Assays for the detection of IL-33

Abstract

The present disclosure relates to methods for detecting antigenically distinct forms of IL-33 in biological samples, kits for carrying out such methods, and binding molecules and reporter molecules for use in such methods.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 371,932, filed August 19, 2022, and U.S. Provisional Patent Application No. 63 / 385,817, filed December 2, 2022, which are hereby incorporated by reference in their entireties for all purposes.

[0002] Reference to an electronically submitted sequence listing This application incorporates by reference the Sequence Listing submitted with this application in computer readable format (CRF) as a text file entitled "IL33-208-WO-PCT Sequence Listing," created on August 17, 2023, and having a size of 46,433 bytes.

[0003] The present disclosure relates to methods for detecting antigenically distinct forms of IL-33 in biological samples, kits for carrying out such methods, and binding molecules and reporter molecules for use in such methods. [Background technology]

[0004] Interleukin-33 (IL-33), also known as IL-1F11, is a member of the IL-1 cytokine family. IL-33 is a 270-amino acid protein consisting 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 two distinct 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, forming at least one disulfide bond in the oxidized form, and that the two forms likely have different binding patterns and effects.

[0005] Furthermore, reduced forms of IL-33 have been shown to bind to ST2 and, in fact, are the only known ligands 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 NFκB and MAP kinase pathways, which leads to the production of cytokines and chemokines, such as IL-4, IL-5, and IL-13, promoting inflammation. Soluble ST2 (sST2) is thought to be a decoy receptor that inhibits reduced IL-33 signaling.

[0006] More recently, it has been shown that oxidized IL-33 forms also have physiological effects: they do not bind to ST2 but instead bind to the receptor for advanced glycation end products (RAGE) and signal through this alternative pathway.

[0007] IL-33 is therefore of great interest as a therapeutic target, primarily due to the ability of what is currently known as the reduced form of IL-33 to stimulate ST2 and exert its potent inflammatory effects, but now also due to the ability of the oxidized form to stimulate RAGE, which is involved in a wide range of diseases.

[0008] 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 biological samples.

[0009] However, to date, there has been no means to reliably distinguish between different forms of IL-33. Several assays have been developed to detect IL-33. However, the readouts from these assays typically do not distinguish between different forms and therefore cannot inform the user about biological pathways that may be inhibited or activated in a given biological sample. For example, the user has no way of knowing whether the results indicate activation of the ST2 pathway or the RAGE pathway. Furthermore, because these assays do not distinguish between different forms, it is often unclear which form is being measured in a given assay, leading to a lack of consistency and reliability in the results.

[0010] The present inventors have developed a highly sensitive assay that can detect not only one of the different forms of IL-33 in a biological sample, but each of the different forms of IL-33, namely, reduced IL-33, oxidized IL-33, and bound IL-33 / sST2. This assay is based on the development of novel binding molecules that can bind to each of the different forms of IL-33. The present inventors believe that such an assay will be useful for analyzing antigenically distinct forms of IL-33 present in patient-derived samples to guide medical treatment.

[0011] Accordingly, one or more aspects of the present disclosure and further features thereof are defined below.

[0012] Aspects of the Disclosure According to a first aspect 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: (a) contacting the sample with one or more binding molecules each capable of binding to antigenically distinct forms of IL-33 selected from reduced IL-33, oxidized IL-33, and / or IL-33 / sST2 under conditions sufficient to form complexes; (b) detecting the level of one or more of reduced IL-33 complex, oxidized IL-33 complex, and / or IL-33 / sST2 complex in the sample; (i) A binding molecule that binds to reduced IL-33 is a. a variable heavy domain having VH CDRs 1-3 of SEQ ID NOS: 1, 2, and 3, respectively, and a variable light domain having VL CDRs 1-3 of SEQ ID NOS: 5, 6, and 7, respectively, wherein one or more CDRs have three or fewer single amino acid substitutions, deletions, or insertions; b. A variable heavy domain having VH CDRs 1-3 of SEQ ID NOs: 25, 26, and 27, respectively, and a variable light domain having VL CDRs 1-3 of SEQ ID NOs: 29, 30, and 31, respectively, wherein one or more CDRs have no more than three single amino acid substitutions, deletions, or insertions; or c. A variable heavy domain having VH CDRs 1-3 of SEQ ID NOs: 35, 36, and 37, respectively, and a variable light domain having VL CDRs 1-3 of SEQ ID NOs: 39, 40, and 41, respectively, wherein one or more CDRs have no more than three single amino acid substitutions, deletions, or insertions; (ii) a binding molecule that binds 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 no more than three single amino acid substitutions, deletions, or insertions; (iii) a 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 no more than three single amino acid substitutions, deletions, or insertions; A method is provided.

[0013] In one example, the method of the first embodiment further comprises contacting the complexes with one or more reporter molecules capable of binding to one or more of the complexes, in such case detecting step (b) comprises detecting the level of bound reporter molecules in the sample.

[0014] In one example, the reporter molecule is as defined below.

[0015] In one example, step (a) comprises contacting the sample with one or more binding molecules capable of binding to reduced IL-33. Preferably, in such cases, the one or more binding molecules comprise those defined in (i). Preferably, in such cases, the one or more binding molecules consist of those defined in (i). Preferably, in such cases, step (b) comprises detecting the level of reduced IL-33-binding molecule complexes.

[0016] In one example, step (a) comprises contacting the sample with one or more binding molecules capable of binding to oxidized IL-33. Preferably, in such cases, the one or more binding molecules comprise those defined in (ii). Preferably, in such cases, the one or more binding molecules consist of those defined in (ii). Preferably, in such cases, step (b) comprises detecting the level of oxidized IL-33-binding molecule complexes.

[0017] In one example, step (a) comprises contacting the sample with one or more binding molecules capable of binding to IL-33 / sST2. Preferably, in such cases, the one or more binding molecules comprise those defined in (iii). Preferably, in such cases, the one or more binding molecules consist of those defined in (iii). Preferably, in such cases, step (b) comprises detecting the level of an IL-33 / sST2-binding molecule complex.

[0018] 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. Preferably, in such cases, at least one of the binding molecules is as defined in (i) or (iii). Preferably, in such cases, the binding molecules include those defined in (i) and (iii). Preferably, in such cases, the binding molecules consist of those defined in (i) and (iii). Preferably, in such cases, step (b) comprises detecting the levels of reduced IL-33-binding molecule complexes and IL-33 / sST2-binding molecule complexes.

[0019] 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. Preferably, in such cases, at least one of the binding molecules is as defined in (ii) or (iii). Preferably, in such cases, the binding molecules include those defined in (ii) and (iii). Preferably, in such cases, the binding molecules consist of those defined in (ii) and (iii). Preferably, in such cases, step (b) comprises detecting the levels of oxidized IL-33-binding molecule complexes and IL-33 / sST2-binding molecule complexes.

[0020] 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. Preferably, in such cases, at least one of the binding molecules is as defined in (i) or (ii). Preferably, in such cases, the binding molecules include those defined in (i) and (ii). Preferably, in such cases, the binding molecules consist of those defined in (i) and (ii). Preferably, in such cases, step (b) comprises detecting the levels of reduced IL-33-binding molecule complexes and oxidized IL-33-binding molecule complexes.

[0021] 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. Preferably, in such cases, at least one of the binding molecules is as defined in (i), (ii), or (iii). Preferably, in such cases, the binding molecules include those defined in (i), (ii), and (iii). Preferably, in such cases, the binding molecules consist of those defined in (i), (ii), and (iii). Preferably, in such cases, 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.

[0022] According to a second aspect of the present disclosure, there is provided a method for detecting antigenically distinct forms of IL-33 in a biological sample, comprising: (a) contacting the sample with one or more binding molecules capable of binding reduced IL-33, oxidized IL-33, and IL-33 / sST2 under conditions sufficient to form complexes; (b) detecting the level of reduced IL-33 complex, oxidized IL-33 complex, and / or IL-33 / sST2 complex in the sample.

[0023] In one example, the binding molecule is as defined in the fourth, fifth and sixth aspects.

[0024] In one example, the method of the second embodiment further comprises contacting the complex with one or more reporter molecules capable of binding to the complex, in such case detecting step (b) comprises detecting the level of bound reporter molecules in the sample.

[0025] In one example, the reporter molecule is as defined below.

[0026] According to a third aspect of the present disclosure, there is provided an assay kit for detecting antigenically distinct forms of IL-33 in a biological sample, comprising: (a) a binding molecule capable of binding to and forming a complex with an antigenically distinct form of IL-33 selected from reduced IL-33, oxidized IL-33, and / or IL-33 / sST2; (b) a reporter molecule capable of binding to a binding molecule complex selected from a reduced IL-33 binding molecule complex, an oxidized IL-33 binding molecule complex, and / or an IL-33 / sST2 binding molecule complex; An assay kit is provided, comprising:

[0027] 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.

[0028] In one example, the reporter molecule is as defined below: In a further example, the reporter molecule may be as defined in the seventh aspect.

[0029] According to a fourth aspect of the present disclosure, there is provided a binding molecule that binds to reduced IL-33, comprising: a. a variable heavy domain having VH CDRs 1-3 of SEQ ID NOS: 1, 2, and 3, respectively, and a variable light domain having VL CDRs 1-3 of SEQ ID NOS: 5, 6, and 7, respectively, wherein one or more CDRs have three or fewer single amino acid substitutions, deletions, or insertions; b. A variable heavy domain having VH CDRs 1-3 of SEQ ID NOs: 25, 26, and 27, respectively, and a variable light domain having VL CDRs 1-3 of SEQ ID NOs: 29, 30, and 31, respectively, wherein one or more CDRs have no more than three single amino acid substitutions, deletions, or insertions; or c. A variable heavy domain having VH CDRs 1-3 of SEQ ID NOs: 35, 36, and 37, respectively, and a variable light domain having VL CDRs 1-3 of SEQ ID NOs: 39, 40, and 41, respectively, wherein one or more CDRs have no more than three single amino acid substitutions, deletions, or insertions; A binding molecule is provided.

[0030] According to a fifth aspect of the present disclosure, there is provided a binding molecule that binds to oxidized IL-33, the binding molecule comprising a variable heavy domain having VHCDRs1-3 of SEQ ID NOs: 9, 10, and 11, respectively, and a variable light domain having VLCDRs1-3 of SEQ ID NOs: 13, 14, and 15, respectively, wherein one or more CDRs have no more than three single amino acid substitutions, deletions, or insertions.

[0031] According to a sixth aspect of the present disclosure, there is provided a binding molecule 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 no more than three single amino acid substitutions, deletions, or insertions.

[0032] In one example, any of the binding molecules defined above may be considered as a capture molecule or a probe. Preferably, each capture molecule or probe may be used together with a reporter molecule, preferably as defined below. Preferably, in the method for detecting antigenically distinct forms of IL-33 in a biological sample as defined in the second aspect.

[0033] 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, in one example, the or each binding molecule herein may suitably be used as a reporter molecule.

[0034] According to a seventh aspect of the present disclosure, there is provided a reporter molecule that binds to a reduced IL33-binding molecule complex, the reporter molecule comprising: (a) a variable heavy domain having VHCDRs1-3 of SEQ ID NOs: 1, 2, and 3, respectively, and a variable light domain having VLCDRs1-3 of SEQ ID NOs: 5, 6, and 7, respectively, wherein one or more CDRs have no more than three single amino acid substitutions, deletions, or insertions; or (b) a variable heavy domain having VHCDRs1-3 of SEQ ID NOs: 25, 26, and 27, respectively, and a variable light domain having VLCDRs1-3 of SEQ ID NOs: 29, 30, and 31, respectively, wherein one or more CDRs have no more than three single amino acid substitutions, deletions, or insertions; or (b) a variable heavy domain having VHCDRs1-3 of SEQ ID NOs: 35, 36, and 37, respectively, and a variable light domain having VLCDRs1-3 of SEQ ID NOs: 39, 40, and 41, respectively, wherein one or more CDRs have no more than three 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.

[0035] 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, the reporter molecule comprising a variable heavy domain having VHCDR1-3 of SEQ ID NOs: 9, 10, and 11, respectively, and a variable light domain having VLCDR1-3 of SEQ ID NOs: 13, 14, and 15, respectively, wherein one or more CDRs have no more than three 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.

[0036] According to an alternative seventh aspect of the present disclosure, there is provided a reporter molecule that binds to an IL-33 / sST2-binding molecule complex, the reporter molecule 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 no more than three single amino acid substitutions, deletions, or insertions. In one example, the or each reporter molecule can comprise a detectable label. In one example, the or each reporter molecule is conjugated to a detectable label.

[0037] In one example of the fourth to seventh aspects, the binding molecule and the reporter molecule are antibodies or antigen-binding fragments thereof.

[0038] In one example, a molecular pair may be provided, comprising a binding molecule selected from the fourth to sixth aspects and a reporter molecule (optionally selected from one of the seventh aspects). Preferably, the binding molecule is according to the fourth aspect and the reporter molecule is according to one of the seventh aspects. Preferably, the binding molecule is according to the fifth aspect and the reporter molecule is according to one of the seventh aspects. Preferably, 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 may be selected from the fourth to sixth aspects and the reporter molecule may be selected from commercially available reporter molecules. Preferably, any commercially available reporter molecule capable of binding to a reduced IL33-binding molecule complex, an oxidized IL33-binding molecule complex, or an IL-33 / sST2-binding molecule complex may be used. Preferably, such commercially available reporter molecules are described elsewhere herein. Alternatively, the binding molecule may be selected from commercially available binding molecules and the reporter molecule may be selected from one of the seventh aspect. Suitably, any commercially available binding molecule capable of binding to reduced IL33, oxidized IL33, or IL-33 / sST2 may be used. Suitable such commercially available binding molecules are described elsewhere herein.

[0039] Advantageously, a person skilled in the art can select a suitable pair of binding molecule and reporter molecule for detecting reduced IL-33, oxidized IL-33 or IL33 / sST2 from those described in the fourth to seventh aspects and / or from commercially available molecules.

[0040] For example, in one example, there is provided a molecular pair comprising a binding molecule according to the sixth aspect that binds to IL-33 / sST2, the binding molecule comprising a variable heavy domain having VHCDR1-3 of SEQ ID NOs: 17, 18, and 19, respectively, and a variable light domain having VLCDR1-3 of SEQ ID NOs: 21, 22, and 23, respectively, wherein one or more CDRs have three or fewer single amino acid substitutions, deletions, or insertions, and a reporter molecule capable of binding to an IL-33 / sST2-binding molecule complex, the reporter molecule optionally comprising a detectable label. The reporter molecule is preferably for use in detecting reduced IL-33 / sST2 in a biological sample.

[0041] For example, in one example, a molecular pair is provided that includes a binding molecule capable of binding to IL-33 / sST2 and a reporter molecule that includes a variable heavy domain having VHCDR1-3 of SEQ ID NOs: 17, 18, and 19, respectively, and a variable light domain having VLCDR1-3 of SEQ ID NOs: 21, 22, and 23, respectively, wherein one or more CDRs have no more than three single amino acid substitutions, deletions, or insertions, and that optionally includes a detectable label. Preferably, the molecular pair is also for use in detecting reduced IL-33 / sST2 in a biological sample.

[0042] In one example, the molecular pair is for use in detecting reduced IL-33 in a biological sample. In one example, the molecular pair is for use in detecting oxidized IL-33 in a biological sample. In one example, the molecular pair is for use in detecting reduced IL-33 / sST2 in a biological sample.

[0043] Preferably, as described above, when used in a molecular pair with a reporter molecule, the binding molecule may be considered a capture molecule or a probe.

[0044] In an eighth aspect of the present disclosure, there is provided a polynucleotide encoding the VH domain or VL domain of any of the binding molecules of the fourth, fifth or sixth aspects, or a polynucleotide encoding the VH domain or VL domain of the reporter molecule of the seventh aspect. In a ninth aspect of the present disclosure, there is provided a polynucleotide encoding any of the binding molecules of the fourth, fifth or sixth aspects, or a polynucleotide encoding the reporter molecule of the seventh aspect.

[0045] In a tenth aspect, there is provided a vector comprising one or more polynucleotides of the eighth or ninth aspect.

[0046] In an eleventh aspect, there is provided a host cell comprising one or more polynucleotides of the eighth or ninth aspect or one or more vectors of the tenth aspect. In one embodiment, the host cell may comprise a first vector comprising a polynucleotide encoding a VH domain of a binding molecule of the fourth, fifth, or sixth aspect, and a second vector comprising a polynucleotide encoding a corresponding VL domain of a binding molecule of the fourth, fifth, or sixth aspect.

[0047] In a twelfth aspect, there is provided a method of producing a binding molecule of the fourth, fifth or sixth aspect, the method comprising culturing a 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.

[0048] In a thirteenth aspect, there is provided a binding molecule produced according to the twelfth aspect.

[0049] In a fourteenth aspect, there is provided a composition comprising a binding molecule of the fourth, fifth or sixth aspect.

[0050] In a fifteenth aspect, there is provided the use of a binding molecule of the fourth, fifth or sixth aspect, or a 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.

[0051] Further features and examples of the above-defined aspects are described in the following heading sections, each of which may be combined in any compatible combination with any of the above-mentioned aspects. [Brief explanation of the drawings]

[0052] The present disclosure will now be described with reference to the following drawings: [Figure 1A] MSD assay (top) and MSD S-PLEX assay® (bottom) for recombinant protein standards of reduced and oxidized IL-33 are shown. AB1070012 was used as the capture antibody and AB1070019 was used as the detection antibody. [Figure 1B] MSD assay (top) and MSD S-PLEX assay® (bottom) for recombinant protein standards of reduced and oxidized IL-33 are shown. AB1070141 was used as the capture antibody, and AF3625 (R&D Systems) was used as the detection antibody. [Figure 1C] MSD assay (top) and MSD S-PLEX assay (bottom) for IL-33 / sST2 recombinant protein standard are shown. AB1070008 was used as the capture antibody, and MAB5232 (R&D Systems) was used as the detection antibody. [Figure 2A]Examples of the measurement of released endogenous human IL-33 forms in healthy individual and patient samples, and in an in vitro human epithelial cell culture system using the assays of the present disclosure are shown. (A) An example of the MSD S-PLEX assay® used to detect different IL-33 forms in commercially available human serum samples (n=50) from healthy individuals (n=20), asthma (GINA 2-3 severity, n=14), and COPD patients (GOLD 2-3 severity, n=16). The concentrations of IL-33 forms in the samples were extrapolated using the relevant purified recombinant protein standard curves. [Figure 2B] An example is shown using the MSD assay to measure reduced IL-33 (n=14), oxidized IL-33 (n=4), and IL-33 / sST2 (n=6) in nasal lining fluid at baseline (top) and after intranasal grass pollen allergen challenge (30 min before to 480 min after challenge) (bottom). The concentrations of IL-33 forms in the samples were extrapolated using the relevant purified recombinant protein standard curves. [Figure 2C] An example is shown using the MSD S-PLEX assay® to measure reduced and oxidized IL-33 released into culture supernatants from perfused healthy human bronchial epithelial cells (NHBE) (time course of release 1-1440 min). The concentrations of IL-33 forms in samples were extrapolated using relevant purified recombinant protein standard curves. [Figure 2D] An example is shown using the MSD assay to measure reduced and oxidized IL-33 released from human epithelial air-liquid interfaces (ALI) into culture supernatants (n=7 donors, 24 h post-treatment). The concentrations of IL-33 forms in the samples were extrapolated using relevant purified recombinant protein standard curves. [Figure 3] Showing two ACCORD recruitment periods during the pandemic. [Figure 4]1 shows the measurement of IL33-sST2 in patients in the ACCORD Phase 2a trial using an assay of the present disclosure. The number of patients treated with SoC and with SoC in combination with tozolaximab treatment who died or experienced respiratory failure by day 29 of the trial is shown for (a) overall, (b) those with low baseline levels of IL-33 / sST2 as measured by the assay, or (c) those with high baseline levels of IL-33 / sST2 as measured by the assay. [Figure 5] Both AB1070012 and AB1070069, when paired with AB1070019, show comparable performance in the MSD assay format for detecting reduced IL-33. DETAILED DESCRIPTION OF THE INVENTION

[0053] definition As used herein, "IL-33" protein refers to interleukin-33, particularly mammalian interleukin-33 protein, e.g., the human protein deposited under UniProt number 095760. However, the inventors' findings indicate that this entity is not a single species, but exists in reduced and oxidized forms. The terms "IL-33" and "IL-33 polypeptide" are used interchangeably. In certain instances, IL-33 is full-length. In other instances, 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 amino acids 72-270, 79-270, 95-270, 99-270, 107-270, 109-270, 111-270, and 112-270, may have enhanced activity (Lefrancais 2012, 2014). In another example, IL-33 can include full-length IL-33, a fragment thereof, or an IL-33 mutant or variant polypeptide, where a fragment of IL-33 or an IL-33 variant polypeptide retains some or all of the functional properties of active IL-33.

[0054] As used herein, "oxidized IL-33" or "oxIL-33" refers to a form of IL-33 that binds to RAGE and initiates RAGE-EGFR-mediated signaling. Oxidized IL-33 is a protein that appears as a distinct band, for example, by Western blot analysis under non-reducing conditions, and in particular has a mass that is 4 Da less than the corresponding reduced form. In particular, it refers to a protein that has one or two disulfide bonds between cysteines independently selected from cysteines 208, 227, 232, and 259. In one example, oxidized IL-33 does not exhibit binding to ST2.

[0055] As used herein, "reduced IL-33" or "redIL-33" refers to a form of IL-33 that binds to ST2 and initiates ST2-mediated signaling. In particular, cysteines 208, 227, 232, and 259 in the reduced form are not disulfide bonded. In one example, reduced IL-33 does not exhibit binding to RAGE.

[0056] As used herein, "IL-33 / sST2" refers to the form of reduced IL-33 bound to the soluble ST2 receptor, which form may also be referred to as a complex.

[0057] As used herein, "antigenically distinct forms of IL-33" refers to any form of IL-33 that can act as an antigen and be recognized by an antibody or a binding fragment thereof, and in the context of the present disclosure typically refers to oxidized IL-33, reduced IL-33, and reduced IL-33 / sST2 complex.

[0058] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, with no limit on the maximum number of amino acids that a protein or peptide sequence can contain. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains (commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers) and longer chains (commonly referred to in the art as proteins, of which there are many varieties). "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. A polypeptide includes natural peptides, recombinant peptides, or combinations thereof.

[0059] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, in addition to the sequence explicitly indicated. 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)).

[0060] The term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or alter 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 an antibody or antibody fragment of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0061] It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "an anti-IL-33 antibody" is understood to refer to one or more anti-IL-33 antibodies. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0062] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or treatment is desired, except where 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, dairy cows, etc.

[0063] sample The present disclosure relates to assaying a biological sample by measuring the levels of antigenically distinct forms of IL-33 present in the sample.

[0064] Preferably, the sample is a biological sample. Preferably, the sample is a body fluid sample. Alternatively, a suitable sample may include a tissue sample, such as a biopsy tissue.

[0065] Suitable bodily 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 derived from 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).

[0066] Preferably, the supernatant sample is an epithelial cell supernatant sample, preferably derived from a bronchial epithelial culture, and preferably derived from the cells or the air-liquid interface of the culture.

[0067] Preferably, different antigenically distinct forms of IL-33 may be present in different body fluids, and therefore preferably different antigenically distinct forms of IL-33 may be measured in different body fluid samples.

[0068] Preferably, oxidized IL-33 is measured in mucosal lining fluid samples or lavage samples.

[0069] Preferably, reduced IL-33 is measured in mucosal lining fluid samples or lavage samples.

[0070] Preferably, the IL-33 / sST2 complex is measured in a serum sample.

[0071] Preferably, the mucosal lining fluid sample or lavage sample contains biological cells, and preferably, these samples contain epithelial cells.

[0072] Preferably, the method of the present disclosure may further comprise the step of obtaining a biological sample. Preferably, the biological sample is obtained from the subject. Preferably, this step is performed before the contacting step (a). Alternatively, the sample may be obtained previously.

[0073] Preferably, the subject may be a healthy subject or may have a disease. Thus, preferably, the subject may be a patient. Thus, preferably, the method of the present disclosure may be performed on a biological sample obtained from a patient having a disease. Thus, preferably, the method of the present disclosure may further comprise the step of obtaining a biological sample from the patient.

[0074] Preferably, the blood sample may be obtained by drawing blood from the subject.

[0075] Preferably, the mucosal lining fluid sample may be obtained from the subject by nasal swab.

[0076] Preferably, the lavage sample may be obtained by pulmonary lavage from the subject.

[0077] Suitably, the supernatant sample may be obtained by culturing cells from the subject, suitably by culturing epithelial cells isolated from the epithelium of the subject, suitably isolated from the respiratory epithelium of the subject.

[0078] Preferably, the sample represents cytokine levels for the subject on whom the method is being performed. Preferably, the sample represents interleukin levels for the subject on whom the method is being performed. Preferably, the sample represents IL-33 levels for the subject on whom the method is being performed.

[0079] Preferably, the sample is a mucosal lining fluid sample from the respiratory tract, preferably from the epithelium of the respiratory tract. Preferably, it may be isolated from the upper or lower respiratory tract, preferably from the epithelium of the upper or lower respiratory tract. Preferably, it is isolated from the upper respiratory tract, preferably from the epithelium of the upper respiratory tract. Preferably, the sample is nasal lining fluid, preferably nasal lining fluid isolated from the epithelium of the nasal canal.

[0080] Nasal 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 readily available. In addition, obtaining such samples involves much less risk and discomfort than samples such as lung tissue samples.

[0081] The sample may be processed to enrich for antigenically distinct forms of IL-33. Suitable techniques for such enrichment can be determined based on the nature of the sample. Generally, examples of suitable techniques (e.g., techniques for isolating biological molecules such as cytokines from a sample) are well known to those skilled in the art.

[0082] subject The methods of the present disclosure can be performed on a sample obtained from a subject.

[0083] Preferably, the subject may be a human. The subject may be receiving or seeking medical care. Preferably, the subject is male or female. Preferably, the subject is an adult or a child.

[0084] As explained above, the subject may be healthy or diseased. Preferably, the subject may be a patient.

[0085] Preferably, in the methods of the present disclosure, a suitable subject may be one suspected of having an abnormal level of an antigenically distinct form of IL-33. Preferably, the subject may be one believed to have a physiological condition or disease associated with an abnormal level of an antigenically distinct form of IL-33, or a subject whose prognosis or diagnosis of a condition or disease depends on the level of one or more antigenically distinct forms of IL-33. Preferably, the abnormal level of an antigenically distinct form of IL-33 may be the result or cause of the disease or condition.

[0086] Preferably, the subject may exhibit symptoms of such a condition or disease.

[0087] Alternatively, a suitable subject in the context of the disclosed methods may be a subject who is considered to be at risk of developing such a condition or disease, for example, such a subject may have come into contact with an individual suffering from such a disease or condition, may be suffering from a related condition, or may meet risk factors associated with the condition, such as smoking, advanced age, allergies, etc.

[0088] contact The methods of the disclosure involve contacting a biological sample with one or more binding molecules capable of specifically binding to one or more antigenically distinct forms of IL-33 selected from reduced IL-33, oxidized IL-33, and / or IL-33 / sST2 under conditions sufficient to form a complex.

[0089] Suitably, the methods of the disclosure involve contacting a biological sample with one or more binding molecules capable of specifically binding to one or more antigenically distinct forms of IL-33 selected from reduced IL-33, oxidized IL-33, and / or IL-33 / sST2.

[0090] Suitably, "specifically binds" means that each binding molecule is capable of binding to a specific form of IL-33.

[0091] Preferably, the disclosed method may also include contacting the complex with one or more reporter molecules capable of binding to one or more complexes. In such a case, the detecting step (b) includes detecting the level of bound reporter molecules in the sample. In such a case, preferably, the method further includes contacting the biological sample with one or more reporter molecules capable of binding to one or more complexes therein, preferably for a time sufficient to allow the reporter molecules to bind to one or more complexes.

[0092] Preferably, the method of the present disclosure may also comprise contacting the complexes with one or more reporter molecules capable of specifically binding to the one or more complexes. Preferably, contacting the complexes with the one or more reporter molecules may comprise contacting a biological sample with the one or more reporter molecules.

[0093] Suitably, "specifically bind" means that each reporter molecule is capable of binding to a particular form of the IL-33-binding molecule complex.

[0094] Suitable binding molecules and reporter molecules are defined elsewhere herein.

[0095] Optionally, the method 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 distinct forms of IL-33, which further step comprises immobilizing the antigenically distinct isoforms of IL-33 by contacting the biological sample with a solid surface under conditions suitable for binding of the antigenically distinct isoforms of IL-33 to the solid surface, preferably by adsorption or covalent binding. Preferably, the solid surface may comprise a plate, such as a microtiter plate or a multiwell plate, a well, a slide, such as a glass slide, paper, such as filter paper, a cuvette, or the like. Preferably, the solid surface is contained within a container in which the method is performed. Thus, preferably, a solid surface comprising antigenically distinct isoforms of IL-33 is produced. Preferably, in such a case, the binding molecule is then contacted with the solid surface comprising the antigenically distinct isoforms of IL-33 for a time sufficient to form a complex.

[0096] Preferably, the binding molecule is contacted with the biological sample for a time sufficient to form complexes. Preferably, the reporter molecule is contacted with the biological sample for a time sufficient to bind to one or more complexes.

[0097] Preferably, this time may be referred to as the incubation time. Preferably, the incubation time varies depending on the binding molecule and reporter molecule used, and the biological sample.

[0098] Preferably, a sufficient incubation time may be from 5 minutes to 180 minutes, preferably from 10 minutes to 150 minutes, preferably from 20 minutes to 120 minutes, preferably from 30 minutes to 100 minutes, preferably from 45 minutes to 90 minutes, preferably about 60 minutes.

[0099] Preferably, the 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. Preferably, the one or more binding molecules are contacted with the biological sample for about 9 minutes.

[0100] Preferably, the one or more reporter molecules are in contact 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. Preferably, the one or more reporter molecules are in contact with the complex / biological sample and are in contact with the biological sample for about 9 minutes.

[0101] 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.

[0102] Suitably, the biological sample may be contacted with the binding molecule or reporter molecule in any suitable manner using known assay techniques.

[0103] The sample may be contacted with one or more binding molecules by use of a solid phase support or carrier. Suitably, either the sample or the binding molecules may be immobilized on the support or carrier.

[0104] Preferably, the support may then be washed. Preferably, the support is washed with a suitable buffer solution. Preferably, the method of the present disclosure may include one or more washing steps. Preferably, washing is performed after the addition of one or more binding molecules and before the addition of one or more reporter molecules. Preferably, washing is performed after the addition of one or more reporter molecules and before the measurement step.

[0105] Preferably, at least one washing step is carried out after step (a) and preferably before step (b).

[0106] Preferably, the support may be contacted with one or more reporter molecules after step (a). Thus, preferably, the step of contacting the complex with one or more reporter molecules capable of binding to the one or more complexes may comprise contacting the complex immobilized on the support with one or more reporter molecules capable of binding to the one or more complexes. Preferably, the support may then be washed a second time to remove unbound reporter molecules. Thus, preferably, a second washing step may be performed after contacting the complex with one or more reporter molecules.

[0107] Optionally, the binding molecule or reporter molecule may comprise a detectable label or may be subsequently labeled. Suitable labels are discussed elsewhere herein. The amount of bound label that may be bound on the solid support may then be detected by conventional means, preferably as described in step (b) of the methods of the present disclosure.

[0108] Preferably, in some embodiments where the assay is an immunoassay, the binding molecule may be biotinylated. Preferably, in some embodiments where the assay is an immunoassay, the reporter molecule may be labeled with a chemiluminescent compound.

[0109] "Solid phase 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 celluloses, polyacrylamide, and magnetite. The nature of the support, for purposes of this disclosure, can be soluble to some extent or insoluble. The support material can have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the shape of the support can be spherical, such as a bead, or cylindrical, such as the interior surface of a test tube or the exterior surface of a rod. Alternatively, the surface can be flat, such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those of skill in the art will know of many other suitable carriers for binding antibodies or antigens, or will be able to identify them by routine experimentation.

[0110] detection The methods of the disclosure involve detecting the level of one or more of reduced IL-33, oxidized IL-33, and / or IL-33 / sST2 complex in a biological sample.

[0111] Preferably, detecting the level of one or more of 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. Preferably, such complexes are formed in step (a) from 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.

[0112] Suitably, the complexes detected in step (b) correspond to the binding molecules used in the method, and thus suitably, when the method is carried out using only oxidized IL-33 binding molecules, only oxidized IL-33 complexes are detected in step (b).

[0113] Suitably, detection of one or more of reduced IL-33, oxidized IL-33, and / or IL-33 / sST2 complex in a sample may be carried out by any suitable assay technique known in the art.

[0114] Preferably, as described above, detecting one or more of 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, preferably detecting the level of one or more reporter molecules bound to one or more of reduced IL-33, oxidized IL-33, and / or IL-33 / sST2 complexes in the sample.

[0115] Suitably, the reporter molecules are detected by detection of 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.

[0116] Alternatively, 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 binding molecules in the sample. In such cases, preferably the binding molecules are detected directly by detecting a detectable label associated with each binding molecule. Preferably, in such cases, a reporter molecule is not required. Preferably, the detectable label may be part of the binding molecule, or may be added separately, preferably after the binding molecule has bound to one or more reduced IL-33, oxidized IL-33, and / or IL-33 / sST2 complexes in the sample.

[0117] Suitable detectable labels are discussed elsewhere herein.

[0118] Therefore, the method of the present disclosure may preferably further comprise the step of adding a detectable label to the sample. Preferably, step (b) may comprise detecting the detectable label associated with one or more reporter molecules.

[0119] Alternatively, step (b) may comprise detecting a detectable label associated with one or more binding molecules.

[0120] In one example, the reporter molecule includes a detectable label conjugated thereto, in which case the reporter molecule is detected by detection of the conjugated detectable label.

[0121] In one example, the binding molecule includes a detectable label conjugated thereto, in which case the binding molecule is detected by detection of the conjugated detectable label.

[0122] Preferably, the assays used are highly sensitive, and thus the methods of the present disclosure are highly sensitive: preferably, the methods of the present disclosure are capable of detecting antigenically distinct forms of IL-33 in a sample at sub-microgram / ml, sub-nanogram / ml, sub-picogram / ml, or sub-femtogram / ml concentrations.

[0123] Preferably, the detection limit is a concentration of microgram / ml or less, nanogram / ml or less, picogram / ml or less, or femtogram / ml or less. Preferably, the detection limit is a concentration of femtogram / ml.

[0124] In one example, the method is capable of detecting antigenically distinct forms of IL-33 in a sample at concentrations of at least femtograms / ml.

[0125] Assay Suitably, the methods of the present disclosure may involve detecting one or more antigenically distinct forms of IL-33 in a biological sample in a single assay.

[0126] Alternatively, the method of the present disclosure may comprise detecting one or more antigenically distinct forms of IL-33 in a biological sample in two or more assays. Thus, the method of the present disclosure may also preferably comprise two or more biological samples. Thus, the method of the present disclosure may preferably comprise two or more steps within step (a) and two or more steps within step (b).

[0127] Suitably, the methods of the present disclosure may comprise separately contacting one or more biological samples with one or more binding molecules capable of binding to reduced IL-33, oxidized IL-33, and / or IL-33 / sST2.

[0128] Suitably, step (a) of the method of the present disclosure may comprise: (ai) contacting a first sample with a binding molecule capable of binding to oxidized IL-33; (aii) contacting a second sample with a binding molecule capable of binding to reduced IL-33; and / or (aiii) contacting a third sample with a binding molecule capable of binding to IL-33 / sST2. Suitably, the first, second and third samples may be the same or different samples.

[0129] Suitably, the methods of the present disclosure may also comprise separately detecting the levels of one or more of reduced IL-33, oxidized IL-33, and / or IL-33 / sST2 complex in one or more samples.

[0130] Suitably, step (b) of the method of the present disclosure may comprise (bi) detecting the level of oxidized IL-33-binding molecule complexes in a first sample, (bii) detecting the level of reduced IL-33-binding molecule complexes in a second sample, and / or (biii) detecting the level of IL-33 / sST2-binding molecule complexes in a third sample. Suitably, the first, second or third sample may be the same or different samples.

[0131] As noted above, different antigenically distinct forms of IL-33 may be present in the same sample or in different samples and therefore suitably different antigenically distinct forms of IL-33 may require different assays for different samples, and therefore suitably the method may comprise one or more assays.

[0132] Suitably, step (a) of the methods of the present disclosure may comprise: (ai) contacting the mucosal lining fluid or lung lavage sample with an oxidized IL-33 binding molecule; (aii) contacting the mucosal lining fluid or lung lavage sample with a reduced IL-33 binding molecule; and / or (aiii) contacting the serum sample with an IL-33 / sST2 binding molecule.

[0133] Suitably, any of the antigenically distinct forms of IL-33 may be detected in the same sample or in any combination of different samples.

[0134] For example, the disclosed method may suitably comprise (bi) detecting the level of oxidized IL-33-binding molecule complexes and the level of reduced IL-33-binding molecule complexes in a first sample, and / or (bii) detecting the level of IL-33 / sST2-binding molecule complexes in a second sample. For example, the disclosed method may suitably comprise (bi) detecting the level of oxidized IL-33-binding molecule complexes and the level of reduced IL-33-binding molecule complexes in a mucosal lining fluid or lung lavage sample, and / or (bii) detecting the level of IL-33 / sST2-binding molecule complexes in a serum sample.

[0135] Prior to step (a), the method of the present disclosure may further comprise isolating one or more antigenically distinct forms of IL-33 from the biological sample. Preferably, the isolating step may comprise isolation by sample fractionation. Suitable isolation techniques are known in the art and may include chromatography, centrifugation, affinity purification, filtration, etc. Preferably, the isolating step may comprise immobilizing one or more antigenically distinct forms of IL-33 on a solid surface, as described elsewhere herein.

[0136] Examples of suitable samples are described elsewhere herein. It will be appreciated, then, that the nature of the sample may determine the nature of the assay techniques that may be used in practicing the methods of the present disclosure.

[0137] The various forms of IL-33 are all proteins, therefore preferably the assay technique is a protein assay technique.

[0138] Protein levels can be determined by a variety of assay techniques, such as S-plex, ELISA, ECLIA, CLIA, radioimmunoassay, immunoprecipitation, Western blot and mass spectrometry.

[0139] Suitably, the level of one or more antigenically distinct forms of IL-33 is determined by immunoassay.

[0140] Immunoassays typically 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 include: ELISA (enzyme-linked immunosorbent assay), ECLIA (enzyme-based electrochemiluminescence immunoassay), e.g., Roche's Elecsys®, S-plex, CLIA (chemiluminescence immunoassay), Western blotting, immunocytochemistry, immunoprecipitation, affinity chromatography, Biolayer Interferometry (Octet, ForteBio), and biochemical assays such as dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA®, Perkin Elmer), Förster resonance energy transfer (FRET) assays (e.g., homogeneous time-resolved fluorescence (HTRF®, Cis Biointernational), and radioimmuno / radioligand binding assays.

[0141] Western blot analysis generally involves preparing a protein sample, electrophoresing the protein sample in a polyacrylamide gel (e.g., 8-20 SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF, or nylon, blocking the membrane in a blocking solution (e.g., PBS containing 3% BSA or nonfat milk), washing the membrane in a wash buffer (e.g., PBS-Tween 20), blocking the membrane with a primary antibody (antibody of interest) diluted in the blocking buffer, washing the membrane in the wash buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an 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 the blocking buffer, washing the membrane in the wash buffer, and detecting the presence of the antigen. Those skilled in the art will be familiar with parameters that can be modified to increase the detected signal and reduce background noise. For further discussion of Western blot protocols, see, e.g., Ausubel et al., eds. (1994) Current Protocols in Molecular Biology (John Wiley & Sons, Inc., NY) Vol. 1 at 10.8.1.

[0142] ELISA involves 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, incubating for a certain period of time, and detecting the presence of the antigen. In ELISA, the antibody does not need to be conjugated to a detectable compound; instead, a second antibody (that recognizes the first antibody) conjugated to a detectable compound may be added to the well. Both the first and second antibodies bind to the antigen, forming a "sandwich." Furthermore, instead of coating the wells with the antigen, an antibody may be coated to the well. In this case, the second antibody conjugated to a detectable compound may be added after the antigen of interest has been added to the coated well. Those skilled in the art will be familiar with parameters that can be modified to increase the signal detected, as well as other variations of ELISA known in the art. For further discussion of ELISA, see, e.g., Ausubel et al., eds. (1994) Current Protocols in Molecular Biology (John Wiley & Sons, Inc., NY) Vol. 1 at 13.2.1.

[0143] Preferably, the level of one or more antigenically distinct forms of IL-33 is determined by immunoassay, preferably by using a modified ELISA called the S-plex assay, which is available from Meso Scale Diagnostics LLC, along with suitable instructions for use.

[0144] Alternatively, the level of one or more antigenically distinct forms of IL-33 is determined by ECLIA. Preferably, the ECLIA assay involves contacting a sample containing an antigen with a first antibody specific to the antigen conjugated to biotin and incubating the sample for a period of time to allow the first antibody to bind to the antigen. A second antibody specific to the antigen but conjugated to a labeled molecule is then added. Typically, the labeled molecule is a ruthenium compound, preferably ruthenium bipyridine. Both the first and second antibodies bind to the antigen, forming 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 on the first antibody to form an antibody complex. Preferably, the beads or microparticles are magnetic. The sample is then passed over an electrode, to which the antibody complex binds via the streptavidin beads or microparticles. After washing away unbound antibody with a wash buffer such as ProCell, a reactive agent is added and a current is passed through the electrodes to induce chemiluminescence of the labeled molecule. Preferably, the reactive agent is an amine, such as tripropylamine or dibutylethanolamine. The level of chemiluminescence is measured with 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 Elecsys® assay available from Roche, which can be preferably performed on a Cobas e immunoassay analyzer.

[0145] 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, preferably using one or more of the binding molecules and / or reporter molecules described herein.

[0146] Thus, 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: (a) contacting the sample with one or more binding molecules each capable of specifically binding to an antigenically distinct form of IL-33 selected from reduced IL-33, oxidized IL-33, and / or IL-33 / sST2 under conditions sufficient to form a complex, wherein the binding molecules each comprise a tag; (b) contacting the sample with one or more reporter molecules each capable of binding to the complexes produced in (a), under conditions sufficient to form a sandwich complex, wherein the reporter molecules each comprise a label; (c) contacting the sample with one or more capture partners, each capable of binding to a tag included in each binding molecule, under conditions sufficient to form a captured sandwich complex; (d) detecting the amount of captured sandwich complex using the label; A method is provided that includes:

[0147] Suitably, the binding molecule and reporter molecule are as defined elsewhere herein.

[0148] Preferably, the tag is biotin. Therefore, preferably, the capture partner is streptavidin. However, preferably, other known tags and capture partners can be used. Preferably, the capture molecule can be attached to a support, preferably a solid support, preferably a bead or a microparticle. Therefore, preferably, step (c) comprises contacting the sample with one or more capture beads containing streptavidin, each capable of binding to the biotin tag contained in each binding molecule. Therefore, preferably, step (c) produces beads containing one or more sandwich complexes.

[0149] Preferably, the beads are formed from a magnetic material, preferably a magnetic metal such as iron, nickel or cobalt. Thus, preferably, step (c) comprises contacting the sample with one or more magnetic capture beads comprising, preferably coated with, streptavidin, each capable of binding to a biotin tag contained on each binding molecule.

[0150] Preferably the label is a fluorescent or luminescent label, or a chemiluminescent label as specified above. Preferably the label is a chemiluminescent label, and preferably the label is a ruthenium compound, preferably ruthenium pyridine.

[0151] Preferably, detecting the captured sandwich complex comprises detecting the label, preferably detecting the level of luminescence produced by the label. Preferably, the level of luminescence corresponds to the level of antigen present in the sample, preferably the level of an antigenically distinct form of IL-33 selected from reduced IL-33, oxidized IL-33, and / or IL-33 / sST2, in the sample.

[0152] Preferably, chemiluminescent labels require activation to emit light and be detected. Therefore, preferably, the method includes a step of activating the label attached to each reporter molecule in the captured sandwich complex, preferably before the detection step. Preferably, the activation step includes contacting the label with a stimulus capable of activating the label. Suitable stimuli include a chemical reactant and / or a current / voltage. Preferably, in the case of a chemiluminescent label, the activation step includes both a reactant and a current / voltage, preferably in close proximity. Therefore, preferably, the method may include a step of contacting the captured sandwich complex with a reactant. Preferably, the reactant may be an electron donor, such as an amine compound, e.g., tripropylamine or dibutylethanolamine. Preferably, the step may further include exposing the captured sandwich complex to a current / voltage, preferably simultaneously with contacting with the reactant. Therefore, preferably, the method includes a step of contacting the captured sandwich complex with a reactant and passing a current through an electrode to activate the chemiluminescent label on each reporter molecule.

[0153] Preferably, the method may include immobilizing the captured sandwich complexes to ensure activation of the chemiluminescent label. Preferably, they are immobilized on a surface. In one embodiment, this can be achieved by using magnetic beads, as described above, in combination with a metal surface. Preferably, the method may include binding or immobilizing the captured sandwich complexes to a magnetic surface, preferably via the magnetic beads. Preferably, the surface is also capable of receiving a current / voltage. Thus, preferably, the surface is an electrode, preferably a magnetic electrode. Thus, in one embodiment, the method includes immobilizing the captured sandwich complexes to a magnetic electrode, preferably via the magnetic beads of the captured sandwich complexes. Preferably, this step is performed before the step of activating the chemiluminescent label. Preferably, this step is performed before contacting the captured sandwich complexes with a reactant and applying a current to the electrode.

[0154] Preferably, upon activation, the label on each reporter molecule of each captured sandwich complex can be detected. Preferably, in the case of a chemiluminescent label, the luminescence can be detected. Preferably, the detection of such luminescence can be carried out using any known technique, for example, by using a photomultiplier. Preferably, the level of luminescence detected corresponds to the amount of captured sandwich complex, which preferably corresponds to the level of antigen present in the sample. In the context of the present invention, preferably, the level of luminescence corresponds to the level of reduced IL-33, oxidized IL-33, and / or IL-33 / sST2 in the sample.

[0155] Thus, 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: (a) contacting the sample with one or more binding molecules each capable of specifically binding to an antigenically distinct form of IL-33 selected from reduced IL-33, oxidized IL-33, and / or IL-33 / sST2, under conditions sufficient to form a complex, wherein the binding molecules each comprise a biotin tag; (b) contacting the sample with one or more reporter molecules each capable of binding to the complexes produced in (a) under conditions sufficient to form sandwich complexes, wherein the reporter molecules each comprise a chemiluminescent ruthenium label; (c) contacting the sample with one or more streptavidin-coated magnetic capture beads each capable of binding to a biotin tag contained in each binding molecule under conditions sufficient to form a captured sandwich complex; (d) immobilizing the captured sandwich complex on a magnetic electrode via magnetic capture beads; (e) contacting the captured sandwich complex with a reactive agent and applying a current to the electrode to activate the chemiluminescent label on each reporter molecule; (d) detecting the level of chemiluminescence, thereby detecting one or more antigenically distinct forms of IL-33 in the sample; A method is provided that includes:

[0156] Immunoassays for measuring antigenically distinct forms of IL-33 typically involve incubating a sample in the presence of one or more detectably labeled molecules capable of specifically binding to each of the antigenically distinct forms of IL-33 of interest or peptide fragments thereof, and detecting the bound molecules by any of several 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.

[0157] Binding molecules and reporter molecules The present disclosure relates to the use of binding molecules and reporter molecules to detect antigenically distinct forms of IL-33 in biological samples.

[0158] Although many techniques are known in the art for measuring and detecting proteins such as IL-33 in a sample, preferably the methods of the present disclosure are carried out by immunoassay as described above.

[0159] 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 one or more of the complexes formed.

[0160] The term "binding molecule" as used herein refers in its broadest sense to a molecule that specifically binds to an antigenic determinant. Preferably, one or more binding molecules specifically bind to IL-33, particularly reduced IL-33, oxidized IL-33, or IL-33 / sST2. In some examples, binding molecules of the present disclosure may be considered capture molecules or probes.

[0161] In the present disclosure, the term "reporter molecule" refers in its broadest sense to a molecule capable of specifically binding to and indicating an antigenic determinant. Preferably, one or more reporter molecules specifically bind to a complex of IL-33 and a binding molecule, particularly a reduced IL-33-binding molecule complex, an oxidized IL-33-binding molecule complex, or an IL-33 / sST2-binding molecule complex.

[0162] Alternatively, the reporter molecule and the binding molecule are the same entity. In such cases, the binding molecule can directly bind and indicate an antigenic determinant (e.g., reduced IL-33, oxidized IL-33, or IL-33 / sST2).

[0163] Suitably, the molecules described herein, suitably the antibodies and antigen-binding fragments described herein, may be used as binding molecules or reporter molecules, respectively, suitably as described in the fourth to seventh aspects.

[0164] Preferably, one or more reporter molecules are capable of indicating their presence and / or binding. Preferably, one or more reporter molecules are labelled. Preferably, one or more reporter molecules are detectably labelled.

[0165] Alternatively, one or more binding molecules can indicate their presence and / or binding. Preferably, in such cases, one or more binding molecules are labeled. Preferably, one or more binding molecules are detectably labeled.

[0166] Suitable detectable labels are known in the art and may include radioactive labels, fluorescent labels, enzymes, and chromophores. Preferably, the label may be detectable only upon stimulation. The suitable stimulation source varies depending on the label used; for example, when using an enzyme label, the stimulation source may be a substrate, and when using a chromophore label, the stimulation source may be radiation of a specific wavelength. Such a stimulation source may be considered a stimulating agent.

[0167] Suitable fluorescent labels may include rhodamine, fluorescein, Cy5, ruthenium compounds such as pyridine complexes, diimine complexes, and phosphorescent porphyrin dyes.

[0168] Suitable enzyme labels may include peroxidase, glucose oxidase, alkaline phosphatase (AP), β-galactosidase, catalase, or luciferase. Substrates used with particular enzymes are generally selected to produce a detectable color change upon hydrolysis 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 commonly used. Preferably, fluorogenic substrates that produce a fluorescent product may be used.

[0169] Preferably, in some embodiments where the assay is an immunoassay, the binding molecule may be biotinylated. Preferably, in some embodiments where the assay is an immunoassay, the reporter molecule may be labelled with a chemiluminescent compound, preferably a ruthenium compound, preferably ruthenium bipyridine.

[0170] Thus, in a further aspect the present invention may provide a binding molecule according to any of the fourth to sixth aspects, wherein the binding molecule is biotinylated.

[0171] Thus, in a further aspect of the invention there may be provided a reporter molecule according to the seventh aspect, wherein the reporter molecule is ruthenylated.

[0172] Alternatively, the binding molecules or reporter molecules may be unlabeled. In such instances, the binding molecules or reporter molecules may be attached by a label, preferably a detectable label. Preferably, the detectable label is capable of binding to and indicating the binding molecule or reporter molecule. Preferably, the detectable label is specific for the corresponding binding molecule or reporter molecule.

[0173] Preferably, the methods of the present disclosure may utilize a detectable label of an oxidized IL-33 complex, a detectable label of a reduced IL-33 complex, and / or a detectable label of an IL-33 / sST2 complex. Preferably, such one or more detectable labels may be added to the assay methods of the present disclosure after the one or more reporter molecules. Preferably, the detecting step (b) of the method may comprise detecting one or more bound detectable labels.

[0174] Alternatively, the methods of the present disclosure may utilize a detectable label for oxidized IL-33, and / or a detectable label for reduced IL-33, and / or a detectable label for IL-33 / sST2. Preferably, such one or more detectable labels may be added to the assay methods of the present disclosure after the one or more binding molecules. Preferably, detecting step (b) of the method may comprise detecting one or more bound detectable labels.

[0175] In one example, one or more reporter molecules are detectably labeled. In one example, one or more reporter molecules include a detectable label conjugated thereto. In one example, one or more reporter molecules are sulfo-tagged.

[0176] Suitably, the method involves 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 several complexes.

[0177] Suitably, the method comprises 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.

[0178] Suitably, the method comprises 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.

[0179] Suitably, the method comprises 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.

[0180] Suitably, the method comprises 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.

[0181] Preferably, each binding molecule has a corresponding reporter molecule, and preferably, each reporter molecule may comprise a corresponding detectable label.

[0182] Preferably, the reporter molecule and the binding molecule are combined as a pair, for example, if 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.

[0183] 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 CDR or light chain CDR of a reference antibody molecule, and at least six CDRs from one or more reference antibody molecules.

[0184] Preferably, one or more of the binding or reporter molecules is an antibody or binding fragment thereof.

[0185] Preferably, one or more of the binding molecules is an anti-IL-33 antibody or a binding fragment thereof. Preferably, at least one of the binding molecules is an anti-oxidized IL-33 antibody or a binding fragment thereof. Preferably, at least one of the binding molecules is an anti-reduced IL-33 antibody or a binding fragment thereof. Preferably, at least one of the binding molecules is an anti-IL-33 / sST2 antibody or a binding fragment thereof.

[0186] Preferably, at least one of the reporter molecules is an anti-reduced IL-33-binding molecule complex antibody or a binding fragment thereof. Preferably, at least one of the reporter molecules is an anti-oxidized IL-33-binding molecule complex antibody or a binding fragment thereof. Preferably, at least one of the reporter molecules is an anti-IL-33 / sST2-binding molecule complex antibody or a binding fragment thereof.

[0187] As used herein, "antibody" refers to immunoglobulin molecules, as discussed in more detail below, particularly full length antibodies or molecules comprising full length antibodies, such as DVD-Ig molecules.

[0188] "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, in particular comprising six CDRs, e.g., three CDRs of the heavy chain variable region and three CDRs of the light chain variable region.

[0189] Preferably, one or more of the binding or reporter molecules is an antibody or antigen-binding fragment thereof.

[0190] The term "antibody or antigen-binding fragment thereof" encompasses any antibody or antigen-binding fragment thereof that competes with an antibody of the invention for specific binding to an antigen. Thus, preferably, the binding molecules or reporter molecules referred to herein also encompass molecules that bind to the relevant antigen in competition with the binding molecule or reporter molecule. Thus, preferably, the binding molecules or reporter molecules referred to herein also encompass molecules that specifically bind to reduced IL-33, oxidized IL-33, or IL-33 / sST2 in competition with the binding molecule or reporter molecule. Such competing antibodies or antigen-binding fragments can be identified in competitive binding assays well known in the art. Preferably, the competing antibodies or antigen-binding fragments substantially inhibit the adhesion of a receptor to its cognate receptor, such as the binding of reduced IL-33 to the ST2 receptor. Preferably, excess competing antibodies or antigen-binding fragments reduce the amount of receptor bound to its cognate receptor by at least about 20%, 40%, 60%, or 80%, more usually by more than about 85%, as measured in an in vitro competitive binding assay.

[0191] As used herein, the terms "complementarity determining region" and "CDR" refer to the amino acid residues of an antibody or antigen-binding fragment which are responsible for antigen binding.

[0192] Preferably, the antibody or binding fragment thereof is selected from natural, polyclonal, monoclonal, multispecific, murine, human, humanized, primatized, or chimeric. Preferably, the antibody or binding fragment thereof is an epitope-binding fragment, such as Fab' and F(ab')2, Fd, Fv, single-chain Fv (scFv), disulfide-linked Fv (sdFv), a fragment comprising either the VL domain or the VH domain, or a fragment produced by a Fab expression library. Preferably, the antibody or binding fragment thereof is a minibody, diabody, triabody, tetrabody, or single-chain antibody. Preferably, the antibody or binding fragment thereof 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.

[0193] The phrases "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to polypeptides, including antibodies, bispecific antibodies, etc., having substantially identical amino acid sequences or that are derived from the same genetic source. The terms also include preparations of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0194] 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, IgA2, etc.), or subclass of immunoglobulin molecule.

[0195] Preferably, the or each antibody or binding fragment thereof used as binding molecule is a capture antibody.

[0196] Suitably the or each antibody or binding fragment thereof used as a reporter molecule is a probe antibody.

[0197] Preferably, as described above, the binding molecules and reporter molecules are paired together so that each capture antibody has a corresponding probe antibody. Preferably, the capture antibodies are paired with the probe antibodies.

[0198] Thus, preferably, one or more of the binding molecules is an anti-IL-33 capture antibody. Preferably, at least one of the binding molecules is an anti-oxidized IL-33 capture antibody. Preferably, at least one of the binding molecules is an anti-reduced IL-33 capture antibody. Preferably, at least one of the binding molecules is an anti-IL-33 / sST2 capture antibody.

[0199] Preferably, at least one of the reporter molecules is an anti-reduced IL-33 probe antibody. Preferably, at least one of the reporter molecules is an antioxidant IL-33 probe antibody. Preferably, at least one of the reporter molecules is an anti-IL-33 / sST2 probe antibody or a binding fragment thereof.

[0200] Preferably, the contacting step of the method 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, and preferably, the detecting step comprises the use of one or more probe antibodies capable of binding to one or more complexes.

[0201] Preferably, the one or more binding molecules comprise a specific sequence that enables the binding molecule to one of oxidized IL-33, reduced IL-33, or IL-33 / sST2. Preferably, the one or more reporter molecules comprise a specific sequence that enables the binding to one of oxidized IL-33-binding molecule complexes, reduced IL-33-binding molecule complexes, or IL-33 / sST2-binding molecule complexes.

[0202] Suitable binding molecules and reporter molecules with such capabilities, eg antibodies, are available in the art or can be ordered from reagent companies such as R&D systems.

[0203] Preferably, however, the binding molecules and / or reporter molecules used in the methods and kits of the present disclosure comprise the following sequences:

[0204] Reduced IL-33 binding molecule - AB1070012 Preferably, a binding molecule that binds reduced IL-33 comprises a heavy chain variable region or domain (VH) and a light chain variable region or domain (VL). Preferably, a binding molecule that binds reduced IL-33 comprises three CDRs in the heavy chain variable region or domain (VH) and three CDRs in the light chain variable region or domain (VL). Preferably, a binding molecule that binds reduced IL-33 may comprise three CDRs, for example, three CDRs in the heavy chain variable region set forth in SEQ ID NO: 4 and / or three CDRs in the light chain variable region set forth in SEQ ID NO: 8.

[0205] Suitably, the binding molecule that binds reduced IL-33 comprises a heavy chain variable region (VH) comprising three CDRs having an amino acid sequence at least 85%, at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VH set forth in SEQ ID NO:4, and / or a light chain variable region (VL) comprising three CDRs having an amino acid sequence at least 85%, at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VL set forth in SEQ ID NO:8.

[0206] Preferably, a binding molecule that binds reduced IL-33 may comprise a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises VHCDRs 1 to 3 of SEQ ID NOs: 1, 2, and 3, and one or more VHCDRs have no more than three single amino acid substitutions, deletions, or insertions. Preferably, such substitutions may be conservative substitutions.

[0207] Suitably, one or more VHCDRs have three, two, one or none single amino acid substitutions, deletions or insertions. Suitably, the VHCDRs consist of SEQ ID NOs: 1, 2 and 3.

[0208] Preferably, a binding molecule that binds reduced IL-33 may comprise a variable heavy domain (VH) and a variable light domain (VL), wherein the VL comprises VLCDRs 1 to 3 of SEQ ID NOs: 5, 6, and 7, and one or more VLCDRs have no more than three single amino acid substitutions, deletions, or insertions. Preferably, such substitutions may be conservative substitutions.

[0209] Preferably, one or more VLCDRs have three, two, one or none single amino acid substitutions, deletions or insertions. Preferably, the VLCDRs consist of SEQ ID NOs: 5, 6, and 7.

[0210] Preferably, a binding molecule that binds reduced IL-33 may comprise a VH having VH CDRs 1-3 of SEQ ID NOs: 1, 2, and 3, and a VL having VL CDRs of SEQ ID NOs: 5, 6, and 7, wherein one or more of the CDRs have three or fewer single amino acid substitutions, deletions, or insertions. Preferably, one or more of the CDRs have three, two, one, or none single amino acid substitutions, deletions, or insertions. Preferably, such substitutions may be conservative substitutions.

[0211] Preferably, a binding molecule that binds to reduced IL-33 may comprise a VH having VHCDR1 to 3 of SEQ ID NOs: 1, 2, and 3, and a VL having VLCDRs of SEQ ID NOs: 5, 6, and 7. Preferably, a binding molecule that binds to reduced IL-33 may comprise a VH having VHCDR1 to 3 of SEQ ID NOs: 1, 2, and 3, and a VL having VLCDRs of SEQ ID NOs: 5, 6, and 7.

[0212] Suitably, a binding molecule that binds reduced IL-33 may comprise a CDR selected from one or more of: a VHCDR1 having the sequence of SEQ ID NO: 1, a VHCDR2 having the sequence of SEQ ID NO: 2, a VHCDR3 having the sequence of SEQ ID NO: 3, a VLCDR1 having the sequence of SEQ ID NO: 5, a VLCDR2 having the sequence of SEQ ID NO: 6, and a VLCDR3 having the sequence of SEQ ID NO: 7.

[0213] Suitably, a binding molecule that binds 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.

[0214] Thus, preferably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence at least 85%, at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VH set forth in SEQ ID NO:4.

[0215] Thus, preferably, the binding molecule comprises a VH as disclosed above, wherein one, two, three or four amino acids in the framework have been deleted, inserted and / or independently replaced with different amino acids, i.e., a substituted sequence. Preferably, such substitutions may be conservative substitutions.

[0216] Thus, preferably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VH set forth in SEQ ID NO: 4, and comprises VHCDRs 1 to 3 of SEQ ID NOs: 1, 2, and 3, wherein one or more of the VHCDRs have three or fewer single amino acid substitutions, deletions, or insertions. Preferably, the one or more VHCDRs have three, two, one, or none of the VHCDRs have a single amino acid substitution, deletion, or insertion.

[0217] Therefore, preferably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VH set forth in SEQ ID NO: 4, and comprises VH CDRs 1 to 3 of SEQ ID NOs: 1, 2, and 3.

[0218] Thus, preferably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VL has an amino acid sequence at least 85%, at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VL set forth in SEQ ID NO:8.

[0219] Thus, preferably, the binding molecule comprises a VL as disclosed above, wherein one, two, three or four amino acids in the framework have been deleted, inserted and / or independently replaced with different amino acids, i.e., substituted sequences. Preferably, such substitutions may be conservative substitutions.

[0220] Thus, preferably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VL has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VL set forth in SEQ ID NO: 8, and comprises VLCDRs 1 to 3 of SEQ ID NOs: 5, 6, and 7, wherein one or more VLCDRs have three or fewer single amino acid substitutions, deletions, or insertions. Preferably, one or more VLCDRs have three, two, one, or none single amino acid substitutions, deletions, or insertions.

[0221] Therefore, preferably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VL has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VL set forth in SEQ ID NO: 8, and comprises VLCDR1 to 3 of SEQ ID NOs: 5, 6, and 7.

[0222] Therefore, preferably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VH set forth in SEQ ID NO: 4 and comprises VH CDRs 1 to 3 of SEQ ID NOs: 1, 2, and 3, and the VL has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VL set forth in SEQ ID NO: 8 and comprises VL CDRs 1 to 3 of SEQ ID NOs: 5, 6, and 7.

[0223] Thus, preferably, a binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VH, wherein the VH comprises the amino acid sequence of SEQ ID NO: 4. Thus, preferably, a binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VL, wherein the VL comprises the amino acid sequence of SEQ ID NO: 8. Thus, preferably, a binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VH and VL, wherein the VH has the amino acid sequence of SEQ ID NO: 4 and the VL has the amino acid sequence of SEQ ID NO: 8.

[0224] Reduced IL-33 binding molecule 2 - AB1070019 Preferably, a binding molecule that binds reduced IL-33 comprises a heavy chain variable region or domain (VH) and a light chain variable region or domain (VL). Preferably, a binding molecule that binds reduced IL-33 comprises three CDRs in the heavy chain variable region or domain (VH) and three CDRs in the light chain variable region or domain (VL). Preferably, a binding molecule that binds reduced IL-33 may comprise three CDRs, for example, three CDRs in the heavy chain variable region set forth in SEQ ID NO: 28 and / or three CDRs in the light chain variable region set forth in SEQ ID NO: 32.

[0225] Suitably, a binding molecule that binds reduced IL-33 comprises a heavy chain variable region (VH) comprising three CDRs having an amino acid sequence at least 85%, at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VH set forth in SEQ ID NO: 28, and / or a light chain variable region (VL) comprising three CDRs having an amino acid sequence at least 85%, at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VL set forth in SEQ ID NO: 32.

[0226] Preferably, a binding molecule that binds reduced IL-33 may comprise a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises VHCDRs 1 to 3 of SEQ ID NOs: 25, 26, and 27, and one or more VHCDRs have no more than three single amino acid substitutions, deletions, or insertions.

[0227] Suitably, one or more of the VHCDRs may contain three, two, one or none single amino acid substitutions, deletions or insertions. Suitably, the VHCDRs consist of SEQ ID NOs: 25, 26 and 27.

[0228] Preferably, a binding molecule that binds reduced IL-33 may comprise a variable heavy domain (VH) and a variable light domain (VL), wherein the VL comprises VLCDRs 1 to 3 of SEQ ID NOs: 29, 30, and 31, and one or more VLCDRs have no more than three single amino acid substitutions, deletions, or insertions.

[0229] Suitably, one or more VLCDRs may contain three, two, one or none single amino acid substitutions, deletions or insertions. Suitably, the VLCDRs consist of SEQ ID NOs: 29, 30 and 31.

[0230] Preferably, a binding molecule that binds reduced IL-33 may comprise a VH having VH CDRs 1-3 of SEQ ID NOs: 25, 26, and 27, and a VL having VL CDRs of SEQ ID NOs: 29, 30, and 31, wherein one or more of the CDRs have three or fewer single amino acid substitutions, deletions, or insertions. Preferably, one or more of the CDRs have three, two, one, or none single amino acid substitutions, deletions, or insertions. Preferably, such substitutions may be conservative substitutions.

[0231] Preferably, a binding molecule that binds to reduced IL-33 may comprise a VH having VHCDRs1 to 3 of SEQ ID NOs: 25, 26, and 27, and a VL having VLCDRs of SEQ ID NOs: 29, 30, and 31. Preferably, a binding molecule that binds to reduced IL-33 may comprise a VH having VHCDRs1 to 3 of SEQ ID NOs: 25, 26, and 27, and a VL having VLCDRs of SEQ ID NOs: 29, 30, and 31.

[0232] Suitably, a binding molecule that binds reduced IL-33 may comprise a CDR selected from a VHCDR1 having the sequence of SEQ ID NO: 25, a VHCDR2 having the sequence of SEQ ID NO: 26, a VHCDR3 having the sequence of SEQ ID NO: 27, a VLCDR1 having the sequence of SEQ ID NO: 29, a VLCDR2 having the sequence of SEQ ID NO: 30, and a VLCDR3 having the sequence of SEQ ID NO: 31.

[0233] Preferably, a binding molecule that binds reduced IL-33 may comprise the following CDRs: a VH CDR1 having the sequence of SEQ ID NO: 25, a VH CDR2 having the sequence of SEQ ID NO: 26, a VH CDR3 having the sequence of SEQ ID NO: 27, a VL CDR1 having the sequence of SEQ ID NO: 29, a VL CDR2 having the sequence of SEQ ID NO: 30, and a VL CDR3 having the sequence of SEQ ID NO: 31. Preferably, therefore, the binding molecule that binds reduced IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence at least 85%, at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VH set forth in SEQ ID NO: 28.

[0234] Thus, preferably, the binding molecule comprises a VH as disclosed above, wherein one, two, three or four amino acids in the framework have been deleted, inserted and / or independently replaced with different amino acids, i.e., a substituted sequence. Preferably, such substitutions may be conservative substitutions.

[0235] Thus, preferably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VH set forth in SEQ ID NO: 28, and comprises VHCDRs 1 to 3 of SEQ ID NOs: 25, 26, and 27, wherein one or more VHCDRs have three or fewer single amino acid substitutions, deletions, or insertions. Preferably, the one or more VHCDRs have three, two, one, or none single amino acid substitutions, deletions, or insertions.

[0236] Therefore, preferably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VH set forth in SEQ ID NO: 28, and comprises VH CDRs 1 to 3 of SEQ ID NOs: 25, 26, and 27.

[0237] Thus, preferably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VL has an amino acid sequence at least 85%, at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VL set forth in SEQ ID NO: 32.

[0238] Thus, preferably, the binding molecule comprises a VL as disclosed above, wherein one, two, three or four amino acids in the framework have been deleted, inserted and / or independently replaced with different amino acids, i.e., substituted sequences. Preferably, such substitutions may be conservative substitutions.

[0239] Thus, preferably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VL has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VL set forth in SEQ ID NO: 32, and comprises VLCDRs 1 to 3 of SEQ ID NOs: 29, 30, and 31, wherein one or more VLCDRs have three or fewer single amino acid substitutions, deletions, or insertions. Preferably, one or more VLCDRs have three, two, one, or none single amino acid substitutions, deletions, or insertions.

[0240] Therefore, preferably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VL has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VL set forth in SEQ ID NO: 32, and comprises VLCDR1 to 3 of SEQ ID NOs: 29, 30, and 31.

[0241] Thus, preferably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VH set forth in SEQ ID NO: 28 and comprises VH CDRs 1 to 3 of SEQ ID NOs: 25, 26, and 27, and the VL has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VL set forth in SEQ ID NO: 32 and comprises VL CDRs 1 to 3 of SEQ ID NOs: 29, 30, and 31.

[0242] Thus, preferably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VH, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 28. Thus, preferably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VL, wherein the VL comprises the amino acid sequence set forth in SEQ ID NO: 32.

[0243] Therefore, preferably, 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 the amino acid sequence of SEQ ID NO: 28 and the VL has the amino acid sequence of SEQ ID NO: 32.

[0244] Reduced IL-33 binding molecule 3 - AB1070069 Preferably, a binding molecule that binds reduced IL-33 comprises a heavy chain variable region or domain (VH) and a light chain variable region or domain (VL). Preferably, a binding molecule that binds reduced IL-33 comprises three CDRs in the heavy chain variable region or domain (VH) and three CDRs in the light chain variable region or domain (VL). Preferably, a binding molecule that binds reduced IL-33 may comprise three CDRs, for example, three CDRs in the heavy chain variable region set forth in SEQ ID NO: 38 and / or three CDRs in the light chain variable region set forth in SEQ ID NO: 42.

[0245] Suitably, a binding molecule that binds reduced IL-33 comprises a heavy chain variable region (VH) comprising three CDRs having an amino acid sequence at least 85%, at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VH set forth in SEQ ID NO: 38, and / or a light chain variable region (VL) comprising three CDRs having an amino acid sequence at least 85%, at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VL set forth in SEQ ID NO: 42.

[0246] Preferably, a binding molecule that binds reduced IL-33 may comprise a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises VHCDRs 1 to 3 of SEQ ID NOs: 35, 36, and 37, and one or more VHCDRs have no more than three single amino acid substitutions, deletions, or insertions.

[0247] Suitably, one or more of the VHCDRs may contain three, two, one or none single amino acid substitutions, deletions or insertions. Suitably, the VHCDRs consist of SEQ ID NOs: 35, 36 and 37.

[0248] Preferably, a binding molecule that binds reduced IL-33 may comprise a variable heavy domain (VH) and a variable light domain (VL), wherein the VL comprises VLCDRs 1 to 3 of SEQ ID NOs: 39, 40, and 41, and one or more VLCDRs have no more than three single amino acid substitutions, deletions, or insertions.

[0249] Suitably, one or more VLCDRs may contain three, two, one or none single amino acid substitutions, deletions or insertions. Suitably, the VLCDRs consist of SEQ ID NOs: 39, 40 and 41.

[0250] Preferably, a binding molecule that binds reduced IL-33 may comprise a VH having VH CDRs 1-3 of SEQ ID NOs: 35, 36, and 37, and a VL having VL CDRs of SEQ ID NOs: 39, 40, and 41, wherein one or more of the CDRs have three or fewer single amino acid substitutions, deletions, or insertions. Preferably, one or more of the CDRs have three, two, one, or none single amino acid substitutions, deletions, or insertions. Preferably, such substitutions may be conservative substitutions.

[0251] Preferably, a binding molecule that binds to reduced IL-33 may comprise a VH having VHCDRs1 to 3 of SEQ ID NOs: 35, 36, and 37, and a VL having VLCDRs of SEQ ID NOs: 39, 40, and 41. Preferably, a binding molecule that binds to reduced IL-33 may comprise a VH having VHCDRs1 to 3 of SEQ ID NOs: 35, 36, and 37, and a VL having VLCDRs of SEQ ID NOs: 39, 40, and 41.

[0252] Suitably, a binding molecule that binds reduced IL-33 may comprise a CDR selected from a VHCDR1 having the sequence of SEQ ID NO: 35, a VHCDR2 having the sequence of SEQ ID NO: 36, a VHCDR3 having the sequence of SEQ ID NO: 37, a VLCDR1 having the sequence of SEQ ID NO: 39, a VLCDR2 having the sequence of SEQ ID NO: 40, and a VLCDR3 having the sequence of SEQ ID NO: 41.

[0253] Suitably, a binding molecule that binds 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.

[0254] Thus, preferably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence at least 85%, at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VH set forth in SEQ ID NO: 38.

[0255] Thus, preferably, the binding molecule comprises a VH as disclosed above, wherein one, two, three or four amino acids in the framework have been deleted, inserted and / or independently replaced with different amino acids, i.e., a substituted sequence. Preferably, such substitutions may be conservative substitutions.

[0256] Thus, preferably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VH set forth in SEQ ID NO: 38, and comprises VHCDRs 1 to 3 of SEQ ID NOs: 35, 36, and 37, wherein one or more VHCDRs have three or fewer single amino acid substitutions, deletions, or insertions. Preferably, the one or more VHCDRs have three, two, one, or none single amino acid substitutions, deletions, or insertions.

[0257] Therefore, preferably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VH set forth in SEQ ID NO: 38, and comprises VH CDRs 1 to 3 of SEQ ID NOs: 35, 36, and 37.

[0258] Thus, preferably, the binding molecule that binds reduced IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VL has an amino acid sequence at least 85%, at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VL set forth in SEQ ID NO: 42.

[0259] Thus, preferably, the binding molecule comprises a VL as disclosed above, wherein one, two, three or four amino acids in the framework have been deleted, inserted and / or independently replaced with different amino acids, i.e., substituted sequences. Preferably, such substitutions may be conservative substitutions.

[0260] Thus, preferably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VL has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VL set forth in SEQ ID NO: 42, and comprises VLCDRs 1 to 3 of SEQ ID NOs: 39, 40, and 41, wherein one or more VLCDRs have three or fewer single amino acid substitutions, deletions, or insertions. Preferably, one or more VLCDRs have three, two, one, or none single amino acid substitutions, deletions, or insertions.

[0261] Therefore, preferably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VL has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VL set forth in SEQ ID NO: 42, and comprises VLCDR1 to 3 of SEQ ID NOs: 39, 40, and 41.

[0262] Therefore, preferably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VH set forth in SEQ ID NO: 38 and comprises VH CDRs 1 to 3 of SEQ ID NOs: 35, 36, and 37, and the VL has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VL set forth in SEQ ID NO: 42 and comprises VL CDRs 1 to 3 of SEQ ID NOs: 39, 40, and 41.

[0263] Thus, preferably, a binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising a VH, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 38. Thus, preferably, a binding molecule that binds to reduced IL-33 is an antibody or a binding fragment thereof comprising a VL, wherein the VL comprises the amino acid sequence set forth in SEQ ID NO: 42.

[0264] Therefore, preferably, 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 the amino acid sequence of SEQ ID NO: 38 and the VL has the amino acid sequence of SEQ ID NO: 42.

[0265] Oxidized IL-33 binding molecule-AB1070141 Preferably, a 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). Preferably, a binding molecule that binds to oxidized IL-33 comprises three CDRs in the heavy chain variable region or domain (VH) and three CDRs in the light chain variable region or domain (VL). Preferably, a binding molecule that binds to oxidized IL-33 may comprise three CDRs, for example, three CDRs in the heavy chain variable region set forth in SEQ ID NO: 12 and / or three CDRs in the light chain variable region set forth in SEQ ID NO: 16.

[0266] Suitably, a binding molecule that binds oxidized IL-33 comprises a heavy chain variable region (VH) comprising three CDRs having an amino acid sequence at least 85%, at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VH set forth in SEQ ID NO: 12, and / or a light chain variable region (VL) comprising three CDRs having an amino acid sequence at least 85%, at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VL set forth in SEQ ID NO: 16.

[0267] Preferably, a binding molecule that binds 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, and one or more VHCDRs have no more than three single amino acid substitutions, deletions, or insertions.

[0268] Suitably, one or more VHCDRs may contain three, two, one or none single amino acid substitutions, deletions or insertions. Suitably, the VHCDRs consist of SEQ ID NOs: 9, 10 and 11.

[0269] Preferably, a binding molecule that binds 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, and one or more VLCDRs have no more than three single amino acid substitutions, deletions, or insertions.

[0270] Suitably, one or more VLCDRs may contain three, two, one or none single amino acid substitutions, deletions or insertions. Suitably, the VLCDRs consist of SEQ ID NOs: 13, 14 and 15.

[0271] Suitably, a binding molecule that binds oxidized IL-33 may comprise a VH having VH CDRs 1-3 of SEQ ID NOs: 9, 10, and 11, and a VL having VL CDRs of SEQ ID NOs: 13, 14, and 15, wherein one or more of the CDRs have no more than three single amino acid substitutions, deletions, or insertions. Suitably, one or more of the CDRs have three, two, one, or none single amino acid substitutions, deletions, or insertions. Suitably, such substitutions may be conservative substitutions.

[0272] Preferably, a binding molecule that binds to oxidized IL-33 may comprise a VH having VHCDR1 to 3 of SEQ ID NOs: 9, 10, and 11, and a VL having VLCDRs of SEQ ID NOs: 13, 14, and 15. Preferably, a binding molecule that binds to oxidized IL-33 may comprise a VH having VHCDR1 to 3 of SEQ ID NOs: 9, 10, and 11, and a VL having VLCDRs of SEQ ID NOs: 13, 14, and 15.

[0273] Suitably, a binding molecule that binds oxidized IL-33 may comprise one or more CDRs selected from 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.

[0274] Suitably, a binding molecule that binds 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.

[0275] Thus, preferably, 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 at least 85%, at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VH set forth in SEQ ID NO: 12.

[0276] Thus, preferably, the binding molecule comprises a VH as disclosed above, wherein one, two, three or four amino acids in the framework have been deleted, inserted and / or independently replaced with different amino acids, i.e., a substituted sequence. Preferably, such substitutions may be conservative substitutions.

[0277] Thus, preferably, 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 at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VH set forth in SEQ ID NO: 12, and comprises VHCDRs 1 to 3 of SEQ ID NOs: 9, 10, and 11, wherein one or more of the VHCDRs have three or fewer single amino acid substitutions, deletions, or insertions. Preferably, the one or more VHCDRs have three, two, one, or none of the VHCDRs have a single amino acid substitution, deletion, or insertion.

[0278] Therefore, preferably, the binding molecule that binds to reduced IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VH set forth in SEQ ID NO: 12, and comprises VH CDRs 1 to 3 of SEQ ID NOs: 9, 10, and 11.

[0279] Thus, preferably, the binding molecule that binds oxidized IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VL has an amino acid sequence at least 85%, at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VL set forth in SEQ ID NO: 16.

[0280] Thus, preferably, the binding molecule comprises a VL as disclosed above, wherein one, two, three or four amino acids in the framework have been deleted, inserted and / or independently replaced with different amino acids, i.e., substituted sequences. Preferably, such substitutions may be conservative substitutions.

[0281] Thus, preferably, the binding molecule that binds to oxidized IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VL has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VL set forth in SEQ ID NO: 16, and comprises VLCDRs 1 to 3 of SEQ ID NOs: 13, 14, and 15, wherein one or more VLCDRs have three or fewer single amino acid substitutions, deletions, or insertions. Preferably, one or more VLCDRs have three, two, one, or none single amino acid substitutions, deletions, or insertions.

[0282] Therefore, preferably, the binding molecule that binds to oxidized IL-33 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VL has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VL set forth in SEQ ID NO: 16, and comprises VLCDR1 to 3 of SEQ ID NOs: 13, 14, and 15.

[0283] Thus, preferably, 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 at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VH set forth in SEQ ID NO: 12 and comprises VH CDRs 1 to 3 of SEQ ID NOs: 9, 10, and 11, and the VL has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VL set forth in SEQ ID NO: 16 and comprises VL CDRs 1 to 3 of SEQ ID NOs: 13, 14, and 15.

[0284] Thus, preferably, a binding molecule that binds to oxidized IL-33 is an antibody or binding fragment thereof comprising a VH, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 12. Thus, preferably, a binding molecule that binds to oxidized IL-33 is an antibody or binding fragment thereof comprising a VL, wherein the VL comprises the amino acid sequence set forth in SEQ ID NO: 16.

[0285] Therefore, preferably, 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 the amino acid sequence of SEQ ID NO: 12 and the VL has the amino acid sequence of SEQ ID NO: 16.

[0286] IL33 / sST2 binding molecule-AB1070008 Preferably, a 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). Preferably, a binding molecule that binds to IL-33 / sST2 comprises three CDRs in the heavy chain variable region or domain (VH) and three CDRs in the light chain variable region or domain (VL). Preferably, a binding molecule that binds to IL-33 / sST2 may comprise three CDRs, for example, three CDRs in the heavy chain variable region set forth in SEQ ID NO: 20 and / or three CDRs in the light chain variable region set forth in SEQ ID NO: 24.

[0287] Suitably, a binding molecule that binds to IL-33 / sST2 comprises a heavy chain variable region (VH) comprising three CDRs having an amino acid sequence at least 85%, at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VH set forth in SEQ ID NO: 20, and / or a light chain variable region comprising three CDRs having an amino acid sequence at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VL set forth in SEQ ID NO: 24.

[0288] Preferably, a 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 to 3 of SEQ ID NOs: 17, 18, and 19, and one or more VHCDRs have no more than three single amino acid substitutions, deletions, or insertions.

[0289] Suitably, one or more VHCDRs contain 3, 2, 1 or none single amino acid substitutions, deletions or insertions. Suitably, the VHCDRs consist of SEQ ID NOs: 17, 18 and 19.

[0290] Preferably, a 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, and one or more VLCDRs have no more than three single amino acid substitutions, deletions, or insertions.

[0291] Preferably, one or more VLCDRs contain 3, 2, 1 or none single amino acid substitutions, deletions or insertions. Preferably, the VLCDRs consist of SEQ ID NOs: 21, 22 and 23.

[0292] Preferably, a binding molecule that binds to IL-33 / sST2 may comprise a VH having VH CDRs 1-3 of SEQ ID NOs: 17, 18, and 19, and a VL having VL CDRs of SEQ ID NOs: 21, 22, and 23, wherein one or more of the CDRs have no more than three single amino acid substitutions, deletions, or insertions. Preferably, one or more of the CDRs have three, two, one, or none single amino acid substitutions, deletions, or insertions. Preferably, such substitutions may be conservative substitutions.

[0293] Preferably, a binding molecule that binds to IL-33 / sST2 may comprise a VH having VHCDRs 1 to 3 of SEQ ID NOs: 17, 18, and 19, and a VL having VLCDRs of SEQ ID NOs: 21, 22, and 23. Preferably, a binding molecule that binds to IL-33 / sST2 may comprise a VH having VHCDRs 1 to 3 of SEQ ID NOs: 17, 18, and 19, and a VL having VLCDRs of SEQ ID NOs: 21, 22, and 23.

[0294] Suitably, a binding molecule that binds to IL-33 / sST2 may comprise any of the following CDRs: VHCDR1 having the sequence of SEQ ID NO: 17, VHCDR2 having the sequence of SEQ ID NO: 18, VHCDR3 having the sequence of SEQ ID NO: 19, VLCDR1 having the sequence of SEQ ID NO: 21, VLCDR2 having the sequence of SEQ ID NO: 22, and VLCDR3 having the sequence of SEQ ID NO: 23.

[0295] Suitably, a binding molecule that binds to IL-33 / sST2 may comprise a VHCDR1 having the sequence of SEQ ID NO: 17, a VHCDR2 having the sequence of SEQ ID NO: 18, a VHCDR3 having the sequence of SEQ ID NO: 19, a VLCDR1 having the sequence of SEQ ID NO: 21, a VLCDR2 having the sequence of SEQ ID NO: 22, and a VLCDR3 having the sequence of SEQ ID NO: 23.

[0296] Thus, preferably, the binding molecule that binds to IL-33 / sST2 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence at least 85%, at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VH set forth in SEQ ID NO: 20.

[0297] Thus, preferably, the binding molecule comprises a VH as disclosed above, wherein one, two, three or four amino acids in the framework have been deleted, inserted and / or independently replaced with different amino acids, i.e., a substituted sequence. Preferably, such substitutions may be conservative substitutions.

[0298] Thus, preferably, the binding molecule that binds to IL-33 / sST2 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VH set forth in SEQ ID NO: 20, and comprises VHCDRs 1 to 3 of SEQ ID NOs: 17, 18, and 19, wherein one or more VHCDRs have three or fewer single amino acid substitutions, deletions, or insertions. Preferably, one or more VHCDRs have three, two, one, or none single amino acid substitutions, deletions, or insertions.

[0299] Therefore, preferably, 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 at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VH set forth in SEQ ID NO: 20, and comprises VH CDRs 1 to 3 of SEQ ID NOs: 17, 18, and 19.

[0300] Thus, preferably, 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 VL has an amino acid sequence at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, identical to the VL set forth in SEQ ID NO:24.

[0301] Thus, preferably, the binding molecule comprises a VL as disclosed above, wherein one, two, three or four amino acids in the framework have been deleted, inserted and / or independently replaced with different amino acids, i.e., substituted sequences. Preferably, such substitutions may be conservative substitutions.

[0302] Thus, preferably, the binding molecule that binds to IL-33 / sST2 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VL has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VL set forth in SEQ ID NO: 24, and comprises VLCDRs 1 to 3 of SEQ ID NOs: 21, 22, and 23, wherein one or more VLCDRs have three or fewer single amino acid substitutions, deletions, or insertions. Preferably, one or more VLCDRs have three, two, one, or none single amino acid substitutions, deletions, or insertions.

[0303] Therefore, preferably, 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 VL has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VL set forth in SEQ ID NO: 24, and comprises VLCDR1 to 3 of SEQ ID NOs: 21, 22, and 23.

[0304] Thus, preferably, the binding molecule that binds to IL-33 / sST2 is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VH set forth in SEQ ID NO: 20 and comprises VH CDRs 1 to 3 of SEQ ID NOs: 17, 18, and 19, and the VL has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VL set forth in SEQ ID NO: 24 and comprises VL CDRs 1 to 3 of SEQ ID NOs: 21, 22, and 23.

[0305] Thus, preferably, a binding molecule that binds to oxidized IL-33 is an antibody or binding fragment thereof comprising a VH, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 20. Thus, preferably, a binding molecule that binds to oxidized IL-33 is an antibody or binding fragment thereof comprising a VL, wherein the VL comprises the amino acid sequence set forth in SEQ ID NO: 24.

[0306] Therefore, preferably, 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 the amino acid sequence of SEQ ID NO: 20 and the VL has the amino acid sequence of SEQ ID NO: 24.

[0307] Reduced IL-33 reporter molecule - AB1070019 Optionally, the reduced IL-33 binding molecule may be used as a reduced IL-33 reporter molecule. Suitably, the binding molecule of step (b)(i)b of the first aspect or the binding molecule of step (b) of the fourth aspect may also be used as a reporter molecule. Thus, suitably, in some assays for detecting reduced IL-33, the binding molecule and the reporter molecule may be the same. Thus, suitably, in some assays for detecting reduced IL-33, the binding molecule and the reporter molecule may both be the molecule defined in step (b)ib of the first aspect or step (b) of the fourth aspect.

[0308] Preferably, 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). Preferably, the reporter molecule that binds to the reduced IL33-binding molecule complex comprises three CDRs in the heavy chain variable region or domain (VH) and three CDRs in the light chain variable region or domain (VL). Preferably, the reporter molecule that binds to the reduced IL33-binding molecule complex may comprise three CDRs, for example, three CDRs in the heavy chain variable region set forth in SEQ ID NO: 28 and / or three CDRs in the light chain variable region set forth in SEQ ID NO: 32.

[0309] Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex comprises a heavy chain variable region (VH) comprising three CDRs having an amino acid sequence at least 85%, at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VH set forth in SEQ ID NO: 28, and / or a light chain variable region (VL) comprising three CDRs having an amino acid sequence at least 85%, at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VL set forth in SEQ ID NO: 32.

[0310] Preferably, 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) having VHCDRs 1 to 3 of SEQ ID NOs: 25, 26, and 27, wherein one or more VHCDRs have no more than three single amino acid substitutions, deletions, or insertions.

[0311] Suitably one or more of the VHCDRs contain 3, 2, 1 or none single amino acid substitutions, deletions or insertions. Suitably the VHCDRs consist of SEQ ID NOs: 25, 26 and 27.

[0312] Preferably, 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) having VLCDRs 1 to 3 of SEQ ID NOs: 29, 30, and 31, wherein one or more VLCDRs have no more than three single amino acid substitutions, deletions, or insertions.

[0313] Preferably, one or more of the VLCDRs contain none, three, two, one or a single amino acid substitution, deletion or insertion. Preferably, the VLCDRs consist of SEQ ID NOs: 29, 30, and 31.

[0314] Preferably, a reporter molecule that binds to a reduced IL33-binding molecule complex may comprise a VH having VH CDRs 1 to 3 of SEQ ID NOs: 25, 26, and 27, and a VL having VL CDRs of SEQ ID NOs: 29, 30, and 31, wherein one or more of the CDRs have three or fewer single amino acid substitutions, deletions, or insertions. Preferably, one or more of the CDRs have three, two, one, or none single amino acid substitutions, deletions, or insertions. Preferably, such substitutions may be conservative substitutions.

[0315] Preferably, a reporter molecule that binds to a reduced IL33-binding molecule complex may comprise a VH having VHCDR1 to 3 of SEQ ID NOs: 25, 26, and 27, and a VL having VLCDRs of SEQ ID NOs: 29, 30, and 31. Preferably, a reporter molecule that binds to a reduced IL33-binding molecule complex may comprise a VH having VHCDR1 to 3 of SEQ ID NOs: 25, 26, and 27, and a VL having VLCDRs of SEQ ID NOs: 29, 30, and 31.

[0316] Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex may comprise any 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.

[0317] Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex may comprise a VHCDR1 having the sequence of SEQ ID NO: 25, a VHCDR2 having the sequence of SEQ ID NO: 26, a VHCDR3 having the sequence of SEQ ID NO: 27, a VLCDR1 having the sequence of SEQ ID NO: 29, a VLCDR2 having the sequence of SEQ ID NO: 30, and a VLCDR3 having the sequence of SEQ ID NO: 31.

[0318] Therefore, preferably, 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%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VH set forth in SEQ ID NO: 28.

[0319] Thus, suitably, the reporter molecule comprises a VH as disclosed above, wherein one, two, three or four amino acids in the framework have been deleted, inserted and / or independently replaced with different amino acids, i.e., a substituted sequence. Suitably, such substitutions may be conservative substitutions.

[0320] Therefore, preferably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to that of the VH set forth in SEQ ID NO: 28, and comprises VHCDRs 1 to 3 of SEQ ID NOs: 25, 26, and 27, wherein one or more VHCDRs have three or fewer single amino acid substitutions, deletions, or insertions. Preferably, the one or more VHCDRs have three, two, one, or none single amino acid substitutions, deletions, or insertions.

[0321] Therefore, preferably, 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%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VH set forth in SEQ ID NO: 28, and comprises VH CDRs 1 to 3 of SEQ ID NOs: 25, 26, and 27.

[0322] Therefore, preferably, 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 VL has an amino acid sequence that is at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, identical to the VL set forth in SEQ ID NO: 32.

[0323] Preferably, the reporter molecule therefore comprises a VL as disclosed above, wherein one, two, three or four amino acids in the framework have been deleted, inserted and / or independently replaced with different amino acids, i.e., a substituted sequence. Preferably, such substitutions may be conservative substitutions.

[0324] Therefore, preferably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VL has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VL set forth in SEQ ID NO: 32, and comprises VLCDRs 1 to 3 of SEQ ID NOs: 29, 30, and 31, wherein one or more VLCDRs have three or fewer single amino acid substitutions, deletions, or insertions. Preferably, one or more VLCDRs have three, two, one, or none single amino acid substitutions, deletions, or insertions.

[0325] Therefore, preferably, 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 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 set forth in SEQ ID NO: 32, and comprises VLCDR1 to 3 of SEQ ID NOs: 29, 30, and 31.

[0326] Therefore, preferably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VH set forth in SEQ ID NO: 28 and comprises VH CDRs 1 to 3 of SEQ ID NOs: 25, 26, and 27, and the VL has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VL set forth in SEQ ID NO: 32 and comprises VL CDRs 1 to 3 of SEQ ID NOs: 29, 30, and 31.

[0327] Therefore, preferably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or a binding fragment thereof comprising a VH, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 28. Therefore, preferably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or a binding fragment thereof comprising a VL, wherein the VL comprises the amino acid sequence set forth in SEQ ID NO: 32.

[0328] Therefore, preferably, 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 the amino acid sequence of SEQ ID NO: 28 and the VL has the amino acid sequence of SEQ ID NO: 32.

[0329] Reduced IL-33 reporter molecule 2 - AB1070069 Optionally, the reduced IL-33 binding molecule may be used as a reduced IL-33 reporter molecule. Suitably, the binding molecule of step (b)(i)b of the first aspect or the binding molecule of step (b) of the fourth aspect may also be used as a reporter molecule. Thus, suitably, in some assays for detecting reduced IL-33, the binding molecule and the reporter molecule may be the same. Thus, suitably, in some assays for detecting reduced IL-33, the binding molecule and the reporter molecule may both be the molecule defined in step (b)ib of the first aspect or step (b) of the fourth aspect.

[0330] Preferably, 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). Preferably, the reporter molecule that binds to the reduced IL33-binding molecule complex comprises three CDRs in the heavy chain variable region or domain (VH) and three CDRs in the light chain variable region or domain (VL). Preferably, the reporter molecule that binds to the reduced IL33-binding molecule complex may comprise three CDRs, for example, three CDRs in the heavy chain variable region set forth in SEQ ID NO: 38 and / or three CDRs in the light chain variable region set forth in SEQ ID NO: 42.

[0331] Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex comprises a heavy chain variable region (VH) comprising three CDRs with an amino acid sequence at least 85%, at least 90%, such as 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VH set forth in SEQ ID NO: 38, and / or a light chain variable region (VL) comprising three CDRs with an amino acid sequence at least 85%, at least 90%, such as 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VL set forth in SEQ ID NO: 42.

[0332] Preferably, 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) having VHCDRs 1 to 3 of SEQ ID NOs: 35, 36, and 37, wherein one or more VHCDRs have no more than three single amino acid substitutions, deletions, or insertions.

[0333] Suitably, one or more of the VHCDRs contain none, three, two, one or even none single amino acid substitutions, deletions or insertions. Suitably, the VHCDRs consist of SEQ ID NOs: 35, 36 and 37.

[0334] Preferably, 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) having VLCDRs 1 to 3 of SEQ ID NOs: 39, 40, and 41, wherein one or more VLCDRs have no more than three single amino acid substitutions, deletions, or insertions.

[0335] Preferably, one or more of the VLCDRs contain none, three, two, one or a single amino acid substitution, deletion or insertion. Preferably, the VLCDRs consist of SEQ ID NOs: 39, 40, and 41.

[0336] Preferably, a reporter molecule that binds to a reduced IL33-binding molecule complex may comprise a VH having VH CDRs 1 to 3 of SEQ ID NOs: 35, 36, and 37, and a VL having VL CDRs of SEQ ID NOs: 39, 40, and 41, wherein one or more of the CDRs have three or fewer single amino acid substitutions, deletions, or insertions. Preferably, one or more of the CDRs have three, two, one, or none single amino acid substitutions, deletions, or insertions. Preferably, such substitutions may be conservative substitutions.

[0337] Preferably, a reporter molecule that binds to a reduced IL33-binding molecule complex may comprise a VH having VHCDR1 to 3 of SEQ ID NOs: 35, 36, and 37, and a VL having VLCDRs of SEQ ID NOs: 39, 40, and 41. Preferably, a reporter molecule that binds to a reduced IL33-binding molecule complex may comprise a VH having VHCDR1 to 3 of SEQ ID NOs: 35, 36, and 37, and a VL having VLCDRs of SEQ ID NOs: 39, 40, and 41.

[0338] Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex may comprise any 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.

[0339] Suitably, the reporter molecule that binds to the reduced IL33-binding molecule complex may comprise a VHCDR1 having the sequence of SEQ ID NO: 35, a VHCDR2 having the sequence of SEQ ID NO: 36, a VHCDR3 having the sequence of SEQ ID NO: 37, a VLCDR1 having the sequence of SEQ ID NO: 39, a VLCDR2 having the sequence of SEQ ID NO: 40, and a VLCDR3 having the sequence of SEQ ID NO: 41.

[0340] Therefore, preferably, the reporter molecule that binds to the reduced IL33-binding molecule complex 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%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VH set forth in SEQ ID NO: 38.

[0341] Thus, suitably, the reporter molecule comprises a VH as disclosed above, wherein one, two, three or four amino acids in the framework have been deleted, inserted and / or independently replaced with different amino acids, i.e., a substituted sequence. Suitably, such substitutions may be conservative substitutions.

[0342] Therefore, preferably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence at least 85%, at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VH set forth in SEQ ID NO: 28, and comprises VHCDRs 1 to 3 of SEQ ID NOs: 35, 36, and 37, wherein one or more VHCDRs have three or fewer single amino acid substitutions, deletions, or insertions. Preferably, the one or more VHCDRs have three, two, one, or none single amino acid substitutions, deletions, or insertions.

[0343] Therefore, preferably, 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%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VH set forth in SEQ ID NO: 38, and comprises VH CDRs 1 to 3 of SEQ ID NOs: 35, 36, and 37.

[0344] Therefore, preferably, 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 VL has an amino acid sequence that is at least 90%, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, identical to the VL set forth in SEQ ID NO: 42.

[0345] Preferably, the reporter molecule therefore comprises a VL as disclosed above, wherein one, two, three or four amino acids in the framework have been deleted, inserted and / or independently replaced with different amino acids, i.e., a substituted sequence. Preferably, such substitutions may be conservative substitutions.

[0346] Therefore, preferably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VL has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VL set forth in SEQ ID NO: 32, and comprises VLCDRs 1 to 3 of SEQ ID NOs: 39, 40, and 41, wherein one or more VLCDRs have three or fewer single amino acid substitutions, deletions, or insertions. Preferably, one or more VLCDRs have three, two, one, or none single amino acid substitutions, deletions, or insertions.

[0347] Therefore, preferably, 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 VL has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VL set forth in SEQ ID NO: 42, and comprises VLCDR1 to 3 of SEQ ID NOs: 39, 40, and 41.

[0348] Therefore, preferably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VH set forth in SEQ ID NO: 38 and comprises VH CDRs 1 to 3 of SEQ ID NOs: 35, 36, and 37, and the VL has an amino acid sequence at least 85%, at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the VL set forth in SEQ ID NO: 42 and comprises VL CDRs 1 to 3 of SEQ ID NOs: 39, 40, and 41.

[0349] Therefore, preferably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or a binding fragment thereof comprising a VH, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 38. Therefore, preferably, the reporter molecule that binds to the reduced IL33-binding molecule complex is an antibody or a binding fragment thereof comprising a VL, wherein the VL comprises the amino acid sequence set forth in SEQ ID NO: 42.

[0350] Therefore, preferably, 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 the amino acid sequence of SEQ ID NO: 38 and the VL has the amino acid sequence of SEQ ID NO: 42.

[0351] Oxidized IL-33 reporter molecule Preferably, the reporter molecule that binds to the oxidized IL33-binding molecule complex can be obtained from any suitable supplier, for example, preferably, the reporter molecule that binds to the oxidized IL33-binding molecule complex is AF3625 manufactured by R&D systems.

[0352] However, optionally, an oxidized IL-33 binding molecule as described herein may be used as an oxidized IL-33 reporter molecule. Suitably, the binding molecule of (b)(ii) of the first aspect or the binding molecule of the fifth aspect may also be used as a reporter molecule. Thus, suitably, in some assays for detecting oxidized IL-33, the binding molecule and the reporter molecule may be the same. Thus, suitably, in some assays for detecting oxidized IL-33, the binding molecule and the reporter molecule may both be the molecule defined in step (b)(ii) of the first or fifth aspect. Suitably, details of oxidized IL-33 binding molecules that may be used as reporter molecules are described herein above under the heading "Oxidized IL-33 Binding Molecule - AB1070141."

[0353] Thus, suitably, there is provided a reporter molecule that binds to an oxidized IL33-binding molecule complex, the reporter molecule comprising a variable heavy domain having VHCDR1-3 of SEQ ID NOs: 9, 10, and 11, respectively, and a variable light domain having VLCDR1-3 of SEQ ID NOs: 13, 14, and 15, respectively, wherein one or more CDRs have no more than three single amino acid substitutions, deletions, or insertions. Suitably, 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.

[0354] IL-33 / sST2 reporter molecule Preferably, the reporter molecule that binds to the IL-33 / sST2-binding molecule complex can be obtained from any suitable supplier, for example, preferably, the reporter molecule that binds to the IL-33 / sST2-binding molecule complex is MAB5232 manufactured by R&D systems.

[0355] However, optionally, an IL-33 / sST2 binding molecule described herein may be used as an IL-33 / sST2 reporter molecule. Suitably, the binding molecule of (b)(iii) of the first aspect or the binding molecule of the sixth aspect may also be used as a reporter molecule. Thus, suitably, in some assays for detecting IL-33 / sST2, the binding molecule and the reporter molecule may be the same. Thus, suitably, in some assays for detecting IL-33 / sST2, both the binding molecule and the reporter molecule may be the molecule defined in step (b)(iii) of the first aspect or the sixth aspect. Suitably, details of IL-33 / sST2 binding molecules that may be used as reporter molecules are described herein above under the heading "IL33 / sST2 binding molecule - AB1070008."

[0356] Thus, suitably, there is provided a reporter molecule that binds to an IL-33 / sST2-binding molecule complex, the reporter molecule comprising a variable heavy domain having VHCDR1-3 of SEQ ID NOs: 17, 18, and 19, respectively, and a variable light domain having VLCDR1-3 of SEQ ID NOs: 21, 22, and 23, respectively, wherein one or more CDRs have no more than three single amino acid substitutions, deletions, or insertions. Suitably, further sequences defining such reporter molecules are described elsewhere herein in connection with 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.

[0357] Polynucleotides encoding binding molecules and reporter molecules The present disclosure also provides polynucleotide molecules encoding the binding molecules and reporter molecules of the invention, preferably isolated polynucleotide molecules encoding the binding molecules and reporter molecules of the invention.

[0358] Preferably, the isolated polynucleotide comprises, consists essentially of, or consists of a nucleic acid encoding an immunoglobulin heavy chain variable domain (VH domain), wherein the sequences of the CDRs of the VH domain are a VHCDR1 sequence comprising an amino acid sequence selected from SEQ ID NOs: 1, 9, 17, 25 and 35, a VHCDR2 sequence comprising an amino acid sequence selected from SEQ ID NOs: 2, 10, 18, 26 and 36, and a VHCDR3 sequence comprising an amino acid sequence selected from SEQ ID NOs: 3, 11, 19, 27 and 37.

[0359] 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 NOs: 4, 12, 20, 28 and 38.

[0360] Preferably, the isolated polynucleotide comprises, consists essentially of, or consists of a nucleic acid encoding an immunoglobulin light chain variable domain (VL domain), wherein the sequences of the CDRs of the VL domain are a VLCDR1 sequence comprising an amino acid sequence selected from SEQ ID NOs: 5, 13, 21, 29 and 39, a VLCDR2 sequence comprising an amino acid sequence selected from SEQ ID NOs: 6, 14, 22, 30 and 40, and a VLCDR3 sequence comprising an amino acid sequence selected from SEQ ID NOs: 7, 15, 23, 31 and 41.

[0361] 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 NOs: 8, 16, 24, 32 and 42.

[0362] Any of the above polynucleotides may further comprise additional nucleic acid encoding, for example, a signal peptide that directs secretion of the encoded polypeptide, an antibody constant region as described herein, or other heterologous polypeptide as described herein. Also, as described in more detail elsewhere herein, the present disclosure includes compositions comprising one or more of the above polynucleotides.

[0363] In one embodiment, a composition is provided comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a VH domain described herein and the second polynucleotide encodes a VL domain described herein.

[0364] The present disclosure also includes fragments of the polynucleotides of the present disclosure, as described elsewhere. Additionally, polynucleotides encoding the Fab fragments and other derivatives described herein are also contemplated by the present disclosure.

[0365] Polynucleotides can be produced or manufactured by any method known in the art. For example, if the nucleotide sequence of a binding molecule is known, a polynucleotide encoding the binding molecule can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which briefly involves synthesizing overlapping oligonucleotides containing portions of the sequence encoding the binding molecule, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0366] Alternatively, polynucleotides encoding binding molecules or reporter molecules, such as antibodies of the present disclosure, or antigen-binding fragments, variants, or derivatives thereof, 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, nucleic acid encoding the antibody can be chemically synthesized by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for the particular gene sequence, e.g., by identifying cDNA clones from a cDNA library encoding the antibody or other anti-IL-33 antibodies, or can be obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell that expresses the antibody or other anti-IL-33 antibody (e.g., hybridoma cells selected to express the antibody), or nucleic acid, preferably polyA+ RNA, isolated from any tissue or cell that expresses the antibody or other anti-IL-33 antibody). Amplified nucleic acids generated by PCR can then be cloned into a replicable cloning vector using any method well known in the art.

[0367] Once the nucleotide sequence and corresponding amino acid sequence of a binding molecule or reporter molecule has been determined, the nucleotide sequence can be manipulated using methods well known in the art for the manipulation of nucleotide sequences, such as recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (see, e.g., 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), both of which are incorporated herein by reference in their entireties), to generate binding molecules with different amino acid sequences, e.g., to make amino acid substitutions, deletions, and / or insertions.

[0368] Polynucleotides encoding binding or reporter molecules, such as antibodies, or antigen-binding fragments, variants, or derivatives thereof, can be composed of any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides encoding antibodies, or antigen-binding fragments, variants, or derivatives thereof, can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules containing DNA and RNA, which can be single-stranded, or more typically double-stranded, or a mixture of single- and double-stranded regions. In addition, polynucleotides encoding binding molecules, such as antibodies, or antigen-binding fragments, variants, or derivatives thereof, can be composed of RNA, DNA, or triple-stranded regions containing both RNA and DNA. Polynucleotides encoding binding molecules, such as antibodies, or antigen-binding fragments, variants, or derivatives thereof, 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 unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.

[0369] Isolated polynucleotides encoding non-naturally occurring variants of polypeptides derived from immunoglobulins (e.g., immunoglobulin heavy or light chain portions) can be made by introducing one or more nucleotide substitutions, additions, or deletions into the nucleotide sequence of the immunoglobulin, such 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.

[0370] Expression of binding molecules and reporter molecules Polynucleotides encoding the binding molecules or reporter molecules of the invention are typically inserted into expression vectors for introduction into host cells, which can be used to produce desired quantities of the binding molecules or reporter molecules. Thus, expression vectors containing one or more polynucleotides encoding the binding molecules or reporter molecules of the invention, and host cells containing such expression vectors, are encompassed by the invention.

[0371] Recombinant expression of a binding molecule or reporter molecule, e.g., the heavy and / or light chain of an antibody that binds to a target molecule described herein (e.g., 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 disclosure, e.g., the heavy and / or light chain of an antibody, or a portion thereof (preferably containing the heavy chain variable domain or light chain variable domain), is obtained, a vector for the production of the molecule can be produced by recombinant DNA technology using techniques well known in the art.

[0372] DNA sequences encoding binding or reporter molecules can be generated simultaneously or separately using reverse transcriptase and DNA polymerase according to well-known methods. PCR can be initiated by consensus constant region primers or by more specific primers based on published DNA and amino acid sequences. PCR can also be used to isolate DNA clones encoding antibody light and heavy chains. In this case, libraries can be screened with consensus primers or larger homologous probes, such as mouse constant region probes.

[0373] Thus, methods for preparing proteins by expressing polynucleotides containing nucleotide sequences encoding binding molecules or reporter molecules are described herein. Expression vectors containing binding molecule- or reporter molecule-coding sequences and appropriate transcriptional and translational control signals can be constructed using methods well known to those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Thus, the present disclosure provides replicable vectors containing a nucleotide sequence encoding a binding molecule or reporter molecule of the present disclosure (e.g., a heavy or light chain, or a heavy or light chain variable domain) operably linked to a promoter. Such vectors may include nucleotide sequences encoding the constant region of an antibody molecule (see, e.g., WO 86 / 05807, WO 89 / 01036, and U.S. Pat. No. 5,122,464), and antibody variable domains may be cloned into such vectors for expression of the entire heavy and / or light chain.

[0374] As used herein, the term "vector" or "expression vector" refers to a vector used in accordance with the present disclosure as a vehicle for introducing and expressing a desired polynucleotide in a host cell. As known to those skilled in the art, such vectors may be easily selected from the group consisting of plasmids, phages, viruses, and retroviruses. Generally, vectors compatible with the present disclosure may contain a selectable marker, appropriate restriction sites to facilitate cloning of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0375] For purposes of the present disclosure, numerous expression vector systems may be used. For example, one class of vectors utilizes DNA elements derived from animal viruses, such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV, or MOMLV), or SV40 virus. Other vectors involve the use of polycistronic systems containing internal ribosome binding sites. Additionally, cells that have integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow for selection of transfected host cells. Markers may provide prototrophy for auxotrophic hosts, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene may be directly linked to the DNA sequence to be expressed or introduced into the same cell by cotransformation. Additional elements may be required for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals.

[0376] In one embodiment for antibody expression, the cloned variable region genes are inserted into an expression vector along with heavy and light chain constant region genes (preferably human) synthesized as discussed above. Of course, any expression vector capable of eliciting expression in eukaryotic cells may be used in the present disclosure. Examples of suitable vectors include, but are not limited to, the plasmids pcDNA3, pHCMV / Zeo, pCR3.1, pEF1 / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAX1, and pZeoSV2 (available from Invitrogen, San Diego, Calif.), and the plasmid pCI (available from Promega, Madison, Wis.). Generally, screening large numbers of transformants for those expressing suitably high levels of immunoglobulin heavy and light chains is routine experimentation that can be performed, for example, by robotic systems.

[0377] More generally, once a vector or DNA sequence encoding a binding molecule or reporter molecule has been prepared, the expression vector can be introduced into a suitable host cell. Introduction of the plasmid into the host cell can be accomplished by a variety of techniques well known to those skilled in the art, including, but not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See Ridgway (1988) "Mammalian Expression Vectors" in Vectors, ed. Rodriguez and Denhardt (Butterworths, Boston, Mass.), Chapter 24.2, pp. 470-472. Typically, plasmid introduction into the host is by electroporation. Host cells harboring the expression construct are grown under conditions appropriate for production of the binding molecule or reporter molecule and assayed for protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorter analysis (FACS), immunohistochemistry, and the like.

[0378] The expression vector is transferred into a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the binding or reporter molecules used in the methods described herein. Thus, the present disclosure includes host cells containing a polynucleotide encoding a binding or reporter molecule of the disclosure, e.g., a heavy and / or light chain, or a variable heavy and / or variable light chain, operably linked to a heterologous promoter.

[0379] In one embodiment, a culture medium comprising a host cell of the invention is provided, hi one embodiment, a fermentation vessel comprising the culture medium is provided.

[0380] Preferably, the culture medium and fermentation vessel are suitable for carrying out the method for producing a binding molecule or reporter molecule of the invention.

[0381] As used herein, "host cells" refer to cells that have been constructed using recombinant DNA techniques and that contain a vector encoding at least one heterologous polynucleotide. In describing the process of isolating binding molecules or reporter molecules from a recombinant host, the terms "cells" and "cell culture" are used interchangeably to indicate the source of the binding molecules or reporter molecules, unless otherwise specified. In other words, recovery of polypeptides from "cells" can mean either recovery from spun-down whole cells or recovery from the cell culture containing both the medium and suspended cells.

[0382] A variety of host-expression vector systems can be utilized to express binding or reporter molecules for use in the methods described herein. Such host-expression systems are vehicles by which coding sequences of interest can be produced and subsequently purified, but are also cells that can express the molecules of the present disclosure in situ when transformed or transfected with the appropriate nucleotide coding sequence. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the coding sequence; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the coding sequence; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the coding sequence; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the coding sequence; or mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0383] Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, particularly for the expression of whole recombinant antibody molecules, are used for the expression of binding molecules or reporter molecules. For example, mammalian cells such as Chinese hamster ovary cells (CHO) in combination with vectors such as the major immediate 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)).

[0384] Host cell lines used for protein expression are often mammalian in origin, and those skilled in the art are believed to be able to preferentially determine the particular host cell line that is 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 minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVI with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, 293, WI38, R1610 (Chinese hamster fibroblast), BALBC / 3T3 (mouse fibroblast), HAK (hamster kidney line), SP2 / 0 (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-lclBPT (bovine endothelial cells), RAJI (human lymphocytes), and 293 (human kidney). Host cell lines are typically available from commercial services, the American Tissue Culture Collection, or from published literature.

[0385] Additionally, a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems may be selected to ensure the correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.

[0386] Stable expression is preferred for long-term, high-yield production of recombinant proteins. For example, cell lines that stably express the binding molecules or reporter molecules herein can be engineered. Rather than using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the foreign DNA, engineered cells can be grown in an enriched medium for 1-2 days and then switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci, which can then be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that stably express the binding molecules or reporter molecules.

[0387] Any number of selection systems can be used, including, but not limited to, 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 in tk-, hgprt-, or aprt-cells, respectively. Additionally, antimetabolite resistance can be used as a selection criterion for the following genes: dhfr, which confers methotrexate resistance (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 mycophenolic acid resistance (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2012 (1981)); neo, which confers aminoglycoside G-418 resistance (Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 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); TIB TECH 13(5):155-215 (May, 1993); and hygro, which confers hygromycin resistance (Santerre et al., Gene 30:141 (1984)).Methods that may be used, as generally known in the art of recombinant DNA technology, 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 (incorporated herein by reference in their entireties).

[0388] The expression level of a binding molecule or reporter molecule can be increased by vector amplification (for a 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) Vol. 3). If the marker in the vector system expressing the binding molecule or reporter molecule is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Because the amplified region is associated with the binding molecule or reporter molecule gene, antibody production will also increase (Crouse et al., Mol. Cell. Biol. 3:251 (1983)).

[0389] In vitro production can be scaled up to obtain large quantities of the desired polypeptide. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include, for example, homogeneous suspension culture in airlift or continuously stirred reactors, or culturing cells immobilized or entrapped in, for example, hollow fibers, microcapsules, agarose microbeads, or ceramic cartridges. If necessary and / or desired, the polypeptide solution can be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose, or (immuno)affinity chromatography, for example, after preferential biosynthesis of the synthetic hinge region polypeptide, or before or following the HIC chromatography step described herein.

[0390] Genes encoding the binding molecules or reporter molecules of the present disclosure, or portions thereof, such as VH or VL regions, can also be expressed in non-mammalian cells, such as insect, bacterial, or yeast cells, or in plant cells. Bacteria that readily take up nucleic acids include members of the Enterobacteriaceae family, such as Escherichia coli or Salmonella; Bacillaceae, such as strains of Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. It is further understood that when expressed in bacteria, the heterologous polypeptide typically becomes part of inclusion bodies. This heterologous polypeptide must be isolated, purified, and then assembled into a functional molecule. If a tetravalent form of the antibody is desired, the subunits then self-assemble into a tetravalent antibody (WO 02 / 096948 A2).

[0391] In bacterial systems, when large quantities of such proteins are to be produced, several expression vectors can be advantageously selected, and vectors that direct the expression of high-level 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)) (individual coding sequences can be ligated in-frame with the lacZ coding region 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 foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0392] In addition to prokaryotes, eukaryotic microbes can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used eukaryotic microorganism, although several other strains, such as Pichia pastons, are commonly available.

[0393] For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used (Stinchcomb et al., Nature 282:39 (1979); Kingsman et al., Gene 7:141 (1979); Tschemper et al., Gene 10:151 (1980)). This plasmid already contains the TRP1 gene, which provides a selection marker for yeast mutants lacking the ability to grow in tryptophan, such as accession number 44076 or PEP4-1 (Jones, Genetics 85:12 (1977)). The presence of the trp1 lesion as a characteristic of the yeast host cell genome thus provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0394] In insect systems, Autographa californica nuclear polyhedrosis virus (AcNPV) is typically used as a vector to express foreign genes. This virus is grown in Spodoptera frugiperda cells. The antibody coding sequence can be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0395] Once recombinantly expressed, a binding molecule or reporter molecule of the disclosure can be purified by any method known in the art for purifying immunoglobulin molecules, such as chromatography (e.g., ion exchange, affinity, particularly for a specific antigen following Protein A, and size exclusion column chromatography), centrifugation, differential solubility, or any other standard protein purification technique. Alternatively, a preferred method for increasing the affinity of antibodies of the disclosure is disclosed in U.S. Patent Application Publication No. 2002 0123057(A1).

[0396] kit The present disclosure further provides kits for detecting antigenically distinct forms of IL-33 in a biological sample.

[0397] Preferably, the kit comprises at least one binding molecule and at least one reporter molecule. Preferably, the binding molecule and the reporter molecule are selected from any of those defined above.

[0398] Preferably the kit comprises: (i) an oxidized IL-33 binding molecule and a reporter molecule that binds to the oxidized IL-33-binding molecule complex, and / or (ii) a reduced IL-33 binding molecule and a reporter molecule that binds to the reduced IL-33-binding molecule complex, and / or (iii) an IL-33 / sST2-binding molecule and a reporter molecule that binds to the IL-33 / sST2-binding molecule complex; Includes.

[0399] Preferably, the kit comprises (i) and (ii). Preferably, the kit comprises (i) and (iii). Preferably, the kit comprises (i) and (ii). Preferably, the kit comprises (i), (ii) and (iii).

[0400] Preferably, the reporter molecules are detectably labeled, and preferably each comprise a label as described hereinabove.

[0401] Alternatively, in a further example, there is provided an assay kit for detecting antigenically distinct forms of IL-33 in a biological sample, comprising: (i) a binding molecule capable of binding to and forming a complex with an antigenically distinct form of IL-33 selected from reduced IL-33, oxidized IL-33, and / or IL-33 / sST2; An assay kit is provided comprising:

[0402] Preferably, in such cases, 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. Preferably, in such cases, the kit comprises a reduced IL-33-binding molecule and an oxidized IL-33-binding molecule and an IL-33 / sST2-binding molecule. Preferably, in such cases, the or each binding molecule is detectably labeled.

[0403] Alternatively, a detectable label may be provided separately in the kit, preferably for attachment to a binding molecule or reporter molecule. Thus, preferably the kit may comprise a detectable label, suitable detectable labels being identified above.

[0404] Therefore, preferably the kit further comprises a stimulating agent capable of stimulating or activating the detectable label of each binding molecule or reporter molecule.

[0405] Suitable biological samples for use with the kit are also defined hereinabove.

[0406] Preferably, the kit is adapted to perform an immunoassay as described hereinabove. Preferably, the kit is adapted to perform an ELISA or an ECLIA or a CLIA. Preferably, the kit is adapted to perform an S-plex assay. Preferably, the kit is adapted to perform an ECLIA, such as the Elecsys® assay.

[0407] Preferably, the kit may further comprise a solid support as described hereinabove. Preferably, one or more binding molecules may be disposed on the solid support. Preferably, one or more binding molecules may be bound to the support or impregnated within the support. Preferably, the solid support may be glass or a polymer, including, but not limited to, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene, as described above. Preferably, the solid support may be in the form of a tube, bead, disk, microplate, column, or any other surface suitable for carrying out the assay method described above. In some embodiments, the kit may comprise beads or microparticles, preferably magnetic beads or microparticles.

[0408] Preferably, the kit may further comprise a tag and a capture partner. Preferably, the kit may comprise biotin and streptavidin. In some embodiments, the kit may comprise streptavidin-coated beads or microparticles, preferably streptavidin-coated magnetic beads or microparticles.

[0409] Optionally, the kit may also include reagents such as buffers, wash solutions, distilled water, blocking agents, stop solutions, and the like.

[0410] Preferably, the kit may further include a stimulating agent as previously described herein. Preferably, it is for stimulating the detectable label of one or more reporter molecules or binding molecules. A suitable stimulating agent is a molecule that stimulates a detectable label, which may preferably be part of one or more reporter molecules or binding molecules. A suitable stimulating agent 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 for use with a particular enzyme label are generally selected to produce a detectable color change upon hydrolysis 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 commonly used. Preferably, a fluorogenic substrate that generates a fluorescent product may be used. Preferably, the stimulating agent may be a reactive agent such as an electron donor. Preferably, it is an amine compound such as tripropylamine or dibutylethanolamine.

[0411] Alternatively, the kit may further comprise one or more labels for detecting the one or more reporter molecules or binding molecules. Preferably, the labels are detectable. Preferably, the one or more labels specifically bind to the one or more reporter molecules or binding molecules. Suitable labels are discussed herein above. Preferably, the kit may comprise a detectable label for the oxidized IL-33 complex, and / or a detectable label for the reduced IL-33 complex, and / or a detectable label for the IL-33 / sST2 complex.

[0412] Preferably, the kit may further comprise a means for performing the assay. The means for performing the assay may preferably comprise a container such as a test tube, an Eppendorf® tube, or a plate. Preferably, when the kit comprises a solid support, the solid support may also function as a container for performing the assay. Preferably, the kit comprises one or more microwell plates. Preferably, the kit comprises one or more microwell plates to which one or more binding molecules are immobilized.

[0413] In one example, there is further provided a support comprising one or more immobilized binding molecules, suitably the one or more binding molecules being as defined according to the fourth, fifth or sixth aspect of the present disclosure.

[0414] Suitably, the kit may further comprise a separation means. Suitably, the separation means is for purifying the sample and / or isolating one or more antigenically distinct forms of IL-33 from the sample. Suitable means for purification or isolation may include filters, columns, beads, etc.

[0415] Preferably, the kit may further comprise instructions, preferably instructions to instruct the user how to use the kit to perform the assay, preferably instructions to instruct the user how to perform the method of the present disclosure.

[0416] In one embodiment, the kit comprises: (i) an oxidized IL-33 binding molecule and a reporter molecule that binds to the oxidized IL-33-binding molecule complex, and / or (ii) a reduced IL-33 binding molecule and a reporter molecule that binds to the reduced IL-33-binding molecule complex, and / or (iii) an IL-33 / sST2 binding molecule and a reporter molecule that binds to an IL-33 / sST2-binding molecule complex, wherein each binding molecule comprises a biotin tag and each reporter molecule comprises a chemiluminescent label; (iv) streptavidin-coated magnetic beads; (v) a reactive agent capable of activating a chemiluminescent label in the presence of a current / voltage; Includes.

[0417] Preferably, the chemiluminescent label is a ruthenium compound and the reactant is tripropylamine or dibutylethanolamine.

[0418] Preferably, the kit may further comprise a magnetic electrode.

[0419] The present disclosure will now be described with reference to the following non-limiting examples. [Example]

[0420] Hybridoma campaign for the production of binding and reporter molecules Monoclonal antibodies were produced by hybridoma technology from wild-type outbred mice. 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 in Fab format complexed with MEDI3506. AB1070012 (Mouse IgG1) AB1070019 (Mouse IgG2b) AB1070141 (Mouse IgG1) AB1070008 (Mouse IgG1)

[0421] Using a repetitive immunization at multiple sites (RIMS) strategy, CD1 mice were immunized as follows. -4 days before: Preliminary blood collection Day -0: Primary immunization Day -7: Second booster Day -13: First blood draw Day -15: 3rd booster Day -20: Second blood draw Day -22: 4th booster Day -24: 5th booster Day -28: Final blood draw and collection of spleen and lymph nodes

[0422] For immunization, 12 mice were divided into two groups of 6. Animals were immunized with human recombinant IL-33 or human recombinant IL-33 complexed with MEDI3506 Fab.

[0423] Lymphoid cells were collected from immunized mice and mixed with Sp2 / 0 Ag14 myeloma cells to form hybridomas using electrofusion. After fusion, the cells were resuspended in semisolid medium containing the selection reagents hypoxanthine / azaserine and anti-mouse IgG FITC-conjugated antibody.

[0424] Hybridoma clones were grown for 13–17 days, and then FITC-positive IgG-expressing clones were harvested from the semi-solid medium using a ClonePixFL robot and transferred into liquid culture medium. After 3–7 days of growth, IgG-containing supernatants were screened for binding using a homogeneous time-resolved fluorescence (HTRF) binding assay. Positive hybridomas were overgrown in 24-well plates, and the supernatants were purified using ProPlus Phynexus chips to confirm their binding properties. cDNA was prepared from selected hybridomas, and the antibody variable heavy (VH) and light (VL) chain sequences were determined. The VH domain was cloned into an expression vector containing a mouse heavy chain constant domain equivalent to that of the original hybridoma cell line (i.e., mouse IgG1, except for AB1070019, which was mouse IgG2b). The VL domain was cloned into an expression vector containing a mouse kappa constant domain. To produce purified recombinant IgG, CHO cells were transiently transfected with the heavy and light chain IgG expression vectors. IgG was purified from the culture supernatant using a MabSelect Sure column on an AKTAxpress. The amino acid sequences of each of the antibody variable domains are shown below.

[0425] [Table 1]

[0426] Development of assays for the detection of different IL-33 forms Reduced IL-33 Briefly, recombinant reduced IL-33 (112-270 amino acids) was expressed in Escherichia coli and purified to homogeneity (as described in WO 216 / 156440(A1) and Cohen et al., 2015).

[0427] Oxidized IL-33 Oxidized IL-33 (oxIL-33) was produced from a stock of reduced IL-33 with an N-terminal His-Avi tag. Reduced IL-33 was diluted to a final concentration of 0.5 mg / ml in 60% IMDM medium (phenol red-free), 40% DPBS, and oxidized by overnight incubation at 37°C. Aggregates generated during the oxidation process were removed from the sample by loading the sample onto a HiTrap Q HP anion exchange column. Under these loading conditions, aggregates bound to the column, while monomeric oxIL-33 flowed through and was collected. The tag was cleaved from oxIL-33 by incubation with factor Xa at a final concentration of 10 U per mg of oxIL-33 for 120 minutes at room temperature. To deplete the remaining reduced IL-33 from the sample, soluble human ST2 extracellular domain fused to human IgG1 Fc was incubated with the sample and allowed to bind to the 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, which separated 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 ) was determined.

[0428] IL-33 / sST2 complex The hST2 extracellular domain sequence with a C-terminal Flag-His10 tag was expressed in suspension HEK Expi293F cells by transient transfection with a plasmid encoding the hST2Flag His10 sequence using Expifectamine. After 6–7 days, cells were harvested by centrifugation, and the supernatant was clarified by filtration. Protein was purified from the clarified supernatant using affinity capture chromatography (HisTrap Excel). Eluted monomeric fractions with a molecular weight of approximately 38.7 kDa were pooled. Protein concentration was determined from its OD280 nm reading on a Nanodrop using an extinction coefficient (unit) of 1 AU = 0.77 mg / ml.

[0429] The IL-33 / sST2 complex was prepared by incubating reduced IL-33 with sST2-FH at a molar ratio of 1:10 for 15 minutes.

[0430] MSD Assay - Standard Curve MSD standard binding plates were coated with 50 μl of 5 μg / ml capture antibody in PBS for approximately 18 hours, washed in 300 μl of 3× PBS / 0.05% Tween-20 (PBS-T), incubated in 200 μl of PBS / 1% BSA for 1 hour, and washed in 3× PBS-T. Recombinant human IL-33 standards were diluted in PBS-T containing 0.1% BSA, and 25 μl was added to wells and incubated for 2 hours. Plates were washed in 3× PBS-T, incubated in 50 μl of 1 μg / ml sulfo-tagged detection antibody for 2 hours, washed in 3× PBS-T, incubated in 150 μl of MSD 1× read buffer for 10 minutes, and read.

[0431] All standards and samples were performed in duplicate. Incubation steps were performed at room temperature on a shaking table (450 rpm). All assays were read on an MSD Discovery Workbench Sector S600 reader. Graphs of concentration-dependent binding curves are shown in Figure 1. For reduced IL-33 (Figure 1A) and oxidized IL-33 (Figure 1B), the assay reagents showed no detectable binding to the IL33 / sST2 complex (data not shown). Both AB1070012 and AB1070069 were found to have comparable performance for detecting reduced IL-33 (Figure 5).

[0432] [Table 2]

[0433] MSD S-PLEX assays® for reduced IL-33, oxIL-33, and IL-33 / sST2 complexes The MSD S-PLEX assay® was performed at MSD (Gaithersburg, USA) using S-PLEX® technology with the same antibody pairs against each form of IL-33 as described above for the standard MSD assay.

[0434] [Table 3] ND - Not Detected

[0435] The estimated LLOD is calculated as the concentration from the standard curve that produces a signal 2.5 standard deviations above the diluent alone (buffer only).

[0436] MSD Assays - Biological Samples All biological samples were stored in aliquots at -80°C after collection.

[0437] serum Serum samples from healthy individuals, asthmatics (GINA stages 1-2), and COPD patients (GOLD stages 1-2) were obtained from a commercial supplier (Seralabs / BioIVT, UK).

[0438] Nasal mucosal lining fluid Mucosal lining fluid (MLF) was collected from atopic subjects at baseline (-30 min, -15 min) and after a single nasal challenge with Timothy grass pollen (Aquagen, Denmark) (5–480 min) as previously described (Leaker et al., 2016). MLF was collected on a synthetic absorbent matrix (SAM, Hunts, UK) and eluted from the SAM with 330 μl of PBS / 0.1% Tween-20 / 0.1% BSA.

[0439] Normal human bronchial epithelial cell (NHBE) culture supernatant NHBE cells (Cambrex, UK) were cultured as previously described (Scott et al., 2018). NHBE cells were plated overnight in 96-well plates (50K) and supernatants were collected between 1 and 1440 minutes after scratch wounding (Woundmaker, Essen Bioscience).

[0440] Air-liquid interface (ALI) culture supernatant Basolateral supernatants of ALI cultures were collected 24 h after changing the culture medium.

[0441] MSD standard binding plates were coated with 50 μl of 5 μg / ml capture antibody in PBS for approximately 18 hours, washed with 300 μl of 3×PBS / 0.05% Tween-20 (PBS-T), incubated in 200 μl of PBS / 1% BSA for 1 hour, and washed with 3×PBS-T. Biological samples were diluted in PBS-T containing 0.1% BSA (minimum dilution 1:2), and 25 μl was added to wells and incubated for 2 hours. The plates were washed with 3×PBS-T, incubated in 50 μl of 1 μg / ml sulfo-tagged detection antibody for 2 hours, washed with 3×PBS-T, and incubated in 150 μl of MSD 1× read buffer for 10 minutes and read. The concentrations of IL-33 forms in the biological samples were extrapolated from the recombinant protein standard curve.

[0442] The results demonstrate that the assay can be used to detect red IL-33, oxidized IL-33, and IL33 / sST2 from a variety of surrogate biological samples (Figures 2A-2D).

[0443] Use of the assay to detect IL-33 / sST2 complexes in patients and monitor response to treatment Serum samples from patients infected with SARS-CoV-2 were obtained from patients enrolled in the Phase 2a ACCORD trial (EudracT number: 2020-001736-95, Wilkinson et al., 2020). Patients were recruited over two time periods during the pandemic, as shown in Figure 3.

[0444] Patients were randomized to receive either standard of care (SoC) alone or in combination with anti-IL-33 antibody (tozolaximab) treatment (300 mg intravenously (IV) with an optional second dose if invasively ventilated on day 15) for 29 days. The 300 mg IV dose was administered by the following procedure: 2 ml of tozolaximab 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 an IV flush with 5 ml of saline.

[0445] The SoC changed over the two periods of the ACCORD trial as our understanding of the SARS-CoV-2 virus evolved. The SoC used during each period is shown in Table 4 below. The SoC treatment in the second period of the ACCORD trial more accurately reflects current best practices.

[0446] [Table 4]

[0447] Sera from patients receiving tozolaximab plus standard of care (n=46 at baseline, n=37 at day 5, n=10 at day 10) and patients receiving SoC alone (n=37 at baseline, n=23 at day 5, n=10 at day 10) were measured for IL-33 / sST2 at a 1:4 dilution.

[0448] All serum samples were stored in aliquots at -80°C after collection.

[0449] To measure levels of IL-33 / sST2 in serum, the MSD S-PLEX assay was performed on serum samples obtained from patients at MSD (Gaithersburg, USA) using S-PLEX technology, which uses the antibody pair against IL-33 / sST2 as described above.

[0450] 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. All incubations required plate shaking at room temperature (RT) unless otherwise noted. Plates were washed three times with wash buffer (phosphate-buffered saline (PBS) / 0.05% Tween-20) as described. Biotin-coated capture mAb (Table 5) was diluted in Diluent 100 (MSD) with S-Plex Coating Reagent (MSD) to form the coating solution. Assay plates (MSD) were coated with 50 μl / well of the coating solution and incubated for 1 hour. Blocking reagent / solution was prepared by diluting S-Plex Blocker Reagent (MSD) in Diluent 101 (MSD). A standard curve for recombinant IL-33 / sST2 complex was generated by diluting the stock to the highest standard concentration (Table 5) in Diluent 100 (MSD) and then performing four-fold serial dilutions. 25 μl of blocking reagent was added to all wells, and 25 μl of standards and samples were added to the wells and incubated for 1.5 hours. TURBO-boost solution was prepared by diluting TURBO-boost-labeled detection mAb (Table 5) to the working concentration in Diluent 3 (MSD). The plate was washed, and 50 μl of TURBO-boost detection mAb solution was added per well and incubated for 1 hour. The plate was washed, and 50 μl of enhancement solution (MSD) was added per well and incubated for 30 minutes. The plate was washed, and 50 μl of detection solution (MSD) was added per well and incubated for 1 hour at 26°C. The plate was washed, and 150 μl of 1× read buffer A (MSD) was added to each well. Plates were read on an MSD MESO SECTOR S600 instrument.

[0451] [Table 5] *Data are expressed in units / ml for IL-33 / sST2 complex, which can be approximated to pg / ml if assuming 100% complex formation for the IL-33 / sST2 standard.

[0452] The MSD S-PLEX assay was sensitive enough to determine pg / ml levels of IL-33 / sST2 in patient serum.

[0453] Subgroup analysis of the endpoint "death or respiratory failure on day 29" was performed based on the median baseline IL-33 / sST2 level. The median baseline level was 30.15 U / ml, and subgroups were defined as low IL-33: baseline IL-33 / sST2 < 30 / 15 U / ml; high IL-33: baseline IL-33 / sST2 ≥ 30.15 U / ml.

[0454] Within each subgroup, the proportion of subjects who died by day 29 or experienced respiratory failure at day 29 was calculated for each treatment, and the relative risk (tozolaximab:placebo) was calculated for each subgroup, along with its 80% confidence interval. Additionally, a logistic regression model was applied to each subgroup, adjusting for age (continuous variable) and baseline severity (WHO score of 3 or 4 vs. 5). From this logistic regression model, the odds ratio (tozolaximab:placebo) of experiencing death or respiratory failure at day 29, along with its 80% confidence interval, was calculated for each subgroup.

[0455] Using this assay, it was determined that patients with elevated baseline serum IL-33 / sST2 levels of 30.15 pg / ml or greater were significantly less likely to die or experience respiratory failure after 29 days when treated with tozolaximab, as shown in Table 6 and Figure 4.

[0456] Such patients form a subgroup that is likely to respond well to IL-33 axis antagonist treatment, providing a method for stratifying and selecting patients for treatment with IL-33 axis antagonists.

[0457]

Table 6

[0458]

Table 7-1

[0459]

Table 7-2

Claims

1. A method for detecting one or more antigenically distinct forms of IL-33 in a biological sample, (a) The sample is brought into contact with a binding molecule capable of binding to IL-33 / sST2 under conditions sufficient to form a complex, (b) detecting the level of the IL-33 / sST2 complex in the sample, The binding molecule that binds to IL-33 / ST2 comprises variable heavy domains having VHCDR1-3 of SEQ ID NOs. 17, 18, and 19, and variable light domains having VLCDR1-3 of SEQ ID NOs. 21, 22, and 23, respectively, wherein one or more CDRs have three or fewer single amino acid substitutions, deletions, or insertions.

2. The method according to claim 1, wherein the binding molecule that binds to IL-33 / ST2 comprises a variable heavy domain having VHCDR1-3 of SEQ ID NOs. 17, 18, and 19, and a variable light domain having VLCDR1-3 of SEQ ID NOs. 21, 22, and 23, respectively.

3. The method according to claim 1 or 2, wherein the binding molecule is a capture probe.

4. The method according to claim 3, further comprising the step of contacting the complex with one or more reporter molecules capable of binding to one or more of the complexes.

5. The method according to claim 1 or 2, wherein, prior to step (a), a step is performed to immobilize the IL-33 / sST2 by bringing the sample into contact with the solid surface under conditions suitable for bonding the IL-33 / sST2 to the solid surface.

6. The method according to claim 5, wherein the detection step (b) includes detecting the level of a bound reporter molecule in the sample.

7. An assay kit for detecting IL-33 / sST2 in biological samples, (a) IL-33 / sST2 binding molecule, and (b) A reporter molecule that can bind to the IL-33 / sST2 binding molecule complex, Assay kit including

8. The kit according to claim 7, wherein the binding molecule that binds to IL-33 / ST2 comprises a variable heavy domain having VHCDR1-3 of SEQ ID NOs. 17, 18, and 19, and a variable light domain having VLCDR1-3 of SEQ ID NOs. 21, 22, and 23, and one or more CDRs have three or fewer single amino acid substitutions, deletions, or insertions.

9. The kit according to claim 7 or 8, wherein the binding molecule that binds to IL-33 / ST2 comprises a variable heavy domain having VHCDR1-3 of SEQ ID NOs. 17, 18, and 19, and a variable light domain having VLCDR1-3 of SEQ ID NOs. 21, 22, and 23, respectively.

10. An antibody or antigen-binding fragment thereof that binds to IL-33 / sST2, comprising a variable heavy domain having VHCDR1-3 of SEQ ID NOs. 17, 18, and 19, and a variable light domain having VLCDR1-3 of SEQ ID NOs. 21, 22, and 23, respectively, wherein one or more CDRs have three or fewer single amino acid substitutions, deletions, or insertions.

11. An antibody or antigen-binding fragment thereof that binds to IL-33 / sST2, comprising a variable heavy domain having VHCDR1-3 of SEQ ID NOs. 17, 18, and 19, and a variable light domain having VLCDR1-3 of SEQ ID NOs. 21, 22, and 23, respectively, according to claim 10.

12. The antibody or antigen-binding fragment thereof according to claim 10 or 11, conjugated with a detectable label.

13. The kit according to claim 7 or 8, the method according to claim 1 or 2, and the antibody or antigen-binding fragment according to claim 10 or 11, wherein the binding molecule that binds to IL-33 / sST2, or the antibody or antigen-binding fragment thereof, comprises a variable heavy domain having at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% the same amino acid sequence as the VH described in SEQ ID NO: 20, and / or a variable light domain having at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% the same amino acid sequence as the VL described in SEQ ID NO: 24.

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