal such as iron, nickel or cobalt. Thus, suitably, step (c) comprises contacting the sample with one or more magnetic capture beads comprising streptavidin (suitably, coated in streptavidin), each of which is capable of binding to the biotin tag contained in each binding molecule.

[0195] Suitably, the label is a fluorescent or luminescent or chemiluminescent label as identified above. Suitably, the label is a chemiluminescent label, suitably, the label is a ruthenium compound, suitably ruthenium pyridine.

[0196] Suitably, detecting the captured sandwich complex comprises detecting a label, suitably detecting a level of luminescence produced by the label. Suitably, the level of luminescence corresponds to the level of antigen present in the sample, suitably corresponding to the level of antigenically distinct forms of IL-33 in the sample selected from the group consisting of reduced IL-33, oxidized IL-33 and / or IL-33 / sST2.

[0197] Suitably, the chemiluminescent label needs to be activated in order to emit light and be detected. Therefore, suitably, the method includes the step of activating the label of each reporter molecule attached to the captured sandwich complex. Suitably, before the detection step. Suitably, the activation step includes contacting the label with a stimulus, wherein the stimulus is capable of activating the label. Suitable stimuli include chemical reactants and / or current / voltage. Suitably, for chemiluminescent labels, the activation step includes both reactants and current / voltage. Suitably, nearby. Therefore, suitably, the method may include the step of contacting the captured sandwich complex with a reactant. Suitable reactants may be electron donors, such as amine compounds, for example tripropylamine or dibutylethanolamine. Suitably, the step may also include exposing the captured sandwich complex to current / voltage, suitably, while in contact with the reactant. Therefore, suitably, the method includes contacting the captured sandwich complex with a reactant and passing a current through an electrode to activate the chemiluminescent label on each reporter molecule.

[0198] Suitably, in order to ensure that the chemiluminescent label is activated, the method may include fixing the captured sandwich complex. Suitably, they are fixed on a surface. In one embodiment, this can be achieved by using magnetic beads as described above in combination with a metal surface. Suitably, the method may include the step of binding or fixing the captured sandwich complex to a magnetic surface, suitably, by means of magnetic beads. Suitably, the surface is also capable of delivering current / voltage. Therefore, suitably, the surface is an electrode, suitably, a magnetic electrode. Therefore, in one embodiment, the method includes the step of fixing the captured sandwich complex to a magnetic electrode, suitably, by means of magnetic beads of the captured sandwich complex. Suitably, this step occurs before the step of activating the chemiluminescent label. Suitably, before contacting the captured sandwich complex with a reactant and passing a current through the electrode.

[0199] Suitably, once activated, the label on each reporter molecule of each captured sandwich complex can be detected. Suitably, in the case of a chemiluminescent label, its luminescence can be detected. Suitably, detection of such luminescence can be performed using any known technique, for example by using a photomultiplier tube. Suitably, the level of luminescence detected corresponds to the amount of sandwich complex captured, which suitably corresponds to the level of antigen present in the sample. In the context of the present invention, suitably, the level of luminescence corresponds to the level of reduced IL-33, oxidized IL-33 and / or IL-33 / sST2 in the sample.

[0200] Therefore, in one embodiment of the present disclosure, there is provided a method for detecting one or more antigenically different forms of IL-33 in a biological sample, comprising:

[0201] (a) contacting the sample with one or more binding molecules, each of which is capable of specifically binding to an antigenically different form of IL-33 selected from the group consisting of reduced IL-33, oxidized IL-33 and / or IL-33 / sST2, under conditions sufficient to form a complex, wherein each of the binding molecules comprises a biotin tag;

[0202] (b) contacting the sample with one or more reporter molecules each capable of binding to the complex produced in (a) under conditions sufficient to form a sandwich complex, wherein each of the reporter molecules comprises a chemiluminescent ruthenium label;

[0203] (c) contacting the sample with one or more magnetic capture beads coated in streptavidin, each capable of binding to the biotin tag contained in each binding molecule, under conditions sufficient to form a captured sandwich complex;

[0204] (d) Immobilizing the captured sandwich complex on a magnetic electrode via magnetic capture beads;

[0205] (e) contacting the captured sandwich complex with a reactant and passing an electric current through the electrodes to activate the chemiluminescent label on each reporter molecule;

[0206] (d) detecting the level of chemiluminescence, thereby detecting one or more antigenically distinct forms of IL-33 in the sample.

[0207] An immunoassay for measuring antigenic different forms of IL-33 will generally include a step of incubating the sample in the presence of one or more detectably labeled molecules that are capable of specifically binding to each of the antigenic different forms of IL-33 or peptide fragments thereof of interest, and a step of detecting the bound molecules by any of a variety of techniques well known in the art. The methods of the present disclosure follow this general format. Suitable binding molecules, labels, and detection methods are described elsewhere herein.

[0208] Binding molecules and reporter molecules

[0209] The present disclosure relates to the use of binding molecules and reporter molecules for detecting antigenically distinct forms of IL-33 in biological samples.

[0210] While many techniques are known in the art for measuring and detecting proteins such as IL-33 in a sample, suitably the methods of the present disclosure are performed by an immunoassay as described above.

[0211] Suitably, the immunoassay comprises the use of one or more binding molecules capable of binding to one or more antigenically distinct forms of IL-33 to form one or more complexes; and one or more reporter molecules capable of binding to the formed one or more complexes.

[0212] "Binding molecules" of the present disclosure refer in their broadest sense to molecules that specifically bind to antigenic determinants. Suitably, one or more binding molecules specifically bind to IL-33, in particular reduced IL-33, oxidized IL-33 or IL-33 / sST2. In some examples, the binding molecules of the present disclosure may be considered as capture molecules or probes.

[0213] "Reporter molecule" in the present disclosure refers in its broadest sense to a molecule that specifically binds to an antigenic determinant and is capable of such indication. Suitably, one or more reporter molecules specifically bind to a complex of IL-33 and a binding molecule, in particular a reduced IL-33-binding molecule, an oxidized IL-33-binding molecule or an IL-33 / sST2-binding molecule complex.

[0214] Alternatively, the reporter molecule and the binding molecule are the same entity. In such examples, the binding molecule directly binds to the antigenic determinant (e.g., reduced IL-33, oxidized IL-33, or IL-33 / sST2) and is able to indicate so.

[0215] Suitably, the molecules described herein, suitably the antibodies and antigen binding fragments described herein, are each capable of being used as a binding molecule or a reporter molecule. Suitably, as described in the fourth to seventh aspects.

[0216] Suitably, the one or more reporter molecules are capable of indicating their presence and / or binding. Suitably, the one or more reporter molecules are labelled. Suitably, the one or more reporter molecules are detectably labelled.

[0217] Alternatively, one or more binding molecules are capable of indicating their presence and / or binding. Suitably, in such examples, one or more binding molecules are labeled. Suitably, one or more binding molecules are detectably labeled.

[0218] Suitable detectable labels are known in the art and may include radioactive labels, fluorescent labels, enzymes, chromophores. Suitably, the label may be detectable only upon stimulation. Suitable stimuli will vary depending on the label used, for example when an enzyme label is used, the stimuli may be a substrate, when a chromophore label is used, the stimuli may be radiation of a specific wavelength. Such stimuli may be considered as irritants.

[0219] Suitable fluorescent labels may include: rhodamine, fluorescein, Cy5, ruthenium compounds such as pyridine complexes, diimine complexes, and phosphorescent porphyrin dyes.

[0220] Suitable enzyme labels may include: peroxidase, glucose oxidase, alkaline phosphatase (AP), β-galactosidase, catalase or luciferase. The substrate to be used with a particular enzyme is generally selected to produce a detectable color change when hydrolyzed by the corresponding enzyme. For example, p-nitrophenyl phosphate is suitable for use with alkaline phosphatase conjugates; for peroxidase conjugates, 1,2-phenylenediamine or toluidine are generally used. Suitable fluorescent substrates that produce fluorescent products can be used.

[0221] Suitably, in some embodiments where the assay is an immunoassay, the binding molecule may be biotinylated. Suitably, in some embodiments where the assay is an immunoassay, the reporter molecule may be labelled with a chemiluminescent compound, suitably a ruthenium compound, suitably ruthenium bipyridine.

[0222] Thus, in another aspect of the invention, there may be provided a binding molecule according to any one of the fourth to sixth aspects, wherein the binding molecule is biotinylated.

[0223] Thus, in another aspect of the present invention there may be provided a reporter molecule according to the seventh aspect, wherein the reporter molecule is ruthenated.

[0224] Alternatively, the binding molecule or reporter molecule may not be labeled. In such examples, the binding molecule or reporter molecule may be able to be labeled and combined, suitably combined with a detectable label. Suitably, the detectable label can be combined with these binding molecules or reporter molecules and so indicate. Suitably, the detectable label is specific for the corresponding binding molecule or reporter molecule.

[0225] Suitably, the methods of the present disclosure may utilize an oxidized IL-33 complex detectable label, and / or a reduced IL-33 complex detectable label and / or an IL-33 / sST2 complex detectable label. Suitably, such one or more detectable labels may be added to the assay methods of the present disclosure after one or more reporter molecules. Suitably, the detection step (b) of these methods may include detecting one or more bound detectable labels.

[0226] Alternatively, the methods of the present disclosure may utilize an oxidized IL-33 detectable label, and / or a reduced IL-33 detectable label and / or an IL-33 / sST2 detectable label. Suitably, such one or more detectable labels may be added to the assay methods of the present disclosure after one or more binding molecules. Suitably, the detection step (b) of these methods may include detecting one or more bound detectable labels.

[0227] In one example, the one or more reporter molecules are detectably labeled. In one example, the one or more reporter molecules comprise a detectable label conjugated thereto. In one example, the one or more reporter molecules are sulfo-tagged.

[0228] Suitably, these methods comprise the use of a combination of binding molecules capable of binding to several antigenically distinct forms of IL-33 to form several complexes; and a combination of corresponding reporter molecules capable of binding to the several complexes.

[0229] Suitably, these methods comprise the use of a combination of binding molecules capable of binding to oxidized IL-33, reduced IL-33 and / or IL-33 / sST2 to form several complexes; and a combination of reporter molecules capable of binding to several complexes.

[0230] Suitably, these methods comprise the use of a binding molecule capable of binding to oxidized IL-33 to form a complex; and a reporter molecule capable of binding to the oxidized IL-33-binding molecule complex.

[0231] Suitably, these methods comprise the use of a binding molecule capable of binding to reduced IL-33 to form a complex; and a reporter molecule capable of binding to the oxidized IL-33-binding molecule complex.

[0232] Suitably, these methods comprise the use of a binding molecule capable of binding to IL-33 / sST2 to form a complex; and a reporter molecule capable of binding to the IL-33 / sST2-binding molecule complex.

[0233] Suitably, each binding molecule has a corresponding reporter molecule. Suitably, each reporter molecule may comprise a corresponding detectable label.

[0234] Suitably, the reporter molecule and the binding molecule are paired. For example, when a binding molecule capable of binding to oxidized IL-33 is used, a reporter molecule capable of binding to the oxidized IL-33-binding molecule complex is used.

[0235] Suitably, the one or more binding molecules or reporter molecules may be selected from: an antibody, an antigen binding fragment thereof, an aptamer, at least one heavy chain or light chain CDR of a reference antibody molecule, and at least six CDRs from one or more reference antibody molecules.

[0236] Suitably, the one or more binding molecules or reporter molecules are antibodies or binding fragments thereof.

[0237] Suitably, one or more binding molecules are anti-IL-33 antibodies or binding fragments thereof. Suitably, at least one binding molecule is an anti-oxidized IL-33 antibody or binding fragment thereof. Suitably, at least one binding molecule is an anti-reduced IL-33 antibody or binding fragment thereof. Suitably, at least one binding molecule is an anti-IL-33 / sST2 antibody or binding fragment thereof.

[0238] Suitably, at least one reporter molecule is an anti-reduced IL-33-binding molecule complex antibody or a binding fragment thereof. Suitably, at least one reporter molecule is an anti-oxidized IL-33-binding molecule complex antibody or a binding fragment thereof. Suitably, at least one reporter molecule is an anti-IL-33 / sST2-binding molecule complex antibody or a binding fragment thereof.

[0239] As used herein, "antibody" refers to an immunoglobulin molecule, particularly a full-length antibody or a molecule comprising a full-length antibody, such as a DVD-Ig molecule, etc., as discussed in more detail below.

[0240] "Binding fragment thereof" is interchangeable with "antigen binding fragment thereof" and refers to an epitope / antigen binding fragment of an antibody fragment, e.g., comprising the binding region, particularly comprising 6 CDRs, such as 3 CDRs in the heavy variable region and 3 CDRs in the light variable region.

[0241] Suitably, the one or more binding molecules or reporter molecules are antibodies or antigen-binding fragments thereof.

[0242] The term "antibody or antigen binding fragment thereof" encompasses any antibody or antigen binding fragment thereof that competes with the antibody of the present invention for specific binding to an antigen. Thus, suitably, the binding molecules or reporter molecules mentioned herein also encompass molecules that compete with the binding molecules or reporter molecules for binding to the relevant antigen. Thus, suitably, the binding molecules or reporter molecules mentioned herein also encompass molecules that compete with the binding molecules or reporter molecules for specific binding to reduced IL-33, oxidized IL-33 or IL-33 / sST2. Such competitive antibodies or antigen binding fragments can be identified in competitive binding assays well known in the art. Suitably, the competitive antibodies or antigen binding fragments substantially inhibit the adhesion of receptors to counter-receptors, such as the binding of reduced IL-33 to ST2 receptors. Suitably, an excess of competitive antibodies or antigen binding fragments reduces the amount of receptors bound to the counter-receptors by at least about 20%, 40%, 60% or 80%, and more typically greater than about 85% (as measured in an in vitro competitive binding assay).

[0243] The terms "complementarity determining region" and "CDR" as used herein refer to the amino acid residues of an antibody or antigen-binding fragment that are responsible for antigen binding.

[0244] Suitably, the antibody or its binding fragment is selected from: naturally occurring, polyclonal, monoclonal, multispecific, mouse, human, humanized, primatized or chimeric. Suitably, the antibody or its binding fragment can be an epitope binding fragment, for example, Fab' and F(ab')2, Fd, Fv, single-chain Fv (scFv), disulfide-linked Fv (sdFv), a fragment comprising a VL or VH domain, or a fragment produced by a Fab expression library. Suitably, the antibody or its binding fragment can be a mini-antibody, a double antibody, a three-antibody, a four-antibody or a single-chain antibody. Suitably, the antibody or its binding fragment is a monoclonal antibody. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019. In one example, one or more binding molecules or reporter molecules are Fab' or F(ab')2 epitope binding fragments.

[0245] As used herein, the phrase "monoclonal antibody" or "monoclonal antibody composition" refers to polypeptides, including antibodies, bispecific antibodies, etc., which have substantially the same amino acid sequence or are derived from the same genetic source. The term also includes preparations of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0246] The immunoglobulin or antibody molecules of the present disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, etc.), or subclass of immunoglobulin molecules.

[0247] Suitably, the or each antibody or binding fragment thereof used as a binding molecule is a capture antibody.

[0248] Suitably, the or each antibody or binding fragment thereof used as a reporter molecule is a probe antibody.

[0249] Suitably, as described above, the binding molecule and the reporter molecule are paired so that each capture antibody has a corresponding probe antibody. Suitably, the capture antibody is paired with the probe antibody.

[0250] Thus, suitably, one or more binding molecules are anti-IL-33 capture antibodies. Suitably, at least one binding molecule is an anti-oxidized IL-33 capture antibody. Suitably, at least one binding molecule is an anti-reduced IL-33 capture antibody. Suitably, at least one binding molecule is an anti-IL-33 / sST2 capture antibody.

[0251] Suitably, at least one reporter molecule is an anti-reduced IL-33 probe antibody. Suitably, at least one reporter molecule is an anti-oxidized IL-33 probe antibody. Suitably, at least one reporter molecule is an anti-IL-33 / sST2 probe antibody or a binding fragment thereof.

[0252] Suitably, the contacting step of these methods comprises the use of one or more capture antibodies capable of binding to one or more antigenically distinct forms of IL-33 to form one or more complexes. Suitably, the detecting step comprises the use of one or more probe antibodies capable of binding to one or more complexes.

[0253] Suitably, one or more binding molecules comprise a specific sequence that allows the binding molecule to bind to one of oxidized IL-33, reduced IL-33 or IL-33 / sST2. Suitably, one or more reporter molecules comprise a specific sequence that allows the reporter molecule to bind to one of oxidized IL-33, reduced IL-33 or IL-33 / sST2 binding molecule complexes.

[0254] Suitable binding and reporter molecules, such as antibodies, having such capabilities are available in the art and can be ordered from reagent companies such as, for example, R&D systems.

[0255] However, suitably, the binding molecules and / or reporter molecules for use in the methods and kits of the present disclosure comprise the following sequences.

[0256] Reduced IL-33 binding molecule - AB1070012

[0257] Suitably, the binding molecule that binds to reduced IL-33 comprises a heavy chain variable region or domain (VH) and a light chain variable region or domain (VL). Suitably, the binding molecule that binds to reduced IL-33 comprises 3 CDRs in the heavy chain variable region or domain (VH) and 3 CDRs in the light chain variable region or domain (VL). Suitably, the binding molecule that binds to reduced IL-33 may comprise 3 CDRs, for example in the heavy chain variable region according to SEQ ID NO: 4, and / or may comprise 3 CDRs in the light chain variable region according to SEQ ID NO: 8.

[0258] Suitably, the binding molecule that binds to reduced IL-33 comprises 3 CDRs in the heavy chain variable region (VH), wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH according to SEQ ID NO: 4, and / or comprises 3 CDRs in the light chain variable region (VL), wherein VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 8.

[0259] Suitably, the binding molecule that binds to reduced IL-33 may comprise a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises VHCDRs 1-3 of SEQ ID NOs: 1, 2, and 3, wherein one or more VHCDRs have 3 or fewer single amino acid substitutions, deletions, or insertions. Suitably, such substitutions may be conservative substitutions.

[0260] Suitably, one or more of the VHCDRs have 3, 2, 1 or no single amino acid substitutions, deletions or insertions. Suitably, the VHCDRs consist of SEQ ID NO: 1, 2 and 3.

[0261] Suitably, the binding molecule that binds to reduced IL-33 may comprise a variable heavy domain (VH) and a variable light domain (VL), wherein the VL comprises VLCDRs 1-3 of SEQ ID NOs: 5, 6 and 7, wherein one or more VLCDRs have 3 or fewer single amino acid substitutions, deletions or insertions. Suitably, such substitutions may be conservative substitutions.

[0262] Suitably, one or more VLCDRs have 3, 2, 1 or no single amino acid substitutions, deletions or insertions. Suitably, the VLCDRs consist of SEQ ID NO: 5, 6 and 7.

[0263] Suitably, the binding molecule that binds to reduced IL-33 may comprise a VH having VHCDR1-3 of SEQ ID NOs: 1, 2 and 3 and a VL having VLCDR of SEQ ID NOs: 5, 6 and 7, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions. Suitably, one or more CDRs have 3, 2, 1 or no single amino acid substitutions, deletions or insertions. Suitably, such substitutions may be conservative substitutions.

[0264] Suitably, the binding molecule that binds to reduced IL-33 may comprise a VH having VHCDR1-3 of SEQ ID NOs: 1, 2 and 3 and a VL having VLCDR of SEQ ID NOs: 5, 6 and 7. Suitably, the binding molecule that binds to reduced IL-33 may comprise a VH having VHCDR 1-3 consisting of SEQ ID NOs: 1, 2 and 3 and a VL having VLCDR consisting of SEQ ID NOs: 5, 6 and 7.

[0265] Suitably, the binding molecule that binds to reduced IL-33 may comprise one or more CDRs selected from the group consisting of: VHCDR1 having a sequence of SEQ ID NO: 1, VHCDR2 having a sequence of SEQ ID NO: 2, VHCDR3 having a sequence of SEQ ID NO: 3, VLCDR1 having a sequence of SEQ ID NO: 5, VLCDR2 having a sequence of SEQ ID NO: 6, and VLCDR3 having a sequence of SEQ ID NO: 7.

[0266] Suitably, the binding molecule that binds to reduced IL-33 may comprise the following CDRs: VHCDR1 having the sequence of SEQ ID NO: 1, VHCDR2 having the sequence of SEQ ID NO: 2, VHCDR3 having the sequence of SEQ ID NO: 3, VLCDR1 having the sequence of SEQ ID NO: 5, VLCDR2 having the sequence of SEQ ID NO: 6, and VLCDR3 having the sequence of SEQ ID NO: 7.

[0267] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VH according to SEQ ID NO: 4.

[0268] Thus, suitably, the binding molecule comprises a VH, wherein the VH disclosed above has a sequence in which 1, 2, 3 or 4 amino acids in the framework are deleted, inserted and / or independently replaced (i.e. substituted) with different amino acids. Suitably, such substitutions may be conservative substitutions.

[0269] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VH according to SEQ ID NO: 4, and comprises VHCDRs 1-3 of SEQ ID NO: 1, 2 and 3, wherein one or more VHCDRs have 3 or fewer single amino acid substitutions, deletions or insertions. Suitably, one or more VHCDRs have 3, 2, 1 or no single amino acid substitutions, deletions or insertions.

[0270] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VH according to SEQ ID NO: 4, and comprises VHCDRs 1-3 of SEQ ID NOs: 1, 2 and 3.

[0271] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising VH and VL, wherein VL has an amino acid sequence that is at least 85%, at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 8.

[0272] Thus, suitably, the binding molecule comprises a VL, wherein the VL disclosed above has a sequence in which 1, 2, 3 or 4 amino acids in the framework are deleted, inserted and / or independently replaced (i.e. substituted) with different amino acids. Suitably, such substitutions may be conservative substitutions.

[0273] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising VH and VL, wherein VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 8, and comprises VLCDRs 1-3 of SEQ ID NO: 5, 6 and 7, wherein one or more VLCDRs have 3 or fewer single amino acid substitutions, deletions or insertions. Suitably, one or more VLCDRs have 3, 2, 1 or no single amino acid substitutions, deletions or insertions.

[0274] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising VH and VL, wherein VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 8, and comprises VLCDRs 1-3 of SEQ ID NOs: 5, 6 and 7.

[0275] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH according to SEQ ID NO: 4 and comprises the VHCDRs 1-3 of SEQ ID NOs: 1, 2 and 3, and wherein the VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 8 and comprises the VLCDRs 1-3 of SEQ ID NOs: 5, 6 and 7.

[0276] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising VH, wherein VH comprises an amino acid sequence according to SEQ ID NO: 4. Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising VL, wherein VL comprises an amino acid sequence according to SEQ ID NO: 8. Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence consisting of SEQ ID NO: 4, and VL has an amino acid sequence consisting of SEQ ID NO: 8.

[0277] Reduced IL-33 binding molecule 2-AB1070019

[0278] Suitably, the binding molecule that binds to reduced IL-33 comprises a heavy chain variable region or domain (VH) and a light chain variable region or domain (VL). Suitably, the binding molecule that binds to reduced IL-33 comprises 3 CDRs in the heavy chain variable region or domain (VH) and 3 CDRs in the light chain variable region or domain (VL). Suitably, the binding molecule that binds to reduced IL-33 may comprise 3 CDRs, for example in the heavy chain variable region according to SEQ ID NO: 28, and / or may comprise 3 CDRs in the light chain variable region according to SEQ ID NO: 32.

[0279] Suitably, the binding molecule that binds to reduced IL-33 comprises 3 CDRs in the heavy chain variable region (VH), wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH according to SEQ ID NO: 28, and / or comprises 3 CDRs in the light chain variable region (VL), wherein VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 32.

[0280] Suitably, the binding molecule that binds to reduced IL-33 may comprise a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises VHCDRs 1-3 of SEQ ID NOs: 25, 26 and 27, wherein one or more of the VHCDRs have 3 or fewer single amino acid substitutions, deletions or insertions.

[0281] Suitably, one or more VHCDRs may comprise 3, 2, 1 or no single amino acid substitutions, deletions or insertions. Suitably, the VHCDRs consist of SEQ ID NOs: 25, 26 and 27.

[0282] Suitably, the binding molecule that binds to reduced IL-33 may comprise a variable heavy domain (VH) and a variable light domain (VL), wherein the VL comprises VLCDRs 1-3 of SEQ ID NOs: 29, 30 and 31, wherein one or more VLCDRs have 3 or fewer single amino acid substitutions, deletions or insertions.

[0283] Suitably, one or more VLCDRs may comprise 3, 2, 1 or no single amino acid substitutions, deletions or insertions. Suitably, the VLCDRs consist of SEQ ID NOs: 29, 30 and 31.

[0284] Suitably, the binding molecule that binds to reduced IL-33 may comprise a VH having VHCDRs 1-3 of SEQ ID NOs: 25, 26 and 27 and a VL having VLCDRs of SEQ ID NOs: 29, 30 and 31, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions. Suitably, one or more CDRs have 3, 2, 1 or no single amino acid substitutions, deletions or insertions. Suitably, such substitutions may be conservative substitutions.

[0285] Suitably, the binding molecule that binds to reduced IL-33 may comprise a VH having VHCDRs 1-3 of SEQ ID NOs: 25, 26 and 27 and a VL having VLCDRs of SEQ ID NOs: 29, 30 and 31. Suitably, the binding molecule that binds to reduced IL-33 may comprise a VH having VHCDRs 1-3 consisting of SEQ ID NOs: 25, 26 and 27 and a VL having VLCDRs consisting of SEQ ID NOs: 29, 30 and 31.

[0286] Suitably, the binding molecule that binds to reduced IL-33 may comprise a CDR selected from the group consisting of a VHCDR1 having a sequence of SEQ ID NO: 25, a VHCDR2 having a sequence of SEQ ID NO: 26, a VHCDR3 having a sequence of SEQ ID NO: 27, a VLCDR1 having a sequence of SEQ ID NO: 29, a VLCDR2 having a sequence of SEQ ID NO: 30, and a VLCDR3 having a sequence of SEQ ID NO: 31.

[0287] Suitably, the binding molecule that binds to reduced IL-33 may comprise the following CDRs: VHCDR1 having the sequence of SEQ ID NO: 25, VHCDR2 having the sequence of SEQ ID NO: 26, VHCDR3 having the sequence of SEQ ID NO: 27, VLCDR1 having the sequence of SEQ ID NO: 29, VLCDR2 having the sequence of SEQ ID NO: 30, and VLCDR3 having the sequence of SEQ ID NO: 31. Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VH according to SEQ ID NO: 28.

[0288] Thus, suitably, the binding molecule comprises a VH, wherein the VH disclosed above has a sequence in which 1, 2, 3 or 4 amino acids in the framework are deleted, inserted and / or independently replaced (i.e. substituted) with different amino acids. Suitably, such substitutions may be conservative substitutions.

[0289] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VH according to SEQ ID NO: 28, and comprises VHCDRs 1-3 of SEQ ID NOs: 25, 26 and 27, wherein one or more VHCDRs have 3 or fewer single amino acid substitutions, deletions or insertions. Suitably, one or more VHCDRs have 3, 2, 1 or no single amino acid substitutions, deletions or insertions.

[0290] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VH according to SEQ ID NO: 28, and comprises VH CDRs 1-3 of SEQ ID NOs: 25, 26 and 27.

[0291] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising VH and VL, wherein VL has an amino acid sequence that is at least 85%, at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 32.

[0292] Thus, suitably, the binding molecule comprises a VL, wherein the VL disclosed above has a sequence in which 1, 2, 3 or 4 amino acids in the framework are deleted, inserted and / or independently replaced (i.e. substituted) with different amino acids. Suitably, such substitutions may be conservative substitutions.

[0293] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising VH and VL, wherein VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 32, and comprises VLCDRs 1-3 of SEQ ID NOs: 29, 30 and 31, wherein one or more VLCDRs have 3 or fewer single amino acid substitutions, deletions or insertions. Suitably, one or more VLCDRs have 3, 2, 1 or no single amino acid substitutions, deletions or insertions.

[0294] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising VH and VL, wherein VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 32, and comprises VLCDRs 1-3 of SEQ ID NOs: 29, 30 and 31.

[0295] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH according to SEQ ID NO: 28 and comprises the VHCDRs 1-3 of SEQ ID NOs: 25, 26 and 27, and wherein the VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 32 and comprises the VLCDRs 1-3 of SEQ ID NOs: 29, 30 and 31.

[0296] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising VH, wherein VH comprises an amino acid sequence according to SEQ ID NO: 28. Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising VL, wherein VL comprises an amino acid sequence according to SEQ ID NO: 32.

[0297] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence consisting of SEQ ID NO: 28 and VL has an amino acid sequence consisting of SEQ ID NO: 32.

[0298] Reduced IL-33 binding molecule 3-AB1070069

[0299] Suitably, the binding molecule that binds to reduced IL-33 comprises a heavy chain variable region or domain (VH) and a light chain variable region or domain (VL). Suitably, the binding molecule that binds to reduced IL-33 comprises 3 CDRs in the heavy chain variable region or domain (VH) and 3 CDRs in the light chain variable region or domain (VL). Suitably, the binding molecule that binds to reduced IL-33 may comprise 3 CDRs, for example in the heavy chain variable region according to SEQ ID NO: 38, and / or may comprise 3 CDRs in the light chain variable region according to SEQ ID NO: 42.

[0300] Suitably, the binding molecule that binds to reduced IL-33 comprises 3 CDRs in the heavy chain variable region (VH), wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH according to SEQ ID NO: 38, and / or comprises 3 CDRs in the light chain variable region (VL), wherein VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 42.

[0301] Suitably, the binding molecule that binds to reduced IL-33 may comprise a variable heavy domain (VH) and a variable light domain (VL), wherein VH comprises VHCDRs 1-3 of SEQ ID NOs: 35, 36 and 37, wherein one or more VHCDRs have 3 or fewer single amino acid substitutions, deletions or insertions.

[0302] Suitably, one or more VHCDRs may comprise 3, 2, 1 or no single amino acid substitutions, deletions or insertions. Suitably, the VHCDRs consist of SEQ ID NOs: 35, 36 and 37.

[0303] Suitably, the binding molecule that binds to reduced IL-33 may comprise a variable heavy domain (VH) and a variable light domain (VL), wherein the VL comprises VLCDRs 1-3 of SEQ ID NOs: 39, 40 and 41, wherein one or more VLCDRs have 3 or fewer single amino acid substitutions, deletions or insertions.

[0304] Suitably, one or more VLCDRs may comprise 3, 2, 1 or no single amino acid substitutions, deletions or insertions. Suitably, the VLCDRs consist of SEQ ID NO: 39, 40 and 41.

[0305] Suitably, the binding molecule that binds to reduced IL-33 may comprise a VH having VHCDRs 1-3 of SEQ ID NOs: 35, 36 and 37 and a VL having VLCDRs of SEQ ID NOs: 39, 40 and 41, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions. Suitably, one or more CDRs have 3, 2, 1 or no single amino acid substitutions, deletions or insertions. Suitably, such substitutions may be conservative substitutions.

[0306] Suitably, the binding molecule that binds to reduced IL-33 may comprise a VH having VHCDRs 1-3 of SEQ ID NOs: 35, 36 and 37 and a VL having VLCDRs of SEQ ID NOs: 39, 40 and 41. Suitably, the binding molecule that binds to reduced IL-33 may comprise a VH having VHCDRs 1-3 consisting of SEQ ID NOs: 35, 36 and 37 and a VL having VLCDRs consisting of SEQ ID NOs: 39, 40 and 41.

[0307] Suitably, the binding molecule that binds to reduced IL-33 may comprise a CDR selected from the group consisting of a VHCDR1 having a sequence of SEQ ID NO: 35, a VHCDR2 having a sequence of SEQ ID NO: 36, a VHCDR3 having a sequence of SEQ ID NO: 37, a VLCDR1 having a sequence of SEQ ID NO: 39, a VLCDR2 having a sequence of SEQ ID NO: 40, and a VLCDR3 having a sequence of SEQ ID NO: 41.

[0308] Suitably, the binding molecule that binds to reduced IL-33 may comprise the following CDRs: VHCDR1 having the sequence of SEQ ID NO: 35, VHCDR2 having the sequence of SEQ ID NO: 36, VHCDR3 having the sequence of SEQ ID NO: 37, VLCDR1 having the sequence of SEQ ID NO: 39, VLCDR2 having the sequence of SEQ ID NO: 40, and VLCDR3 having the sequence of SEQ ID NO: 41.

[0309] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VH according to SEQ ID NO: 38.

[0310] Thus, suitably, the binding molecule comprises a VH, wherein the VH disclosed above has a sequence in which 1, 2, 3 or 4 amino acids in the framework are deleted, inserted and / or independently replaced (i.e. substituted) with different amino acids. Suitably, such substitutions may be conservative substitutions.

[0311] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VH according to SEQ ID NO: 38, and comprises VHCDRs 1-3 of SEQ ID NOs: 35, 36 and 37, wherein one or more VHCDRs have 3 or fewer single amino acid substitutions, deletions or insertions. Suitably, one or more VHCDRs have 3, 2, 1 or no single amino acid substitutions, deletions or insertions.

[0312] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VH according to SEQ ID NO: 38, and comprises VHCDRs 1-3 of SEQ ID NOs: 35, 36 and 37.

[0313] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising VH and VL, wherein VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 42.

[0314] Thus, suitably, the binding molecule comprises a VL, wherein the VL disclosed above has a sequence in which 1, 2, 3 or 4 amino acids in the framework are deleted, inserted and / or independently replaced (i.e. substituted) with different amino acids. Suitably, such substitutions may be conservative substitutions.

[0315] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising VH and VL, wherein VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 42, and comprises VLCDRs 1-3 of SEQ ID NOs: 39, 40 and 41, wherein one or more VLCDRs have 3 or fewer single amino acid substitutions, deletions or insertions. Suitably, one or more VLCDRs have 3, 2, 1 or no single amino acid substitutions, deletions or insertions.

[0316] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising VH and VL, wherein VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 42, and comprises VLCDRs 1-3 of SEQ ID NOs: 39, 40 and 41.

[0317] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH according to SEQ ID NO: 38 and comprises the VHCDRs 1-3 of SEQ ID NOs: 35, 36 and 37, and wherein the VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 42 and comprises the VLCDRs 1-3 of SEQ ID NOs: 39, 40 and 41.

[0318] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising VH, wherein VH comprises an amino acid sequence according to SEQ ID NO: 38. Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising VL, wherein VL comprises an amino acid sequence according to SEQ ID NO: 42.

[0319] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence consisting of SEQ ID NO: 38 and VL has an amino acid sequence consisting of SEQ ID NO: 42.

[0320] Oxidized IL-33 binding molecule - AB1070141

[0321] Suitably, the binding molecule that binds to oxidized IL-33 comprises a heavy chain variable region or domain (VH) and a light chain variable region or domain (VL). Suitably, the binding molecule that binds to oxidized IL-33 comprises 3 CDRs in the heavy chain variable region or domain (VH) and 3 CDRs in the light chain variable region or domain (VL). Suitably, the binding molecule that binds to oxidized IL-33 may comprise 3 CDRs, for example in a heavy chain variable region according to SEQ ID NO: 12, and / or may comprise 3 CDRs in a light chain variable region according to SEQ ID NO: 16.

[0322] Suitably, the binding molecule that binds to oxidized IL-33 comprises 3 CDRs in the heavy chain variable region (VH), wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH according to SEQ ID NO: 12, and / or comprises 3 CDRs in the light chain variable region (VL), wherein VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 16.

[0323] Suitably, the binding molecule that binds to oxidized IL-33 may comprise a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises VHCDRs 1-3 of SEQ ID NOs: 9, 10 and 11, wherein one or more of the VHCDRs have 3 or fewer single amino acid substitutions, deletions or insertions.

[0324] Suitably, one or more of the VHCDRs may comprise 3, 2, 1 or no single amino acid substitutions, deletions or insertions. Suitably, the VHCDRs consist of SEQ ID NOs: 9, 10 and 11.

[0325] Suitably, the binding molecule that binds to oxidized IL-33 may comprise a variable heavy domain (VH) and a variable light domain (VL), wherein the VL comprises VLCDRs 1-3 of SEQ ID NOs: 13, 14 and 15, wherein one or more VLCDRs have 3 or fewer single amino acid substitutions, deletions or insertions.

[0326] Suitably, one or more VLCDRs may comprise 3, 2, 1 or no single amino acid substitutions, deletions or insertions. Suitably, the VLCDRs consist of SEQ ID NO: 13, 14 and 15.

[0327] Suitably, the binding molecule that binds to oxidized IL-33 may comprise a VH having VHCDR1-3 of SEQ ID NOs: 9, 10 and 11 and a VL having VLCDR of SEQ ID NOs: 13, 14 and 15, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions. Suitably, one or more CDRs have 3, 2, 1 or no single amino acid substitutions, deletions or insertions. Suitably, such substitutions may be conservative substitutions.

[0328] Suitably, the binding molecule that binds to oxidized IL-33 may comprise a VH having VHCDR1-3 of SEQ ID NOs: 9, 10 and 11 and a VL having VLCDR of SEQ ID NOs: 13, 14 and 15. Suitably, the binding molecule that binds to oxidized IL-33 may comprise a VH having VHCDR 1-3 consisting of SEQ ID NOs: 9, 10 and 11 and a VL having VLCDR consisting of SEQ ID NOs: 13, 14 and 15.

[0329] Suitably, the binding molecule that binds to oxidized IL-33 may comprise one or more CDRs selected from the group consisting of a VHCDR1 having a sequence of SEQ ID NO: 9, a VHCDR2 having a sequence of SEQ ID NO: 10, a VHCDR3 having a sequence of SEQ ID NO: 11, a VLCDR1 having a sequence of SEQ ID NO: 13, a VLCDR2 having a sequence of SEQ ID NO: 14, and a VLCDR3 having a sequence of SEQ ID NO: 15.

[0330] Suitably, the binding molecule that binds to reduced IL-33 may comprise the following CDRs: VHCDR1 having the sequence of SEQ ID NO: 9, VHCDR2 having the sequence of SEQ ID NO: 10, VHCDR3 having the sequence of SEQ ID NO: 11, VLCDR1 having the sequence of SEQ ID NO: 13, VLCDR2 having the sequence of SEQ ID NO: 14, and VLCDR3 having the sequence of SEQ ID NO: 15.

[0331] Thus, suitably, the binding molecule that binds to oxidized IL-33 is an antibody or a binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VH according to SEQ ID NO: 12.

[0332] Thus, suitably, the binding molecule comprises a VH, wherein the VH disclosed above has a sequence in which 1, 2, 3 or 4 amino acids in the framework are deleted, inserted and / or independently replaced (i.e. substituted) with different amino acids. Suitably, such substitutions may be conservative substitutions.

[0333] Thus, suitably, the binding molecule that binds to oxidized IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH according to SEQ ID NO: 12, and comprises VHCDRs 1-3 of SEQ ID NOs: 9, 10 and 11, wherein one or more VHCDRs have 3 or fewer single amino acid substitutions, deletions or insertions. Suitably, one or more VHCDRs have 3, 2, 1 or no single amino acid substitutions, deletions or insertions.

[0334] Thus, suitably, the binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VH according to SEQ ID NO: 12, and comprises VHCDRs 1-3 of SEQ ID NOs: 9, 10 and 11.

[0335] Thus, suitably, the binding molecule that binds to oxidized IL-33 is an antibody or a binding fragment thereof comprising VH and VL, wherein VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 16.

[0336] Thus, suitably, the binding molecule comprises a VL, wherein the VL disclosed above has a sequence in which 1, 2, 3 or 4 amino acids in the framework are deleted, inserted and / or independently replaced (i.e. substituted) with different amino acids. Suitably, such substitutions may be conservative substitutions.

[0337] Thus, suitably, the binding molecule that binds to oxidized IL-33 is an antibody or binding fragment thereof comprising VH and VL, wherein VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VL according to SEQ ID NO: 16, and comprises VLCDRs 1-3 of SEQ ID NO: 13, 14 and 15, wherein one or more VLCDRs have 3 or fewer single amino acid substitutions, deletions or insertions. Suitably, one or more VLCDRs have 3, 2, 1 or no single amino acid substitutions, deletions or insertions.

[0338] Thus, suitably, the binding molecule that binds to oxidized IL-33 is an antibody or binding fragment thereof comprising VH and VL, wherein VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 16, and comprises VLCDR 1-3 of SEQ ID NO: 13, 14 and 15.

[0339] Thus, suitably, the binding molecule that binds to oxidized IL-33 is an antibody or a binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH according to SEQ ID NO: 12 and comprises the VHCDRs 1-3 of SEQ ID NOs: 9, 10 and 11, and wherein the VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 16 and comprises the VLCDRs 1-3 of SEQ ID NOs: 13, 14 and 15.

[0340] Thus, suitably, the binding molecule that binds to oxidized IL-33 is an antibody or a binding fragment thereof comprising VH, wherein VH comprises the amino acid sequence according to SEQ ID NO: 12. Thus, suitably, the binding molecule that binds to oxidized IL-33 is an antibody or a binding fragment thereof comprising VL, wherein VL comprises the amino acid sequence according to SEQ ID NO: 16.

[0341] Thus, suitably, the binding molecule that binds to oxidized IL-33 is an antibody or a binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence consisting of SEQ ID NO: 12 and VL has an amino acid sequence consisting of SEQ ID NO: 16.

[0342] IL33 / sST2 Binding Molecule - AB1070008

[0343] Suitably, the binding molecule that binds to IL-33 / sST2 comprises a heavy chain variable region or domain (VH) and a light chain variable region or domain (VL). Suitably, the binding molecule that binds to IL-33 / sST2 comprises 3 CDRs in the heavy chain variable region or domain (VH) and 3 CDRs in the light chain variable region or domain (VL). Suitably, the binding molecule that binds to IL-33 / sST2 may comprise 3 CDRs, for example in a heavy chain variable region according to SEQ ID NO: 20, and / or may comprise 3 CDRs in a light chain variable region according to SEQ ID NO: 24.

[0344] Suitably, the binding molecule that binds to IL-33 / sST2 comprises 3 CDRs in the heavy chain variable region (VH), wherein the VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH according to SEQ ID NO: 20, and / or comprises 3 CDRs in the light chain variable region, wherein the VL has an amino acid sequence that is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 24.

[0345] Suitably, the binding molecule that binds to IL-33 / sST2 may comprise a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises VHCDRs 1-3 of SEQ ID NOs: 17, 18 and 19, wherein one or more of the VHCDRs have 3 or fewer single amino acid substitutions, deletions or insertions.

[0346] Suitably, one or more VHCDRs comprise 3, 2, 1 or no single amino acid substitutions, deletions or insertions. Suitably, the VHCDRs consist of SEQ ID NOs: 17, 18 and 19.

[0347] Suitably, the binding molecule that binds to IL-33 / sST2 may comprise a variable heavy domain (VH) and a variable light domain (VL), wherein the VL comprises VLCDRs 1-3 of SEQ ID NOs: 21, 22 and 23, wherein one or more VLCDRs have 3 or fewer single amino acid substitutions, deletions or insertions.

[0348] Suitably, one or more VLCDRs comprise 3, 2, 1 or no single amino acid substitutions, deletions or insertions. Suitably, the VLCDRs consist of SEQ ID NO: 21, 22 and 23.

[0349] Suitably, the binding molecule that binds to IL-33 / sST2 may comprise a VH having VHCDR1-3 of SEQ ID NOs: 17, 18 and 19 and a VL having VLCDR of SEQ ID NOs: 21, 22 and 23, wherein one or more CDRs have 3 or less single amino acid substitutions, deletions or insertions. Suitably, one or more CDRs have 3, 2, 1 or no single amino acid substitutions, deletions or insertions. Suitably, such substitutions may be conservative substitutions.

[0350] Suitably, the binding molecule that binds to IL-33 / sST2 may comprise a VH having VHCDR1-3 of SEQ ID NOs: 17, 18 and 19 and a VL having VLCDR of SEQ ID NOs: 21, 22 and 23. Suitably, the binding molecule that binds to IL-33 / sST2 may comprise a VH having VHCDR 1-3 consisting of SEQ ID NOs: 17, 18 and 19 and a VL having VLCDR consisting of SEQ ID NOs: 21, 22 and 23.

[0351] Suitably, the binding molecule that binds to IL-33 / sST2 may comprise any one of the following CDRs: VHCDR1 having a sequence of SEQ ID NO: 17, VHCDR2 having a sequence of SEQ ID NO: 18, VHCDR3 having a sequence of SEQ ID NO: 19, VLCDR1 having a sequence of SEQ ID NO: 21, VLCDR2 having a sequence of SEQ ID NO: 22, and VLCDR3 having a sequence of SEQ ID NO: 23.

[0352] Suitably, the binding molecule that binds to IL-33 / sST2 may comprise a VHCDR1 having a sequence of SEQ ID NO: 17, a VHCDR2 having a sequence of SEQ ID NO: 18, a VHCDR3 having a sequence of SEQ ID NO: 19, a VLCDR1 having a sequence of SEQ ID NO: 21, a VLCDR2 having a sequence of SEQ ID NO: 22, and a VLCDR3 having a sequence of SEQ ID NO: 23.

[0353] Thus, suitably, the binding molecule that binds to IL-33 / sST2 is an antibody or a binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VH according to SEQ ID NO: 20.

[0354] Thus, suitably, the binding molecule comprises a VH, wherein the VH disclosed above has a sequence in which 1, 2, 3 or 4 amino acids in the framework are deleted, inserted and / or independently replaced (i.e. substituted) with different amino acids. Suitably, such substitutions may be conservative substitutions.

[0355] Thus, suitably, the binding molecule that binds to IL-33 / sST2 is an antibody or binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VH according to SEQ ID NO: 20, and comprises VHCDRs 1-3 of SEQ ID NO: 17, 18 and 19, wherein one or more VHCDRs have 3 or fewer single amino acid substitutions, deletions or insertions. Suitably, one or more VHCDRs have 3, 2, 1 or no single amino acid substitutions, deletions or insertions.

[0356] Thus, suitably, the binding molecule that binds to IL-33 / sST2 is an antibody or a binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VH according to SEQ ID NO: 20, and comprises VHCDRs 1-3 of SEQ ID NOs: 17, 18 and 19.

[0357] Thus, suitably, the binding molecule that binds to IL-33 / sST2 is an antibody or a binding fragment thereof comprising VH and VL, wherein VL has an amino acid sequence that is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 24.

[0358] Thus, suitably, the binding molecule comprises a VL, wherein the VL disclosed above has a sequence in which 1, 2, 3 or 4 amino acids in the framework are deleted, inserted and / or independently replaced (i.e. substituted) with different amino acids. Suitably, such substitutions may be conservative substitutions.

[0359] Thus, suitably, the binding molecule that binds to IL-33 / sST2 is an antibody or binding fragment thereof comprising VH and VL, wherein VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 24, and comprises VLCDRs 1-3 of SEQ ID NO: 21, 22 and 23, wherein one or more VLCDRs have 3 or less single amino acid substitutions, deletions or insertions. Suitably, one or more VLCDRs have 3, 2, 1 or no single amino acid substitutions, deletions or insertions.

[0360] Thus, suitably, the binding molecule that binds to IL-33 / sST2 is an antibody or a binding fragment thereof comprising VH and VL, wherein VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 24, and comprises VLCDR 1-3 of SEQ ID NO: 21, 22 and 23.

[0361] Thus, suitably, the binding molecule that binds to IL-33 / sST2 is an antibody or a binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH according to SEQ ID NO: 20 and comprises the VHCDRs 1-3 of SEQ ID NOs: 17, 18 and 19, and wherein the VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 24 and comprises the VLCDRs 1-3 of SEQ ID NOs: 21, 22 and 23.

[0362] Thus, suitably, the binding molecule that binds to oxidized IL-33 is an antibody or a binding fragment thereof comprising VH, wherein VH comprises the amino acid sequence according to SEQ ID NO: 20. Thus, suitably, the binding molecule that binds to oxidized IL-33 is an antibody or a binding fragment thereof comprising VL, wherein VL comprises the amino acid sequence according to SEQ ID NO: 24.

[0363] Thus, suitably, the binding molecule that binds to IL-33 / sST2 is an antibody or a binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence consisting of SEQ ID NO:20 and VL has an amino acid sequence consisting of SEQ ID NO:24.

[0364] Reduced IL-33 reporter - AB1070019

[0365] Optionally, the reduced IL-33 binding molecule can be used as a reduced IL-33 reporter molecule. Suitably, the binding molecule of (b)(i)b of the first aspect or the binding molecule of (b) of the fourth aspect can also be used as a reporter molecule. Therefore, suitably, in some assays for detecting reduced IL-33, the binding molecule and the reporter molecule may be the same. Therefore, suitably, in some assays for detecting reduced IL-33, the binding molecule and the reporter molecule may both be molecules as defined in step (b)ib of the first aspect or (b) of the fourth aspect.

[0366] Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex comprises a heavy chain variable region or domain (VH) and a light chain variable region or domain (VL). Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex comprises 3 CDRs in the heavy chain variable region or domain (VH) and 3 CDRs in the light chain variable region or domain (VL). Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex may comprise 3 CDRs, for example in the heavy chain variable region according to SEQ ID NO: 28, and / or may comprise 3 CDRs in the light chain variable region according to SEQ ID NO: 32.

[0367] Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex comprises 3 CDRs in the heavy chain variable region (VH), wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH according to SEQ ID NO: 28, and / or comprises 3 CDRs in the light chain variable region (VL), wherein VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 32.

[0368] Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex may comprise a variable heavy domain (VH) and a variable light domain (VL), the VH having VHCDRs 1-3 of SEQ ID NOs: 25, 26 and 27, wherein one or more VHCDRs have 3 or fewer single amino acid substitutions, deletions or insertions.

[0369] Suitably, one or more VHCDRs comprise 3, 2, 1 or no single amino acid substitutions, deletions or insertions. Suitably, the VHCDRs consist of SEQ ID NOs: 25, 26 and 27.

[0370] Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex may comprise a variable heavy domain (VH) and a variable light domain (VL), the VL having VLCDRs 1-3 of SEQ ID NOs: 29, 30 and 31, wherein one or more VLCDRs have 3 or fewer single amino acid substitutions, deletions or insertions.

[0371] Suitably, one or more VLCDRs comprise 3, 2, 1 or no single amino acid substitutions, deletions or insertions. Suitably, the VLCDRs consist of SEQ ID NO: 29, 30 and 31.

[0372] Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex may comprise a VH having VHCDRs 1-3 of SEQ ID NOs: 25, 26 and 27 and a VL having VLCDRs of SEQ ID NOs: 29, 30 and 31, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions. Suitably, one or more CDRs have 3, 2, 1 or no single amino acid substitutions, deletions or insertions. Suitably, such substitutions may be conservative substitutions.

[0373] Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex may comprise a VH having VHCDRs 1-3 of SEQ ID NOs: 25, 26 and 27 and a VL having VLCDRs of SEQ ID NOs: 29, 30 and 31. Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex may comprise a VH having VHCDRs 1-3 consisting of SEQ ID NOs: 25, 26 and 27 and a VL having VLCDRs consisting of SEQ ID NOs: 29, 30 and 31.

[0374] Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex may comprise any one of the following CDRs: VHCDR1 having the sequence of SEQ ID NO:25, VHCDR2 having the sequence of SEQ ID NO:26, VHCDR3 having the sequence of SEQ ID NO:27, VLCDR1 having the sequence of SEQ ID NO:29, VLCDR2 having the sequence of SEQ ID NO:30, and VLCDR3 having the sequence of SEQ ID NO:31.

[0375] Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex may comprise VHCDR1 having the sequence of SEQ ID NO:25, VHCDR2 having the sequence of SEQ ID NO:26, VHCDR3 having the sequence of SEQ ID NO:27, VLCDR1 having the sequence of SEQ ID NO:29, VLCDR2 having the sequence of SEQ ID NO:30, and VLCDR3 having the sequence of SEQ ID NO:31.

[0376] Thus, suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or a binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VH according to SEQ ID NO: 28.

[0377] Thus, suitably, the reporter comprises a VH, wherein the VH disclosed above has a sequence in which 1, 2, 3 or 4 amino acids in the framework are deleted, inserted and / or independently replaced (i.e. substituted) with different amino acids. Suitably, such substitutions may be conservative substitutions.

[0378] Thus, suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VH according to SEQ ID NO: 28, and comprises VHCDRs 1-3 of SEQ ID NO: 25, 26 and 27, wherein one or more VHCDRs have 3 or fewer single amino acid substitutions, deletions or insertions. Suitably, one or more VHCDRs have 3, 2, 1 or no single amino acid substitutions, deletions or insertions.

[0379] Thus, suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VH according to SEQ ID NO: 28, and comprises VHCDRs 1-3 of SEQ ID NOs: 25, 26 and 27.

[0380] Thus, suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or a binding fragment thereof comprising VH and VL, wherein VL has an amino acid sequence that is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 32.

[0381] Thus, suitably, the reporter comprises a VL, wherein the VL disclosed above has a sequence in which 1, 2, 3 or 4 amino acids in the framework are deleted, inserted and / or independently replaced (i.e. substituted) with different amino acids. Suitably, such substitutions may be conservative substitutions.

[0382] Thus, suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or binding fragment thereof comprising VH and VL, wherein VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VL according to SEQ ID NO: 32, and comprises VLCDRs 1-3 of SEQ ID NO: 29, 30 and 31, wherein one or more VLCDRs have 3 or fewer single amino acid substitutions, deletions or insertions. Suitably, one or more VLCDRs have 3, 2, 1 or no single amino acid substitutions, deletions or insertions.

[0383] Thus, suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or binding fragment thereof comprising VH and VL, wherein VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 32, and comprises VLCDR 1-3 of SEQ ID NO: 29, 30 and 31.

[0384] Thus, suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or a binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH according to SEQ ID NO: 28 and comprises the VHCDRs 1-3 of SEQ ID NOs: 25, 26 and 27, and wherein the VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 32 and comprises the VLCDRs 1-3 of SEQ ID NOs: 29, 30 and 31.

[0385] Thus, suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or a binding fragment thereof comprising VH, wherein VH comprises the amino acid sequence according to SEQ ID NO: 28. Thus, suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or a binding fragment thereof comprising VL, wherein VL comprises the amino acid sequence according to SEQ ID NO: 32.

[0386] Thus, suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence consisting of SEQ ID NO:28 and VL has an amino acid sequence consisting of SEQ ID NO:32.

[0387] Reduced IL-33 reporter molecule 2-AB1070069

[0388] Optionally, the reduced IL-33 binding molecule can be used as a reduced IL-33 reporter molecule. Suitably, the binding molecule of (b)(i)b of the first aspect or the binding molecule of (b) of the fourth aspect can also be used as a reporter molecule. Therefore, suitably, in some assays for detecting reduced IL-33, the binding molecule and the reporter molecule may be the same. Therefore, suitably, in some assays for detecting reduced IL-33, the binding molecule and the reporter molecule may both be molecules as defined in step (b)ib of the first aspect or (b) of the fourth aspect.

[0389] Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex comprises a heavy chain variable region or domain (VH) and a light chain variable region or domain (VL). Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex comprises 3 CDRs in the heavy chain variable region or domain (VH) and 3 CDRs in the light chain variable region or domain (VL). Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex may comprise 3 CDRs, for example in the heavy chain variable region according to SEQ ID NO: 38, and / or may comprise 3 CDRs in the light chain variable region according to SEQ ID NO: 42.

[0390] Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex comprises 3 CDRs in the heavy chain variable region (VH), wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH according to SEQ ID NO: 38, and / or comprises 3 CDRs in the light chain variable region (VL), wherein VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 42.

[0391] Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex may comprise a variable heavy domain (VH) and a variable light domain (VL), the VH having VHCDRs 1-3 of SEQ ID NOs: 35, 36 and 37, wherein one or more VHCDRs have 3 or fewer single amino acid substitutions, deletions or insertions.

[0392] Suitably, one or more VHCDRs comprise 3, 2, 1 or no single amino acid substitutions, deletions or insertions. Suitably, the VHCDRs consist of SEQ ID NOs: 35, 36 and 37.

[0393] Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex may comprise a variable heavy domain (VH) and a variable light domain (VL), wherein the VL has VLCDRs 1-3 of SEQ ID NOs: 39, 40 and 41, wherein one or more VLCDRs have 3 or fewer single amino acid substitutions, deletions or insertions.

[0394] Suitably, one or more VLCDRs comprise 3, 2, 1 or no single amino acid substitutions, deletions or insertions. Suitably, the VLCDRs consist of SEQ ID NO: 39, 40 and 41.

[0395] Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex may comprise a VH having VHCDRs 1-3 of SEQ ID NOs: 35, 36 and 37 and a VL having VLCDRs of SEQ ID NOs: 39, 40 and 41, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions. Suitably, one or more CDRs have 3, 2, 1 or no single amino acid substitutions, deletions or insertions. Suitably, such substitutions may be conservative substitutions.

[0396] Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex may comprise a VH having VHCDRs 1-3 of SEQ ID NOs: 35, 36 and 37 and a VL having VLCDRs of SEQ ID NOs: 39, 40 and 41. Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex may comprise a VH having VHCDRs 1-3 consisting of SEQ ID NOs: 35, 36 and 37 and a VL having VLCDRs consisting of SEQ ID NOs: 39, 40 and 41.

[0397] Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex may comprise any one of the following CDRs: VHCDR1 having the sequence of SEQ ID NO:35, VHCDR2 having the sequence of SEQ ID NO:36, VHCDR3 having the sequence of SEQ ID NO:37, VLCDR1 having the sequence of SEQ ID NO:39, VLCDR2 having the sequence of SEQ ID NO:40, and VLCDR3 having the sequence of SEQ ID NO:41.

[0398] Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex may comprise VHCDR1 having the sequence of SEQ ID NO:35, VHCDR2 having the sequence of SEQ ID NO:36, VHCDR3 having the sequence of SEQ ID NO:37, VLCDR1 having the sequence of SEQ ID NO:39, VLCDR2 having the sequence of SEQ ID NO:40, and VLCDR3 having the sequence of SEQ ID NO:41.

[0399] Thus, suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or a binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VH according to SEQ ID NO: 38.

[0400] Thus, suitably, the reporter comprises a VH, wherein the VH disclosed above has a sequence in which 1, 2, 3 or 4 amino acids in the framework are deleted, inserted and / or independently replaced (i.e. substituted) with different amino acids. Suitably, such substitutions may be conservative substitutions.

[0401] Thus, suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VH according to SEQ ID NO: 28, and comprises VHCDRs 1-3 of SEQ ID NO: 35, 36 and 37, wherein one or more VHCDRs have 3 or fewer single amino acid substitutions, deletions or insertions. Suitably, one or more VHCDRs have 3, 2, 1 or no single amino acid substitutions, deletions or insertions.

[0402] Thus, suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or a binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VH according to SEQ ID NO: 38, and comprises VHCDRs 1-3 of SEQ ID NOs: 35, 36 and 37.

[0403] Thus, suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or a binding fragment thereof comprising VH and VL, wherein VL has an amino acid sequence that is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 42.

[0404] Thus, suitably, the reporter comprises a VL, wherein the VL disclosed above has a sequence in which 1, 2, 3 or 4 amino acids in the framework are deleted, inserted and / or independently replaced (i.e. substituted) with different amino acids. Suitably, such substitutions may be conservative substitutions.

[0405] Thus, suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or binding fragment thereof comprising VH and VL, wherein VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VL according to SEQ ID NO: 32, and comprises VLCDRs 1-3 of SEQ ID NO: 39, 40 and 41, wherein one or more VLCDRs have 3 or fewer single amino acid substitutions, deletions or insertions. Suitably, one or more VLCDRs have 3, 2, 1 or no single amino acid substitutions, deletions or insertions.

[0406] Thus, suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or binding fragment thereof comprising VH and VL, wherein VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 42, and comprises VLCDR 1-3 of SEQ ID NO: 39, 40 and 41.

[0407] Thus, suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or a binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a VH according to SEQ ID NO: 38 and comprises the VHCDRs 1-3 of SEQ ID NOs: 35, 36 and 37, and wherein the VL has an amino acid sequence that is at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a VL according to SEQ ID NO: 42 and comprises VLCDRs 1-3 of SEQ ID NOs: 39, 40 and 41.

[0408] Thus, suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or a binding fragment thereof comprising VH, wherein VH comprises the amino acid sequence according to SEQ ID NO: 38. Thus, suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or a binding fragment thereof comprising VL, wherein VL comprises the amino acid sequence according to SEQ ID NO: 42.

[0409] Thus, suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or a binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence consisting of SEQ ID NO:38 and VL has an amino acid sequence consisting of SEQ ID NO:42.

[0410] Oxidized IL-33 reporter

[0411] Suitably, the reporter molecule that binds to the oxidized IL33-binding molecule complex may be obtained from any suitable supplier. For example, suitably, the reporter molecule that binds to the oxidized IL33-binding molecule complex is AF3625 from R&D systems.

[0412] Optionally, however, the oxidized IL-33 binding molecules described herein may also be used as oxidized IL-33 reporter molecules. Suitably, the binding molecules of (b)(ii) of the first aspect or the binding molecules of the fifth aspect may also be used as reporter molecules. Thus, suitably, in some assays for detecting oxidized IL-33, the binding molecules and the reporter molecules may be the same. Thus, suitably, in some assays for detecting oxidized IL-33, the binding molecules and the reporter molecules may both be molecules as defined in step (b)(ii) of the first aspect or the fifth aspect. Suitable details of oxidized IL-33 binding molecules that can be used as reporter molecules are described above under the heading "Oxidized IL-33 Binding Molecules-AB1070141".

[0413] Thus, suitably, there is provided a reporter molecule that binds to an oxidized IL33-binding molecule complex, comprising a variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 9, 10 and 11, respectively, and a variable light domain having VLCDRs 1-3 of SEQ ID NOs: 13, 14 and 15, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions. Suitable further sequences defining such reporter molecules are described elsewhere herein in relation to molecule AB1070141. In one example, the or each reporter molecule may comprise a detectable label. In one example, the or each reporter molecule is conjugated to a detectable label.

[0414] IL-33 / sST2 reporter

[0415] Suitably, the reporter molecule that binds to the IL-33 / sST2-binding molecule complex can be obtained from any suitable supplier. For example, suitably, the reporter molecule that binds to the IL-33 / sST2-binding molecule complex is MAB5232 from R&D systems.

[0416] Optionally, however, the IL-33 / sST2 binding molecules described herein may also be used as IL-33 / sST2 reporter molecules. Suitably, the binding molecules of (b) (iii) of the first aspect or the binding molecules of the sixth aspect may also be used as reporter molecules. Thus, suitably, in some assays for detecting IL-33 / sST2, the binding molecules and the reporter molecules may be the same. Thus, suitably, in some assays for detecting IL-33 / sST2, the binding molecules and the reporter molecules may both be molecules as defined in step (b) (iii) of the first aspect or the sixth aspect. Suitable details of IL-33 / sST2 binding molecules that can be used as reporter molecules are described above under the heading "IL33 / sST2 binding molecules-AB1070008".

[0417] Therefore, suitably, a reporter molecule combined with an IL-33 / sST2-binding molecule complex is provided, comprising a variable heavy domain with VHCDR 1-3 of SEQ ID NO: 17, 18 and 19 accordingly and a variable light domain with VLCDR 1-3 of SEQ ID NO: 21, 22 and 23 accordingly, wherein one or more CDRs have 3 or less single amino acid substitutions, deletions or insertions. Suitable additional sequences defining such reporter molecules are described elsewhere herein about molecule AB1070008. In one example, the or each reporter molecule may comprise a detectable label. In one example, the or each reporter molecule is conjugated to a detectable label.

[0418] Polynucleotides encoding binding and reporter molecules

[0419] The present disclosure also provides polynucleotide molecules encoding the binding molecules and reporter molecules of the present invention. Suitably, isolated polynucleotide molecules encoding the binding molecules and reporter molecules of the present invention.

[0420] Suitably, the isolated polynucleotide comprises, consists essentially of or consists of a nucleic acid encoding an immunoglobulin heavy chain variable domain (VH domain), wherein the sequence of the CDR of the VH domain is: a VHCDR1 sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 9, 17, 25 and 35, a VHCDR2 sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 10, 18, 26 and 36, a VHCDR3 sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11, 19, 27 and 37.

[0421] Suitably, the isolated polynucleotide comprises, consists essentially of or consists of a nucleic acid encoding an immunoglobulin heavy chain variable domain (VH domain) having an amino acid sequence that is at least 85%, at least 90%, at least 95%, such as 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NO: 4, 12, 20, 28 and 38.

[0422] Suitably, the isolated polynucleotide comprises, consists essentially of or consists of a nucleic acid encoding an immunoglobulin light chain variable domain (VL domain), wherein the sequence of the CDR of the VL domain is: a VLCDR1 sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29 and 39, a VLCDR2 sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 14, 22, 30 and 40, a VLCDR3 sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 15, 23, 31 and 41.

[0423] Suitably, the isolated polynucleotide comprises, consists essentially of or consists of a nucleic acid encoding an immunoglobulin light chain variable domain (VL domain) having an amino acid sequence that is at least 85%, at least 90%, at least 95%, such as 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NO: 8, 16, 24, 32 and 42.

[0424] Any of the above polynucleotides may also include additional nucleic acids encoding, for example, a signal peptide that directs secretion of the encoded polypeptide, an antibody constant region as described herein, or other heterologous polypeptides as described herein. In addition, as described in more detail elsewhere herein, the present disclosure includes compositions comprising one or more of the above polynucleotides.

[0425] In one embodiment, a composition comprising a first polynucleotide and a second polynucleotide is provided, wherein the first polynucleotide encodes a VH domain as described herein and wherein the second polynucleotide encodes a VL domain as described herein.

[0426] The present disclosure also includes fragments of the polynucleotides of the present disclosure, as described elsewhere. Additionally, the present disclosure also contemplates polynucleotides encoding Fab fragments and other derivatives as described herein.

[0427] Polynucleotides can be produced or manufactured by any method known in the art. For example, if the nucleotide sequence of the binding molecule is known, polynucleotides encoding the binding molecule can be assembled by chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which briefly include the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the binding molecule, annealing and connection of those oligonucleotides, and then amplification of the connected oligonucleotides by PCR.

[0428] Alternatively, polynucleotides encoding binding or reporter molecules (such as antibodies, or antigen-binding fragments, variants, or derivatives thereof, of the present disclosure) can be generated from nucleic acids from a suitable source. If a clone containing nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, the nucleic acid encoding the antibody can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library produced from any tissue or cell expressing the antibody or other anti-IL-33 antibody (such as a hybridoma cell selected to express the antibody), or nucleic acid isolated therefrom, preferably poly A+RNA), by PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for a particular gene sequence to identify, for example, a cDNA clone encoding the antibody or other anti-IL-33 antibody from the cDNA library. The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method well known in the art.

[0429] Once the nucleotide sequence and corresponding amino acid sequence of the binding molecule or reporter molecule are determined, the nucleotide sequence thereof can be manipulated to produce binding molecules with different amino acid sequences, for example, to produce amino acid substitutions, deletions and / or insertions, using methods well known in the art for manipulating nucleotide sequences, such as recombinant DNA technology, site-directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al. (1990) Molecular Cloning, A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) and Ausubel et al., eds. (1998) Current Protocols in Molecular Biology (John Wiley & Sons, NY), each of which is incorporated herein by reference in its entirety).

[0430] The polynucleotide encoding binding molecules or reporter molecules (e.g., antibodies or their antigen-binding fragments, variants or derivatives) can be composed of any polyribonucleotides or polydeoxyribonucleotides, which can be unmodified RNA or DNA or modified RNA or DNA. For example, the polynucleotide encoding antibodies or their antigen-binding fragments, variants or derivatives can be composed of single-stranded and double-stranded DNA, DNA for a mixture of single-stranded regions and double-stranded regions, single-stranded and double-stranded RNA, and RNA for a mixture of single-stranded regions and double-stranded regions, comprising DNA and RNA hybrid molecules that can be single-stranded or more generally double-stranded or a mixture of single-stranded regions and double-stranded regions. In addition, the polynucleotide encoding binding molecules such as antibodies or their antigen-binding fragments, variants or derivatives can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. The polynucleotide encoding binding molecules (e.g., antibodies or their antigen-binding fragments, variants or derivatives) can also contain one or more modified bases or DNA or RNA backbones modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and rare bases such as inosine. A variety of modifications may be made to DNA and RNA; thus, "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms.

[0431] The isolated polynucleotides encoding non-natural variants of polypeptides derived from immunoglobulins (e.g., immunoglobulin heavy chain portions or light chain portions) can be produced by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of the immunoglobulin so that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more non-essential amino acid residues.

[0432] Binding and reporter expression

[0433] The polynucleotides encoding the binding molecules of the present invention or reporter molecules are usually inserted into expression vectors to be introduced into host cells, and the host cells can be used to produce the binding molecules or reporter molecules of the desired amount. Therefore, expression vectors comprising one or more polynucleotides encoding the binding molecules of the present invention or reporter molecules and host cells comprising the expression vectors are encompassed in the present invention.

[0434] Recombinant expression of a binding molecule or reporter molecule (e.g., a heavy chain and / or light chain of an antibody that binds to a target molecule described herein, such as reduced IL-33, oxidized IL-33, or IL-33 / sST2) requires construction of an expression vector containing a polynucleotide encoding the binding molecule or reporter molecule. Once a polynucleotide encoding a binding molecule or reporter molecule of the present disclosure (e.g., a heavy chain and / or light chain of an antibody or a portion thereof (preferably containing a heavy chain or light chain variable domain)) is obtained, a vector for producing the molecule can be generated by recombinant DNA technology using techniques well known in the art.

[0435] The DNA sequence encoding the binding molecule or reporter molecule can be prepared simultaneously or separately using reverse transcriptase and DNA polymerase according to well-known methods. PCR can be initiated by a common constant region primer or by a more specific primer based on the published DNA and amino acid sequence. PCR can also be used to separate DNA clones encoding antibody light chain and heavy chain. In this case, the library can be screened by a common primer or a larger homologous probe (such as a mouse constant region probe).

[0436] Therefore, the method for preparing protein by expressing polynucleotides containing binding molecules or reporter molecule encoding nucleotide sequences is described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing binding molecule encoding sequences or reporter molecule encoding sequences and appropriate transcription and translation control signals. These methods include, for example, in vitro recombinant DNA technology, synthetic technology, and in vivo genetic recombination. Therefore, the present disclosure provides a reproducible vector, which includes a nucleotide sequence encoding the binding molecules or reporter molecules (such as heavy chain or light chain, or heavy chain or light chain variable domains) of the present disclosure that are operably connected to a promoter. Such vectors may include nucleotide sequences encoding the constant region of an antibody molecule (see, for example, PCT Publication WO 86 / 05807; PCT Publication WO 89 / 01036; and U.S. Patent No. 5,122,464), and the variable domains of an antibody may be cloned into such vectors for expression of the entire heavy chain and / or light chain.

[0437] The term "vector" or "expression vector" is used herein to refer to a vector used as a vehicle for introducing a desired polynucleotide into a host cell and expressing the desired polynucleotide in the host cell according to the present disclosure. As known to those skilled in the art, such vectors can be easily selected from the group consisting of plasmids, phages, viruses and retroviruses. In general, vectors compatible with the present disclosure will contain a selection marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0438] For the purpose of the present disclosure, many expression vector systems can be used. For example, a class of vectors utilizes DNA elements derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV or MOMLV) or SV40 virus. Others relate to the use of polycistronic systems with internal ribosome binding sites. In addition, the cells that have been integrated into their chromosomes by DNA can be selected by introducing one or more markers of the selection of the host cells that allow transfection. The marker can provide the prototrophy of the auxotrophic host, biocide resistance (for example, antibiotics) or resistance to heavy metals such as copper. The selective marker gene can be directly connected to the DNA sequence to be expressed, or introduced into the same cell by co-transformation. The optimal synthesis of mRNA can also require other elements. These elements can include signal sequences, splicing signals and transcription promoters, enhancers and termination signals.

[0439] In one embodiment for expressing antibodies, the cloned variable region genes are inserted into an expression vector together with the heavy chain and light chain constant region genes (preferably, people) synthesized as described above. Of course, any expression vector that can cause expression in eukaryotic cells can be used for the present disclosure. The example of a suitable vector includes but is not limited to plasmids pcDNA3, pHCMV / Zeo, pCR3.1, pEF 1 / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAX1 and pZeoSV2 (available from Invitrogen, San Diego, Calif.) and plasmid pCI (available from Promega, Madison, Wis.). Typically, screening cells expressing appropriate high levels of immunoglobulin heavy and light chains in a large number of transformed cells is a routine experiment that can be performed, for example, by a robotic system.

[0440] More generally, once a vector or DNA sequence encoding a binding molecule or a reporter molecule is prepared, the expression vector can be introduced into a suitable host cell. The introduction of a plasmid into a host cell can be achieved by various techniques well known to those skilled in the art. These techniques include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with envelope DNA, microinjection, and infection with a complete virus. See Ridgway (1988) "Mammalian Expression Vectors", in Vectors, Rodriguez and Denhardt edited (Butterworths, Boston, Mass.), Chapter 24.2, pages 470-472. Typically, a plasmid is introduced into a host by electroporation. The host cell carrying the expression construct is grown under conditions suitable for producing a binding molecule or a reporter molecule, and protein synthesis is determined. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or fluorescence activated cell sorter analysis (FACS), immunohistochemistry, etc.

[0441] The expression vector is transferred to a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce binding molecules or reporter molecules for use in the methods described herein. Therefore, the present disclosure includes host cells containing polynucleotides encoding binding molecules or reporter molecules (e.g., heavy chains and / or light chains, or variable heavy chains and / or variable light chains) of the present disclosure that are operably linked to heterologous promoters.

[0442] In one embodiment, a culture medium comprising the host cell of the present invention is provided. In one embodiment, a fermentation vessel comprising the culture medium is provided.

[0443] Suitably, the culture medium and fermentation vessel are suitable for carrying out the method of producing a binding molecule or reporter molecule of the invention.

[0444] As used herein, "host cell" refers to a cell carrying a vector constructed using recombinant DNA technology and encoding at least one heterologous polynucleotide. In the description of a method for isolating a binding molecule or a reporter molecule from a recombinant host, the terms "cell" and "cell culture" are used interchangeably to indicate the source of the binding molecule or reporter molecule, unless otherwise expressly specified. In other words, recovering a polypeptide from a "cell" can mean from whole cells that have been centrifuged, or from a cell culture containing culture medium and suspended cells.

[0445] A variety of host-expression vector systems can be used to express binding molecules or reporter molecules for use in the methods described herein. Such host-expression systems represent vehicles by which coding sequences of interest can be produced and subsequently purified, but also represent cells that, when transformed or transfected with appropriate nucleotide coding sequences, can express the molecules of the present disclosure in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with a recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vector containing the coding sequence; yeast (e.g., Saccharomyces, Pichia) transformed with a recombinant yeast expression vector containing the coding sequence; insect cell systems infected with a recombinant virus expression vector (e.g., baculovirus) containing the coding sequence; plant cell systems infected with a recombinant virus expression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) containing the coding sequence; or mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3 cells) carrying a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0446] Preferably, bacterial cells such as Escherichia coli are used, and more preferably, eukaryotic cells, particularly eukaryotic cells for expressing complete recombinant antibody molecules, are used to express binding molecules or reporter molecules. For example, mammalian cells such as Chinese hamster ovary cells (CHO) together with vectors such as the major intermediate early gene promoter element from human cytomegalovirus are effective expression systems for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio / Technology 8:2 (1990)).

[0447] Host cell lines used for protein expression are generally of mammalian origin; those skilled in the art are considered to be able to preferentially determine the specific host cell lines that are most suitable for expressing the desired gene product therein. Exemplary host cell lines include, but are not limited to, CHO (Chinese hamster ovary), DG44 and DUXB13 (Chinese hamster ovary lines, DHFR negative), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (derivative of CVI with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, 293, WI38, R1610 (Chinese hamster fibroblasts), BALBC / 3T3 (mouse fibroblasts), HAK (hamster kidney line), SP2 / 0 (mouse myeloma), P3.times.63-Ag3.653 (mouse myeloma), BFA-lclBPT (bovine endothelial cells), RAJI (human lymphocytes) and 293 (human kidney). Host cell lines are generally available from commercial services, American Tissue Culture Collection or published literature.

[0448] In addition, host cell strains that regulate the expression of inserted sequences or modify and process gene products in a desired specific manner can be selected. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be critical for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure the correct modification and processing of expressed foreign proteins. For this reason, eukaryotic host cells with cellular machinery for proper processing, glycosylation, and phosphorylation of primary transcripts of gene products can be used.

[0449] For the long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, the cell line of the binding molecule or reporter molecule of stable expression herein can be engineered. The DNA and selective marker controlled by appropriate expression control elements (for example, promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.) can be used to transform the host cell, rather than using an expression vector comprising a viral origin of replication. After introducing foreign DNA, the engineered cells can be grown in a rich culture medium for 1-2 days, and then switched to the selection culture medium. The selective marker in the recombinant plasmid confers resistance to selection, and the cell is stably integrated into its chromosome by the plasmid and grown to form a gathering point (foci), which can then be cloned and expanded into the cell line. The method can be advantageously used to engineer the cell line of the binding molecule or reporter molecule of stable expression.

[0450] A number of selection systems are available, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 13:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes, which can be used for tk-, hgprt-, or aprt- cells, respectively. In addition, antimetabolite resistance can be used as the basis for selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1521 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2012 (1981)); neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 12:577-611 (1981)); 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 52:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); TIBTECH 13(5):155-215 (May 1993); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:141 (1984).Methods well known in the art of recombinant DNA technology that can be used are described in Ausubel et al. (1993) Current Protocols in Molecular Biology (John Wiley & Sons, NY); Kriegler (1990) "Gene Transfer and Expression" in A Laboratory Manual (Stockton Press, NY); Dracopoli et al. (eds.) (1994) Current Protocols in Human Genetics (John Wiley & Sons, NY), Chapters 12 and 13; Colberre-Garapin et al. (1981) J. Mol. Biol. 150:1, which are incorporated herein by reference in their entirety.

[0451] The expression level of the binding molecule or reporter molecule can be increased by vector amplification (for review, see Bebbington and Hentschel (1987) "The Use of Vectors Based on Gene Amplification for the Expression of Cloned Genes in Mammalian Cells in DNA Cloning" (Academic Press, NY) Volume 3). When the marker in the vector system expressing the binding molecule or reporter molecule is amplifiable, the increase in the level of inhibitors present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the binding molecule gene or the reporter molecule gene, the production of antibodies will also increase (Crouse et al., Mol. Cell. Biol. 3: 251 (1983)).

[0452] In vitro production allows for scaling up to produce a large amount of desired polypeptides. The technology for cultivating mammalian cells under tissue culture conditions is known in the art, and includes, for example, homogeneous suspension culture carried out in an airlift reactor or in a continuous agitator reactor, or immobilization or entrapped cell culture carried out in a hollow fiber, microcapsule, agarose microbeads or ceramic cylinders. If necessary and / or desired, polypeptide solutions can be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, DEAE-cellulose chromatography or (immunity) affinity chromatography, for example, after the preferential biosynthesis of synthetic hinge region polypeptides or before or after HIC chromatography steps as described herein.

[0453] Genes encoding binding molecules or reporter molecules of the present disclosure or portions thereof (such as VH or VL regions) may also be expressed in non-mammalian cells such as insects, bacteria or yeast or plant cells. Bacteria that readily take up nucleic acids include members of the family Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. It should also be understood that when expressed in bacteria, heterologous polypeptides typically become part of inclusion bodies. Heterologous polypeptides must be separated, purified, and then assembled into functional molecules. When a tetravalent form of the antibody is desired, the subunits will then self-assemble into a tetravalent antibody (WO 02 / 096948A2).

[0454] In bacterial systems, many expression vectors can be advantageously selected, and when large amounts of such proteins are to be produced, vectors that direct high-level expression of fusion protein products that are easily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2: 1791 (1983)), in which the coding sequence can be individually ligated into a frame with the lacZ coding region in the vector to produce a fusion protein; pIN vectors (Inouye and Inouye, Nucleic Acids Res. iJ: 3101-3109 (1985); Van Heeke and Schuster, J. Biol. Chem. 24: 5503-5509 (1989)); and the like. pGEX vectors can also be used to express exogenous polypeptides as fusion proteins with glutathione S-transferase (GST). Typically, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0455] In addition to prokaryotes, eukaryotic microorganisms may also be used. Saccharomyces cerevisiae or common baker's yeast is the most commonly used of the eukaryotic microorganisms, but many other strains are also commonly available, such as Pichia pastoris.

[0456] For expression in yeast, plasmid YRp7 (Stinchcomb et al., Nature 282:39 (1979) Kingsman et al., Gene 7:141 (1979); Tschemper et al., Gene 10:151 (1980)) is usually used. This plasmid already contains the TRP1 gene, which provides a selection marker for yeast mutants lacking the ability to grow in tryptophan (e.g., ATCC No. 44076 or PEP4-1) (Jones, Genetics 85:12 (1977)). The presence of the trpl lesion, a characteristic of the yeast host cell genome, provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0457] In insect systems, Autographa californica nuclear polyhedrosis virus (AcNPV) is commonly used as a vector for expressing foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence can be cloned separately into a non-essential region of the virus (e.g., the polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0458] Once the binding molecules or reporter molecules of the present disclosure have been recombinantly expressed, they can be purified by any method known in the art for purifying immunoglobulin molecules, for example, by chromatography {e.g., ion exchange chromatography, affinity chromatography (particularly by affinity for a specific antigen after protein A) and size-sieving column chromatography), centrifugation, differential solubility or by any other standard technique for purifying proteins. Alternatively, preferred methods for increasing the affinity of the antibodies of the present disclosure are disclosed in U.S. Patent Application Publication No. 2002 0123057A1.

[0459] Reagent test kit

[0460] The present disclosure also provides a kit for detecting antigenically different forms of IL-33 in a biological sample.

[0461] Suitably, the kit comprises at least one binding molecule and at least one reporter molecule.Suitably, the binding molecule and the reporter molecule are selected from any of those as defined above.

[0462] Suitably, the kit comprises:

[0463] (i) an oxidized IL-33 binding molecule and a reporter molecule that binds to the oxidized IL-33-binding molecule complex; and / or

[0464] (ii) a reduced IL-33 binding molecule and a reporter molecule that binds to the reduced IL-33-binding molecule complex; and / or

[0465] (iii) IL-33 / sST2 binding molecule and a reporter molecule that binds to the IL-33 / sST2-binding molecule complex.

[0466] Suitably, the kit comprises (i) and (ii). Suitably, the kit comprises (i) and (iii). Suitably, the kit comprises (i) and (ii). Suitably, the kit comprises (i), (ii) and (iii).

[0467] Suitably, the reporter molecules are detectably labelled, suitably each of the reporter molecules comprises a label as described above.

[0468] Alternatively, in another example, an assay kit for detecting antigenic different forms of IL-33 in a biological sample is provided, comprising:

[0469] (i) A binding molecule capable of binding to an antigenically different form of IL-33 selected from the group consisting of reduced IL-33, oxidized IL-33 and / or IL-33 / sST2 to form a complex.

[0470] Suitably, in such examples, the kit comprises a reduced IL-33 binding molecule and / or an oxidized IL-33 binding molecule and / or an IL-33 / sST2 binding molecule. Suitably, in such examples, the kit comprises a reduced IL-33 binding molecule and an oxidized IL-33 binding molecule and an IL-33 / sST2 binding molecule. Suitably, in such examples, the or each binding molecule is detectably labeled.

[0471] Alternatively, the detectable label may be provided separately in the kit, suitable for attachment to a binding molecule or a reporter molecule. Thus, suitably, the kit may include a detectable label, suitable detectable labels being as identified above.

[0472] Thus, suitably, the kit further comprises a stimulatory agent capable of stimulating or activating the detectable label of the or each binding molecule or reporter molecule.

[0473] Suitable biological samples for use with the kit are also defined above.

[0474] Suitably, the kit is suitable for performing an immunoassay as described above. Suitably, the kit is suitable for performing an ELISA or an ECLIA or a CLIA. Suitably, the kit is suitable for performing an S-plex assay. Suitably, the kit is suitable for performing an ECLIA, such as Determination.

[0475] Suitably, the test kit may also include a solid support as described above. Suitably, one or more binding molecules may be located on the solid support. Suitably, one or more binding molecules may be attached to the support or impregnated in the support. Suitably, the solid support may be glass or polymer, including but not limited to cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene as explained above. Suitably, the solid support may be a tube, a bead, a dish, a microplate, a column or the form of any other surface suitable for carrying out the assay method as explained above. In some embodiments, the test kit may include a bead or a microparticle, suitably, a magnetic bead or a microparticle.

[0476] Suitably, the kit may further comprise a label and a capture partner. Suitably, the kit may comprise biotin and streptavidin. In some embodiments, the kit may comprise streptavidin-coated beads or microparticles, suitably streptavidin-coated magnetic beads or microparticles.

[0477] Optionally, the kit may further contain reagents such as buffer, washing solution, distilled water, blocking agent, stop solution and the like.

[0478] Suitably, the kit may also include a stimulator as described herein. Suitably, a detectable label for stimulating one or more reporter molecules or binding molecules. A suitable stimulator is a molecule that stimulates a detectable label, which may suitably be a part of one or more reporter molecules or binding molecules. A suitable stimulator may include a substrate that can be acted upon by a detectable label. For example, an enzyme label to provide a detectable signal, or a dye that can react with a fluorophore label to provide a detectable signal. Suitable substrates to be used with a specific enzyme label are typically selected to produce a detectable color change when hydrolyzed by the corresponding enzyme. For example, p-nitrophenyl phosphate is suitable for use with alkaline phosphatase conjugates; for peroxidase conjugates, 1,2-phenylenediamine or toluidine are typically used. Suitable fluorescent substrates that produce fluorescent products may be used. Suitably, the stimulator may be a reactant, such as an electron donor. Suitably, an amine compound, such as tripropylamine or dibutylethanolamine.

[0479] Alternatively, the kit may further include one or more labels for detecting one or more reporter molecules or binding molecules. Suitably, the label is detectable. Suitably, the one or more labels specifically bind to one or more reporter molecules or binding molecules. Suitable labels are discussed above. Suitably, the kit may include: an oxidized IL-33 complex detectable label, and / or a reduced IL-33 complex detectable label and / or an IL-33 / sST2 complex detectable label.

[0480] Suitably, the kit may also include a device for performing the assay. The device for performing the assay may suitably include a container, such as a test tube, Tubes, plates, etc. Suitably, if the kit comprises a solid support, the solid support may also serve as a container for carrying out the assay. Suitably, the kit comprises one or more microwell plates. Suitably, the kit comprises one or more microwell plates on which one or more binding molecules are immobilized.

[0481] In one example, a support comprising one or more binding molecules immobilized thereon is also provided. Suitably, the one or more binding molecules are as defined according to the fourth, fifth or sixth aspect of the present disclosure.

[0482] Suitably, the kit may further comprise a separation device. Suitably, the separation device is used to purify the sample and / or separate one or more antigenically distinct forms of IL-33 from the sample. Suitable devices for purification or separation may include filters, columns, beads, and the like.

[0483] Suitably, the kit may further comprise instructions, suitably for instructing the user how to use the kit to perform an assay. Suitably, for instructing the user how to perform the method of the present disclosure.

[0484] In one embodiment, the kit comprises:

[0485] (i) an oxidized IL-33 binding molecule and a reporter molecule that binds to the oxidized IL-33-binding molecule complex; and / or

[0486] (ii) a reduced IL-33 binding molecule and a reporter molecule that binds to the reduced IL-33-binding molecule complex; and / or

[0487] (iii) an IL-33 / sST2 binding molecule and a reporter molecule that binds to the IL-33 / sST2-binding molecule complex, wherein each binding molecule comprises a biotin tag, and wherein each reporter molecule comprises a chemiluminescent label;

[0488] (iv) streptavidin-coated magnetic beads;

[0489] (v) Able to activate chemiluminescent labeled reactants in the presence of current / voltage

[0490] Suitably, wherein the chemiluminescent label is a ruthenium compound and the reactant is tripropylamine or dibutylethanolamine.

[0491] Suitably, the kit may further comprise a magnetic electrode.

[0492] The present disclosure will now be described with reference to the following non-limiting examples, in which:

[0493] Example

[0494] Hybridoma Campaign for the production of binding and reporter molecules

[0495] Monoclonal antibodies were generated from wild-type outbred mice by hybridoma technology. Antibodies AB1070019 and AB1070141 were isolated from mice immunized with human IL-33 and antibodies AB1070008, AB1070069 and AB1070012 were isolated from mice immunized with human IL-33 complexed with MEDI3506 in Fab form.

[0496] AB1070012 (mouse IgG1)

[0497] AB1070019 (mouse IgG2b)

[0498] AB1070141 (mouse IgG1)

[0499] AB1070008 (mouse IgG1)

[0500] A repeated immunization at multiple sites (RIMS) strategy was used, in which CD1 mice were immunized as follows:

[0501] - Minus 4 days: before bloodletting

[0502] - Day 0: Primary immunization

[0503] -7 days: Second reinforcement

[0504] - Day 13: First bloodletting

[0505] -15 days: The third reinforcement

[0506] -20 days: Second bloodletting

[0507] -22 days: Fourth reinforcement

[0508] -24 days: Fifth reinforcement

[0509] - Day 28: Terminal bleeding and harvesting of spleen and lymph nodes

[0510] For immunization, twelve mice were divided into 2 groups, each containing 6 animals. Animals were immunized with human recombinant IL-33 or human recombinant IL-33 complexed with MEDI3506 fab.

[0511] Lymphocytes from immunized mice were harvested and mixed with Sp2 / 0Ag14 myeloma cells to form hybridomas using electrofusion. After fusion, cells were resuspended in semisolid medium containing the selection reagents hypoxanthine / diazaserine and anti-mouse IgG FITC-conjugated antibody.

[0512] Hybridoma clones were grown for 13-17 days, and then FITC-positive IgG-expressing clones were picked from semi-solid culture medium into liquid culture medium using the ClonePixFL robot. After 3-7 days of growth, the supernatant containing IgG was screened for binding using a homogeneous time-resolved fluorescence (HTRF) binding assay. Positive hybridomas were overgrown in 24-well plates, the supernatant was purified using the ProPlus Phynexus tips, and the binding properties were confirmed. cDNA from the selected hybridomas was prepared and the antibody variable heavy chain (VH) and light chain (VL) sequences were determined. The VH domain was cloned into an expression vector containing a mouse heavy constant domain of mouse IgG1 that was equivalent to the original hybridoma cell line, i.e., except for AB1070019 (which is mouse IgG2b). The VL domain was cloned into an expression vector containing a mouse kappa constant domain. To produce purified recombinant IgG, the heavy and light chain IgG expression vectors were transiently transfected into CHO cells. IgG was purified from culture supernatants using MabSelect Sure columns on AKTAxpress. The amino acid sequence of each antibody variable domain is shown below.

[0513] Table 1 - Affinity measurements of hybridoma-derived mAbs (BIAcore)

[0514]

[0515] Development of an assay for detecting different IL-33 forms

[0516] Reduced IL-33

[0517] Briefly, recombinant reduced IL-33 (112-270 amino acids) was expressed in E. coli and purified to homogeneity (as described in WO216 / 156440A1 and Cohen et al., 2015).

[0518] Oxidized IL-33

[0519] Oxidized IL-33 (oxIL-33) was generated from a stock solution of reduced IL-33 with an N-terminal His-Avi tag. Reduced IL-33 was oxidized by diluting to a final concentration of 0.5 mg / ml in 60% IMDM medium (without phenol red), 40% DPBS and incubating overnight at 37°C. Aggregates generated during oxidation were removed from the sample by loading it onto a HiTrap Q HP anion exchange column under loading conditions, the aggregates bound to the column, and monomeric oxIL-33 flowed through and was collected. The tag was cleaved from oxIL-33 by incubating with factor Xa at a final concentration of 10 U / mg oxIL-33 at room temperature for 120 minutes. In order to deplete any remaining reduced IL-33 in the sample, a soluble human ST2 extracellular domain fused to human IgG1 Fc was incubated with the sample and bound to reduced IL-33. The sample was concentrated in a centrifugal concentrator with a 5,000 Da cutoff and loaded onto a Superdex 75 26 / 600 column to separate monomeric oxIL-33 from other components. Fractions containing oxIL-33 were pooled and concentrated, and the final concentration of the sample was determined by UV absorption spectroscopy (EC = 14,690 M - 1 cm -1 ).

[0520] IL-33 / sST2 complex

[0521] By using Expifectamine, utilizing the plasmid transient transfection of encoding hST2Flag His10 sequence, the hST2 extracellular domain sequence with C-terminal Flag-His10 tag is expressed in suspension HEK Expi293F cells. After 6-7 days, the cells are harvested by centrifugation and the supernatant is clarified by filtration. Affinity capture chromatography (HisTrap Excel) is used to purify the protein from the clarified supernatant. The monomer fraction of the elution of MW being about 38.7kDa is collected. Determine the protein concentration from its OD280nm reading on Nanodrop, wherein the extinction coefficient (unit) is 1AU=0.77mg / ml

[0522] IL-33 / sST2 complex was prepared by incubating reduced IL-33 and sST2-FH together at a molar ratio of 1:10 for 15 minutes.

[0523] MSD assay - standard curve

[0524] MSD standard binding plates were coated with 50 μl of 5 μg / ml capture Ab in PBS for ~18 h, washed in 300 μl of 3x PBS / 0.05% Tween-20 (PBS-T), incubated in 200 μl of PBS / 1% BSA for 1 h, and washed in 3x PBS-T. Recombinant human IL-33 standards were diluted in PBS-T containing 0.1% BSA, 25 μl was added to the wells and incubated for 2 h. Plates were washed in 3x PBS-T, incubated in 50 μl of 1 μg / ml detection Ab with sulfo tag for 2 h, washed 3x PBS-T, and incubated in 150 μl MSD 1x read buffer for 10 min and read.

[0525] All standards and samples were run in duplicate. Incubation steps were performed at room temperature on an oscillating platform (450 rpm). All assays were read on an MSD Discovery Workbench Sector S600 reader. A graphical representation of the concentration-dependent binding curves is shown in Figure 1. For reduced IL-33 ( Figure 1A ) and oxidized IL-33 ( Figure 1B ), the assay reagent showed no detectable binding to the IL33 / sST2 complex (data not shown). AB1070012 and AB1070069 were found to have equivalent performance for the detection of reduced IL-33 ( Figure 5 ).

[0526] Table 2 - Antibody pairs used for IL-33 assay

[0527] Determination Binding molecule (capture Ab) Reporter molecule (detection Ab) Reduced IL-33 AB1070012 AB1070019 Oxidized IL-33 AB1070141 R&D systems AF3625 IL-33 / sST2 complex AB1070008 R&D systems MAB5232 Reduced IL-33 AB1070069 AB1070019

[0528] MSD S-PLEX for reduced, oxIL-33, and IL-33 / sST2 complex

[0529] MSD S-PLEX Used at MSD (Gaithersburg, USA) The same Ab pairs were used for each form of IL-33 using the same technique as described above for the standard MSD assay.

[0530] Table 3 - MSD determination and MSD S-PLEX Estimated lower limit of detection (LLOD)

[0531]

[0532] ND = Not Determined

[0533] The estimated LLOD was calculated as the concentration off the standard curve that produced a signal 2.5 standard deviations higher than diluent alone (buffer only).

[0534] MSD Assay - Biological Samples

[0535] After collection, all biological samples were stored in aliquots at -80°C

[0536] serum

[0537] Serum samples from healthy individuals, asthmatic patients (GINA stage 1-2) and COPD patients (GOLD stage 1-2) were obtained from a commercial supplier (Seralabs / BioIVT, UK).

[0538] Nasal mucosal lining fluid

[0539] Mucosal lining fluid (MLF) was collected at baseline (-30 min, -15 min) and after a single nasal challenge with Timothy grass pollen (Aquagen, Denmark) in atopic patients (5 min-480 min) as previously described (Leaker et al., 2016). MLF was collected onto a synthetic adsorbent matrix (SAM, Hunts, UK) and eluted from SAM using 330 μl PBS / 0.1% Tween-20 / 0.1% BSA.

[0540] Normal human bronchial epithelial cell (NHBE) culture supernatant

[0541] NHBE cells (Cambrex, UK) were cultured as previously described (Scott et al., 2018). NHBE were plated in 96-well plates (50K) overnight and supernatants were collected from 1 minute to 1440 minutes after scratch injury (Woundmaker, Essen Bioscience).

[0542] Air-liquid interface (ALI) culture supernatant

[0543] The basolateral supernatant of ALI cultures was collected 24 h after medium replacement.

[0544] MSD standard binding plates were coated with 50 μl of 5 μg / ml capture Ab in PBS for ~18 h, washed in 300 μl of 3x PBS / 0.05% Tween-20 (PBS-T), incubated in 200 μl of PBS / 1% BSA for 1 h, and washed in 3x PBS-T. Biological samples were diluted in PBS-T containing 0.1% BSA (minimum dilution 1:2), 25 μl was added to the wells and incubated for 2 h. The plates were washed in 3xPBS-T, incubated in 50 μl of 1 μg / ml detection Ab with sulfo tag for 2 h, washed 3xPBS-T, and incubated in 150 μl MSD 1x read buffer for 10 minutes and read. The concentration of IL-33 forms in biological samples was extrapolated from the recombinant protein standard curve.

[0545] The results showed that these assays can be used to detect redIL-33, oxidized IL-33, and IL33 / sST2 ( Figure 2A-2D ).

[0546] Use this assay to detect IL-33 / sST2 complexes in patients and monitor response to treatment

[0547] Serum samples from patients with SARS-CoV-2 infection were obtained from patients participating in the Phase 2a ACCORD study (EudraCT number: 2020-001736-95, Wilkinson et al., 2020). Figure 3 As shown, patients were recruited during two time periods spanning the pandemic.

[0548] Patients were randomized and received standard of care (SoC) alone or in combination with anti-IL-33 antibody (torezumab) treatment (300 mg IV and optional second dose if invasive ventilation was performed on day 15) over a period of 29 days. The 300 mg IV dose was administered by the following procedure: 2 ml of torelezumab was diluted with 8 ml of saline to a total volume of 10 ml and administered to the patient via IV push over 1-2 minutes using an IV-line filter, followed by a 5 ml saline IV flush.

[0549] As the understanding of the SARS-CoV-2 virus deepened, SoC changed in the two time periods of the ACCORD study. The SoC used during each time period is shown in Table 4 below. The SoC therapy in the second time period of the ACCORD study more accurately reflects current best practices.

[0550] Table 4 - SoC

[0551]

[0552] Sera from patients dosed with torelizumab + standard of care (n=46 as baseline, n=37 on day 5, n=10 on day 10) and SoC alone (n=37 as baseline, n=23 on day 5, n=10 on day 10) were measured for IL-33 / sST2 at a 1:4 dilution.

[0553] All serum samples were stored in aliquots at -80°C after collection.

[0554] At MSD (Gaithersburg, USA), we used Serum samples obtained from patients were subjected to MSD S-PLEX using the antibodies for IL-33 / sST2 as described above. To measure the levels of IL-33 / sST2 in serum.

[0555] The custom MSD S-PLEX IL-33 / sST2 complex assay was prepared and validated by Mesoscale Discovery (MSD) and performed according to the manufacturer's instructions. Unless otherwise specified, all incubations required plate shaking at room temperature (RT). As specified, the plates were washed 3x with wash buffer (phosphate buffered saline (PBS) / 0.05% Tween-20). The biotin-coated capture mAb (Table 5) was diluted in diluent 100 (MSD) with S-Plex coating reagent (MSD) to form a coating solution. The assay plate (MSD) was coated with 50 μl / well of the coating solution and incubated for 1 h. The blocking reagent / solution was prepared by diluting the S-Plex Blocker reagent (MSD) in diluent 101 (MSD). The recombinant IL-33 / sST2 complex standard curve was generated by diluting the stock solution in diluent 100 (MSD) to the highest standard concentration (Table 5) and performing a further 4-fold serial dilution. 25 μl blocking reagent was added to all wells and 25 μl standards and samples were added to the wells and incubated for 1.5 h. TURBO-enhanced solution was prepared by diluting the TURBO-enhanced labeled detection mAb (Table 5) to a working concentration in diluent 3 (MSD). Wash the plate, add 50 μl of TURBO-enhanced detection mAb solution to each well and incubate for 1 h. Wash the plate, add 50 μl of enhanced solution (MSD) to each well and incubate for 30 minutes. Wash the plate, add 50 μl of detection solution (MSD) to each well and incubate for 1 h at 26 ° C. Wash the plate and add 150 μl of 1x read buffer A (MSD) to each well. Read the plate on an MSD MESO SECTOR S600 instrument.

[0556] Table 5 - Concentrations of antibodies and IL-33 / sST2 standards

[0557] Reagents IL-33 / ST2 complex assay Biotin-coated capture Ab (μg / ml) 0.25 TURBO-enhanced detection Ab (μg / ml) 0.1 Highest Standard (Unit / ml)* 1000

[0558] *Data for IL-33 / sST2 complexes are expressed in units / ml; for the IL-33 / sST2 standard this can be approximated as pg / ml if 100% complex formation is assumed.

[0559] The MSD S-PLEX assay is sensitive enough to determine pg / ml levels of IL-33 / sST2 in patient serum.

[0560] Subgroup analysis of the endpoint "death or respiratory failure on day 29" was performed based on the median value of baseline IL-33 / sST2. The median value at baseline was 30.15 U / ml, and the subgroups were defined as IL-33 low: baseline IL-33 / sST2 <30 / 15 U / ml; IL-33 high: baseline IL-33 / sST2>=30.15 U / ml.

[0561] For each treatment in each subgroup, the proportion of subjects who died or were in respiratory failure on day 29 was calculated, and the relative risk (torezumab: placebo) and 80% confidence interval were calculated for each subgroup. In addition, a logistic regression model was fitted to each subgroup, adjusting for age (continuous) and baseline severity (WHO score of "3 or 4" versus "5"). From the logistic regression model, the odds ratio (torezumab: placebo) of death or respiratory failure on day 29 and 80% confidence interval were calculated for each subgroup.

[0562] Using this assay, it was determined that those patients with high baseline serum levels of IL-33 / sST2 equal to or above 30.15 pg / ml were significantly less likely to die or have respiratory failure after 29 days when treated with torelizumab, as shown in Tables 6 and Figure 4 as shown in .

[0563] Such patients form a subgroup that may respond well to treatment with IL-33 axis antagonists, thereby providing a means to stratify and select patients for treatment with IL-33 axis antagonists.

[0564] Table 6

[0565]

[0566] sequence

[0567]

[0568]

[0569]

Claims

1. A method for detecting one or more antigenically distinct forms of IL-33 in a biological sample, comprising: (a) contacting the sample with one or more binding molecules, each of which is capable of binding to an antigenically different form of IL-33 selected from the group consisting of IL-33 / sST2, reduced IL-33 and / or oxidized IL-33, under conditions sufficient to form a complex; and (b) detecting the level of one or more IL-33 / sST2, reduced IL-33 and / or oxidized IL-33 complexes in the sample, wherein: (i) The binding molecules that bind to IL-33 / ST2 comprise molecules having SEQ ID NO: a variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 17, 18 and 19 and a variable light domain having VLCDRs 1-3 of SEQ ID NOs: 21, 22 and 23, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions; (ii) the binding molecule that binds to reduced IL-33 comprises: a. A variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 1, 2 and 3, respectively, and a variable light domain having VLCDRs 1-3 of SEQ ID NOs: 5, 6 and 7, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions; b. a variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 25, 26 and 27, respectively, and a variable light domain having VLCDRs 1-3 of SEQ ID NOs: 29, 30 and 31, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions; or c. a variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 35, 36 and 37, respectively, and a variable light domain having VLCDRs 1-3 of SEQ ID NOs: 39, 40 and 41, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions; and (iii) the binding molecule that binds to oxidized IL-33 comprises a variable heavy domain having VHCDR1-3 of SEQ ID NOs: 9, 10 and 11, respectively, and a variable light domain having VLCDR 1-3 of SEQ ID NOs: 13, 14 and 15, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions.

2. The method of claim 1, wherein step (a) comprises contacting the sample with the binding molecule capable of binding to IL-33 / sST2, and step (b) comprises detecting the level of the IL-33 / sST2 complex.

3. The method of claim 1, wherein step (a) comprises contacting the sample with the binding molecule capable of binding to reduced IL-33, and step (b) comprises detecting the level of reduced IL-33 complex.

4. The method of claim 1, wherein step (a) comprises contacting the sample with the binding molecule capable of binding to oxidized IL-33, and step (b) comprises detecting the level of oxidized IL-33 complex.

5. The method of claim 1, wherein step (a) comprises contacting the sample with the binding molecule capable of binding to reduced IL-33 and IL-33 / sST2, and step (b) comprises detecting the levels of reduced IL-33 complex and IL-33 / sST2 complex.

6. The method of claim 1, wherein step (a) comprises contacting the sample with the binding molecule capable of binding to oxidized IL-33 and IL-33 / sST2, and step (b) comprises detecting the levels of oxidized IL-33 complex and IL-33 / sST2 complex.

7. The method of claim 1, wherein step (a) comprises contacting the sample with the binding molecule capable of binding to reduced IL-33 and oxidized IL-33, and step (b) comprises detecting the levels of reduced IL-33 complex and oxidized IL-33 complex.

8. The method of claim 1, wherein step (a) comprises contacting the sample with the binding molecule capable of binding to reduced IL-33, oxidized IL-33, and IL-33 / sST2, and step (b) comprises detecting the levels of reduced IL-33 complex, oxidized IL-33 complex, and IL-33 / sST2 complex.

9. The method according to any preceding claim, wherein the one or more binding molecules are capture probes.

10. The method of claim 9, wherein the method further comprises the step of contacting the complexes with one or more reporter molecules capable of binding to the one or more complexes.

11. The method according to any one of claims 1 to 8, wherein: Prior to step (a), the following step is performed: immobilizing the antigenically different isoforms of IL-33 by contacting the sample with the solid surface under conditions suitable for the antigenically different isoforms of IL-33 to bind to the solid surface.

12. The method of claim 11, wherein the detecting step (b) comprises detecting the level of bound reporter molecule in the sample.

13. An assay kit for detecting antigenic different forms of IL-33 in a biological sample, comprising: (a) a binding molecule capable of binding to an antigenically different form of IL-33 selected from the group consisting of IL-33 / sST2, reduced IL-33 and / or oxidized IL-33 to form a complex; (b) a reporter molecule capable of binding to a binding molecule complex selected from the group consisting of an IL-33 / sST2, a reduced IL-33 and / or an oxidized IL-33 binding molecule complex.

14. The kit according to claim 13, wherein the binding molecule that binds to reduced IL-33 comprises: a. A variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 1, 2 and 3, respectively, and a variable light domain having VLCDRs 1-3 of SEQ ID NOs: 5, 6 and 7, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions; b. a variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 25, 26 and 27, respectively, and a variable light domain having VLCDRs 1-3 of SEQ ID NOs: 29, 30 and 31, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions; or c. a variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 35, 36 and 37, respectively, and a variable light domain having VLCDRs 1-3 of SEQ ID NOs: 39, 40 and 41, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions.

15. The kit of claim 13, wherein the binding molecule that binds to oxidized IL-33 comprises a variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 9, 10, and 11, respectively, and a variable light domain having VLCDRs 1-3 of SEQ ID NOs: 13, 14, and 15, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions, or insertions.

16. A kit according to claim 13, wherein the binding molecule that binds to IL-33 / ST2 comprises a variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 17, 18 and 19, respectively, and a variable light domain having VLCDRs 1-3 of SEQ ID NOs: 21, 22 and 23, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions.

17. The kit according to any one of claims 13 to 16, wherein the kit comprises: (i) an IL-33 / sST2 binding molecule and a reporter molecule that binds to the IL-33 / sST2-binding molecule complex; and / or (ii) an oxidized IL-33 binding molecule and a reporter molecule that binds to the oxidized IL-33-binding molecule complex; and / or (iii) a reduced IL-33 binding molecule and a reporter molecule that binds to the reduced IL-33-binding molecule complex.

18. An antibody or antigen-binding fragment thereof that binds to reduced IL-33, comprising: a. a variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 1, 2 and 3, respectively, and a variable light domain having VLCDRs 1-3 of SEQ ID NOs: 5, 6 and 7, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions; or b. a variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 25, 26 and 27, respectively, and a variable light domain having VLCDRs 1-3 of SEQ ID NOs: 29, 30 and 31, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions; or c. a variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 35, 36 and 37, respectively, and a variable light domain having VLCDRs 1-3 of SEQ ID NOs: 39, 40 and 41, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions.

19. An antibody or antigen-binding fragment thereof that binds to oxidized IL-33, comprising a variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 9, 10 and 11, respectively, and a variable light domain having VLCDRs 1-3 of SEQ ID NOs: 13, 14 and 15, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions.

20. An antibody or antigen-binding fragment thereof that binds to IL-33 / sST2, comprising a variable heavy domain having VHCDRs 1-3 of SEQ ID NOs: 17, 18 and 19, respectively, and a variable light domain having VLCDRs 1-3 of SEQ ID NOs: 21, 22 and 23, respectively, wherein one or more CDRs have 3 or fewer single amino acid substitutions, deletions or insertions.

21. The antibody or antigen-binding fragment thereof according to any one of claims 18 to 20, which is conjugated to a detectable label.

22. The kit according to any one of claims 13 to 17, the method according to any one of claims 1 to 12, and the antibody or antigen-binding fragment thereof according to claim 18, wherein the binding molecule or antibody or antigen-binding fragment thereof that binds to reduced IL-33 comprises: a. a variable heavy domain (VH), wherein the VH has an amino acid sequence that is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH according to SEQ ID NO: 4, and / or a variable light domain (VL), wherein the VL has an amino acid sequence that is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 8; b. a variable heavy domain (VH), wherein the VH has an amino acid sequence that is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH according to SEQ ID NO: 28, and / or a variable light domain (VL), wherein the VL has an amino acid sequence that is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO: 32; or c. a variable heavy domain (VH), wherein the VH has an amino acid sequence that is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH according to SEQ ID NO: 38, and / or a variable light domain (VL), wherein the VL has an amino acid sequence that is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO:

42.

23. The kit according to any one of claims 13 to 17, the method according to any one of claims 1 to 12, and the antibody or antigen-binding fragment thereof according to claim 18, wherein the binding molecule or antibody or antigen-binding fragment thereof that binds to reduced IL-33 comprises a VHCDR1 having a sequence of SEQ ID NO: 1, a VHCDR2 having a sequence of SEQ ID NO: 2, a VHCDR3 having a sequence of SEQ ID NO: 3, a VLCDR1 having a sequence of SEQ ID NO: 5, a VLCDR2 having a sequence of SEQ ID NO: 6, and a VLCDR3 having a sequence of SEQ ID NO:

7.

24. The kit according to any one of claims 13 to 17, the method according to any one of claims 1 to 12, and the antibody or antigen-binding fragment thereof according to claim 18, wherein the binding molecule or antibody or antigen-binding fragment thereof that binds to reduced IL-33 comprises a VHCDR1 having a sequence of SEQ ID NO: 25, a VHCDR2 having a sequence of SEQ ID NO: 26, a VHCDR3 having a sequence of SEQ ID NO: 27, a VLCDR1 having a sequence of SEQ ID NO: 29, a VLCDR2 having a sequence of SEQ ID NO: 30, and a VLCDR3 having a sequence of SEQ ID NO:

31.

25. The kit according to any one of claims 13 to 17, the method according to any one of claims 1 to 12, and the antibody or antigen-binding fragment thereof according to claim 18, wherein the binding molecule or antibody or antigen-binding fragment thereof that binds to reduced IL-33 comprises a VHCDR1 having a sequence of SEQ ID NO: 35, a VHCDR2 having a sequence of SEQ ID NO: 36, a VHCDR3 having a sequence of SEQ ID NO: 37, a VLCDR1 having a sequence of SEQ ID NO: 39, a VLCDR2 having a sequence of SEQ ID NO: 40, and a VLCDR3 having a sequence of SEQ ID NO:

41.

26. The kit according to any one of claims 13 to 17, the method according to any one of claims 1 to 12, and the antibody or antigen-binding fragment thereof according to claim 19, wherein the binding molecule or antibody or antigen-binding fragment thereof that binds to oxidized IL-33 comprises a variable heavy domain, wherein the VH has an amino acid sequence that is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH according to SEQ ID NO: 12, and / or a variable light domain, wherein the VL has an amino acid sequence that is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO:

16.

27. The kit according to any one of claims 13 to 17, the method according to any one of claims 1 to 12, and the antibody or antigen-binding fragment thereof according to claim 19, wherein the binding molecule or antibody or antigen-binding fragment thereof capable of binding to oxidized IL-33 comprises a VHCDR1 having a sequence of SEQ ID NO: 9, a VHCDR2 having a sequence of SEQ ID NO: 10, a VHCDR3 having a sequence of SEQ ID NO: 11, a VHCDR4 having a sequence of SEQ ID NO: 12, a VHCDR5 having a sequence of SEQ ID NO: 13, a VHCDR6 having a sequence of SEQ ID NO: 14, a VHCDR7 having a sequence of SEQ ID NO: 15, a VHCDR8 having a sequence of SEQ ID NO: 16, a VHCDR9 having a sequence of SEQ ID NO: 17, a VHCDR11 having a sequence of SEQ ID NO: 18, a VHCDR12 having a sequence of SEQ ID NO: 19, a VHCDR13 having a sequence of SEQ ID NO: 20, a VHCDR14 having a sequence of SEQ ID NO: 21, a VHCDR15 having a sequence of SEQ ID NO: 23, a VHCDR16 having a sequence of SEQ ID NO: 24, a VHCDR17 having a sequence of SEQ ID NO: 25 A VHCDR2 having the sequence of SEQ ID NO:10, a VHCDR3 having the sequence of SEQ ID NO:11, a VLCDR1 having the sequence of SEQ ID NO:13, a VLCDR2 having the sequence of SEQ ID NO:14, and a VLCDR3 having the sequence of SEQ ID NO:

15.

28. The kit according to any one of claims 13 to 17, the method according to any one of claims 1 to 12, and the antibody or antigen-binding fragment thereof according to claim 20, wherein the binding molecule or antibody or antigen-binding fragment thereof that binds to IL-33 / sST2 comprises a variable heavy domain, wherein the VH has an amino acid sequence that is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH according to SEQ ID NO: 20, and / or a variable light domain, wherein the VL has an amino acid sequence that is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL according to SEQ ID NO:

24.

29. The kit according to any one of claims 13 to 17, the method according to any one of claims 1 to 12, and the antibody or antigen-binding fragment thereof according to claim 20, wherein the binding molecule or antibody or antigen-binding fragment thereof that binds to IL-33 / sST2 comprises a VHCDR1 having a sequence of SEQ ID NO: 25, a VHCDR2 having a sequence of SEQ ID NO: 26, a VHCDR3 having a sequence of SEQ ID NO: 27, a VLCDR1 having a sequence of SEQ ID NO: 29, a VLCDR2 having a sequence of SEQ ID NO: 30, and a VLCDR3 having a sequence of SEQ ID NO: 31.

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