Anti-interleukin-33 antibodies and uses thereof

Abstract

The present invention provides interleukin-33 (IL-33) antibodies and methods of making and using the same, for example, for the detection of IL-33 in biological samples.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 277,953, filed November 10, 2021, and U.S. Provisional Application No. 63 / 413,206, filed October 4, 2022, the contents of each of which are incorporated by reference in their entirety herein.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy, created on November 1, 2022, is named 50474-267WO3_Sequence_Listing_11_1_22.xml and is 38,330 bytes in size.

[0003] FIELD OF THEINVENTION The present invention relates to anti-interleukin-33 (IL-33) antibodies, and methods of making and using same, including the detection of IL-33 in biological samples. [Background technology]

[0004] Interleukin-33 (IL-33) is a member of the interleukin-1 (IL-1) cytokine family, encoded by the IL33 gene, and is expressed in structural cells such as smooth muscle, epithelium (e.g., retinal pigment epithelial (RPE) cells), endothelial cells (e.g., choroidal endothelial cells), Müller cells, and astrocytes. IL-33 can be induced by inflammatory factors in macrophages and dendritic cells. During the past decades, human IL-33 (hIL-33) has emerged as an important contributor to the pathogenesis of numerous inflammatory diseases. Cellular stress caused by environmental factors, such as allergens, toxins, and pathogens, can lead to IL-33 release. For example, IL-33 is released from Müller cells upon phototoxic stress and induces CCL2- and monocyte-dependent retinal alterations in preclinical models. Bioavailable IL-33 binds to the heterodimeric IL-33 receptor complex composed of suppressor of tumorigenicity 2 (ST2) protein and interleukin-1 receptor accessory protein (IL-1RAcP) to activate the AP-1 and NF-κB pathways through the adaptor proteins myeloid differentiation primary response 88 (MyD88) and potentially MyD88 adaptor-like (Mal) protein. IL-33 stimulates multiple cell types, including innate type II (ILC2) cells, mast cells, basophils, eosinophils, and dendritic cells, to promote type 2 immunity.

[0005] Although there are multiple commercially available hIL-33 assays that span multiple platforms, the ability to provide precise hIL-33 concentration measurements in various matrices and distinguish between active (reduced form) hIL-33 and inactive (oxidized form) hIL-33 remains uncertain. This is particularly true for relatively small sample volumes, matrices with low hIL-33 concentrations, and matrices with elevated levels of interleukin 1 receptor-like 1 (sST2), and for inactive forms of ST2 that compete with membrane-bound ST2 for hIL-33 binding. Precise measurement of active human IL-33 (hIL-33) in biological matrices remains difficult for at least the following reasons: 1) in non-diseased to moderate disease states, hIL-33 is often present at undetectable to low levels (< pg / mL), 2) hIL-33 exists in different structural forms (reduced vs. oxidized), and 3) hIL-33 may be either unbound or bound to human sST2 (hsST2), which hinders its detection.

[0006] There is a need for a precise and sensitive assay to detect all forms of IL-33, whether unbound or bound to hsST2. SUMMARY OF THE INVENTION

[0007] The present invention relates in particular to anti-IL-33 antibodies and methods for their production and use. The present disclosure provides antibodies having high sensitivity (e.g., binding affinity) for all forms of IL-33, both unbound and bound, as well as methods, assays, and kits that include and use said antibodies.

[0008] In one aspect, the invention features an isolated antibody or antigen-binding fragment thereof that specifically binds to IL-33, the antibody comprising a binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, the binding domain comprising the following six complementarity determining regions (CDRs): (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:1; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:2; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:3; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:5; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6.

[0009] In some aspects, the VH domain comprises the following framework regions (FR): (a) FR-H1 comprising the amino acid sequence of SEQ ID NO: 11; (b) FR-H2 comprising the amino acid sequence of SEQ ID NO: 12; (c) FR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and (d) FR-H4 comprising the amino acid sequence of SEQ ID NO: 14.

[0010] In some aspects, the VL domain comprises the following FRs: (a) FR-L1 comprising the amino acid sequence of SEQ ID NO: 15; (b) FR-L2 comprising the amino acid sequence of SEQ ID NO: 16; (c) FR-L3 comprising the amino acid sequence of SEQ ID NO: 17; and (d) FR-L4 comprising the amino acid sequence of SEQ ID NO: 18.

[0011] In some aspects, the antibodies disclosed herein comprise: (a) a VH domain comprising an amino acid sequence having at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 7; (b) a VL domain comprising an amino acid sequence having at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 8; or (c) the VH domain of (a) and the VL domain of (b).

[0012] In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:7.

[0013] In some embodiments, the VL domain comprises the amino acid sequence of SEQ ID NO:8.

[0014] In one aspect, the present invention provides an isolated antibody that specifically binds to IL-33 or an antigen-binding fragment thereof, the antibody comprising a binding domain comprising (a) a VH domain comprising the amino acid sequence of SEQ ID NO:7 and (b) a VL domain comprising the amino acid sequence of SEQ ID NO:8.

[0015] In some aspects, the antibodies disclosed herein comprise: (b) a heavy chain (HC) having at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 9; (b) a light chain (LC) having at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 10; or (c) the HC of (a) and the LC of (b).

[0016] In some embodiments, the HC comprises the amino acid sequence of SEQ ID NO:9.

[0017] In some embodiments, the LC comprises the amino acid sequence of SEQ ID NO:10.

[0018] In one aspect, the present invention provides an isolated antibody, or antigen-binding fragment thereof, that specifically binds to IL-33, the antibody comprising (a) an HC domain comprising the amino acid sequence of SEQ ID NO: 9, and (b) an LC comprising the amino acid sequence of SEQ ID NO: 10.

[0019] In one aspect, the present invention provides an isolated antibody that specifically binds to IL-33 or an antigen-binding fragment thereof, the antibody comprising a binding domain comprising the following six HVRs: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24.

[0020] In some embodiments, the VH domain comprises the following FRs: (a) FR-H1 comprising the amino acid sequence of SEQ ID NO: 29; (b) FR-H2 comprising the amino acid sequence of SEQ ID NO: 30; (c) FR-H3 comprising the amino acid sequence of SEQ ID NO: 31; and (d) FR-H4 comprising the amino acid sequence of SEQ ID NO: 32.

[0021] In some aspects, the VL domain comprises the following FRs: (a) FR-L1 comprising the amino acid sequence of SEQ ID NO: 33; (b) FR-L2 comprising the amino acid sequence of SEQ ID NO: 34; (c) FR-L3 comprising the amino acid sequence of SEQ ID NO: 35; and (d) FR-L4 comprising the amino acid sequence of SEQ ID NO: 36.

[0022] In some aspects, the antibodies disclosed herein comprise: (a) a VH domain having at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 25; (b) a VL domain having at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 26; or (c) the VH domain of (a) and the VL domain of (b).

[0023] In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:25.

[0024] In some embodiments, the VL domain comprises the amino acid sequence of SEQ ID NO:26.

[0025] In one aspect, the present invention provides an isolated antibody that specifically binds to IL-33, or an antigen-binding fragment thereof, the antibody comprising a binding domain comprising (a) a VH domain comprising the amino acid sequence of SEQ ID NO:25 and (b) a VL domain comprising the amino acid sequence of SEQ ID NO:26.

[0026] In some aspects, the antibody comprises (a) a HC having at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 27; (b) a LC having at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 28; or (c) a VH of (a) and a LC of (b).

[0027] In some embodiments, the HC comprises the amino acid sequence of SEQ ID NO:27.

[0028] In some embodiments, the LC comprises the amino acid sequence of SEQ ID NO:28.

[0029] In one aspect, the present invention provides an isolated antibody, or antigen-binding fragment thereof, that specifically binds to IL-33, the antibody comprising (a) an HC domain comprising the amino acid sequence of SEQ ID NO:27, and (b) an LC comprising the amino acid sequence of SEQ ID NO:28.

[0030] In some aspects, the antibody binds to oxidized or reduced IL-33.

[0031] In some aspects, the antibody has a 2-fold (e.g., within 1.5-fold or within 1-fold) K affinity for oxidized IL-33 and reduced IL-33. D In some aspects, the antibody binds oxidized IL-33 and reduced IL-33 with substantially the same K D Combine with.

[0032] In some embodiments, the antibody binds to reduced IL-33. DIn some embodiments, the antibody binds reduced IL-33 with an affinity of between 0.01 nM and 1 nM (e.g., between 0.01 and 0.9 nM, between 0.01 and 0.8 nM, between 0.01 and 0.7 nM, between 0.01 and 0.6 nM, between 0.01 and 0.5 nM, between 0.05 and 0.9 nM, between 0.1 and 0.9 nM, between 0.2 and 0.9 nM, between 0.3 and 0.9 nM, between 0.2 and 0.6 nM, between 0.3 and 0.5 nM, between 0.3 and 0.4 nM, or between 0.4 and 0.5 nM; e.g., about 0.05 nM, about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0.325 nM, about 0.34 nM, about 0.35 nM, about 0.4 nM, about 0.45 nM, about 0.475 nM, about 0.49 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, or about 1 nM). D In some embodiments, the antibody binds reduced IL-33 at K D In some embodiments, the antibody binds reduced IL-33 with a K between 0.1 nM and 0.5 nM (e.g., between 0.2 and 0.5 nM, between 0.3 nM and 0.5 nM, between 0.3 nM and 0.4 nM, or between 0.4 nM and 0.5 nM). D In some embodiments, the antibody binds reduced IL-33 with a K of about 15 pM. D In some embodiments, the antibody binds reduced IL-33 with a K of about 0.338 nM. D Combine with.

[0033] In some embodiments, the antibody binds to human IL-33.

[0034] In some embodiments, the antibody has a K within 2-fold (e.g., within 1.5-fold or within 1-fold) of IL-33 bound to soluble interleukin-1 receptor-like 1 (sST2) protein and IL-33 not bound to sST2. D In some embodiments, the antibody binds to IL-33 bound to sST2 protein with substantially the same K as IL-33 that is not bound to sST2. D In some embodiments, the sST2 is human sST2 (hsST2).

[0035] In some forms, the antibody is a monoclonal antibody.

[0036] In some aspects, the antibody is a chimeric antibody.

[0037] In some embodiments, the antibody is a rat antibody.

[0038] In some embodiments, the antibody is an IgG antibody.

[0039] In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is a human IgG1 antibody.

[0040] In some embodiments, the antibody is an IgG2a antibody. In some embodiments, the antibody is a rat IgG2a antibody.

[0041] In some embodiments, the antibody is an antibody fragment that specifically binds to IL-33.

[0042] In some aspects, the antibody fragment is selected from the group consisting of a Fab, a single chain variable fragment (scFv), Fv, Fab', Fab'-SH, F(ab')2, and a diabody.

[0043] In one aspect, the invention is an isolated nucleic acid encoding any one of the antibodies disclosed herein, or a set of isolated nucleic acids which together encode any one of the antibodies disclosed herein.

[0044] In one aspect, the invention is a vector or a set of vectors comprising an isolated nucleic acid or a set of isolated nucleic acids disclosed herein.

[0045] In one aspect, the present invention provides a host cell comprising any one of the vectors or any one of the sets of vectors described herein. In some aspects, the host cell is a mammalian cell. In some aspects, the cell is a rat cell. In some aspects, the mammalian cell is a Chinese Hamster Ovary (CHO) cell. In some aspects, the host cell is a prokaryotic cell. In some aspects, the prokaryotic cell is E. coli.

[0046] In another aspect, the invention provides a method of producing an antibody that specifically binds to IL-33, the method comprising culturing in a medium any one of the host cells disclosed herein. In some aspects, the method further comprises recovering the antibody from the host cell or the medium.

[0047] In one aspect, the invention provides an immunoconjugate comprising any one of the antibodies described herein.

[0048] In one aspect, the invention provides any one of the antibodies or immunoconjugates described herein for use in detecting the presence or level of IL-33 in a biological sample. In one aspect, the invention provides any one of the antibodies described herein for use in detecting the presence or level of IL-33 in a biological sample. In one aspect, the invention provides any one of the immunoconjugates described herein for use in detecting the presence or level of IL-33 in a biological sample.

[0049] In some embodiments, detection is by immunohistochemistry (IHC), immunofluorescence (IF), flow cytometry, enzyme-linked immunosorbent assay (ELISA), immunoblotting, or immunopolymerase chain reaction (iPCR). In some embodiments, detection is by ELISA. In some embodiments, detection is by iPCR.

[0050] In some aspects, the biological sample is a blood sample, an ocular sample, a nasal sample, or a cell culture sample. In some aspects, the blood sample is a plasma or serum sample. In some aspects, the ocular sample is a vitreous or aqueous humor sample. In some aspects, the ocular sample is a tissue sample. In some aspects, the nasal sample is a nasal absorption fluid sample.

[0051] In some embodiments, the biological sample is from a subject having or at risk for an IL-33 mediated disorder, hi some embodiments, the IL-33 mediated disorder is an eye disorder, an inflammatory condition, an immune disease, a fibrotic disorder, an eosinophilic disorder, an infection, pain, a central nervous system disorder, or a solid tumor.

[0052] In some embodiments, the ocular disorder is age-related macular degeneration (AMD), ocular retinopathy, polypoidal choroidal vasculopathy (PCV), diabetic macular edema, dry eye disease, Behcet's disease, retinal detachment, glaucoma, uveitis, retinitis pigmentosa, Leber's congenital amaurosis, Stargardt's disease, traumatic eye injury, or conjunctivitis. In some embodiments, the AMD is geographic atrophy (GA), wet AMD, or dry AMD. In some embodiments, the AMD is GA. In some embodiments, the AMD is intermediate AMD or advanced AMD. In some embodiments, the ocular retinopathy is diabetic retinopathy (DR) or retinopathy of prematurity (ROP). In some embodiments, the ocular retinopathy is high altitude DR. In some embodiments, the conjunctivitis is infectious conjunctivitis or non-infectious conjunctivitis. In some embodiments, the conjunctivitis is allergic conjunctivitis.

[0053] In some embodiments, the inflammatory condition is asthma, sepsis, septic shock, atopic dermatitis, allergic rhinitis, rheumatoid arthritis, or chronic obstructive pulmonary disease (COPD).

[0054] In some embodiments, the immune disorder is asthma, rheumatoid arthritis, allergy, anaphylaxis, anaphylactic shock, allergic rhinitis, psoriasis, inflammatory bowel disease (IBD), Crohn's disease, diabetes, or liver disease.

[0055] In some embodiments, the fibrotic disease is idiopathic pulmonary fibrosis (IPF).

[0056] In some embodiments, the eosinophilic disorder is eosinophil-associated gastrointestinal disorder (EGID). In some embodiments, the EGID is eosinophilic esophagitis.

[0057] In some embodiments, the subject is a human.

[0058] In some embodiments, the presence or level of IL-33 is the presence or level of total IL-33. In some embodiments, the presence or level of IL-33 is the presence or level of reduced IL-33. In some embodiments, the presence or level of IL-33 is the presence or level of human IL-33. In some embodiments, the presence or level of IL-33 is the presence or level of unbound IL-33. In some embodiments, the presence or level of IL-33 is the presence or level of IL-33 bound to sST2. In some embodiments, sST2 is hsST2.

[0059] In one aspect, the invention provides a method of detecting the presence or level of IL-33 in a biological sample comprising contacting the biological sample with any one of the antibodies or immunoconjugates described herein and detecting the presence of bound antibody or immunoconjugate. In one aspect, the invention provides a method of detecting the presence or level of IL-33 in a biological sample comprising contacting the biological sample with any one of the antibodies described herein and detecting the presence of bound antibody. In one aspect, the invention provides a method of detecting the presence or level of IL-33 in a biological sample comprising contacting the biological sample with any one of the immunoconjugates described herein and detecting the presence of the immunoconjugate.

[0060] In one aspect, the present invention provides a method of detecting the presence or level of IL-33 in a biological sample comprising contacting the biological sample with any one of the immunoconjugates described herein in the presence of a second anti-IL-33 antibody that specifically binds to an epitope on IL-33 that is different from the epitope specifically bound by the anti-IL-33 antibody in the immunoconjugate, and detecting the presence of the bound immunoconjugate. In one aspect, the second anti-IL-33 antibody is 1E1v8. In one aspect, the biological sample is treated with tris(2-carboxyethyl)phosphine (TCEP) prior to said contacting.

[0061] In some embodiments, detecting is by IHC, IF, flow cytometry, ELISA, immunoblotting, or iPCR. In some embodiments, detecting is by ELISA. In some embodiments, detecting is by iPCR.

[0062] In some embodiments, the biological sample is a blood sample, an ocular sample, a nasal sample, or a cell culture sample. In some embodiments, the blood sample is a plasma or serum sample. In some embodiments, the ocular sample is a vitreous or aqueous humor sample. In some embodiments, the ocular sample is a tissue sample. In some embodiments, the nasal sample is a nasal fluid (NF) sample.

[0063] In some embodiments, the biological sample is from a subject having or at risk for an IL-33 mediated disorder, hi some embodiments, the IL-33 mediated disorder is an eye disorder, an inflammatory condition, an immune disease, a fibrotic disorder, an eosinophilic disorder, an infection, pain, a central nervous system disorder, or a solid tumor.

[0064] In some embodiments, the ocular disorder is AMD, ocular retinopathy, PCV, diabetic macular edema, dry eye disease, Behcet's disease, retinal detachment, glaucoma, uveitis, retinitis pigmentosa, Leber's congenital amaurosis, Stargardt's disease, traumatic eye injury, or conjunctivitis. In some embodiments, the AMD is GA, wet AMD, or dry AMD. In some embodiments, the AMD is GA. In some embodiments, the AMD is intermediate AMD or advanced AMD. In some embodiments, the ocular retinopathy is DR or ROP. In some embodiments, the ocular retinopathy is high altitude DR. In some embodiments, the conjunctivitis is infectious conjunctivitis or non-infectious conjunctivitis. In some embodiments, the conjunctivitis is allergic conjunctivitis.

[0065] In some embodiments, the inflammatory condition is asthma, sepsis, septic shock, atopic dermatitis, allergic rhinitis, rheumatoid arthritis, or COPD.

[0066] In some embodiments, the immune disorder is asthma, rheumatoid arthritis, allergy, anaphylaxis, anaphylactic shock, allergic rhinitis, psoriasis, IBD, Crohn's disease, diabetes, or liver disease.

[0067] In some embodiments, the fibrotic disease is IPF.

[0068] In some embodiments, the eosinophilic disorder is EGID. In some embodiments, the EGID is eosinophilic esophagitis.

[0069] In some embodiments, the subject is a human.

[0070] In some embodiments, the presence or level of IL-33 is the presence or level of total IL-33. In some embodiments, the presence or level of IL-33 is the presence or level of reduced IL-33. In some embodiments, the presence or level of IL-33 is the presence or level of human IL-33. In some embodiments, the presence or level of IL-33 is the presence or level of unbound IL-33. In some embodiments, the presence or level of IL-33 is the presence or level of IL-33 bound to sST2. In some embodiments, the cytokine is hsST2.

[0071] In some aspects, the methods further include selecting a treatment comprising an IL-33 axis binding antagonist for the subject based on the presence or level of IL-33 in the biological sample.

[0072] In some embodiments, the methods further comprise administering to the subject a therapeutically effective amount of an IL-33 axis binding antagonist.

[0073] In some embodiments, the IL-33 axis binding antagonist is an IL-33 binding antagonist. In some embodiments, the IL-33 binding antagonist is an anti-IL-33 antibody.

[0074] In one aspect, the invention provides an assay for identifying a subject having an IL-33 mediated disorder who is a candidate for a treatment comprising an IL-33 axis binding antagonist, the assay comprising determining the presence or level of IL-33 in a biological sample obtained from the subject using any one of the antibodies or immunoconjugates described herein. In one aspect, the invention provides an assay for identifying a subject having an IL-33 mediated disorder who is a candidate for a treatment comprising an IL-33 axis binding antagonist, the assay comprising determining the presence or level of IL-33 in a biological sample obtained from the subject using any one of the antibodies described herein. In one aspect, the invention provides an assay for identifying a subject having an IL-33 mediated disorder who is a candidate for a treatment comprising an IL-33 axis binding antagonist, the assay comprising determining the presence or level of IL-33 in a biological sample obtained from the subject using any one of the immunoconjugates described herein.

[0075] In one aspect, the invention provides a kit comprising: (a) any one of the antibodies described herein; and (b) a package insert containing instructions for using the antibody to detect the presence or level of IL-33 in a biological sample.

[0076] In one aspect, the invention provides a kit comprising: (a) any one of the immunoconjugates described herein; and (b) a package insert containing instructions for using the immunoconjugate to detect the presence or level of IL-33 in a biological sample. [Brief description of the drawings]

[0077] [Figure 1] Graph showing concentrations of hsST2 determined in vitreous fluid (n=22 samples) and serum (n=53 samples) from control donors. [Figure 2A]Images of SDS-PAGE gel analysis of hIL-33 diluted in phosphate buffered saline (PBS) (a) or 60% Iscove's modified Dulbecco's medium (IMDM) (b) and incubated at 37° C. Shown are samples taken at time intervals t=0 and t=18 hours. Protein samples were run under non-reducing and reducing conditions with dithiothreitol (DTT). Molecular weight standards were applied in lanes 3, 6 and 9. [Figure 2B] FIG. 1 is a set of mass spectrograms showing mass spectrometry of hIL-33 incubated at 37° C. in PBS at (a) t=0, (b) PBS at t=18 h, and (c) IMDM medium at t=18 h. The molecular weight (MW) of hIL-33 detected in each spectrum is indicated. The theoretical MW of hIL-33 with all four of its cysteine ​​residues fully reduced is 19857.19 Daltons (Da). For fully oxidized hIL-33, with all four cysteines forming two intradisulfide bonds, the theoretical molecular weight (MW) is 19853.16 Da. Panels (a) and (b) show hIL-33 with its cysteine ​​residues in a fully reduced state, and panel (c) shows the cysteine ​​residues of hIL-33 in an oxidized state. [Diagram 3] Graph showing comparison of hIL-33 standard curves established using eBioscience hIL-33 Platinum ELISA kit and hIL-33 with the kit (circles), in-house reduced hIL-33 (triangles), and oxidized hIL-33 (inverted triangles). Data are the mean ± SD of triplicates. [Figure 4]A is a graph showing a comparison of standard curves obtained for reduced hIL-33, oxidized hIL-33, reduced hIL-33 + 20 ng / mL hsST2, reduced hIL-33 + 40 ng / mL hsST2. The bottom line corresponds to the standard curve obtained for oxidized hIL-33. B is a graph showing a comparison of standard curves for reduced and oxidized hIL-33 in the presence of 20 ng / mL hsST2. Data are representative of three experiments performed in duplicate. C is a graph showing the specificity of the total hIL-33 ELISA determined by immunodepletion of reduced and oxidized hIL-33 diluted in human vitreous humor samples using beads conjugated to hIL-33 Ab. P=0.0019. Data are the mean ± SD of duplicates. [Diagram 5] A) is a graph showing multi-cycle kinetic sensorgrams of hIL-33 binding to rat anti-hIL-33 15.21C7 MAb captured at 25° C. as determined by BIACORE® surface plasmon resonance, and B) is a graph showing multi-cycle kinetic sensorgrams of hIL-33 binding to rat / human chimeric anti-hIL-33 3F10 MAb captured at 25° C. as determined by BIACORE® surface plasmon resonance. [Figure 6] (A) Standard curve of reduced hIL-33 obtained using iPCR in sample buffer. Data are the mean + SD of six independent standard curves, each performed in quadruplicate. (B) Specificity of the iPCR assay for reduced hIL-33, as determined by immunodepletion of reduced hIL-33 diluted in human vitreous samples using rat anti-reduced hIL-33 15.21C7 IgG2a-conjugated beads, rat anti-gp120 IgG2a-conjugated control beads, and sample buffer. P=0.0004 (1:2 vitreous), P=<0.0001 (1:4 vitreous). Data are the mean + SD of triplicates. [Figure 7]A is a graph showing the concentration of reduced hIL-33 in post-mortem human vitreous humor samples from 17 control and 19 AMD donors. P=0.0155. Data are the mean of duplicates. LLOQ=lower limit of quantification. B is a graph showing the concentration of reduced hIL-33 in post-mortem human AH samples from 10 control and 9 AMD donors. ns=not significant. Data are the mean of duplicates. LLOQ=lower limit of quantification. [Figure 8] A is a graph showing the concentration of reduced hIL-33 in human nasal absorbed fluid (NF) from 9 controls and 9 asthmatic patients. ns=not significant. Data are the mean of duplicates or quadruplicates. LLOQ=lower limit of quantification. B is a graph showing the concentration of reduced hIL-33 in human serum from 9 controls and 9 asthmatic patients. ns=not significant. Data are the mean of duplicates or quadruplicates. LLOQ=lower limit of quantification. C is a graph showing the concentration of reduced hIL-33 in human plasma from 9 controls and 9 asthmatic patients. ns=not significant. Data are the mean of duplicates or quadruplicates. LLOQ=lower limit of quantification. D is a graph showing the correlation of the concentration of total vs. reduced hIL-33 in serum (circles) and NF (triangles) samples from asthmatic donors (n=23). R2=0.95, P<0.0001 for NF. Data are the mean of duplicates or quadruplicates. LLOQ = lower limit of quantification. [Figure 9] A is a graph showing the concentration of total hIL-33 in human nasal absorbance fluid (NF) from 9 controls and 9 asthmatic patients. ns=not significant. Data are the mean of duplicates or quadruplicates. LLOQ=lower limit of quantification. B is a graph showing the concentration of total hIL-33 in human serum from 9 controls and 9 asthmatic patients. ns=not significant. Data are the mean of duplicates or quadruplicates. LLOQ=lower limit of quantification. C is a graph showing the concentration of total hIL-33 in human plasma from 9 controls and 9 asthmatic patients. ns=not significant. Data are the mean of duplicates or quadruplicates. LLOQ=lower limit of quantification. [Figure 10]A is a graph showing the concentration of hsST2 in human serum from 9 controls and 9 asthmatic patients. ns=not significant. Data are the mean of quadruplicates. B is a graph showing the concentration of total hIL-33 in human nasal fluid (NF) from 9 controls and 9 asthmatic patients. ns=not significant. Data are the mean of duplicates or quadruplicates. [Figure 11] Schematic diagram showing the steps of an exemplary IL-33 iPCR assay. MM=master mix; SEC=size exclusion chromatography. [Figure 12] 1 is a graph showing the detectability of reduced IL-33 in various body fluid, serum, and plasma samples of normal and disease states using the reduced IL-33 iPCR assay. AMD=age-related macular degeneration; AH=aqueous humor; HS=human serum; Hu=human; N=total number of samples; NH=normal human; NHP=normal human plasma; NHS=normal human serum; NZ=New Zealand; RA=rheumatoid arthritis; %=number of samples>lower limit of quantification (LLOQ) / N×100. [Figure 13] 1 shows the detectability of reduced and total IL-33 in various human body fluid, serum, and plasma samples of normal and disease states using reduced or total IL-33 iPCR assays. AMD=age-related macular degeneration; AH=aqueous humor; HS=human serum; Hu=human; N=total number of samples; NH=normal human; NHP=normal human plasma; NHS=normal human serum; RA=rheumatoid arthritis; %=number of samples>lower limit of quantification (LLOQ) / N×100. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0078] I. Definition The term "about" as used herein refers to the normal error range of the respective value, which is easily understood by a person skilled in the art. Reference herein to a value or parameter marked with "about" includes (describes) an embodiment relating to the value or parameter itself. In some embodiments, the term "about" refers to ±10% of the stated value.

[0079] An "acceptor human framework" for the purposes of this specification is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0080] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). Typically, the affinity of a molecule X for its partner Y is measured by the dissociation constant (K D ) Affinity can be measured by common methods known in the art, including those described herein. Certain exemplary embodiments for measuring binding affinity are described below.

[0081] An "affinity matured" antibody is an antibody with one or more alterations in one or more HVRs and / or framework regions that result in an improvement in the affinity of the antibody for an antigen compared to a parent antibody that does not possess those one or more alterations. Preferred affinity matured antibodies will have nanomolar, and in some cases picomolar, affinities for the target antigen. Affinity matured antibodies are generated by procedures known in the art. For example, Marks et al. Bio / Technology 10:779-783, 1992 describes affinity maturation by shuffling VH and VL domains. Random mutagenesis of HVR and / or framework residues has been described in Barbas et al. Proc. Natl. Acad. Sci. USA 91:3809-3813, 1994; Schier et al. Gene 169:147-155, 1995; Yelton et al. J. Immunol. 155:1994-2004, 1995; Jackson et al. J. Immunol. 154(7):3310-3319, 1995; and Hawkins et al. J. Mol. Biol. 226:889-896, 1992.

[0082] The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0083] The term "interleukin-33 (IL-33)" as used herein refers to any native IL-33 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. IL-33 is also known in the art as nuclear factor of high endothelial venules (NF-HEV; see, e.g., Baekkevold et al. Am. J. Pathol. 163(1):69-79, 2003), DVS27, C9orf26, and interleukin-1 family member 11 (IL-1F11). The term encompasses "full-length," unprocessed IL-33 as well as any form of IL-33 resulting from processing within the cell. Human full-length unprocessed IL-33 contains 270 amino acids (aa) and is expressed as IL-33. 1-270 The processed form of human IL-33 includes, for example, IL-33 95-270 , IL-33 99-270 , IL-33 109-270 , IL-33 112-270 , IL-33 1-178 , and IL-33 179-270 (Lefrancais et al. Proc. Natl. Acad. Sci. 109(5):1673-1678, 2012 and Martin, Semin. Immunol. 25:449-457, 2013). In some embodiments, processed forms of human IL-33, such as IL-33 95-270 , IL-33 99-270 , IL-33 109-270IL-33, or other forms processed by proteases such as calpain, proteinase 3, neutrophil elastase, and cathepsin G, may have increased biological activity compared to full-length IL-33. The term also encompasses naturally occurring variants of IL-33, such as splice variants (e.g., the constitutively active splice variant spIL-33 lacking exon 3, Hong et al. J. Biol. Chem. 286(22):20078-20086, 2011) or allelic variants. IL-33 can exist intracellularly (e.g., within the nucleus) or as a secreted cytokine form. Full-length IL-33 protein contains a helix-turn-helix DNA binding motif that includes a nuclear localization sequence (aa1-75 of human IL-33) that includes a chromatin binding motif (aa40-58 of human IL-33). The processed and secreted form of IL-33 lacks these N-terminal motifs. An exemplary amino acid sequence of human IL-33 can be found, for example, at UniProtKB Accession No. O95760. In some embodiments, IL-33 in vivo can be in oxidized and / or reduced form. In some embodiments, reduced IL-33 can be prepared by incubating IL-33 (e.g., human IL-33 (hIL-33)) with a reducing agent for a period of time. In some embodiments, reducing agents that can be used to reduce IL-33 (e.g., hIL-33) or stabilize reduced IL-33 (e.g., hIL-33) include, but are not limited to, dithiothreitol (DTT; CAS#: 3483-12-3) and tris(2-carboxyethyl)phosphine (TCEP; CAS#: 51805-45-9). In some embodiments, reduced IL-33 binds to ST2. In some embodiments, reduced IL-33 binds to ST2 with higher affinity than oxidized IL-33. In some embodiments, reduced IL-33 differs from oxidized IL-33 in its secondary structure and the absence of disulfide bonds. In some embodiments, reduced IL-33 does not include the C208-C259 and C227-C232 disulfide bonds between the indicated cysteines.In certain embodiments, disulfide bonds in oxidized IL-33 (e.g., at C208-C259 and C227-C232) prevent its binding to ST2. In some embodiments, "unbound IL-33" refers to IL-33 that is not bound to ST2.

[0084] "IL-33 axis" refers to a nucleic acid (e.g., a gene or mRNA transcribed from a gene) or a polypeptide involved in IL-33 signaling. For example, the IL-33 axis can include the ligand IL-33, a receptor (e.g., ST2 and / or IL-1RAcP), an adaptor molecule (e.g., MyD88), or a protein that associates with the receptor molecule and / or the adaptor molecule (e.g., kinases such as interleukin-1 receptor-associated kinase 1 (IRAK1) and interleukin-1 receptor-associated kinase 4 (IRAK4), or an E3 ubiquitin ligase such as TNF receptor-associated factor 6 (TRAF6)).

[0085] The terms "Interleukin 1 Receptor-Like 1 (IL1RL1)" and "ST2", as used interchangeably herein, refer to any native ST2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. ST2 is also referred to in the art as DER4, T1, and FIT-1. The term encompasses "full-length" unprocessed ST2, as well as any form of ST2 resulting from processing within a cell. At least four isoforms of ST2 are known in the art, including soluble (sST2, also known as IL1RL1-a) and transmembrane (ST2L, also known as IL1RL1-b), which result from differential mRNA expression from a dual promoter system, and ST2V and ST2LV, which result from alternative splicing, as described below. In some examples, ST2, including sST2, can be human ST2 or human sST2 (hsST2), respectively. The domain structure of ST2L includes three extracellular immunoglobulin-like C2 domains, a transmembrane domain, and a cytoplasmic Toll / Interleukin 1 receptor (TIR) ​​domain. sST2 lacks the transmembrane and cytoplasmic ST2L internal domains and contains a unique nine amino acid (aa) C-terminal sequence (see, e.g., Kakkar et al. Nat. Rev. Drug Disc. 7:827-840, 2008). sST2 can function as a decoy receptor that inhibits soluble IL-33. The term also encompasses naturally occurring variants of ST2, such as splice variants (e.g., ST2V, which lacks the third immunoglobulin motif and has a unique hydrophobic tail, and ST2LV, which lacks the transmembrane domain of ST2L) or allelic variants (e.g., variants that are protective against asthma risk or confer asthma risk as described herein). An exemplary amino acid sequence of human ST2 can be found, for example, at UniProtKB Accession No. Q01638. ST2 is part of the IL-33 receptor together with the co-receptor protein IL-1RAcP.Binding of IL-33 to ST2 and the co-receptor interleukin-1 receptor accessory protein (IL-1RAcP) forms a 1:1:1 ternary signaling complex to promote downstream signaling, as shown in FIG. 1A (see, e.g., Lingel et al. Structure 17(10):1398-1410, 2009, and Liu et al. Proc. Natl. Acad. Sci. 110(37):14918-14924, 2013). In some embodiments, the "IL-1 receptor accessory protein" or "IL-1RAcP" is a co-receptor for ST2.

[0086] "IL-33 axis binding antagonist" refers to a molecule that inhibits the interaction of an IL-33 axis binding partner with its binding partner. As used herein, IL-33 axis binding antagonists include IL-33 binding antagonists, ST2 binding antagonists, and IL1RAcP binding antagonists.Exemplary IL-33 axis binding antagonists include anti-IL-33 antibodies and antigen-binding fragments thereof (e.g., anti-IL-33 antibodies such as ANB-020 (AnaptysBio, Inc.), or those described in U.S. Patent Application Publication No. 2021-0284725, U.S. Patent No. 10,093,730, EP 1725261, U.S. Patent No. 8,187,596, WO 2011031600, WO 2014164959, WO 201417142 ... any of the antibodies described in WO 2015099175, WO 2015106080, WO 2016077381, or WO 2021183849; a polypeptide that binds to IL-33 and / or its receptor (ST2 and / or IL-1RACP) and blocks the ligand-receptor interaction (e.g., a ST2-Fc protein such as that described in WO 2014 / 152195, which is incorporated by reference in its entirety); munoadhesins, peptibodies, and soluble ST2, or derivatives thereof; anti-IL-33 receptor antibodies (e.g., anti-ST2 antibodies, such as astergolimab (also known as MSTT1041A), AMG-282 (Amgen) or STLM15 (Janssen), or any of the anti-ST2 antibodies described in WO 2013 / 173761 and WO 2013 / 165894, each of which is incorporated by reference in its entirety; or ST2 -Fc proteins, such as those described in WO 2013 / 173761; WO 2013 / 165894; or WO 2014 / 152195, each of which is incorporated by reference in its entirety; and IL-33 receptor antagonists, such as small molecule inhibitors, aptamers that bind IL-33, and nucleic acids that hybridize under stringent conditions to IL-33 axis nucleic acid sequences (e.g., short interfering RNAs (siRNAs) or clustered regularly interspaced short palindromic repeat RNAs (CRISPR-RNAs or crRNAs), including single guide RNAs (sgRNAs) having crRNA and tracrRNA sequences as described in Mali et al. (Science. 339:823-26, 2013), which are incorporated by reference in their entireties.

[0087] The terms "anti-IL-33 antibody," "antibody that binds IL-33," and "antibody that specifically binds IL-33" refer to an antibody that can bind IL-33 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting IL-33. In one embodiment, the extent of binding of an anti-IL-33 antibody to an unrelated, non-IL-33 protein is less than about 10% of the binding of the antibody to IL-33, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antibody that binds IL-33 has an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 Dissociation constant (K D In some embodiments, the anti-IL-33 antibody has a K between 0.01 nM and 1 nM. D In some embodiments, the anti-IL-33 antibody binds to IL-33 with a K between 0.1 nM and 0.5 nM. D In some embodiments, the anti-IL-33 antibody binds to IL-33 in oxidized and reduced forms. In some embodiments, the anti-IL-33 antibody binds to reduced IL-33. In some embodiments, the anti-IL-33 antibody binds to IL-33 with a K D (For example, K within 1.5 times or 1 times D ) binds oxidized IL-33 and reduced IL-33. In some embodiments, the anti-IL-33 antibody binds oxidized IL-33 and reduced IL-33 at substantially the same K DIn some embodiments, the anti-IL-33 antibody binds to an epitope of IL-33 that is conserved among IL-33 from different species. In some embodiments, the anti-IL-33 antibody binds to mammalian IL-33. In certain embodiments, the anti-IL-33 antibody binds to human IL-33 (hIL-33). In some embodiments, the anti-IL-33 antibody binds to IL-33 bound to soluble interleukin-1 receptor-like 1 (sST2) protein and IL-33 that is not bound to sST2 (e.g., unbound IL-33) with a K within 2-fold. D (For example, K within 1.5 times or 1 times D In some embodiments, the anti-IL-33 antibody binds to IL-33 bound to sST2 protein at substantially the same K as IL-33 that is not bound to sST2. D Combine with.

[0088] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competitive assay. Exemplary competitive assays are provided herein.

[0089] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies (see Example 2 of U.S. Pat. No. 5,641,870; Zapata et al. Protein Eng. 8(10):1057-1062, 1995); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0090] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, reflecting their ability to crystallize readily. The Fab fragment contains the entire L chain as well as the variable region domain (VH) of the H chain and the first constant domain (C) of one heavy chain. H1). Pepsin treatment of an antibody produces a single large F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments that have bivalent antigen-binding activity and are still capable of cross-linking antigen. The Fab' fragment contains one or more cysteines from the antibody hinge region that are not C(ab')2 fragments. H It differs from Fab fragments by having additional few residues at the carboxy terminus of one domain. Fab'-SH is the designation herein for Fab' in which one or more cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments are produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0091] The term "Fc region" as used herein defines a C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues within an Fc region or constant region is according to EU numbering, also referred to as the EU index, as described in Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0092] An "Fv" consists of a tight, non-covalently bound dimer of one heavy and one light chain variable domain. The folding of these two domains results in six hypervariable loops (three loops each from the H and L chain) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or even half of an Fv containing only three Hs specific for an antigen) has the ability to recognize and bind antigen, although often with lower affinity than the entire binding site.

[0093] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising the VH and VL antibody domains connected in a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315, 1994.

[0094] The term "diabody" refers to small antibody fragments prepared by constructing sFv fragments (see previous paragraph) with a short linker (about 5-10 residues) between the VH and VL domains, where interchain rather than intrachain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., a fragment with two antigen binding sites. Bispecific diabodies are heterodimers of two "crossover" Fv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described in further detail, for example, in EP 404,097; WO 93 / 11161; and Hollinger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993.

[0095] "Binding domain" refers to a portion of a compound or molecule that specifically binds to a target epitope, antigen, ligand, or receptor. Binding domains include, but are not limited to, antibodies (e.g., monoclonal, polyclonal, recombinant, humanized, and chimeric antibodies), antibody fragments or portions thereof (e.g., Fab fragments, Fab'2, scFv antibodies, SMIPs, domain antibodies, diabodies, minibodies, scFv-Fc, affibodies, nanobodies, and VH and / or VL domains of antibodies), receptors, ligands, aptamers, and other molecules with identified binding partners.

[0096] A "blocking" or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of an antigen to which it binds. Certain blocking or antagonist antibodies substantially or completely inhibit the biological activity of an antigen.

[0097] "Cell-based blocking assay" refers to an assay that can measure the ability of an antibody to inhibit or reduce the biological activity of an antigen to which it binds. For example, cell-based assays can be used to measure the concentration of an antibody required to inhibit a particular biological or biochemical function. In some embodiments, the half maximal inhibitory concentration (IC50) and / or 90% inhibitory concentration (IC90) of an antibody (e.g., an anti-IL-33 antibody disclosed herein) is measured using a cell-based blocking assay. In some embodiments, a cell-based blocking assay is used to determine whether an antibody blocks the interaction between a ligand (e.g., IL-33) and its receptor (e.g., ST2 and / or co-receptor IL-1RAcP). An exemplary cell-based blocking assay for IL-33 is provided, for example, in Example 2B of U.S. Pat. No. 10,093,730. Further exemplary cell-based blocking assays for IL-33 are provided, for example, in Example 8 of U.S. Pat. No. 10,093,730, and include a primary natural killer (NK) cell assay and a primary basophil assay.

[0098] An "immunopolymerase chain reaction" or "iPCR" assay refers to an assay in which the presence or level of a target molecule (e.g., a target biomolecule, e.g., a protein) is detected by an antibody-DNA conjugate. In some embodiments, an iPCR assay uses a first antibody conjugated to biotin and a second antibody conjugated to a nucleic acid moiety, where the first and second antibodies bind to the same target antigen. Both the first and second antibodies are mixed with a solution containing the target antigen, and the resulting mixture is incubated together, e.g., inside a PCR well. PCR amplification is then used to detect the presence of the nucleic acid moiety, thereby indicating detection of the target antigen. In some embodiments, an iPCR assay is used to detect IL-33 (e.g., human IL-33, e.g., reduced IL-33). In some embodiments, the first and second antibodies are anti-IL33 antibodies. In some embodiments, the first and second antibodies share 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity. In some embodiments, the first and second antibodies do not share the same amino acid sequence (e.g., the amino acid sequences of the first and second antibodies share 1% or less, 2% or less, 3% or less, 4% or less, 5% or less, 10% or less, 15% or less, or 20% or less amino acid sequence identity). Examples of iPCR assays are described in Sano T et.al., Science 1992,258:120-122 and Niemeyer et al., Trends Microbiol.2017,8:65.

[0099] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these are further divided into subclasses (isotypes), such as IgG1, IgG2 (e.g., IgG2a or IgG2b), IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0100] An "effector function" of an antibody refers to a biological activity attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region) and varies with the antibody isotype. Examples of antibody effector functions include C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0101] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) allows these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Antibodies "enable" the cytotoxic cells and are absolutely required for such killing. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch et al. Annu. Rev. Immunol. 9:457-492, 1991. To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay can be performed, such as that described in U.S. Patent No. 5,500,362 or 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of a molecule of interest can be assessed in vivo, for example in an animal model as disclosed in Clynes et al. Proc. Natl. Acad. Sci. USA 95:652-656, 1998.

[0102] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Additionally, a preferred FcR is one that binds IgG antibodies (gamma receptors), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptors") and FcγRIIB ("inhibiting receptors"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see M. in Daeron, Annu. Rev. Immunol. 15:203-234, 1997). FcRs have been reviewed, for example, in Ravetch et al. Annu. Rev. Immunol. 9:457-492, 1991; Capel et al. Immunomethods 4:25-34, 1994; and de Haas et al. J. Lab. Clin. Med. 126:330-41, 1995. Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (see, for example, Guyer et al. J. Immunol. 117:587, 1976; and Kim et al. J. Immunol. 24:249, 1994).

[0103] A "human effector cell" is a leukocyte that expresses one or more FcRs and performs effector function. Preferably, the cell expresses at least FcγRIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils, with PBMCs and NK cells being preferred. Effector cells can be isolated from natural sources, such as blood.

[0104] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) bound to their cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al. J. Immunol. Methods 202:163, 1996, can be performed. An "epitope" is a portion of an antigen to which an antibody selectively binds. In the case of a polypeptide antigen, an epitope is generally a peptide portion of about 4 to 15 amino acid residues.

[0105] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0106] A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human and / or made using any of the techniques for making human antibodies. This definition of a human antibody specifically excludes humanized antibodies which contain non-human antigen-binding residues.

[0107] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is made from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup as in Kabat et al. Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD, vols. 1-3, 1991. In one embodiment, for VL, the subgroup is subgroup kappa III or kappa IV as in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al., supra.

[0108] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired antibody specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can contain residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. In general, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin, and all or substantially all of the FRs being those of human immunoglobulin sequences. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0109] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including, but not limited to, a cytotoxic agent.

[0110] The term "isolated" as used to describe various antibodies disclosed herein means an antibody that has been identified, separated and / or recovered from the cell or cell culture in which it is expressed. Contaminant components of its natural environment are typically substances that would interfere with diagnostic or therapeutic uses of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined, for example, by electrophoretic (e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing (IEF), or capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse-phase HPLC) methods. For a review of methods for assessing antibody purity, see, for example, Flatman et al. J. Chromatogr. B 848:79-87, 2007. In a preferred embodiment, the antibody is purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ inside recombinant cells, since at least one component of the polypeptide's natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.

[0111] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope, except for possible variant antibodies, e.g., variants that are usually present in minor amounts, that may contain, for example, naturally occurring mutations or that arise during the production of the monoclonal antibody preparation. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0112] The term "multispecific antibody" is used in the broadest sense and includes in particular antibodies comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) in which the VH-VL unit has polyepitopic specificity (i.e. can bind to two different epitopes on one biomolecule or to each epitope on a different biomolecule). Such multispecific antibodies include, but are not limited to, full-length antibodies, antibodies with two or more VL and VH domains, antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies and triabodies, covalently or non-covalently linked antibody fragments. "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on one or more of the same or different targets. "Dual specificity" or "bispecificity" refers to the ability to specifically bind to two different epitopes on one or more of the same or different targets. However, in contrast to bispecific antibodies, dual-specific antibodies have two antigen-binding arms that are identical in amino acid sequence, with each Fab arm being capable of recognizing two antigens. Dual specificity allows an antibody to interact with two different antigens as a single Fab or IgG molecule with high affinity. According to one embodiment, a multispecific antibody in the IgG1 format binds each epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0.1 μM to 0.001 pM. "Monospecificity" refers to the ability to bind to only one epitope.

[0113] "Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical composition.

[0114] With respect to antibody binding to a target molecule, the terms "specific binding" or "specifically binds" or "is specific" for a particular polypeptide or epitope on a particular polypeptide target refer to binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule. For example, specific binding can be determined by competition with a control molecule similar to the target, for example, an excess of unlabeled target. In this case, specific binding is indicated when binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. As used herein, the terms "specific binding" or "specifically binds" or "specific" for a particular polypeptide or epitope on a particular polypeptide target refer to binding that is measurably different from non-specific interactions. -4 M or less, alternatively 10 -5 M or less, alternatively 10 -6 M or less, alternatively 10 -7 M or less, alternatively 10 -8 M or less, alternatively 10 -9 M or less, alternatively 10 -10 M or less, alternatively 10 -11 M or less, alternatively 10 -12 K against targets smaller than M D , or 10 -4 M to 10 -6 M or 10 -6 M to 10 -10 M or 10 -7 M~10 -9 K in the M range D As will be appreciated by those skilled in the art, affinity and K D The values ​​are inversely correlated. A high affinity for the antigen corresponds to a low K D In one embodiment, the term "specific binding" refers to the binding of a molecule to a particular polypeptide or an epitope on a particular polypeptide without binding to substantially any other polypeptides or polypeptide epitopes.

[0115] The term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The variable or "V" domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, the variability is not uniformly distributed across the 110 amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches of 15-30 amino acids called framework regions (FRs) separated by short regions of extreme variability called "hypervariable regions", each of which is 9-12 amino acids long. Naturally occurring heavy and light chain variable domains each contain four FRs, mostly in a beta-sheet configuration, connected by three hypervariable regions that form loops that connect and in some cases form part of the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., supra). Thus, the HVR and FR sequences generally appear in VH (or VL) in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. The constant domains are not involved directly in binding the antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0116] The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain that are hypervariable in sequence ("complementarity determining regions" or "CDRs") and / or that form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contacts"). Typically, antibodies contain six HVRs; three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732745 (1996)); and (d) a combination of (a), (b), and / or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Includes:

[0117] Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0118] "Framework" or "FR" refers to variable domain residues other than the complementarity determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the CDR and FR sequences generally appear in the VH (or VL) in the following order: FR1-CDR-H1 (CDR-L1)-FR2-CDR-H2 (CDR-L2)-FR3-CDR-H3 (CDR-L3)-FR4.

[0119] The term "Kabat variable domain residue numbering" or "Kabat amino acid position numbering" and variations thereof refer to the numbering system used for the light chain variable domain or heavy chain variable domain of the compilation of antibodies of Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or an insertion into, the FR or HVR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat). The Kabat numbering of residues may be determined for a given antibody by alignment of the "standard" Kabat numbered sequence with the antibody sequence in regions of homology.

[0120] The Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., supra). The "EU numbering system" or "EU index" is generally used when referring to residues in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The "Kabat EU index" refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise stated herein, reference to a residue number in a variable domain of an antibody refers to the numbering of the residue according to the Kabat numbering system. Unless otherwise stated herein, reference to a residue number in a constant domain of an antibody refers to the numbering of the residue according to the EU numbering system (e.g., see US Provisional Application No. 60 / 640323, drawings for EU numbering).

[0121] As used herein, "administering" refers to a method of giving a dosage of a compound (e.g., an anti-IL-33 axis binding antagonist provided herein (e.g., an anti-IL-33 antibody) or a nucleic acid encoding an anti-IL-33 antibody provided herein) or composition (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition comprising an anti-IL-33 antibody provided herein) to a subject. The compositions utilized in the methods described herein can be administered, for example, intravitreally, intramuscularly, intravenously, intradermally, transdermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesically, mucosally, intrapericardially, subumbilically, intraocularly, intraorbitally, orally, topically, transdermally, periocularly, conjunctivally, subtenonally, anteriorly, subretinally, retrobulbarly, intracanalicularly, by inhalation, by injection, by implantation, by infusion, by continuous infusion, by localized perfusion directly bathing the target cells, by catheter, by perfusion, in a cream, or in a lipid composition. The compositions utilized in the methods described herein can also be administered systemically or locally. The method of administration can vary depending on various factors, such as the compound or composition administered and the severity of the condition, disease, or disorder being treated.

[0122] The term "asthma" as used herein refers to a disorder characterized by variable and recurrent symptoms, reversible airway obstruction (e.g., by bronchodilators), and bronchial hypersensitivity, with or without associated underlying inflammation. Thus, asthma can be inflammatory / inflamed asthma or non-inflammatory / non-inflamed asthma. Examples of asthma include allergic asthma, exercise-induced asthma, aspirin-sensitive / exacerbated asthma, atopic asthma, severe asthma, mild asthma, moderate to severe asthma, corticosteroid-naive asthma, chronic asthma, corticosteroid-resistant asthma, corticosteroid-refractory asthma, newly diagnosed untreated asthma, smoking-induced asthma, corticosteroid-uncontrolled asthma, and other asthma mentioned in Bousquet et al. J. Allergy Clin. Immunol. 126(5):926-938, 2010.

[0123] A "disorder" or "disease" is any condition that would benefit from treatment (with an IL-33 axis binding antagonist provided herein). For example, the disorder can be an IL-33 mediated disorder. This includes chronic and acute disorders or diseases, including pathological conditions that predispose the mammal to the disorder in question. Examples of disorders treated herein include IL-33 mediated disorders (e.g., asthma, allergic rhinitis, atopic dermatitis, and fibrosis (e.g., pulmonary fibrosis, e.g., idiopathic pulmonary fibrosis)).

[0124] The term "IL-33-mediated disorder" as used herein refers to any disorder or condition mediated by or associated with the IL-33 axis. In some embodiments, the IL-33-mediated disorder is associated with excessive IL-33 levels or activity, which may present with atypical symptoms due to local and / or systemic IL-33 levels or activity in the body. Exemplary IL-33-mediated disorders include eye disorders, inflammatory conditions, immune diseases, fibrotic disorders, eosinophilic disorders, infections, pain, central nervous system disorders, and solid tumors. IL-33-mediated disorders are described, for example, in Liew et al. Nature Reviews Immunology 10:103-110, 2010, which is incorporated herein by reference in its entirety.

[0125] The term "ocular disorder" as used herein includes any ocular disorder (also referred to interchangeably herein as "ocular condition") associated with pathological neovascularization and / or atrophy. Ocular disorders may be characterized by altered or uncontrolled proliferation and / or invasion of new blood vessels into structures of ocular tissues such as the retina or cornea. Ocular disorders may be characterized by atrophy of retinal tissues (photoreceptors and the underlying retinal pigment epithelium (RPE) and choriocapillaris). Non-limiting ocular disorders include, for example, AMD (e.g., wet AMD, dry AMD, intermediate AMD, advanced AMD, and geographic atrophy (GA)), macular degeneration, macular edema, diabetic macular edema (DME) (e.g., focal, non-central DME, and diffuse centrally involved DME), retinopathies, diabetic retinopathy (DR) (e.g., proliferative DR (PDR), non-proliferative DR (NPDR), and high altitude DR), other ischemia-related retinopathies, ROP, retinopathies ... Retinal venous occlusion (RVO) (e.g., central (CRVO) and branched (BRVO) forms), choroidal neovascularization (CNV) (e.g., myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases associated with retinal / choroidal neovascularization, central serous retinopathy (CSR), pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, familial exudative vitreoretinopathy (FEVR), Coats' disease, Norrie's disease, osteoporosis Retinal abnormalities associated with opioid pseudoglioma syndrome (OPPG), subconjunctival hemorrhage, skin flushing, ocular neovascular disease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatous proliferation, macular vascular ectasia, iris neovascularization, intraocular neovascularization, retinal alterations, cystoid macular edema (CME), vasculitis, papilledema, retinitis including, but not limited to, CMV retinitis, ocular melanoma, retinoblastoma, conjunctivitis (e.g., infectious conjunctivitis and non-infectious conjunctivitis), In some embodiments, the ocular conditions include, but are not limited to, chronic (e.g., allergic) conjunctivitis, Leber's congenital amaurosis (also known as Leber's congenital amaurosis or LCA), uveitis (including infectious and non-infectious uveitis), choroiditis (e.g., multifocal choroiditis), ocular histoplasmosis, blepharitis, dry eye, traumatic eye injury, Sjogren's disease, and other ocular diseases in which the disease or disorder is associated with ocular neovascularization, vascular leakage, and / or retinal edema or atrophy.Further exemplary eye disorders include diseases associated with retinoschisis (abnormal division of the retinal neurosensory layer), cutaneous flushing (angular neovascularization), and diseases caused by abnormal proliferation of fibrovascular or fibrous tissue (including all forms of proliferative vitreoretinopathy).

[0126] Exemplary diseases involving corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, vitamin A deficiency, contact lens overuse, atopic keratitis, superior limbal keratitis, psoriatic keratitis, Sjogren's syndrome, rosacea abrasion, glomerulitis, syphilis, mycobacterial infection, fatty degeneration, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex infection, herpes zoster infection, protozoal diseases, Kaposi's sarcoma, Mooren's ulcer, Therrien's peripheral corneal degeneration, marginal keratitis, rheumatoid arthritis, systemic lupus, polyarteritis nodosa, trauma, Wegener's sarcoidosis, scleritis, Stevens-Johnson syndrome, pemphigoid radial keratotomy, and corneal graph rejection.

[0127] Exemplary ocular diseases associated with choroidal neovascularization and defects in the retinal vasculature, including increased vascular leakage, aneurysms, and capillary dropout, include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Paget's disease, venous occlusion, arterial occlusion, carotid occlusive disease, chronic uveitis / vitreous inflammation, mycobacterial infections, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, retinal edema (including macular edema), Eales' disease, Behcet's disease, infections causing retinitis or choroiditis (e.g., multifocal choroidal), presumed ocular histoplasmosis, Best's disease (vitreous macular degeneration), myopia, optic disc, pars planitis, retinal detachment (e.g., chronic retinal detachment), hyperviscosity syndrome, toxoplasmosis, trauma, and post-laser complications.

[0128] Exemplary diseases associated with atrophy of retinal tissue (photoreceptors and the underlying RPE) include, but are not limited to, atrophic or non-exudative AMD (e.g., geographic atrophy or advanced dry AMD), macular atrophy (e.g., atrophy associated with neovascularization and / or geographic atrophy), diabetic retinopathy, Stargardt's disease, Sosby's fundus dystrophy, retinoschisis, and retinitis pigmentosa.

[0129] Exemplary inflammatory conditions include asthma (e.g., allergic asthma, exercise-induced asthma, aspirin-sensitive / exacerbated asthma, atopic asthma, severe asthma, mild asthma, moderate to severe asthma, corticosteroid-naive asthma, chronic asthma, corticosteroid-resistant asthma, corticosteroid-refractory asthma, newly diagnosed untreated asthma, smoking-induced asthma, corticosteroid-uncontrolled asthma, etc.), airway inflammation, airway hyperresponsiveness, airway hyperresponsiveness, rhinosinusitis, rhinosinusitis with polyps, nasal polyposis, arthritis (e.g., osteoarthritis, rheumatoid arthritis, collagen-induced arthritis, etc.), and / or other conditions. , traumatic arthritis, etc.), eosinophilic inflammation, mast cell-mediated inflammatory diseases, sepsis, septic shock, seronegative tendonitis and arthropathy (SEA) syndrome, osteoporosis, eosinophilic esophagitis, scleroderma, dermatitis, atopic dermatitis, allergic rhinitis, bullous pemphigoid, chronic urticaria, chondritis, polymyalgia rheumatica, polyarteritis nodosa, Wegener's granulomatosis, Behçet's disease, myositis, polymyolitis, dermatomyolitis, vasculitis, arteritis, diabetic nephropathy, interstitial cystitis, transplantation Graft-versus-host disease (GVHD), gastrointestinal inflammatory conditions (e.g., inflammatory bowel disease (IBD), ulcerative colitis (UC), Crohn's disease (CD), colitis (e.g., colitis caused by external stimuli (e.g., resulting from or associated with therapeutic regimens such as chemotherapy, radiation therapy, etc.), infectious colitis, ischemic colitis, collagenous or lymphocytic colitis, necrotizing enterocolitis, colitis in conditions such as chronic granulomatous disease and celiac disease, food allergies, gastritis, infectious gastritis or enteritis (e.g., chronic active gastritis due to Helicobacter pylori infection), and other forms caused by infectious agents. gastroenteritis), and inflammatory lung diseases (e.g., chronic obstructive pulmonary disease (COPD), eosinophilic pulmonary inflammation, infection-induced lung diseases (viral (such as influenza, parainfluenza, rotavirus, human metapneumovirus, and respiratory syncytial virus), bacterial, fungal (such as Aspergillus), parasitic, or prion infections, allergen-induced lung diseases, pollutant-induced lung diseases (e.g., asbestosis, silicosis, or beryllium disease), gastric aspiration-induced lung diseases, immune dysregulation, inflammatory conditions with genetic predisposition such as cystic fibrosis, lung diseases due to physical trauma (such as ventilator injury), emphysema,Bronchitis, sarcoidosis, histiocytosis, lymphangioleiomyomatosis, acute lung injury, acute respiratory distress syndrome, chronic lung disease, bronchopulmonary dysplasia, pneumonia (e.g., community-acquired pneumonia, hospital-acquired pneumonia, ventilator-associated pneumonia, viral pneumonia, bacterial pneumonia, severe pneumonia), airway exacerbation, and those associated with acute respiratory distress syndrome (ARDS).

[0130] Exemplary immune disorders include those mediated at least in part by mast cells, such as asthma (including allergic asthma), eczema, itch, allergies, atopic allergies, anaphylaxis, anaphylactic shock, allergic bronchopulmonary aspergillosis, allergic rhinitis, allergic conjunctivitis, as well as rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, pancreatitis, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythroderma. Autoimmune diseases including psoriasis, paraneoplastic autoimmune diseases, autoimmune hepatitis, bullous pemphigoid, myasthenia gravis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, celiac disease, thyroiditis (e.g., Graves' disease), Sjogren's syndrome, Guillain-Barré disease, Raynaud's phenomenon, Addison's disease, liver diseases (e.g., primary biliary cirrhosis, primary sclerosing cholangitis, nonalcoholic fatty liver disease, and nonalcoholic steatohepatitis), and diabetes (e.g., type 1 diabetes). The terms "fibrotic disorder" and "fibrosis" as used herein refer to conditions involving the formation of excess fibrous connective tissue in an organ or tissue. Exemplary fibrotic disorders include pulmonary fibrosis, liver fibrosis (e.g., fibrosis associated with cirrhosis (e.g., alcohol-induced cirrhosis, viral-induced cirrhosis, post-hepatitis C cirrhosis, cirrhosis of the liver, and primary biliary cirrhosis), schistosomiasis, cholangitis (e.g., sclerosing cholangitis), and autoimmune-induced hepatitis), kidney fibrosis (e.g., tubulointerstitial fibrosis, scleroderma, diabetic nephritis, and glomerulonephritis), skin fibrosis (e.g., scleroderma, hypertrophic and keloid scars, nephrogenic fibrosing dermopathy, and fibrotic fibrosis of the liver). fibrosis, myelofibrosis, neurofibromatosis, fibromas, intestinal fibrosis, and fibrous adhesions due to surgical procedures), cardiac fibrosis (e.g., fibrosis associated with myocardial infarction), vascular fibrosis (e.g., fibrosis associated with postangioplasty arterial restenosis and atherosclerosis), ocular fibrosis (e.g., fibrosis associated with proliferative vitreoretinopathy after cataract surgery, and retroorbital fibrosis), and myelofibrosis (e.g., idiopathic myelofibrosis and drug-induced myelofibrosis). Fibrosis can be organ-specific or systemic (e.g., systemic sclerosis, and fibrosis associated with GVHD).

[0131] Examples of pulmonary fibrosis include, for example, lung or pulmonary fibrosis associated with idiopathic pulmonary fibrosis, fibrosis associated with collagen vascular disease, Hermansky-Pudlak syndrome, adult respiratory distress syndrome, nonspecific interstitial pneumonia, respiratory bronchiolitis, sarcoidosis, histiocytosis X, bronchiolitis obliterans, and organizing pneumonia of unknown etiology. In one embodiment, the pulmonary fibrosis is idiopathic pulmonary fibrosis.

[0132] As used herein, an "eosinophil disorder" is a disorder associated with excessive eosinophil numbers that may present with atypical symptoms due to local or systemic eosinophil levels or activity in the body. Eosinophilic disorders include, but are not limited to, asthma (including aspirin-sensitive asthma, atopic asthma, severe asthma), eosinophilic inflammation, atopic dermatitis, allergic rhinitis (including seasonal allergic rhinitis), non-allergic rhinitis, chronic eosinophilic pneumonia, allergic bronchopulmonary aspergillosis, celiac disease, Churg-Strauss syndrome (periarteritis nodosa + atopy), eosinophilic myalgia syndrome, hypereosinophilic syndrome, edematous reactions including episodic angioedema, helminth infections in which eosinophils may play a protective role, onchocercal dermatitis; eosinophil-associated gastrointestinal disorders (EGID) including, but not limited to, eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic enteritis and eosinophilic colitis, nasal micropolyposis and polyposis, aspirin intolerance, and obstructive sleep apnea. Eosinophil-derived secretions have also been implicated in promoting angiogenesis and connective tissue formation in tumors, and in fibrotic responses seen in conditions such as chronic asthma, Crohn's disease, scleroderma, and endocardial fibrosis (Munitz et al. Allergy 59:268-275, 2004; Adamko et al. Allergy 60:13-22, 2005; Oldhoff et al. Allergy 60:693-696, 2005). Other examples include cancer (e.g., glioblastoma (such as glioblastoma multiforme) and non-Hodgkin's lymphoma (NHL)), atopic dermatitis, allergic rhinitis, inflammatory bowel disease, fibrosis (e.g., pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis (IPF) and pulmonary fibrosis secondary to sclerosis) and liver fibrosis), and COPD.

[0133] The above list is not exhaustive and one of skill in the art will appreciate that a disease or disorder may fall into various categories, for example, asthma may in some cases be classified as both an inflammatory disorder and an immune disorder and may be considered an autoimmune disease by some clinicians.

[0134] The term "ST2 binding antagonist" refers to a molecule that inhibits the interaction of ST2 with IL-33, IL1RAcP, and / or a second ST2 molecule. An ST2 binding antagonist may be a protein such as an "ST2-Fc protein" that includes an IL-33 binding domain (e.g., all or a portion of an ST2 or IL1RAcP protein) and a multimerization domain (e.g., an Fc domain of an immunoglobulin, e.g., an IgG Fc domain selected from the isotypes lgG1, lgG2, lgG3, and lgG4, and any allotype within each isotype group), directly or indirectly linked to each other through a linker (e.g., a serine-glycerine (SG) linker, a glycine-glycine (GG) linker, or variants thereof (e.g., an SGG, GGS, SGS, or GSG linker)), including, but not limited to, the ST2-Fc proteins and variants thereof described in WO 2013 / 173761, WO 2013 / 165894, and WO 2014 / 152195, each of which is incorporated by reference in its entirety. In some embodiments, the ST2 binding antagonist may be an anti-ST2 antibody, such as estegolimab, AMG-282 (Amgen) or STLM15 (Janssen), or any of the anti-ST2 antibodies described in WO 2013 / 173761 and WO 2013 / 165894.

[0135] "Isolated nucleic acid" refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0136] The term "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0137] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. The host cell may be an "isolated host cell," which refers to a host cell that has been separated from a component of its natural environment. In some embodiments, the host cell may be of human or rate origin.

[0138] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Usually, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0139] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps if necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity for the purpose of alignment. Alignment to determine percent amino acid sequence identity can be accomplished using a variety of methods within the skill of the art, such as publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. Alternatively, percent identity values ​​can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and its source code together with user documentation has been submitted to the U.S. Copyright Office, Washington DC, 20559, where it is registered under U.S. Copyright Registration No. TXU510087, and is described in WO 2001 / 007611.

[0140] Unless otherwise indicated, for purposes herein, percent amino acid sequence identity values ​​are generated using the ggsearch program of the FASTA package version 36.3.8c, or subsequently using the BLOSUM50 comparison matrix. The FASTA program package was created by WR Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:24442448; WR Pearson (1996) "Effective protein sequence comparison" Meth. Enzymol. 266:227-258; and Pearson et.al. (1997) Genomics 46:2436, and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, the sequences can be compared using the public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi, using the ggsearch(globalprotein:protein) program and default options (BLOSUM50; open:-10; ext:-2; Ktup=2), ensuring a global, rather than local, alignment. Percent amino acid identity is given in the output alignment header. Amino acid sequences described herein are contiguous amino acid sequences unless otherwise specified.

[0141] The term "package insert" is used to refer to instructions customarily included in the commercial packaging of a diagnostic or therapeutic product, which contain information regarding the indications, usage, dosage, administration, concomitant therapy, contraindications and / or warnings concerning the use of such product. The term "pharmaceutical composition" refers to a preparation that is in a form that effectively utilizes the biological activity of the active ingredient contained therein and does not contain any additional ingredients that are unacceptably toxic to the subject to which the formulation will be administered.

[0142] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0143] By "reducing or inhibiting" is meant the ability to cause an overall decrease, preferably of 20% or more, more preferably 50% or more, and most preferably 75%, 85%, 90%, 95% or more. Reducing or inhibiting may refer to the symptoms of the disorder being treated, the presence or size of metastases, and / or the size of the primary tumor.

[0144] A "subject" is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, farm animals (e.g., cows and sheep), sport animals, pets (e.g., cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), and rodents (e.g., mice and rats). In some embodiments, the anti-IL-33 antibodies provided herein are used to determine the presence or level of IL-33 in a subject, particularly the presence or level of human IL-33 in a human subject.

[0145] The term "therapeutically effective amount" refers to an amount of a compound (e.g., an anti-IL-33 axis binding antagonist provided herein, e.g., an anti-IL-33 antibody (including an antibody fragment, e.g., a Fab fragment) or a nucleic acid encoding an anti-IL-33 antibody provided herein) or composition (e.g., a pharmaceutical composition provided herein, e.g., a pharmaceutical composition comprising an IL-33 axis binding antagonist, e.g., an anti-IL-33 antibody) to treat a disease or disorder of a subject. In the case of an IL-33-mediated disorder, a therapeutically effective amount of an IL-33 axis binding antagonist, e.g., an antibody or antibody fragment (e.g., an anti-IL-33 antibody, including a bispecific anti-IL-33 antibody that binds IL-33 and a second biological molecule, e.g., IL-13, e.g., a bispecific anti-IL-33 antibody / anti-IL-13 antibody) may ameliorate or treat the disease or prevent, alleviate, ameliorate, or treat a symptom associated with the disease. In the case of a proliferative disease (e.g., solid tumor), a therapeutically effective amount of an antibody or antibody fragment may reduce the number of cells; reduce primary tumor size; inhibit (i.e., slow to some extent, preferably stop) cancer cell invasion into peripheral organs; inhibit (i.e., slow to some extent, preferably stop) tumor metastasis; inhibit (i.e., slow to some extent, preferably stop) tumor growth; and / or alleviate to some extent one or more of the symptoms associated with the disorder. To the extent that the antibody or antibody fragment can prevent proliferation and / or kill existing cancer cells, this amount may be cytostatic and / or cytotoxic. In the case of cancer therapy, efficacy in vivo may be measured, for example, by assessing survival time, time to progression (TTP), disease-free survival (DFS), progression-free survival (PFS), response rate (RR), duration of response, and / or quality of life.

[0146] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing the direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating pathology, and remission or improving prognosis. In some embodiments, the antibodies disclosed herein are used to delay the onset of disease or slow the progression of disease. For example, after receiving treatment for asthma, a patient is said to be successfully "treated" for asthma if the patient exhibits an observable and / or measurable reduction in, or the absence of, one or more of the following: recurrent wheezing, coughing, difficulty breathing, chest tightness, symptoms that occur or worsen at night, symptoms triggered by cold air, exercise, or exposure to allergens.

[0147] "Tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0148] The term "vector" as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector into which additional DNA segments can be ligated into the viral genome. Some vectors can autonomously replicate in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. In addition, some vectors can direct the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "recombinant vectors" or "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" may be used interchangeably.

[0149] The term "detection" includes any means of detecting, including direct and indirect detection.

[0150] The term "diagnosis" is used herein to refer to the identification or classification of a molecular or pathological state, disease, or condition (e.g., an IL-33-mediated disorder). For example, "diagnosis" can refer to the identification of a particular type of IL-33-mediated disorder. "Diagnosis" can also refer to the classification of a particular subtype of an IL-33-mediated disorder, for example, by histopathological criteria or by molecular features (e.g., a subtype characterized by the expression of one or a combination of biomarkers (e.g., particular genes or proteins encoded by said genes)).

[0151] The terms "level of expression" or "expression level" are generally used interchangeably and generally refer to the amount of a polynucleotide, mRNA, or amino acid product or protein in a biological sample. "Expression" generally refers to the process of converting genetic coding information into structures present and operating within a cell. Thus, according to the present invention, "expression" of a gene (IL-33 gene) may refer to transcription into a polynucleotide, translation into a protein, or, in some cases, post-translational modification of a protein. A transcribed polynucleotide, a translated protein, or a post-translationally modified fragment of a protein should also be considered expressed, whether derived from a transcript generated by alternative splicing, or a degraded transcript, or from post-translational processing of a protein, for example, by proteolysis. In some embodiments, "expression level" may be measured by immunohistochemistry (IHC), immunoblotting (e.g., Western blotting), immunofluorescence (IF), flow cytometry, such as fluorescence activated cell sorting (FACS), or other techniques. TM IL-33 refers to the amount of a protein (e.g., IL-33) in a biological sample as determined using methods known in the art or described herein, including, but not limited to, immunoassay (ELISA), enzyme-linked immunosorbent assay (ELISA), or immunopolymerase chain reaction (iPCR).

[0152] "Increased expression," "increased expression level," "elevated level," "increased expression," "elevated expression level," or "elevated level" refers to an increase in expression or an increase in the level of a biomarker in an individual relative to one or more individuals not afflicted with a disease or disorder (e.g., cancer) or a control, such as an internal standard (e.g., a housekeeping biomarker).

[0153] "Decreased expression," "reduced expression levels," "reduced levels," "reduced expression," "reduced levels of expression," or "reduced levels" refer to a decrease in expression or a decrease in the level of a biomarker in an individual compared to one or more individuals not afflicted with a disease or disorder (e.g., cancer) or a control, such as an internal standard (e.g., a housekeeping biomarker). In some embodiments, decreased expression is little or no expression.

[0154] The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent such as a polynucleotide probe or antibody, facilitating detection of the conjugated or fused reagent. The label may be detectable itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze chemical modification of a substrate compound or composition that is detectable. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of a probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody, and end-labeling a DNA probe with biotin for detection with fluorescently labeled streptavidin.

[0155] As used herein, a "reference sample", "reference cell", "reference tissue", "reference level", "control sample", "control cell", or "control tissue" refers to a sample, cell, tissue, standard, or level used for comparison purposes. In one embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or non-disease part of the body (e.g., tissue or cell) of the same subject or individual. For example, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue can be a healthy and / or non-disease cell or tissue adjacent to a diseased cell or tissue (e.g., a cell or tissue adjacent to a tumor). In another embodiment, the reference sample is obtained from an untreated tissue and / or cell of the body of the same subject or individual. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or non-disease part of the body (e.g., tissue or cell) of an individual that is not the subject or individual. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell or control tissue is obtained from untreated tissues and / or cells from the body of an individual who is not the subject or individual.

[0156] II. Compositions and Methods In one aspect, the invention is based, at least in part, on improved antibodies that bind to IL-33. The antibodies provided herein have unexpectedly advantageous properties, including high affinity, particularly to reduced forms of IL-33 (e.g., reduced IL-33; e.g., reduced human IL-33 (hIL-33)) or IL-33 associated with interleukin-1 receptor-like 1 (ST2; e.g., soluble ST2 (sST2); e.g., human sST2 (hsST2)). For example, the antibodies disclosed herein have substantially the same binding affinity (e.g., K ) as oxidized IL-33 (e.g., hIL-33) when assessed using surface plasmon resonance (SPR), ELISA, or immunopolymerase chain reaction (iPCR). D) to reduced IL-33 (e.g., hIL-33). Furthermore, the antibodies disclosed herein may specifically bind IL-33 (e.g., hIL-33) bound to ST2 (e.g., sST2 or hsST2) with substantially the same binding affinity as unbound IL-33 (e.g., hIL-33) when assessed using SPR, ELISA, or iPCR. Furthermore, the antibodies provided herein also bind human and cynomolgus monkey (cyno) IL-33 with high affinity when assessed, for example, using SPR, ELISA, or iPCR. The unexpectedly favorable properties described above (including the ability to bind both reduced and oxidized IL-33 and both unbound and bound IL-33 to ST2) are particularly advantageous in biochemical assays (e.g., for detecting the presence of IL-33 in sensitive physiological conditions; e.g., using ELISA or iPCR). For example, without wishing to be bound by any theory, the antibodies disclosed herein have subnanomolar K D and capable of binding to physiologically relevant forms of hIL-33.

[0157] A. Exemplary anti-IL-33 antibodies In some aspects, the invention provides isolated antibodies or antigen-binding fragments thereof that bind to IL-33. In some embodiments, the antigen-binding molecules of the invention comprise at least one, at least two, at least three, at least four, at least five, or all six CDRs set forth in Table 1. In some cases, the antibodies or antigen-binding fragments thereof comprise a VH and / or a VL of Table 1. In some cases, the antibodies comprise a HC and / or a LC of Table 1.

[0158] Table 1. List of SEQ ID NOs of antigen binding molecules against IL-33 TIFF2024544885000001.tif28170

[0159] In one example, provided herein is a CDR-H1 comprising the amino acid sequence of SQNVH (SEQ ID NO: 1); (b) a CDR-H2 comprising the amino acid sequence of RMRFNGDTSYNSTLKS (SEQ ID NO: 2); (c) a CDR-H3 comprising the amino acid sequence of QRDNYGSYYFDD (SEQ ID NO: 3); (d) a CDR-L1 comprising the amino acid sequence of RASESVLTLLN (SEQ ID NO: 4); (e) a CDR-L2 comprising the amino acid sequence of LASHLES (SEQ ID NO: 5); and (f) a CDR-L3 comprising the amino acid sequence of QQSWIDPWT (SEQ ID NO: 6). or at least one, two, three, four, five, or six CDRs selected from one or a combination of more of the above CDRs, and / or one or more variants thereof having at least about 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 1 to 6, or an antigen-binding fragment thereof. For example, in some cases, the anti-IL-33 antibody comprises at least one, two, three, four, five, or six CDRs selected from: (a) a CDR-H1 comprising the amino acid sequence of SQNVH (SEQ ID NO: 1); (b) a CDR-H2 comprising the amino acid sequence of RMRFNGDTSYNSTLKS (SEQ ID NO: 2); (c) a CDR-H3 comprising the amino acid sequence of QRDNYGSYYFDD (SEQ ID NO: 3); (d) a CDR-L1 comprising the amino acid sequence of RASESVLTLLN (SEQ ID NO: 4); (e) a CDR-L2 comprising the amino acid sequence of LASHLES (SEQ ID NO: 5); and (f) a CDR-L3 comprising the amino acid sequence of QQSWIDPWT (SEQ ID NO: 6).

[0160] In another example, provided herein is a VH domain having an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of QVQLEESGPGLVQPSQTLSLTCTVSGFSLTSQNVHWVRQPPGKGLEWMGRMRFNGDTSYNSTLKSRLSISRDTSKNQVFLRLNSLQTDDTGTYYCARQRDNYGSYYFDDWGQGIMVTVSS (SEQ ID NO: 7), or the sequence of SEQ ID NO: 7; or or (c) an anti-IL-33 antibody or antigen-binding fragment thereof comprising the VH domain of (a) and the VL domain of (b).

[0161] Any of the anti-IL-33 antibodies or antigen-binding fragments thereof provided herein comprise the following heavy chain framework regions: FR-H1 comprising the amino acid sequence of QVQLEESGPGLVQPSQTLSLTCTVSGFSLT (SEQ ID NO: 11); FR-H2 comprising the amino acid sequence of WVRQPPGKGLEWMG (SEQ ID NO: 12); FR-H3 comprising the amino acid sequence of RLSISRDTSKNQVFLRLNSLQTDDTGTYYCAR (SEQ ID NO: 13); and FR-H4 comprising the amino acid sequence of WGQGIMVTVSS (SEQ ID NO: 14). 14), or one, two, three, or four of the FR-H4s comprising the amino acid sequence of SEQ ID NO: 11 to 14, or a combination of one or more of the above FRs, and / or one or more variants thereof having at least about 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 11 to 14. In some instances, an anti-IL-33 antibody may comprise one, two, three, or four of the following heavy chain framework regions: FR-H1 comprising the amino acid sequence of QVQLEESGPGLVQPSQTLSLTCTVSGFSLT (SEQ ID NO: 11); FR-H2 comprising the amino acid sequence of WVRQPPGKGLEWMG (SEQ ID NO: 12); FR-H3 comprising the amino acid sequence of RLSISRDTSKNQVFLRLNSLQTDDTGTYYCAR (SEQ ID NO: 13); and FR-H4 comprising the amino acid sequence of WGQGIMVTVSS (SEQ ID NO: 14). In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 7.

[0162] Any of the anti-IL-33 antibodies, or antigen-binding fragments thereof, provided herein comprise the following light chain framework regions: FR-L1 comprising the amino acid sequence of DTVLTQSPALAVSPGERVTISC (SEQ ID NO: 15); FR-L2 comprising the amino acid sequence of WYQQKPGQHPKLLIY (SEQ ID NO: 16); FR-L3 comprising the amino acid sequence of GVPARFSGRGSGTDFTLTIDPVEADDTASYYC (SEQ ID NO: 17); and FR-L4 comprising the amino acid sequence of FGGGTTLELK (SEQ ID NO: 18). The FR-L4 may comprise one, two, three, or four of the amino acid sequences, or a combination of one or more of the above FRs, and / or one or more variants thereof having at least about 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 15 to 18. In some cases, an anti-IL-33 antibody can comprise one, two, three, or four of the following light chain framework regions: FR-L1 comprising the amino acid sequence of DTVLTQSPALAVSPGERVTISC (SEQ ID NO: 15); FR-L2 comprising the amino acid sequence of WYQQKPGQHPKLLIY (SEQ ID NO: 16); FR-L3 comprising the amino acid sequence of GVPARFSGRGSGTDFTLTIDPVEADDTASYYC (SEQ ID NO: 17); and FR-L4 comprising the amino acid sequence of FGGGTTLELK (SEQ ID NO: 18). In some cases, the VL domain comprises the amino acid sequence of SEQ ID NO: 8.

[0163] In another example, provided herein is a method for detecting a pulmonary circulation disorder, comprising: (a) detecting a pulmonary circulation disorder, the pulmonary circulation disorder, and / or the pulmonary circulation disorder; KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9) or the sequence of SEQ ID NO: 9; (b) a heavy chain (HC) comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO: 9; a light chain (LC) having an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10), or the sequence of SEQ ID NO: 10; or (c) an anti-IL-33 antibody comprising the HC of (a) and the LC of (b).

[0164] For example, an anti-IL-33 antibody may comprise: (a) a CDR-H1 comprising the amino acid sequence of SQNVH (SEQ ID NO: 1); (b) a CDR-H2 comprising the amino acid sequence of RMRFNGDTSYNSTLKS (SEQ ID NO: 2); (c) a CDR-H3 comprising the amino acid sequence of QRDNYGSYYFDD (SEQ ID NO: 3); (d) a CDR-L1 comprising the amino acid sequence of RASESVLTLLN (SEQ ID NO: 4); (e) a CDR-L2 comprising the amino acid sequence of LASHLES (SEQ ID NO: 5); and (f) a CDR-L3 comprising the amino acid sequence of QQSWIDPWT (SEQ ID NO: 6). In some cases, the anti-IL-33 antibody comprises: (a) a heavy chain variable (VH) domain having an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of SEQ ID NO:7; (b) a light chain variable (VL) domain having an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of SEQ ID NO:8; or (c) the VH domain of (a) and the VL domain of (b). In some instances, the anti-IL-33 antibody comprises the following heavy chain framework regions: FR-H1 comprising the amino acid sequence of QVQLEESGPGLVQPSQTLSLTCTVSGFSLT (SEQ ID NO: 11); FR-H2 comprising the amino acid sequence of WVRQPPGKGLEWMG (SEQ ID NO: 12); FR-H3 comprising the amino acid sequence of RLSISRDTSKNQVFLRLNSLQTDDTGTYYCAR (SEQ ID NO: 13); and FR-H4 comprising the amino acid sequence of WGQGIMVTVSS (SEQ ID NO: 14). In some instances, the anti-IL-33 antibody comprises the following light chain framework: FR-L1 comprising the amino acid sequence of DTVLTQSPALAVSPGERVTISC (SEQ ID NO: 15); FR-L2 comprising the amino acid sequence of WYQQKPGQHPKLLIY (SEQ ID NO: 16); FR-L3 comprising the amino acid sequence of GVPARFSGRGSGTDFTLTIDPVEADDTASYYC (SEQ ID NO: 17); and FR-L4 region comprising the amino acid sequence of FGGGTTLELK (SEQ ID NO: 18).In some cases, the anti-IL-33 antibody comprises a binding domain comprising (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 8. In some cases, an exemplary anti-IL-33 antibody is 3F10.

[0165] For example, provided herein is an isolated antibody, or antigen-binding fragment thereof, that specifically binds to IL-33, the antibody comprising (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 8. In some instances, an exemplary anti-IL-33 antibody is 3F10.

[0166] In another example, provided herein is an isolated antibody, or antigen-binding fragment thereof, that specifically binds to IL-33, the antibody comprising (a) a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 9 and (b) a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 10. In some instances, an exemplary anti-IL-33 antibody is 3F10.

[0167] In one example, provided herein is a CDR-H1 comprising the amino acid sequence of DFWMS (SEQ ID NO: 19); (b) a CDR-H2 comprising the amino acid sequence of DIKNDGSHTKYAPSLQN (SEQ ID NO: 20); (c) a CDR-H3 comprising the amino acid sequence of VGGTYTAYY (SEQ ID NO: 21); (d) a CDR-L1 comprising the amino acid sequence of RTSQGINTYLN (SEQ ID NO: 22); (e) a CDR-L2 comprising the amino acid sequence of YTSNLES (SEQ ID NO: 23); and (f) a CDR-L3 comprising the amino acid sequence of QQYASSPWT (SEQ ID NO: 24). and CDR-L3 including at least one, two, three, four, five, or six CDRs selected from the group consisting of CDR-L3, CDR-L4, CDR-L5, CDR-L6, CDR-L7, CDR-L8, CDR-L9, CDR-L10, CDR-L11, CDR-L12, CDR-L13, CDR-L14, CDR-L15, CDR-L16, CDR-L17, CDR-L18, CDR-L19, CDR-L20, CDR-L21, CDR-L22, CDR-L23, CDR-L24, CDR-L25, CDR-L36, CDR-L27, CDR-L38, CDR-L39 ... For example, in some cases, the anti-IL-33 antibody comprises at least one, two, three, four, five, or six CDRs selected from: (a) a CDR-H1 comprising the amino acid sequence of DFWMS (SEQ ID NO: 19); (b) a CDR-H2 comprising the amino acid sequence of DIKNDGSHTKYAPSLQN (SEQ ID NO: 20); (c) a CDR-H3 comprising the amino acid sequence of VGGTYTAYY (SEQ ID NO: 21); (d) a CDR-L1 comprising the amino acid sequence of RTSQGINTYLN (SEQ ID NO: 22); (e) a CDR-L2 comprising the amino acid sequence of YTSNLES (SEQ ID NO: 23); and (f) a CDR-L3 comprising the amino acid sequence of QQYASSPWT (SEQ ID NO: 24).

[0168] In another example, provided herein is a VH domain having an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of EVQLVESGGGLVQPGTSLKLSCVASGFTFSDFWMSWVRQTPGKTMEWIADIKNDGSHTKYAPSLQNRFTISRDNAKNTLYLEVTDVRSEDTATYYCTRVGGTYTAYYWGHGVMVTVSS (SEQ ID NO: 25), or the sequence of SEQ ID NO: 25; or QTPSSMPASLGERVTLSCRTSQGINTYLNWYQQKPDGTITPLIHYTSNLESGVPSRFSGSGFGTDYFLTISSLEPEDFAMYFCQQYASSPWTFGGGTKLELK (SEQ ID NO: 26), or a VL domain having the sequence of SEQ ID NO: 26; or (c) an anti-IL-33 antibody or antigen-binding fragment thereof comprising the VH domain of (a) and the VL domain of (b).

[0169] Any of the anti-IL-33 antibodies or antigen-binding fragments thereof provided herein comprise the following heavy chain framework regions: FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQPGTSLKLSCVASGFTFS (SEQ ID NO: 29); FR-H2 comprising the amino acid sequence of WVRQTPGKTMEWI (SEQ ID NO: 30); FR-H3 comprising the amino acid sequence of RFTISRDNAKNTLYLEVTDVRSEDTATYYCTR (SEQ ID NO: 31); and WGHGVMVTVSS (SEQ ID NO: 32). 2) one, two, three, or four of FR-H4 comprising the amino acid sequence of SEQ ID NO: 29 to 32, or a combination of one or more of the above FRs, and / or one or more variants thereof having at least about 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 29 to 32. In some instances, an anti-IL-33 antibody may comprise one, two, three, or four of the following heavy chain framework regions: FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQPGTSLKLSCVASGFTFS (SEQ ID NO: 29); FR-H2 comprising the amino acid sequence of WVRQTPGKTMEWI (SEQ ID NO: 30); FR-H3 comprising the amino acid sequence of RFTISRDNAKNTLYLEVTDVRSEDTATYYCTR (SEQ ID NO: 31); and FR-H4 comprising the amino acid sequence of WGHGVMVTVSS (SEQ ID NO: 32). In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 25.

[0170] Any of the anti-IL-33 antibodies or antigen-binding fragments thereof provided herein comprise the following light chain framework regions: FR-L1 comprising the amino acid sequence of DIQMTQTPSSMPASLGERVTLSC (SEQ ID NO: 33); FR-L2 comprising the amino acid sequence of WYQQKPDGTITPLIH (SEQ ID NO: 34); FR-L3 comprising the amino acid sequence of GVPSRFSGSGFGTDYFLTISSLEPEDFAMYFC (SEQ ID NO: 35); and FGGGTKLELK (SEQ ID NO: 36). or a combination of one or more of the above FRs, and / or one or more variants thereof having at least about 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 33 to 36. In some cases, an anti-IL-33 antibody can comprise one, two, three, or four of the following light chain framework regions: FR-L1 comprising the amino acid sequence of DIQMTQTPSSMPASLGERVTLSC (SEQ ID NO: 33); FR-L2 comprising the amino acid sequence of WYQQKPDGTITPLIH (SEQ ID NO: 34); FR-L3 comprising the amino acid sequence of GVPSRFSGSGFGTDYFLTISSLEPEDFAMYFC (SEQ ID NO: 35); and FR-L4 comprising the amino acid sequence of FGGGTKLELK (SEQ ID NO: 36). In some cases, the VL domain comprises the amino acid sequence of SEQ ID NO: 26.

[0171] In another example, provided herein is a method for detecting a schizophrenia comprising administering to a patient a schizophrenia patient, comprising administering to the patient a schizophrenia patient, the method comprising administering to the patient a schizophrenia patient, the method comprising administering to the patient a schizophrenia patient, the method comprising administering to the patient a schizophrenia patient, the method comprising administering to the patient a schizophrenia patient, the method comprising administering to the patient a schizophrenia patient The amino acid sequence of VPRECNPCGCTGSEVSSVFIFPPKTKDVLTITLTPKVTCVVVDISQNDPEVRFSWFIDDVEVHTAQTHAPEKQSNSTLRSVSELPIVHRDWLNGKTFKCKVNSGAFPAPIEKSISKPEGTPRGPQVYTMAPPKEEMTQSQVSITCMVKGFYPPDIYTEWKMNGQPQENYKNTPPTMDTDGSYFLYSKLNVKKETWQQGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 27) a heavy chain (HC) comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the sequence set forth in SEQ ID NO: 27; or (c) an anti-IL-33 antibody comprising the HC of (a) and the LC of (b).

[0172] For example, an anti-IL-33 antibody may comprise: (a) a CDR-H1 comprising the amino acid sequence of DFWMS (SEQ ID NO: 19); (b) a CDR-H2 comprising the amino acid sequence of DIKNDGSHTKYAPSLQN (SEQ ID NO: 20); (c) a CDR-H3 comprising the amino acid sequence of VGGTYTAYY (SEQ ID NO: 21); (d) a CDR-L1 comprising the amino acid sequence of RTSQGINTYLN (SEQ ID NO: 22); (e) a CDR-L2 comprising the amino acid sequence of YTSNLES (SEQ ID NO: 23); and (f) a CDR-L3 comprising the amino acid sequence of QQYASSPWT (SEQ ID NO: 24). In some cases, the anti-IL-33 antibody comprises: (a) a heavy chain variable (VH) domain having an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of SEQ ID NO:25; (b) a light chain variable (VL) domain having an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of SEQ ID NO:26; or (c) the VH domain of (a) and the VL domain of (b). In some instances, the anti-IL-33 antibody comprises the following heavy chain framework regions: FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQPGTSLKLSCVASGFTFS (SEQ ID NO: 29); FR-H2 comprising the amino acid sequence of WVRQTPGKTMEWI (SEQ ID NO: 30); FR-H3 comprising the amino acid sequence of RFTISRDNAKNTLYLEVTDVRSEDTATYYCTR (SEQ ID NO: 31); and FR-H4 comprising the amino acid sequence of WGHGVMVTVSS (SEQ ID NO: 32). In some instances, the anti-IL-33 antibody comprises the following light chain framework regions: FR-L1 comprising the amino acid sequence of DIQMTQTPSSMPASLGERVTLSC (SEQ ID NO: 33); FR-L2 comprising the amino acid sequence of WYQQKPDGTITPLIH (SEQ ID NO: 34); FR-L3 comprising the amino acid sequence of GVPSRFSGSGFGTDYFLTISSLEPEDFAMYFC (SEQ ID NO: 35); and FR-L4 comprising the amino acid sequence of FGGGTKLELK (SEQ ID NO: 36).In some instances, the anti-IL-33 antibody comprises a binding domain comprising (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 26. In some instances, an exemplary anti-IL-33 antibody is 15.21C7.

[0173] For example, provided herein is an isolated antibody, or antigen-binding fragment thereof, that specifically binds to IL-33, the antibody comprising (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 26. In some instances, an exemplary anti-IL-33 antibody is 15.21C7.

[0174] In another example, provided herein is an isolated antibody, or antigen-binding fragment thereof, that specifically binds to IL-33, the antibody comprising (a) a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 27 and (b) a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 28. In some instances, an exemplary anti-IL-33 antibody is 15.21C7.

[0175] Any of the antibodies provided herein may specifically bind to IL-33 in an oxidized state (e.g., oxidized IL-33) or to IL-33 in a reduced state (e.g., reduced IL-33). In some cases, the antibody specifically binds to reduced IL-33. In some cases, the anti-IL-33 antibodies provided herein have a K within 2-fold (e.g., within 1.5-fold or within 1-fold) of oxidized IL-33 and reduced IL-33. D In some embodiments, the antibody binds oxidized IL-33 and reduced IL-33 with substantially the same K D In some cases, the antibody binds to reduced IL-33. In some cases, the antibody binds to reduced IL-33 with a K of ≦1 nM. DIn some cases, the antibody binds reduced IL-33 at between 0.01 nM and 1 nM (e.g., between 0.01 and 0.9 nM, between 0.01 and 0.8 nM, between 0.01 and 0.7 nM, between 0.01 and 0.6 nM, between 0.01 and 0.5 nM, between 0.05 and 0.9 nM, between 0.1 and 0.9 nM, between 0.2 and 0.9 nM, between 0.3 and 0.9 nM, between 0.2 and 0.6 nM, between 0.3 and 0.5 nM, between 0.3 and 0.4 nM, or between 0.4 and 0.5 nM; e.g., about 0.05 nM, about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0.325 nM, about 0.34 nM, about 0.35 nM, about 0.4 nM, about 0.45 nM, about 0.475 nM, about 0.49 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, or about 1 nM). D In some cases, the antibody binds to reduced IL-33 at K D In some instances, the antibody binds reduced IL-33 with a K between 0.1 nM and 0.5 nM (e.g., between 0.2 and 0.5 nM, between 0.3 nM and 0.5 nM, between 0.3 nM and 0.4 nM, or between 0.4 nM and 0.5 nM). D In some instances, the antibody binds reduced IL-33 with a K of about 0.487 nM. D In some instances, the antibody binds reduced IL-33 with a K of about 0.338 nM. D The above mentioned K D Both values ​​may be determined by surface plasmon resonance (SPR) (see, for example, Examples 1 and 2).

[0176] Any of the antibodies provided herein may specifically bind to human or cynomolgus monkey (cyno) IL-33. In some cases, the antibody specifically binds to human IL-33. In some cases, the antibody specifically binds to human IL-33. DIn some cases, the antibody binds to human IL-33 with an affinity of between 0.01 nM and 1 nM (e.g., between 0.01 and 0.9 nM, between 0.01 and 0.8 nM, between 0.01 and 0.7 nM, between 0.01 and 0.6 nM, between 0.01 and 0.5 nM, between 0.05 and 0.9 nM, between 0.1 and 0.9 nM, between 0.2 and 0.9 nM, between 0.3 and 0.9 nM, between 0.2 and 0.6 nM, between 0.3 and 0.5 nM, between 0.3 and 0.4 nM, or between 0.4 and 0.5 nM; e.g., about 0.05 nM, about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0.325 nM, about 0.34 nM, about 0.35 nM, about 0.4 nM, about 0.45 nM, about 0.475 nM, about 0.49 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, or about 1 nM). D In some instances, the antibody binds to human IL-33 at K D In some instances, the antibody binds to human IL-33 with a K between 0.1 nM and 0.5 nM (e.g., between 0.2 and 0.5 nM, between 0.3 nM and 0.5 nM, between 0.3 nM and 0.4 nM, or between 0.4 nM and 0.5 nM). D In some instances, the antibody binds to human IL-33 with a K of about 0.487 nM. D In some instances, the antibody binds to human IL-33 with a K of about 0.338 nM. D The above mentioned K D Both values ​​may be determined by surface plasmon resonance (SPR) (see, for example, Examples 1 and 2).

[0177] In some embodiments, the antibody has a K within 2-fold (e.g., within 1.5-fold or within 1-fold) of IL-33 bound to soluble interleukin-1 receptor-like 1 (sST2) protein and IL-33 not bound to sST2. D In some embodiments, the antibody binds to IL-33 bound to sST2 protein with substantially the same K as IL-33 that is not bound to sST2. DIn some embodiments, the sST2 is human sST2 (hsST2). In some cases, the antibody binds to IL-33 bound to an sST2 protein (e.g., hsST2) at a K D In some cases, the antibody binds to IL-33 bound to an sST2 (e.g., hsST2) protein at between 0.01 nM and 1 nM (e.g., between 0.01 and 0.9 nM, between 0.01 and 0.8 nM, between 0.01 and 0.7 nM, between 0.01 and 0.6 nM, between 0.01 and 0.5 nM, between 0.05 and 0.9 nM, between 0.1 and 0.9 nM, between 0.2 and 0.9 nM, between 0.3 and 0.9 nM). , between 0.2 and 0.6 nM, between 0.3 and 0.5 nM, between 0.3 and 0.4 nM, or between 0.4 and 0.5 nM; e.g., about 0.05 nM, about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0.325 nM, about 0.34 nM, about 0.35 nM, about 0.4 nM, about 0.45 nM, about 0.475 nM, about 0.49 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, or about 1 nM). D In some cases, the antibody binds to IL-33 bound to an sST2 protein (e.g., hsST2) at K D In some instances, the antibody binds to IL-33 bound to an sST2 (e.g., hsST2) protein with a K between 0.1 nM and 0.5 nM (e.g., between 0.2 and 0.5 nM, between 0.3 nM and 0.5 nM, between 0.3 nM and 0.4 nM, or between 0.4 nM and 0.5 nM). D In some instances, the antibody binds to IL-33 bound to an sST2 (e.g., hsST2) protein with a K of about 0.487 nM. D In some instances, the antibody binds to IL-33 bound to an sST2 (e.g., hsST2) protein with a K of about 0.338 nM. D Combine with.

[0178] In some cases, any of the anti-IL-33 antibodies described herein (e.g., described above or below) may have one or more (e.g., one, two, or three) of the following characteristics: (i) the antibody has a K D (ii) the antibody specifically binds reduced IL-33 with a K between about 0.1 nM and about 0.5 nM; D and / or (iii) the antibody specifically binds to IL-33 bound to an sST2 (e.g., hsST2) protein with a K of between about 0.1 nM and 0.5 nM. D In some cases, any of the anti-IL-33 antibodies described herein may have one of the aforementioned characteristics. In some cases, any of the anti-IL-33 antibodies described herein may have two of the aforementioned characteristics. In some cases, any of the anti-IL-33 antibodies described herein may have all three of the aforementioned characteristics.

[0179] Any of the anti-IL-33 antibodies provided herein can be a monoclonal antibody, including a chimeric, humanized, or human antibody. In some instances, the anti-IL-33 antibody is a monoclonal antibody. In some instances, the anti-IL-33 antibody is a chimeric antibody. In some instances, the anti-IL-33 antibody is a humanized antibody. In some instances, the anti-IL-33 antibody is a rat antibody. In some embodiments, the anti-IL-33 antibody is an IgG antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is a human IgG1. In some embodiments, the antibody is an IgG2a antibody. In some embodiments, the antibody is a rat IgG2a antibody.

[0180] Any of the anti-IL-33 antibodies provided herein can be an antibody fragment, e.g., an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In certain cases, the antibody fragment is a Fab fragment. In some cases, the antibody fragment specifically binds to IL-33 (e.g., hIL-33; e.g., reduced IL-33; e.g., IL-33 bound to ST2).

[0181] In other cases, any of the anti-IL-33 antibodies provided herein can be a full-length antibody, such as an intact IgG1 antibody, an intact IgG4 antibody, or other antibody class or isotype as defined herein. In some cases, the antibody is an IgG4 antibody that includes a mutation in the hinge region. In some cases, the mutation is a substitution mutation. In some cases, the substitution mutation is at amino acid residue S228 (EU numbering). In some cases, the substitution mutation is an S228P mutation.

[0182] Any of the anti-IL-33 antibodies provided herein can be monospecific antibodies. In other cases, any of the anti-IL-33 antibodies provided herein can be multispecific antibodies (e.g., bispecific antibodies).

[0183] In another aspect, the invention is an antibody that binds to the same epitope of IL-33 as any one of the preceding antibodies.

[0184] In another aspect, provided herein is an antibody that competes with, cross-blocks, or is cross-blocked by any one of the preceding antibodies for binding to IL-33 (e.g., human IL-33).

[0185] In further aspects, any of the anti-IL-33 antibodies disclosed herein can incorporate any of the features described in Sections 1-7 below, either alone or in combination:

[0186] 1. Antibody affinity In some embodiments, the antibodies provided herein have a concentration of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, ≦1 pM, or ≦0.1 pM (e.g., 10 -6 M or less, e.g., 10 -6 M to 10 -9 M or less, e.g., 10 -9 M to 10 -13 Dissociation constant (K D In some instances, the antibody has a K for IL-33 of 1 nM. D In some cases, the antibody specifically binds IL-33 at between 0.01 nM and 1 nM (e.g., between 0.01 and 0.9 nM, between 0.01 and 0.9 nM, between 0.01 and 0.8 nM, between 0.01 and 0.7 nM, between 0.01 and 0.6 nM, between 0.01 and 0.5 nM, between 0.05 and 0.9 nM, between 0.1 and 0.9 nM, between 0.2 and 0.9 nM, between 0.3 and 0.9 nM, between 0.2 ... between 0.6 nM, between 0.3 and 0.5 nM, between 0.3 and 0.4 nM, or between 0.4 and 0.5 nM; e.g., about 0.05 nM, about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0.325 nM, about 0.34 nM, about 0.35 nM, about 0.4 nM, about 0.45 nM, about 0.475 nM, about 0.49 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, or about 1 nM). D In some instances, the antibody binds to IL-33 with a K of ≦0.5 nM. D In some instances, the antibody specifically binds IL-33 with a K between 0.1 nM and 0.5 nM (e.g., between 0.2 and 0.5 nM, between 0.3 nM and 0.5 nM, between 0.3 nM and 0.4 nM, or between 0.4 nM and 0.5 nM). D In some instances, the antibody binds to IL-33 with a K of about 0.487 nM. D In some instances, the antibody binds to IL-33 with a K of about 0.338 nM. D Combine with.

[0187] In one embodiment, K Dis measured by radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is determined by the lowest concentration of ( 125 I) Measurements are made by equilibrating Fab with labeled antigen and then capturing bound antigen on a plate coated with anti-Fab antibody (see, e.g., Chen et al. J. Mol. Biol. 293:865-881, 1999). To establish conditions for the assay, MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 μg / mL of capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), then blocked with 2% (w / v) bovine serum albumin in PBS for 2 to 5 hours at room temperature (approximately 23° C.). Non-adsorbent plates (NUNC TM #269620), 100pM or 26pM [ 125 I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12 in Presta et al. Cancer Res. 57:4593-4599, 1997). The Fab of interest is then incubated overnight, although incubation may continue for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate and incubated at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN®-20) in PBS. Once the plate is dry, 150 μL / well of scintillant (MICROSCINT-20) is added. TM , Packard) and TOPCOUNT TM Plates are counted for 10 minutes in a gamma counter (Packard). Concentrations of each Fab that yielded 20% or less of maximal binding are selected for use in competitive binding assays.

[0188] According to another embodiment, K Dis measured using a BIACORE® surface plasmon resonance assay. In a specific embodiment, a Series S Protein A sensor chip (Cytiva, Marlborough, MA, USA) was used to capture recombinant chimeric human IgG versions of anti-hIL-33 Abs using a BIACORE® 8K instrument (Cytiva, Marlborough, MA, USA). Antibody binding to human histidine tagged hIL-33 (Genentech, South San Francisco, CA, USA) was measured using multi-cycle kinetics. Sensorgrams were recorded using a 2 min injection time at a flow rate of 30 μL / min at 25° C. or 37° C. with a running buffer of 10 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) (pH 7.4), 150 mM sodium chloride (NaCl), 3 mM ethylenediaminetetraacetic acid (EDTA), and 0.005% TWEEN® 20. After injection, dissociation of IL-33 from the antibody was monitored for 10 min in running buffer. The surface was regenerated between binding cycles with 30 μl injections of 10 mM glycine hydrochloride (HCl) pH 1.5. Association rates (k ) were calculated using a simple one-to-one Langmuir binding model (BIAcore® Evaluation Software version 3.2) by simultaneously fitting association and dissociation sensorgrams after subtraction of a blank containing only running buffer. on ) and dissociation rate (k off Calculate the equilibrium dissociation constant (K D ) is k off / k on It is calculated as a ratio (see, e.g., Chen et al. (J. Mol. Biol. 293:865-881, 1999)).

[0189] 2. Antibody fragment Any of the antibodies provided herein may be an antibody fragment (e.g., an antigen-binding (e.g., IL-33-binding) antibody fragment). Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. In some cases, the antibody fragment is a Fab. For a review of some antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994). See also WO 93 / 16185, and U.S. Pat. Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a description of Fab and F(ab')2 fragments which contain salvage receptor binding epitope residues and have extended half-lives in vivo.

[0190] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, e.g., EP 404097; WO 1993 / 01161; Hudson et al. Nat. Med. 9:129-134, 2003; and Hollinger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993. Triabodies and tetrabodies are also described in Hudson et al. Nat. Med. 9:129-134, 2003.

[0191] A single domain antibody is an antibody fragment that contains all or a portion of the heavy chain variable domain of an antibody or all or a portion of the light chain variable domain of an antibody. In some embodiments, a single domain antibody is a human single domain antibody (see, e.g., U.S. Pat. No. 6,248,516).

[0192] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies, as well as production by recombinant host cells (e.g., E. coli or phage) as described herein.

[0193] 3. Chimeric and humanized antibodies In some embodiments, the antibody provided herein is a chimeric antibody. Some chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567, and Morrison et al. Proc. Natl. Acad. Sci. USA, 81:6851-6855, 1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region obtained from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from those of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0194] In some embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In general, a humanized antibody comprises one or more variable domains in which the HVRs (or a portion thereof) are derived from a non-human antibody and the FRs (or a portion thereof) are derived from a human antibody sequence. A humanized antibody will also optionally comprise at least a portion of a human constant region. In some embodiments, FR residues in a humanized antibody are replaced with the corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues were derived), e.g., to restore or improve antibody specificity or affinity.

[0195] Humanized antibodies and methods for making them are reviewed, for example, in Almagro et al. Front. Biosci. 13:1619-1633, 2008, and further described, for example, in Riechmann et al. Nature 332:323-329, 1988; Queen et al. Proc. Natl. Acad. Sci. USA 86:10029-10033, 1989; U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al. Methods 36:25-34, 2005 (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498, 1991 (describing resurfacing); Dall'Acqua et al. Methods 36:43-60, 2005 (describing "FR shuffling"); and Osbourn et al. Methods 36:61-68, 2005 and Klimka et al. Br. J. Cancer, 83:252-260, 2000 (describing a "guided selection" approach to FR shuffling).

[0196] Human framework regions that may be used for humanization include, but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296, 1993); framework regions obtained from consensus sequences of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285, 1992; and Presta et al. J. Immunol., 151:2623, 1993); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro et al. Front. Biosci. 13:1619-1633, 2008); and framework regions obtained from screening of FR libraries (see, e.g., Baca et al. J. Biol. Chem. 272:10678-10684, 1997; and Rosok et al. al. J. Biol. Chem. 271:22611-22618, 1996).

[0197] 4. Human antibodies In some embodiments, the antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in the art.Human antibodies are generally described in van Dijk et al.Curr.Opin.Pharmacol.5:368-74,2001 and Lonberg,Curr.Opin.Immunol.20:450-459,2008.

[0198] Human antibodies can be prepared by administering an immunogen to intact antibodies with human variable regions or to transgenic animals that have been modified to produce intact human antibodies in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus or is present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125, 2005. See, e.g., XENOMOUSE, J. Immunol. 2004, 143:1117-1125, 2005. TM Nos. 6,075,181 and 6,150,584, which describe HUMAB technology; U.S. Patent No. 5,770,429, which describes HUMAB technology; U.S. Patent No. 7,041,870, which describes KM MOUSE technology; and VELOCIMOUSE. TM See also US Patent Application Publication No. 2007 / 0061900, which describes the technology. The human variable regions from intact antibodies produced by such animals may be further modified, for example, by combining with a different human constant region.

[0199] Human antibodies can also be produced by hybridoma-based methods. Human myeloma cell lines and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, for example, Kozbor J.Immunol.133:3001,1984; Brodeur et al. Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al. J.Immunol.147:86,1991). Human antibodies produced via human B-cell hybridoma technology have also been described in Li et al. Proc.Natl.Acad.Sci.USA,103:3557-3562,2006. Further methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268, 2006 (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers et al. Histology and Histopathology 20(3):927-937, 2005 and Vollmers et al. Methods and Findings in Experimental and Clinical Pharmacology 27(3):185-91, 2005.

[0200] Human antibodies can also be made by isolating Fv clone variable domain sequences selected from a phage display library derived from humans. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0201] 5. Library-derived antibodies The antibodies disclosed herein can be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al. Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and can be found, for example, in McCafferty et al. Nature 348:552-554, 1990; Clackson et al. Nature 352:624-628, 1991; Marks et al. J. Mol. Biol. 222:581-597, 1992; Marks et al. in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al. J. Mol. Biol. 338(2):299-310, 2004; Lee et al. al. J. Mol. Biol. 340(5):1073-1093, 2004; Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472, 2004; and Lee et al. J. Immunol. Methods 284(1-2):119-132, 2004.

[0202] In some phage display methods, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be screened for antigen-binding phages as described by Winter et al. Ann. Rev. Immunol., 12:433-455, 1994. Phages typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) without immunization to provide a single source of antibodies against a wide range of non-self antigens and even self antigens, as described by Griffiths et al. EMBO J. 12:725-734, 1993. Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells, using PCR primers containing random sequences to encode the highly variable HVR3 region, and achieving rearrangement in vitro as described in Hoogenboom et al. J. Mol. Biol., 227:381-388, 1992. Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0203] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0204] 6. Multispecific antibodies In some embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In some embodiments, bispecific antibodies can bind to two different epitopes of IL-33. In some embodiments, one of the binding specificities is for IL-33 and the other is for any other antigen. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0205] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein et al. Nature 305:537, 1983; WO 93 / 08829, and Traunecker et al. EMBO J. 10:3655, 1991), and "knobs-in-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can be made by a number of techniques, including the manipulation of electrostatic steering effects to create antibody Fc heterodimeric molecules (WO 2009 / 089004); cross-linking of two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al. Science, 229:81, 1985); the use of leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al. J. Immunol., 148(5):1547-1553, 1992); the use of "diabody" technology to generate bispecific antibody fragments (see, e.g., Hollinger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993); and the use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al. J. Immunol. 152:5368, 1994); and the use of glycerol-based Fv (sFv) dimers (see, e.g., Tutt et al. J. Immunol. 152:5368, 1994). Alternatively, the antibodies may be prepared by the preparation of trispecific antibodies as described in et al. J. Immunol. 147:60,1991.

[0206] Engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies," are also included herein (see, eg, US Patent Application Publication No. 2006 / 0025576).

[0207] The antibodies or fragments herein also include "dual acting FAbs" or "DAFs" that contain antigen binding sites that bind to IL-33 as well as another distinct antigen (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820).

[0208] The use of knobs-into-holes as a method for generating multispecific antibodies is described, for example, in U.S. Pat. No. 5,731,168, WO 2009 / 089004, U.S. Patent Application Publication No. 2009 / 0182127, U.S. Patent Application Publication No. 2011 / 0287009, Marvin and Zhu, Acta Pharmacol. Sin. (2005) 26(6):649-658, and Kontermann (2005) Acta Pharmacol. Sin., 26:1-9.

[0209] 7. Antibody variants In some embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it is desired to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct has the desired properties, e.g., antigen binding.

[0210] a) Substitution, insertion, and deletion variants In some embodiments, antibody variants are provided that have one or more amino acid substitutions. Target sites for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 2 under the heading of "preferred substitutions." More substantial changes are shown in Table 2 under the heading of "exemplary substitutions" and are further described below with reference to classes of amino acid side chains. Amino acid substitutions can be introduced into the antibody of interest and screened for the desired activity, e.g., retaining / improving antigen binding, reducing immunogenicity, or improving ADCC or CDC.

[0211] Table 2. Exemplary Amino Acid Substitutions TIFF2024544885000002.tif157170

[0212] Amino acids can be grouped according to the following common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0213] One type of substitutional variant involves substituting one or more HVR (e.g., CDR) residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variant or variants selected for further study will have altered (e.g., improved) certain biological properties (e.g., improved affinity, reduced immunogenicity) compared to the parent antibody and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which is conveniently generated, for example, using phage display-based affinity maturation techniques as described herein. Briefly, one or more HVR residues are mutated and the mutant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0214] To improve antibody affinity, changes (e.g., substitutions) may be made in HVRs (e.g., CDRs). Such changes can be made in HVR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196, 2008), and / or in residues that contact the antigen, and the resulting variants VH or VL are tested for binding affinity. Affinity maturation by building and reselecting from secondary libraries is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al. ed., Human Press, Totowa, NJ, 2001). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-directed approach, in which multiple HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, HVR-H3 and HVR-L3 are often targeted.

[0215] In some embodiments, substitutions, insertions, or deletions may be made within one or more HVRs (e.g., CDRs) so long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made within an HVR. Such changes may, for example, be made outside the antigen contacting residues within the HVR. In some embodiments of the variant VH and VL sequences provided above, each HVR is unchanged or contains no more than one, two, or three amino acid substitutions.

[0216] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described in Cunningham et al. Science 244:1081-1085, 1989. In this method, a residue or target group of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral or negatively charged amino acids (e.g., Ala or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and the antigen. Such contact and adjacent residues may be targeted as candidates for substitution or may be eliminated. The variants may be screened to determine whether they contain the desired properties.

[0217] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion includes an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which extends the serum half-life of the antibody.

[0218] b) Glycosylation variants In some embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Adding or deleting glycosylation sites from an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0219] If the antibody comprises an Fc region, the carbohydrate attached thereto can be altered. Natural antibodies produced by mammalian cells typically comprise branched, biantennary oligosaccharides attached by N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32, 1997. The oligosaccharides can include various carbohydrates, e.g., mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies disclosed herein can be made to generate antibody variants with specific improved properties.

[0220] In one embodiment, antibody variants are provided with a glycostructure that lacks fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycostructure of Asn297 relative to the sum of all glycostructures (e.g., complex, hybrid, and high mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry, e.g., as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 (Eu numbering of Fc region residues) in the Fc region, although Asn297 may also be located about ±3 amino acids upstream or downstream from position 297, i.e., at positions 294 to 300, due to minor sequence differences in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., U.S. Patent Application Publication Nos. 2003 / 0157108 and 2004 / 0093621. Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: U.S. Patent Application Publication Nos. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; U.S. Patent Application Publication Nos. 2003 / 0115614; 2002 / 0164328; 2004 / 0093621; 2004 / 01 No. 32140; No. 2004 / 0110704; No. 2004 / 0110282; No. 2004 / 0109865; International Publication No. 2003 / 085119; No. 20 03 / 084570; 2005 / 035586; 2005 / 035778; 2005 / 053742; 2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249, 2004; Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614, 2004.Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545, 1986; U.S. Patent Application Publication No. 2003 / 0157108; and WO 2004 / 056312, especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87:614, 2004; Kanda et al. Biotechnol. Bioeng. 94(4):680-688, 2006; and WO 2003 / 085107).

[0221] Further provided are antibody variants with bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878; U.S. Pat. No. 6,602,684; and U.S. Pat. Appl. Pub. No. 2005 / 0123546. Also provided are antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0222] c) Fc domain variants In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0223] In some embodiments, the invention contemplates antibody variants that have some, but not all, effector functions that make them desirable candidates for applications where in vivo antibody half-life is important, but where certain effector functions (e.g., complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII and FcγRIII. Expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch et al. Annu. Rev. Immunol. 9:457-492, 1991. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom et al. Proc. Natl. Acad. Sci. USA 83:7059-7063, 1986 and Hellstrom et al. Proc. Natl. Acad. Sci. USA 82:1499-1502, 1985); U.S. Pat. No. 5,821,337 (see, Bruggemann et al. J. Exp. Med. 166:1351-1361, 1987). Alternatively, non-radioactive assay methods may be used (e.g., ACTI for flow cytometry). TMNon-radioactive cytotoxicity assay (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo in an animal model, e.g., as disclosed in Clynes et al. Proc. Natl. Acad. Sci. USA 95:652-656, 1998. C1q binding assays can also be performed to confirm that the antibody cannot bind C1q and thus lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al. J. Immunol. Methods 202:163, 1996; Cragg et al. Blood 101:1045-1052, 2003; and Cragg et al. Blood 103:2738-2743, 2004). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova et al. Intl. Immunol. 18(12):1759-1769, 2006).

[0224] Antibodies with reduced effector function include those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including so-called "DANA" Fc variants with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0225] Certain antibody variants have been described with improved or diminished binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312, and Shields et al. J. Biol. Chem. 9(2):6591-6604, 2001).

[0226] In some embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0227] In some embodiments, modifications are made in the Fc region that result in altered (i.e., improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184, 2000.

[0228] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), responsible for the transfer of maternal IgG to the fetus (Guyer et al. J. Immunol. 117:587, 1976 and Kim et al. J. Immunol. 24:249, 1994), have been described in U.S. Patent Application Publication No. 2005 / 0014934. These antibodies comprise an Fc region with one or more substitutions that improve binding of the Fc region to FcRn. Such Fc variants include substitutions at one or more of 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434, e.g., substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0229] For other examples of Fc region variants, see also Duncan et al. Nature 322:738-40, 1988; U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351.

[0230] d) Cysteine ​​Engineered Antibody Variants In some embodiments, it may be desirable to generate cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with a cysteine ​​residue. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By substituting these residues with cysteine, reactive thiol groups are thereby placed at accessible sites of the antibody, which may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates, as further described herein. In some embodiments, one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibodies may be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0231] e) Antibody derivative In some embodiments, the antibodies provided herein may be further modified to include additional non-proteinaceous moieties known in the art and readily available. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when multiple polymers are attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, and whether the antibody derivative will be used in therapy under limited conditions.

[0232] In another embodiment, a conjugate of an antibody and a non-protein moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al. Proc. Natl. Acad. Sci. USA 102:11600-11605, 2005). The radiation can be of any wavelength, including but not limited to wavelengths that heat the non-protein moiety to a temperature that is not harmful to normal cells but kills cells proximal to the antibody-non-protein moiety.

[0233] B. Recombinant Methods and Compositions Any of the antibodies described herein (e.g., anti-IL-33 antibodies) may be produced using recombinant methods and compositions described, for example, in U.S. Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-IL-33 antibody described herein, or a set of nucleic acids that together encode the antibody, is provided. Such nucleic acids may encode an amino acid sequence comprising the VL of the antibody, and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising one or more such nucleic acids is provided. In one embodiment, the host cell comprises (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, e.g., a rat cell, a Chinese hamster ovary (CHO) cell, a 293 cell, or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of making an anti-IL-33 antibody is provided, the method comprising culturing a host cell comprising nucleic acid encoding the antibody under conditions suitable for expression of the antibody, as described above, and optionally recovering the antibody from the host cell (or host cell medium).

[0234] For recombinant production of an anti-IL-33 antibody, a nucleic acid encoding the antibody, or a set of nucleic acids which together encode the antibody, is isolated, e.g., as described above, and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using standard procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0235] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0236] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains that have been "humanized" in their glycosylation pathways to produce antibodies with partially or fully human glycosylation patterns. See Gerngross Nat. Biotech. 22:1409-1414, 2004 and Li et al. Nat. Biotech. 24:210-215, 2006.

[0237] Suitable host cells for the expression of glycosylated antibodies can also be obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. A number of baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0238] Plant cell cultures can also be utilized as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (PLANTIBODIES FOR PRODUCING ANTIBODIES IN TRANSGENIC PLANTS). TMSee the following link for a description of the technology.

[0239] Vertebrate cells may also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (e.g., 293 cells or 293 cells described in Graham et al. J. Gen Virol. 36:59, 1977); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather Biol. Reprod. 23:243-251, 1980); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (HepG2); mouse mammary tumor (MMT060562); TRI cells, e.g., as described in Mather et al., Annals NYAcad. Sci. 383:44-68, 1982; MRC5 cells; and FS4 cells. Error! Bookmark not defined. Error! Bookmark not defined. Other useful mammalian host cell lines include DHFR - Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al. Proc. Natl. Acad. Sci. USA 77:4216, 1980); and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, e.g., Yazaki et al. Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268, 2003.

[0240] C. Assay The anti-IL-33 antibodies provided herein may be identified, screened or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0241] 1. Binding Assays and Other Assays In one aspect, the anti-IL-33 antibodies disclosed herein are tested for their antigen binding activity by known methods such as, for example, ELISA and Western blot.

[0242] In an exemplary ELISA assay, a first immobilized anti-IL-33 antibody or immunoconjugate is incubated in a solution containing IL-33 (e.g., reduced IL-33 or IL-33 bound to ST2). After incubation under conditions that allow binding of the first antibody or immunoconjugate to IL-33 (e.g., reduced IL-33 or IL-33 bound to ST2), excess unbound IL-33 is removed. A solution containing a second, biotinylated anti-hIL-33 antibody or immunoconjugate is added and incubated with the first immobilized anti-IL-33 antibody or immunoconjugate and the bound IL-33 (e.g., reduced IL-33 or IL-33 bound to ST2). Bound, reduced hIL-33 is then detected using a readout (e.g., with streptavidin conjugated to horseradish peroxidase (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and its substrate 3,3',5,5'-tetramethylbenzidine (Moss Inc., Pasadena, MD, USA)).

[0243] In another aspect, the binding assay includes iPCR, an assay in which the presence or level of a target molecule (e.g., a target biomolecule, e.g., a protein) is detected by an antibody-DNA conjugate. In some embodiments, the iPCR assay uses a first antibody conjugated to biotin and a second antibody conjugated to a nucleic acid moiety, where the first and second antibodies bind to the same target antigen. Both the first and second antibodies are mixed with a solution containing the target antigen, and the resulting mixture is incubated together, e.g., inside a PCR well. The presence of the nucleic acid moiety is then detected using PCR amplification, thereby indicating detection of the target antigen. In some embodiments, the iPCR assay is used to detect IL-33 (e.g., human IL-33; e.g., reduced IL-33). In some embodiments, the first and second antibodies are anti-IL-33 antibodies. In some embodiments, the first and second antibodies share 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity. In some embodiments, the first and second antibodies do not share the same amino acid sequence (e.g., the amino acid sequences of the first and second antibodies share 1% or less, 2% or less, 3% or less, 4% or less, 5% or less, 10% or less, 15% or less, or 20% or less amino acid sequence identity). Examples of iPCR assays are described in Sano T et.al., Science 1992,258:120-122 and Niemeyer et al., Trends Microbiol.2017,8:65.

[0244] In another embodiment, the present invention may be used in a variety of applications, including, for example, immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (e.g., Western blotting), flow cytometry (e.g., FACS), and the like. TMAssays are provided for using an anti-IL-33 antibody or immunoconjugate, such as an anti-IL-33 antibody or immunoconjugate disclosed herein, to detect the presence or level of IL-33 (e.g., human IL-33, reduced IL-33, oxidized IL-33, total IL-33, unbound IL-33, and IL-33 bound to ST2) in a biological sample in a PCR or enzyme-linked immunosorbent assay (ELISA) assay. In some embodiments, the antibodies of the invention are tested for such activity.

[0245] In another aspect, a competition assay can be used to identify antibodies that compete with the anti-IL-33 antibodies disclosed herein for binding to IL-33. In some embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) bound by the anti-IL-33 antibodies disclosed herein. Detailed exemplary methods for mapping the epitope to which an antibody binds are provided in Morris "Epitope Mapping Protocols," in Methods in Molecular Biology Vol. 66 (Humana Press, Totowa, NJ), 1996.

[0246] In an exemplary competitive assay, immobilized IL-33 is incubated in a solution containing a first labeled antibody that binds to IL-33 and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to IL-33. The second antibody can be present in a hybridoma supernatant. As a control, immobilized IL-33 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to IL-33, excess unbound antibody is removed and the amount of label associated with immobilized IL-33 is measured. If the amount of label associated with immobilized IL-33 is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody competes with the first antibody for binding to IL-33. See Harlow et al. Antibodies: A Laboratory Manual Ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), 1988.

[0247] In another embodiment, the competition of two antibodies for binding to the same epitope is determined by epitope binning. For example, a labeled antigen is immobilized on a solid surface and reacted with a saturating amount of a first antibody. A second competing antibody is then added. Any additional binding by the second antibody, detected, for example, by OCTET® (ForteBio) or other biolayer interferometry (BLI) technology, indicates that the two antibodies bind to distant, non-overlapping epitopes. The absence of additional binding indicates that the two antibodies bind to the same or overlapping epitopes. Several other methods, including ELISA, size exclusion chromatography, crystallography, HDX-MS, mutagenesis, and other SPR methods, can also be utilized to demonstrate that two antibodies bind to the same or overlapping epitopes. Similar techniques can be used to determine whether an antibody cross-blocks or is cross-blocked by an antibody of the invention.

[0248] 2. Activity Assay In one aspect, the present invention provides anti-IL-33 antibodies or immunoconjugates for detecting or measuring biological activities. Biological activities can include, for example, binding to IL-33 (e.g., IL-33 in the bloodstream) or peptide fragments thereof in vivo, in vitro, or ex vivo. In other embodiments, biological activities can include blocking or neutralizing IL-33 or preventing IL-33 from binding to a ligand, such as a receptor (e.g., IL-33 receptor ST2 and / or IL-1RAcP). In some embodiments, biological activities can include binding to site 1 on IL-33 and blocking binding to IL-33 receptors (i.e., ST2 and / or IL-1RAcP). Antibodies for detecting or measuring such biological activities in vivo and / or in vitro are also provided. In some embodiments, the antibodies disclosed herein are used to test for such biological activities. In some embodiments, the anti-IL-33 antibodies disclosed herein are used to test for inhibition in a cell-based IL-33 blocking assay. In some embodiments, the anti-IL-33 antibodies disclosed herein are used to test inhibition of IL-33-induced reporter activity in a cell-based blocking assay.

[0249] D. Immunoconjugates The present invention also provides immunoconjugates comprising an anti-IL-33 antibody provided herein conjugated to one or more labels. Labels include, but are not limited to, labels or moieties that are directly detected (e.g., fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels) as well as moieties that are indirectly detected, e.g., via an enzymatic reaction or molecular interaction, e.g., an enzyme or a ligand. Exemplary labels include, but are not limited to, radioisotopes, 32 P, 14 C. 125 I, 13 H, and 131I, rare earth chelates or fluorophores such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase coupled to enzymes that utilize hydrogen peroxide to oxidize dye precursors such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, and stable free radicals. In another embodiment, the label is a positron emitter. Positron emitters include, but are not limited to: 68 Ga, 18 F, 64 Cu, 86 Y, 76 Br, 89 Zr, and 124 In certain embodiments, the positron emitter is Zr. In some embodiments, the label is a nucleic acid, such as single-stranded DNA.

[0250] In some embodiments, the immunoconjugate comprises an anti-IL-33 antibody provided herein conjugated to a nucleic acid, e.g., DNA. In some embodiments, the immunoconjugate comprises an anti-IL-33 antibody conjugated to a 55-mer DNA. In some embodiments, the immunoconjugate comprises a 15.21C7 antibody conjugated to a DNA having the sequence of SEQ ID NO: 38. In some embodiments, the immunoconjugate is purified by size exclusion column.

[0251] In some embodiments, the immunoconjugate comprises an anti-IL-33 antibody provided herein conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope.

[0252] E. Methods and Compositions for Diagnostics and Detection In some embodiments, any one of the anti-IL-33 antibodies (e.g., 3F10 or 15.21C7) or immunoconjugates (e.g., comprising an anti-IL-33 antibody; e.g., comprising 3F10 or 15.21C7) provided herein is useful for detecting the presence of IL-33 (e.g., human IL-33, reduced IL-33, oxidized IL-33, total IL-33, unbound IL-33, and / or IL-33 bound to ST2) in a biological sample. As used herein, the term "detection" encompasses quantitative or qualitative detection.

[0253] In one embodiment, an anti-IL-33 antibody (e.g., 3F10 or 15.21C7) or immunoconjugate is provided for use in a method of diagnosis or detection. In another instance, the invention provides the use of an anti-IL-33 antibody (e.g., 3F10 or 15.21C7) or immunoconjugate in the manufacture of a reagent for use in a method of diagnosis or detection.

[0254] In one instance, a method is provided for detecting the presence or level of IL-33 (e.g., human IL-33, reduced IL-33, oxidized IL-33, total IL-33, unbound IL-33, and / or IL-33 bound to ST2) in a biological sample, e.g., as described below. In some embodiments, the method includes contacting a biological sample with an anti-IL-33 antibody (e.g., 3F10 or 15.21C7) or immunoconjugate described herein under conditions that allow binding of the anti-IL-33 antibody to IL-33 (e.g., human IL-33, reduced IL-33, oxidized IL-33, total IL-33, unbound IL-33, and / or IL-33 bound to ST2) and detecting whether a complex is formed between the anti-IL-33 antibody and IL-33 (e.g., human IL-33, reduced IL-33, oxidized IL-33, total IL-33, unbound IL-33, and / or IL-33 bound to ST2). Such methods can be in vitro or in vivo methods. The anti-IL-33 antibodies of the invention (e.g., 3F10 and 15.21C7) or immunoconjugates of the invention can be used to detect and / or elucidate the IL-33 expression in a variety of cell types, including, for example, immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (e.g., Western blotting), flow cytometry (e.g., FACS), and the like. TM ), enzyme-linked immunosorbent assay (ELISA), and iPCR.

[0255] In some embodiments, the method comprises contacting a biological sample with an immunoconjugate described herein in the presence of a second anti-IL-33 antibody (e.g., 1E1v8) that specifically binds to an epitope on IL-33 that is different from the epitope specifically bound by the anti-IL-33 antibody in the immunoconjugate, detecting the presence of a bound immunoconjugate under conditions that allow binding of the anti-IL-33 antibody to IL-33 (e.g., human IL-33, reduced IL-33, oxidized IL-33, total IL-33, unbound IL-33, and / or IL-33 bound to ST2), and detecting whether a complex is formed between the anti-IL-33 antibody and IL-33 (e.g., human IL-33, reduced IL-33, oxidized IL-33, total IL-33, unbound IL-33, and / or IL-33 bound to ST2). In some embodiments, the biological sample is treated with TCEP prior to said contacting. In some embodiments, the immunoconjugate is any one of the anti-IL-33 antibodies described herein (e.g., 15.21C7) conjugated to a nucleic acid (e.g., single-stranded DNA). In some embodiments, the immunoconjugate is purified by size exclusion chromatography prior to said contacting. In some embodiments, said detecting is by iPCR.

[0256] The invention further provides for the use of an anti-IL-33 antibody in a method of diagnosing a subject suffering from a disorder (e.g., an IL-33-mediated disorder), the method comprising determining the presence or level of IL-33 (e.g., human IL-33, reduced IL-33, oxidized IL-33, total IL-33, unbound IL-33, and / or IL-33 bound to ST2) in a sample obtained from the subject by contacting the sample with an anti-IL-33 antibody disclosed herein (e.g., 3F10 or 15.21C7) and detecting the presence of bound antibody. The invention further provides for the use of the immunoconjugate in a method of diagnosing a subject suffering from a disorder (e.g., an IL-33-mediated disorder), the method comprising determining the presence or level of IL-33 (e.g., human IL-33, reduced IL-33, oxidized IL-33, total IL-33, unbound IL-33, and / or IL-33 bound to ST2) in a sample obtained from the subject by contacting the sample with an immunoconjugate disclosed herein (e.g., comprising an anti-IL-33 antibody; e.g., comprising 3F10 or 15.21C7) and detecting the presence of bound antibody.

[0257] The invention further provides for the use of an anti-IL-33 antibody in the manufacture of a reagent for use in a method of diagnosing a subject suffering from a disorder (e.g., an IL-33-mediated disorder), comprising determining the presence or level of IL-33 in a sample obtained from a subject by contacting the sample with an anti-IL-33 antibody disclosed herein (e.g., 3F10 or 15.21C7) and detecting the presence of bound antibody. The invention further provides for the use of an immunoconjugate in the manufacture of a reagent for use in a method of diagnosing a subject suffering from a disorder (e.g., an IL-33-mediated disorder), comprising determining the presence or level of IL-33 in a sample obtained from a subject by contacting the sample with an immunoconjugate described herein (e.g., comprising an anti-IL-33 antibody; e.g., comprising 3F10 or 15.21C7) and detecting the presence of bound antibody.

[0258] Any suitable sample can be used. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is a blood sample, an ocular sample, a nasal sample, or a cell culture sample. In some embodiments, the blood sample is a plasma or serum sample. In some embodiments, the ocular sample is a vitreous or aqueous humor sample. In some embodiments, the sample is a tissue sample. In some embodiments, the sample is a nasal absorption fluid sample.

[0259] In one aspect, the anti-IL-33 antibodies or immunoconjugates disclosed herein can be used to select subjects eligible for treatment with an IL-33 axis binding antagonist (e.g., an anti-IL-33 antibody or an anti-ST2 antibody), e.g., where IL-33 is a biomarker for patient selection.

[0260] For example, the invention provides a method for selecting a treatment for a subject suffering from a disorder (e.g., an IL-33-mediated disorder), the method comprising determining the presence or level of IL-33 (e.g., human IL-33, reduced IL-33, oxidized IL-33, total IL-33, unbound IL-33, and / or IL-33 bound to ST2) in a sample obtained from the subject by contacting the sample with an anti-IL-33 antibody disclosed herein (e.g., 3F10 or 15.21C7) and detecting the presence of bound antibody; and selecting an IL-33 axis binding antagonist (e.g., an anti-IL-33 antibody or an anti-ST2 antibody) for the subject based on the presence or level of IL-33 (e.g., human IL-33, reduced IL-33, oxidized IL-33, total IL-33, unbound IL-33, and / or IL-33 bound to ST2) in the sample. In another embodiment, the invention provides a method for selecting a treatment for a subject suffering from a disorder (e.g., an IL-33-mediated disorder), the method comprising determining the presence or level of IL-33 (e.g., human IL-33, reduced IL-33, oxidized IL-33, total IL-33, unbound IL-33, and / or IL-33 bound to ST2) in a sample obtained from the subject by contacting the sample with an immunoconjugate disclosed herein (e.g., comprising an anti-IL-33 antibody; e.g., comprising 3F10 or 15.21C7) and detecting the presence of bound antibody; and selecting an IL-33 axis binding antagonist (e.g., an anti-IL-33 antibody or an anti-ST2 antibody) for the subject based on the presence or level of IL-33 (e.g., human IL-33, reduced IL-33, oxidized IL-33, total IL-33, unbound IL-33, and / or IL-33 bound to ST2) in the sample.

[0261] In any of the aspects described herein, the IL-33 axis binding antagonist includes an IL-33 binding antagonist, an ST2 binding antagonist, and an IL1RAcP binding antagonist.Exemplary IL-33 axis binding antagonists include anti-IL-33 antibodies and antigen-binding fragments thereof (e.g., anti-IL-33 antibodies such as ANB-020 (AnaptysBio, Inc.), or those disclosed in U.S. Patent Application Publication No. 2021-0284725, U.S. Patent No. 10,093,730, EP1725261, U.S. Patent No. 8,187,596, WO2011031600, WO2014164959, WO2015099175, WO201602 ... any of the antibodies described in International Publication No. WO 2015106080, WO 2016077381, or WO 2021183849; polypeptides that bind to IL-33 and / or its receptor (ST2 and / or IL-1RACP) and block ligand-receptor interactions (e.g., ST2-Fc proteins such as those described in WO 2014 / 152195, which is incorporated by reference in its entirety; immunoadhesins, peptibodies, and soluble ST2, or an anti-IL-33 receptor antibody (e.g., an anti-ST2 antibody, such as ategolimab (also known as MSTT1041A), AMG-282 (Amgen) or STLM15 (Janssen), or any of the anti-ST2 antibodies described in WO 2013 / 173761 and WO 2013 / 165894, each of which is incorporated by reference in its entirety; or an ST2-Fc protein, such as an anti-IL-33 receptor antibody, such as ategolimab (also known as MSTT1041A), AMG-282 (Amgen) or STLM15 (Janssen), or any of the anti-ST2 antibodies described in WO 2013 / 173761 and WO 2013 / 165894, each of which is incorporated by reference in its entirety; or those described in WO 2013 / 173761; WO 2013 / 165894; or WO 2014 / 152195; and IL-33 receptor antagonists, e.g., small molecule inhibitors, aptamers that bind IL-33, nucleic acids that hybridize under stringent conditions to IL-33 axis nucleic acid sequences (e.g., short interfering RNAs (siRNAs) or clustered regularly interspaced short palindromic repeats RNAs (CRISPR-RNAs or crRNAs), including single guide RNAs (sgRNAs) having crRNA and tracrRNA sequences described in Mali et al. (Science. 339:823-26, 2013), which are incorporated by reference in their entirety.

[0262] In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is a blood sample, an ocular sample, a nasal sample, or a cell culture sample. In some embodiments, the blood sample is a plasma or serum sample. In some embodiments, the ocular sample is a vitreous or aqueous humor sample. In some embodiments, the sample is a tissue sample. In some embodiments, the sample is a cerebrospinal fluid sample.

[0263] In any of the foregoing methods, the biological sample may have a detectable level of IL-33 (e.g., human IL-33, reduced IL-33, oxidized IL-33, total IL-33, unbound IL-33, and / or IL-33 bound to ST2).

[0264] In some embodiments, the presence or level of IL-33 is the presence or level of total IL-33. In some embodiments, the presence or level of IL-33 is the presence or level of reduced IL-33. In some embodiments, the presence or level of IL-33 is the presence or level of oxidized IL-33. In some embodiments, the presence or level of IL-33 is the presence or level of human IL-33. In some embodiments, the presence or level of IL-33 is the presence or level of unbound IL-33. In some embodiments, the presence or level of IL-33 is the presence or level of IL-33 bound to sST2. In some embodiments, sST2 is hsST2.

[0265] It is understood that any of the above-described methods can include an immunoconjugate disclosed herein instead of or in addition to an anti-IL-33 antibody (e.g., any anti-IL-33 antibody disclosed herein).

[0266] F. Biological Samples In some embodiments, any of the anti-IL-33 antibodies provided herein are useful for detecting the presence of IL-33 in a biological sample using methods known in the art or described herein. In some embodiments, the biological sample comprises a tissue or cell sample. For example, the biological sample may comprise cells or tissues from a normal subject or a subject having, susceptible to, or being tested for an IL-33-mediated disorder.

[0267] In some cases, the source of the tissue or cell sample may be a fresh, frozen, and / or archived organ or tissue sample, or a biopsy or aspirate; blood or any blood component (e.g., whole blood, plasma, or serum); nasal fluid (NF), amniotic fluid, peritoneal fluid, or interstitial fluid; solid tissue from cells at any time during the subject's gestation or development. Examples of tissue samples herein include, but are not limited to, ocular samples, including, but not limited to, vitreous and aqueous humor samples. In some embodiments, the biological sample is obtained from an in vitro tissue or cell culture. Examples of biological samples herein include, but are not limited to, tumor biopsies, circulating tumor cells, serum or plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines obtained from tumors or exhibiting tumor-like properties, and archived tumor samples, such as formalin-fixed paraffin-embedded (FFPE) tumor samples or frozen tumor samples.

[0268] In some embodiments, the biological sample contains compounds that are not naturally mixed with the native tissue, such as preservatives, anticoagulants, buffers, nutrients, or antibiotics. In some embodiments, the biological sample has been exposed to one or more fixatives and / or contains one or more fixatives. Fixatives that can be used in the methods and compositions of the invention include formalin, glutaraldehyde, osmium tetroxide, acetic acid, ethanol, acetone, picric acid, chloroform, potassium dichromate, and mercuric chloride, and / or stabilization by microwave heating or freezing.

[0269] In some embodiments, the biological sample is from a subject having, susceptible to, or being tested for an IL-33-mediated disorder. In any of the foregoing embodiments, the IL-33-mediated disorder can be an eye disorder, an inflammatory condition, an immune disorder, a fibrotic disorder, or an eosinophilic disorder. For example, in some cases, the inflammatory condition can be asthma, airway hyperresponsiveness, airway inflammation, sepsis, septic shock, atopic dermatitis, allergic rhinitis, rheumatoid arthritis, or chronic obstructive pulmonary disease (COPD). In some cases, the immune disorder can be asthma, rheumatoid arthritis, allergy, atopic allergy, anaphylaxis, anaphylactic shock, allergic rhinitis, psoriasis, inflammatory bowel disease (IBD), Crohn's disease, diabetes, or liver disease. In some cases, the fibrotic disorder can be idiopathic pulmonary fibrosis (IPF). In some cases, the eosinophilic disorder can be eosinophil-associated gastrointestinal disorder (EGID). In some cases, the EGID can be eosinophilic esophagitis. In certain cases, the IL-33-mediated disorder can be asthma, allergic rhinitis, atopic dermatitis, COPD, eosinophilic esophagitis, or pulmonary fibrosis (e.g., IPF). For example, in some cases, the IL-33-mediated disorder is asthma. In other cases, the IL-33-mediated disorder is pulmonary fibrosis (e.g., IPF).

[0270] In some instances of any of the foregoing embodiments, the IL-33 mediated disorder can be an ocular disorder (e.g., any of the ocular disorders disclosed herein). Non-limiting ocular disorders include, for example, AMD (e.g., wet AMD, dry AMD, intermediate AMD, advanced AMD, and geographic atrophy (GA)), macular degeneration, macular edema, DME (e.g., focal, non-central DME, and diffuse, centrally involved DME), retinopathies, diabetic retinopathy (DR) (e.g., proliferative DR (PDR), non-proliferative DR (NPDR), and high altitude DR), other ischemia-related retinopathies, ROP, retinal glaucoma, and retinal glaucoma. Retinal vein occlusion (RVO) (e.g., central (CRVO) and branched (BRVO) forms), CNV (e.g., myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases associated with retinal / choroidal neovascularization, central serous retinopathy (CSR), pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, FEVR, Coats' disease, Norrie's disease, osteoporotic pseudoglioma syndrome (OPPG) retinal abnormalities associated with, subconjunctival hemorrhage, skin flushing, ocular neovascular disease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatous proliferation, macular vascular ectasia, iris neovascularization, intraocular neovascularization, retinal alterations, cystoid macular edema (CME), vasculitis, papilledema, retinitis including but not limited to CMV retinitis, ocular melanoma, retinoblastoma, conjunctivitis (e.g., infectious conjunctivitis and non-infectious (e.g., allergy Exemplary eye disorders include ocular diseases such as ocular malformation, ...

[0271] Exemplary diseases associated with corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, vitamin A deficiency, contact lens overuse, atopic keratitis, superior limbal keratitis, psoriatic keratitis, Sjogren's syndrome, rosacea abrasion, glomerulitis, syphilis, mycobacterial infection, fatty degeneration, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex infection, herpes zoster infection, protozoal diseases, Kaposi's sarcoma, Mooren's ulcer, Therrien's peripheral corneal degeneration, marginal keratitis, rheumatoid arthritis, systemic lupus, polyarteritis nodosa, trauma, Wegener's sarcoidosis, scleritis, Stevens-Johnson syndrome, pemphigoid radial keratotomy, and corneal graph rejection.

[0272] Exemplary diseases associated with choroidal neovascularization and defects in the retinal vasculature, including increased vascular leakage, aneurysms and capillary dropout, include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Paget's disease, venous occlusion, arterial occlusion, carotid occlusive disease, chronic uveitis / vitreous inflammation, mycobacterial infections, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, retinal edema (including macular edema), Eales' disease, Behcet's disease, infections causing retinitis or choroiditis (e.g., multifocal choroidal), presumed ocular histoplasmosis, Best's disease (vitreous macular degeneration), myopia, optic disc, pars planitis, retinal detachment (e.g., chronic retinal detachment), hyperviscosity syndrome, toxoplasmosis, trauma and post-laser complications.

[0273] Exemplary diseases associated with atrophy of retinal tissue (photoreceptors and the underlying RPE) include, but are not limited to, atrophic or non-exudative AMD (e.g., geographic atrophy or advanced dry AMD), macular atrophy (e.g., atrophy associated with neovascularization and / or geographic atrophy), diabetic retinopathy, Stargardt's disease, Sosby's fundus dystrophy, retinoschisis, and retinitis pigmentosa.

[0274] In some embodiments, ocular disorders include AMD (including wet AMD, dry AMD, and GA), retinopathy (e.g., DR and ROP), PCV, diabetic macular edema, dry eye disease, Behcet's disease, allergic conjunctivitis, and retinal detachment.

[0275] In other embodiments, ocular disorders include intermediate AMD, advanced AMD, glaucoma, uveitis (e.g., infectious and non-infectious uveitis), retinitis pigmentosa, Leber's congenital amaurosis, Stargardt's disease, high altitude diabetic retinopathy, traumatic eye injury, and conjunctivitis (e.g., infectious and non-infectious conjunctivitis).

[0276] For example, provided herein are methods of detecting the presence or level of IL-33 in a subject having, susceptible to, or being tested for an ocular disorder. In one example, the invention provides a method of detecting the presence or level of IL-33 in a subject having, susceptible to, or being tested for an ocular disorder, which detection is performed prior to administration of an IL-33 axis binding antagonist (e.g., an anti-IL-33 antibody or an anti-ST2 antibody disclosed herein) to the subject. In another example, the invention provides a method of detecting IL-33 in a subject having, susceptible to, or being tested for an ocular disorder and in need of detection of IL-33, which subject is administered a therapeutically effective amount of an IL-33 axis binding antagonist disclosed herein, e.g., an anti-IL-33 antibody or an anti-ST2 antibody. In some cases, the ocular disorder may be selected from the group consisting of age-related macular degeneration (AMD), including wet AMD, dry AMD, intermediate AMD, advanced AMD, and geographic atrophy (GA), retinopathy (e.g., diabetic retinopathy (DR), retinopathy of prematurity (ROP), and high altitude DR), polypoidal choroidal vasculopathy (PCV), diabetic macular edema, dry eye disease, Behcet's disease, retinal detachment, glaucoma, uveitis (e.g., infectious and non-infectious uveitis), retinitis pigmentosa, Leber's congenital amaurosis (also known as Leber's congenital amaurosis), Stargardt's disease, traumatic eye injury, and conjunctivitis (e.g., infectious conjunctivitis, non-infectious conjunctivitis, and allergic conjunctivitis). In some cases, the ophthalmic disorder includes AMD (including wet AMD, dry AMD, and GA), retinopathy (e.g., DR and ROP), PCV, diabetic macular edema, dry eye disease, Behcet's disease, allergic conjunctivitis, and retinal detachment. In other cases, the ophthalmic disorder includes intermediate AMD, advanced AMD, glaucoma, uveitis (e.g., infectious and non-infectious uveitis), retinitis pigmentosa, Leber's congenital amaurosis, Stargardt's disease, high altitude diabetic retinopathy, traumatic eye injury, and conjunctivitis (e.g., infectious and non-infectious conjunctivitis).

[0277] G. Pharmaceutical Preparations Pharmaceutical formulations of the anti-IL-33 axis binding antagonists (e.g., IL-33 binding antagonists or ST2 binding antagonists, e.g., anti-IL-33 antibodies or anti-ST2 antibodies) disclosed herein are prepared by mixing such IL-33 binding antagonists having a desired degree of purity with one or more optional pharma- ceutically acceptable carriers (see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980) in the form of a lyophilized formulation or aqueous solution. Pharmaceutically acceptable carriers are typically nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); small molecule Examples of suitable pharmacopoeitic carriers include polypeptides of a small amount (less than about 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmacopoeitic carriers herein further include interstitial drug dispersion agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rhuPH20 (HYLENEX®, Baxter International, Inc.).Some exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0278] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulations including a histidine acetate buffer.

[0279] The formulations herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.

[0280] The active ingredient may be encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose, or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, of macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980.

[0281] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0282] For delivery to the eye (ocular delivery), the antibodies disclosed herein can be combined with, for example, ophthalmologically acceptable preservatives, co-solvents, surfactants, viscosity enhancers, penetration enhancers, buffers, sodium chloride, and / or water. Preservatives may be included, for example, to inhibit microbial contamination during use. Suitable preservatives include edetate disodium, methylparaben, propylparaben, sorbic acid, phenylethyl alcohol, chlorobutanol, polyquaternium-1, or other agents known in the art. These preservatives are typically used at levels of 0.001 to 1.0% w / v. In some cases, the pharmaceutical formulations disclosed herein do not contain preservatives. In some cases, compositions intended for topical administration to the eye can be prepared as eye drops or eye ointments. In some cases, the total amount of antibody is about 0.001 to 1.0% (w / w) of such formulations, for example about 0.01 to about 1.0% (w / w).

[0283] Formulations to be used for in vivo administration are typically sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0284] The IL-33 axis binding antagonist can be formulated into a polymer formulation.

[0285] H. Kits and Products In another aspect, a kit or article of manufacture is provided that contains materials useful for detecting IL-33 using any of the antibodies (e.g., anti-IL-33 antibodies) or immunoconjugates described above. The kit or article of manufacture may include an anti-IL-33 antibody (e.g., any anti-IL-33 antibody disclosed herein). The kit or article of manufacture may include an immunoconjugate described herein (e.g., including an anti-IL-33 antibody; e.g., including 3F10 or 15.21C7). The kit or article of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV infusion bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container may hold a composition, by itself or in combination with another composition, that is effective for assaying, detecting, and / or measuring the presence or level (e.g., expression level) of IL-33 in a sample (e.g., a biological sample). At least one active agent in the composition is an antibody disclosed herein. The label or package insert can indicate that the composition is used for diagnosis or detection of a selected condition (e.g., an IL-33-mediated disorder). Additionally, the kit or article of manufacture can include (a) a first container housing a composition comprising an antibody or immunoconjugate disclosed herein, and (b) a second container housing a composition comprising one or more reagents. The kit or article of manufacture of this embodiment disclosed herein further includes a package insert indicating that the composition can be used to detect IL-33. Alternatively, or additionally, the kit or article of manufacture can further include a second (or third) container comprising an acceptable buffer, e.g., phosphate buffered saline, Ringer's solution, and dextrose solution. The kit or article of manufacture can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0286] For example, provided herein is a kit that includes (a) any of the antibodies disclosed herein; and (b) a package insert that includes instructions for using the antibody to detect the presence or level of IL-33 in a biological sample. In another example, provided herein is a kit that includes (a) any of the immunoconjugates disclosed herein; and (b) a package insert that includes instructions for using the immunoconjugate to detect the presence or level of IL-33 in a biological sample.

[0287] It is understood that any of the above articles of manufacture can include an immunoconjugate disclosed herein in place of or in addition to an anti-IL-33 antibody (e.g., any anti-IL-33 antibody disclosed herein).

[0288] III. Exemplary Embodiments The following exemplary embodiments are intended to be purely illustrative of the present application, and therefore should not be considered as limiting the present invention in any way.

[0289] The embodiments provided herein are as follows:

[0290] Embodiment 1. An isolated antibody, or antigen-binding fragment thereof, that specifically binds to IL-33, wherein the antibody comprises a binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, and the binding domain comprises the following six complementarity determining regions (CDRs): (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:1; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:2; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:3; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:5; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6 An antibody or an antigen-binding fragment thereof comprising:

[0291] Embodiment 2. The VH domain comprises the following framework regions (FR): (a) FR-H1 comprising the amino acid sequence of SEQ ID NO:11; (b) FR-H2 comprising the amino acid sequence of SEQ ID NO: 12; (c) FR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and (d) FR-H4 comprising the amino acid sequence of SEQ ID NO: 14 2. The antibody of embodiment 1, comprising:

[0292] Embodiment 3. The VL domain comprises the following FR: (a) FR-L1 comprising the amino acid sequence of SEQ ID NO: 15; (b) FR-L2 comprising the amino acid sequence of SEQ ID NO: 16; (c) FR-L3 comprising the amino acid sequence of SEQ ID NO: 17; and (d) FR-L4 comprising the amino acid sequence of SEQ ID NO: 18 3. The antibody of embodiment 1 or 2, comprising:

[0293] Embodiment 4. The antibody of any one of embodiments 1 to 3, comprising: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 7; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 8; or (c) the VH domain of (a) and the VL domain of (b).

[0294] Embodiment 5. The antibody of any one of embodiments 1 to 4, wherein the VH domain comprises the amino acid sequence of SEQ ID NO:7.

[0295] Embodiment 6 The antibody of any one of embodiments 1 to 5, wherein the VL domain comprises the amino acid sequence of SEQ ID NO:8.

[0296] Embodiment 7. An isolated antibody, or antigen-binding fragment thereof, that specifically binds to IL-33, wherein the antibody comprises a binding domain comprising (a) a VH domain comprising the amino acid sequence of SEQ ID NO:7 and (b) a VL domain comprising the amino acid sequence of SEQ ID NO:8.

[0297] Embodiment 8. The antibody of any one of embodiments 1 to 7, comprising: (a) a heavy chain (HC) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:9; (b) a light chain (LC) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:10; or (c) the HC of (a) and the LC of (b).

[0298] Embodiment 9 The antibody of any one of embodiments 1 to 8, wherein the HC comprises the amino acid sequence of SEQ ID NO:9.

[0299] Embodiment 10. The antibody of any one of embodiments 1 to 9, wherein the LC comprises the amino acid sequence of SEQ ID NO:10.

[0300] Embodiment 11. An isolated antibody, or antigen-binding fragment thereof, that specifically binds to IL-33, wherein the antibody comprises (a) a HC comprising the amino acid sequence of SEQ ID NO:9 and (b) a LC comprising the amino acid sequence of SEQ ID NO:10.

[0301] Embodiment 12. An isolated antibody, or antigen-binding fragment thereof, that specifically binds to IL-33, wherein the antibody has the following six CDRs: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24 2. An antibody, or an antigen-binding fragment thereof, comprising:

[0302] Embodiment 13. The VH domain comprises the following FR: (a) FR-H1 comprising the amino acid sequence of SEQ ID NO: 29; (b) FR-H2 comprising the amino acid sequence of SEQ ID NO: 30; (c) FR-H3 comprising the amino acid sequence of SEQ ID NO: 31; and (d) FR-H4 comprising the amino acid sequence of SEQ ID NO: 32 13. The antibody of embodiment 12, comprising:

[0303] Embodiment 14. The VH domain comprises the following FR: (a) FR-L1 comprising the amino acid sequence of SEQ ID NO: 33; (b) FR-L2 comprising the amino acid sequence of SEQ ID NO: 34; (c) FR-L3 comprising the amino acid sequence of SEQ ID NO: 35; and (d) FR-L4 comprising the amino acid sequence of SEQ ID NO: 36 The antibody of claim 12 or 13, comprising:

[0304] Embodiment 15. The antibody of any one of embodiments 12 to 14, comprising: (a) a VH domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 25; (b) a VL domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 26; or (c) the VH domain of (a) and the VL domain of (b).

[0305] Embodiment 16 The antibody of any one of embodiments 12 to 15, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 25.

[0306] Embodiment 17. The antibody of any one of embodiments 12 to 16, wherein the VL domain comprises the amino acid sequence of SEQ ID NO:26.

[0307] Embodiment 18. An isolated antibody, or antigen-binding fragment thereof, that specifically binds to IL-33, wherein the antibody comprises a binding domain comprising (a) a VH domain comprising the amino acid sequence of SEQ ID NO:25 and (b) a VL domain comprising the amino acid sequence of SEQ ID NO:26.

[0308] Embodiment 19. The antibody of any one of embodiments 12 to 18, comprising: (a) a HC having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 27; (b) a LC having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 28; or (c) the HC of (a) and the LC of (b).

[0309] Embodiment 20. The antibody of any one of embodiments 12 to 19, wherein the HC comprises the amino acid sequence of SEQ ID NO:27.

[0310] Embodiment 21 The antibody of any one of embodiments 12 to 20, wherein the LC comprises the amino acid sequence of SEQ ID NO:28.

[0311] Embodiment 22. An isolated antibody, or antigen-binding fragment thereof, that specifically binds to IL-33, wherein the antibody comprises (a) a HC comprising the amino acid sequence of SEQ ID NO:27 and (b) a LC comprising the amino acid sequence of SEQ ID NO:28.

[0312] Embodiment 23 The antibody of any one of embodiments 1 to 22, which binds to oxidized or reduced IL-33.

[0313] Embodiment 24. Oxidized IL-33 and reduced IL-33 have a K within 2-fold D 24. The antibody of embodiment 23, which binds at

[0314] Embodiment 25. Oxidized IL-33 and reduced IL-33 are provided with substantially the same K D 25. The antibody of embodiment 24, which binds at

[0315] Embodiment 26 The antibody of any one of embodiments 1 to 25, which binds to reduced IL-33.

[0316] Embodiment 27. The reduced IL-33 has a K of ≦1 nM D 27. The antibody of embodiment 26, which binds at

[0317] Embodiment 28. The reduced IL-33 has a K between 0.01 nM and 1 nM. D 28. The antibody of embodiment 27, which binds at

[0318] Embodiment 29. Reduced IL-33 has a K of ≦0.5 nM D 28. The antibody of embodiment 27, which binds at

[0319] Embodiment 30. Reduced IL-33 has a K between 0.1 nM and 0.5 nM. D 30. The antibody of embodiment 29, which binds at

[0320] Embodiment 31. Reduced IL-33 has a K of about 0.487 nM D 31. The antibody of embodiment 29 or 30, which binds at

[0321] Embodiment 32. Reduced IL-33 has a K of about 0.338 nM D 31. The antibody of embodiment 29 or 30, which binds at

[0322] Embodiment 33 The antibody of any one of embodiments 1 to 32, which binds to human IL-33.

[0323] Embodiment 34. Soluble interleukin-1 receptor-like 1 (sST2) protein and IL-33 not bound to sST2 have a K within 2-fold. D 34. The antibody of any one of embodiments 1 to 33, wherein the antibody binds at

[0324] 35. IL-33 bound to sST2 protein has substantially the same K as IL-33 not bound to sST2. D 35. The antibody of any one of embodiments 1 to 34, wherein the antibody binds at

[0325] Embodiment 36 The antibody of embodiment 34 or 35, wherein the sST2 is human sST2 (hsST2).

[0326] Embodiment 37 The antibody of any one of embodiments 1 to 36, which is a monoclonal antibody.

[0327] Embodiment 38 The antibody of any one of embodiments 1 to 18 and 23 to 37, which is a chimeric antibody.

[0328] Embodiment 39 The antibody of any one of embodiments 12 to 38, which is a rat antibody.

[0329] Embodiment 40. The antibody of any one of embodiments 1 to 36, which is an IgG antibody.

[0330] Embodiment 41 The antibody of any one of embodiments 1 to 18, 21, and 23 to 37, which is an IgG1 antibody.

[0331] Embodiment 42 The antibody of any one of embodiments 1 to 7, 10, and 12 to 20, which is an IgG2a antibody.

[0332] Embodiment 43 The antibody of any one of embodiments 1 to 36, which is an antibody fragment that specifically binds to IL-33.

[0333] Embodiment 44. The antibody of embodiment 43, wherein the antibody fragment is selected from the group consisting of Fab, single chain variable fragment (scFv), Fv, Fab', Fab'-SH, F(ab')2, and diabody.

[0334] Embodiment 45. An isolated nucleic acid encoding the antibody of any one of embodiments 1 to 44, or a set of isolated nucleic acids which together encode said antibodies.

[0335] Embodiment 46. A vector or set of vectors comprising the isolated nucleic acid or set of isolated nucleic acids of embodiment 45.

[0336] Embodiment 47. A host cell comprising the vector or set of vectors of embodiment 46.

[0337] Embodiment 48. The host cell of embodiment 47, which is a mammalian cell.

[0338] Embodiment 49 The host cell of embodiment 48, wherein the mammalian cell is a rat cell.

[0339] Embodiment 50. The host cell of embodiment 48, wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

[0340] Embodiment 51. The host cell of embodiment 47, which is a prokaryotic cell.

[0341] Embodiment 52 The host cell of embodiment 51, wherein the prokaryotic cell is E. coli.

[0342] Embodiment 53 A method for producing an antibody that specifically binds to IL-33, comprising culturing a host cell of any one of embodiments 47 to 52 in a medium.

[0343] Embodiment 54 The method of embodiment 53, further comprising recovering the antibody from the host cell or medium.

[0344] Embodiment 55. An immunoconjugate comprising the antibody of any one of embodiments 1 to 44.

[0345] Embodiment 56 The immunoconjugate of embodiment 55, which is linked to a label.

[0346] Embodiment 57. The antibody of any one of embodiments 1 to 44 or the immunoconjugate of embodiment 55 or 56 for use in detecting the presence or level of IL-33 in a biological sample.

[0347] Embodiment 58. The antibody or immunoconjugate for use of embodiment 57, wherein detection is by immunohistochemistry (IHC), immunofluorescence (IF), flow cytometry, enzyme-linked immunosorbent assay (ELISA), immunoblotting, or immunopolymerase chain reaction (iPCR).

[0348] Embodiment 59. The antibody or immunoconjugate for use according to embodiment 58, wherein detection is by ELISA.

[0349] Embodiment 60. The antibody or immunoconjugate for use of embodiment 58, wherein detection is by iPCR.

[0350] Embodiment 61. The antibody or immunoconjugate for use of any one of embodiments 57 to 60, wherein the biological sample is a blood sample, an eye sample, a nasal sample, or a cell culture sample.

[0351] Embodiment 62. The antibody or immunoconjugate for use of embodiment 61, wherein the blood sample is a plasma or serum sample.

[0352] Embodiment 63. The antibody or immunoconjugate for use of embodiment 61, wherein the ocular sample is a sample of vitreous humor or aqueous humor.

[0353] Embodiment 64. The antibody or immunoconjugate for use of embodiment 61, wherein the ocular sample is a tissue sample.

[0354] Embodiment 65. The antibody or immunoconjugate for use of embodiment 61, wherein the nasal sample is a nasal absorption sample.

[0355] Embodiment 66. The antibody or immunoconjugate for use of any one of embodiments 57 to 65, wherein the biological sample is from a subject having or at risk for an IL-33 mediated disorder.

[0356] Embodiment 67. The antibody for use or the immunoconjugate for use of embodiment 66, wherein the IL-33 mediated disorder is an eye disorder, an inflammatory condition, an immune disease, a fibrotic disorder, an eosinophilic disorder, an infection, pain, a central nervous system disorder, or a solid tumor.

[0357] Embodiment 68. The antibody for use or the immunoconjugate for use of embodiment 67, wherein the ocular disorder is age-related macular degeneration (AMD), ocular retinopathy, polypoidal choroidal vasculopathy (PCV), diabetic macular edema, dry eye disease, Behcet's disease, retinal detachment, glaucoma, uveitis, retinitis pigmentosa, Leber's congenital amaurosis, Stargardt's disease, traumatic eye injury, or conjunctivitis.

[0358] Embodiment 69. The antibody for use or the immunoconjugate for use of embodiment 68, wherein the AMD is geographic atrophy (GA), wet AMD, or dry AMD.

[0359] Embodiment 70. The antibody for use or the immunoconjugate for use of embodiment 68, wherein AMD is GA.

[0360] Embodiment 71. The antibody for use or the immunoconjugate for use of embodiment 68, wherein the AMD is intermediate AMD or advanced AMD.

[0361] Embodiment 72. The antibody for use or the immunoconjugate for use of embodiment 68, wherein the ocular retinopathy is diabetic retinopathy (DR) or retinopathy of prematurity (ROP).

[0362] Embodiment 73. The antibody for use or the immunoconjugate for use of embodiment 68, wherein the ocular retinopathy is high altitude DR.

[0363] Embodiment 74. The antibody for use or the immunoconjugate for use of embodiment 68, wherein the conjunctivitis is infectious conjunctivitis or non-infectious conjunctivitis.

[0364] Embodiment 75. The antibody for use or the immunoconjugate for use of embodiment 68, wherein the conjunctivitis is allergic conjunctivitis.

[0365] Embodiment 76. The antibody for use or the immunoconjugate for use of embodiment 67, wherein the inflammatory condition is asthma, sepsis, septic shock, atopic dermatitis, allergic rhinitis, rheumatoid arthritis, or chronic obstructive pulmonary disease (COPD).

[0366] Embodiment 77. The antibody or immunoconjugate for use of embodiment 67, wherein the immune disease is asthma, rheumatoid arthritis, allergy, anaphylaxis, anaphylactic shock, allergic rhinitis, psoriasis, inflammatory bowel disease (IBD), Crohn's disease, diabetes, or liver disease.

[0367] Embodiment 78. The antibody for use or the immunoconjugate for use of embodiment 67, wherein the fibrotic disease is idiopathic pulmonary fibrosis (IPF).

[0368] Embodiment 79. The antibody for use or the immunoconjugate for use of embodiment 67, wherein the eosinophilic disorder is eosinophil-associated gastrointestinal disorder (EGID).

[0369] Embodiment 80. The antibody for use or the immunoconjugate for use of embodiment 79, wherein the EGID is eosinophilic esophagitis.

[0370] Embodiment 81. The antibody for use or the immunoconjugate for use of any one of embodiments 66 to 80, wherein the subject is a human.

[0371] Embodiment 82. The antibody for use or the immunoconjugate for use of any one of embodiments 57 to 81, wherein the presence or level of IL-33 is the presence or level of total IL-33.

[0372] Embodiment 83. The antibody for use or the immunoconjugate for use of any one of embodiments 57 to 81, wherein the presence or level of IL-33 is the presence or level of reduced IL-33.

[0373] Embodiment 84. The antibody for use or the immunoconjugate for use of any one of embodiments 57 to 83, wherein the presence or level of IL-33 is the presence or level of human IL-33.

[0374] Embodiment 85. The antibody for use or the immunoconjugate for use of any one of embodiments 57 to 84, wherein the presence or level of IL-33 is the presence or level of unbound IL-33.

[0375] Embodiment 86. The antibody for use or the immunoconjugate for use of any one of embodiments 57 to 84, wherein the presence or level of IL-33 is the presence or level of IL-33 bound to sST2.

[0376] Embodiment 87. The antibody for use or the immunoconjugate for use of embodiment 86, wherein sST2 is hsST2.

[0377] Embodiment 88. A method for detecting the presence or level of IL-33 in a biological sample, comprising: (i) contacting the biological sample with the antibody of any one of embodiments 1 to 44 and detecting the presence of bound antibody; or (ii) contacting the biological sample with the immunoconjugate of embodiment 55 or 56 and detecting the presence of bound immunoconjugate. The method includes:

[0378] Embodiment 89. The method of embodiment 88, wherein detecting is by IHC, IF, flow cytometry, ELISA, immunoblotting, or iPCR.

[0379] Embodiment 90. The method of embodiment 89, wherein the detecting is by ELISA.

[0380] Embodiment 91. The method of embodiment 89, wherein the detecting is by iPCR.

[0381] Embodiment 92. The method of embodiment 91, comprising contacting a biological sample with the immunoconjugate of embodiment 55 or 56 in the presence of a second anti-IL-33 antibody that specifically binds to an epitope on IL-33 that is different from the epitope specifically bound by the anti-IL-33 antibody in the immunoconjugate, and detecting the presence of bound immunoconjugate.

[0382] Embodiment 93 The method of embodiment 92, wherein the anti-IL-33 antibody is 1E1v8.

[0383] Embodiment 94 The method of embodiment 92 or 93, wherein the biological sample is treated with tris(2-carboxyethyl)phosphine (TCEP) prior to said contacting.

[0384] Embodiment 95 The method of any one of embodiments 88 to 94, wherein the biological sample is a blood sample, an eye sample, a nasal sample, or a cell culture sample.

[0385] Embodiment 96 The method of embodiment 95, wherein the blood sample is a plasma or serum sample.

[0386] Embodiment 97. The method of embodiment 95, wherein the ocular sample is a sample of vitreous humor or aqueous humor.

[0387] Embodiment 98 The method of embodiment 95, wherein the eye sample is a tissue sample.

[0388] Embodiment 99. The method of embodiment 95, wherein the nasal sample is a nasal absorption fluid sample.

[0389] Embodiment 100. The method of any one of embodiments 88 to 99, wherein the biological sample is derived from a subject having or at risk for an IL-33 mediated disorder.

[0390] Embodiment 101. The method of embodiment 100, wherein the IL-33 mediated disorder is an ocular disorder, an inflammatory condition, an immune disease, a fibrotic disorder, an eosinophilic disorder, an infection, pain, a central nervous system disorder, or a solid tumor.

[0391] Embodiment 102. The method of embodiment 101, wherein the ocular disorder is AMD, ocular retinopathy, PCV, diabetic macular edema, dry eye disease, Behcet's disease, retinal detachment, glaucoma, uveitis, retinitis pigmentosa, Leber's congenital amaurosis, Stargardt's disease, traumatic eye injury, or conjunctivitis.

[0392] Embodiment 103. The method of embodiment 102, wherein the AMD is GA, wet AMD, or dry AMD.

[0393] Embodiment 104. The method of embodiment 102, wherein the AMD is GA.

[0394] Embodiment 105. The method of embodiment 102, wherein the AMD is intermediate AMD or advanced AMD.

[0395] Embodiment 106 The method of embodiment 102, wherein the ocular retinopathy is DR or ROP.

[0396] Embodiment 107. The method of embodiment 102, wherein the ocular retinopathy is high altitude DR.

[0397] Embodiment 108. The method of embodiment 102, wherein the conjunctivitis is infectious conjunctivitis or non-infectious conjunctivitis.

[0398] Embodiment 109. The method of embodiment 102, wherein the conjunctivitis is allergic conjunctivitis.

[0399] Embodiment 110. The method of embodiment 101, wherein the inflammatory condition is asthma, sepsis, septic shock, atopic dermatitis, allergic rhinitis, rheumatoid arthritis, or COPD.

[0400] Embodiment 111 The method of embodiment 101, wherein the immune disorder is asthma, rheumatoid arthritis, allergy, anaphylaxis, anaphylactic shock, allergic rhinitis, psoriasis, IBD, Crohn's disease, diabetes, or liver disease.

[0401] Embodiment 112 The method of embodiment 101, wherein the fibrotic disease is IPF.

[0402] Embodiment 113 The method of embodiment 101, wherein the eosinophilic disorder is EGID.

[0403] Embodiment 114. The method of embodiment 113, wherein the EGID is eosinophilic esophagitis.

[0404] Embodiment 115. The method of any one of embodiments 100 to 114, wherein the subject is a human.

[0405] Embodiment 116 The method of any one of embodiments 88 to 115, wherein the presence or level of IL-33 is the presence or level of total IL-33.

[0406] Embodiment 117. The method of any one of embodiments 88 to 115, wherein the presence or level of IL-33 is the presence or level of reduced IL-33.

[0407] Embodiment 118. The method of any one of embodiments 88 to 117, wherein the presence or level of IL-33 is the presence or level of human IL-33.

[0408] Embodiment 119. The method of any one of embodiments 88 to 118, wherein the presence or level of IL-33 is the presence or level of unbound IL-33.

[0409] Embodiment 120. The method of any one of embodiments 88 to 118, wherein the presence or level of IL-33 is the presence or level of IL-33 bound to sST2.

[0410] Embodiment 121. The method of embodiment 120, wherein the sST2 is hsST2.

[0411] Embodiment 122. The method of any one of embodiments 88 to 121, further comprising selecting a treatment comprising an IL-33 axis binding antagonist for the subject based on the presence or level of IL-33 in the biological sample.

[0412] Embodiment 123 The method of any one of embodiments 88 to 121, further comprising administering to the subject a therapeutically effective amount of an IL-33 axis binding antagonist.

[0413] Embodiment 124 The method of embodiment 122 or 123, wherein the IL-33 axis binding antagonist is an IL-33 binding antagonist.

[0414] Embodiment 125 The method of embodiment 124, wherein the IL-33 binding antagonist is an anti-IL-33 antibody.

[0415] Embodiment 126. An assay for identifying a subject having an IL-33 mediated disorder who is a candidate for a treatment comprising an IL-33 axis binding antagonist, comprising determining the presence or level of IL-33 in a biological sample obtained from the subject using an antibody of any one of embodiments 1 to 44 or an immunoconjugate of embodiment 55 or 56.

[0416] EMBODIMENT 127. (a) the antibody of any one of embodiments 1 to 44; and (b) a package insert containing instructions for using the antibody to detect the presence or level of IL-33 in a biological sample. Kit including:

[0417] EMBODIMENT 128. (a) the immunoconjugate of embodiment 55 or 56; and (b) a package insert containing instructions for using the immunoconjugate to detect the presence or level of IL-33 in a biological sample. Kit including: EXAMPLES

[0418] IV. Working Examples The following are examples of methods and compositions of the present invention. Given the general description provided above, it will be understood that other various embodiments may be practiced.

[0419] Example 1: Materials and Methods Measurement of human sST2 levels Levels of hsST2 were determined using the human ST2 / hIL-33R QUANTIKINE® kit ELISA according to the manufacturer's instructions (#DST200, R&D Systems, Minneapolis, MN, USA). The standard range of hsST2 is 7.81 pg / mL to 1000 pg / mL, with a sensitivity of 15.6 pg / mL in buffer and lower limits of detectable concentration of 1563 pg / mL in aqueous humor (minimum dilution factor (MRD) is 1:100), 625 pg / mL in vitreous humor (MRD is 1:40), 2500 pg / mL in serum (MRD is 1:160), and 1250 pg / mL in NF (MRD is 1:80).

[0420] Measurement of hIL-33 levels in vitreous humor using a commercially available kit The following kits were used according to the manufacturer's instructions: Human IL-33 QUANTIKINE® Kit ELISA (#D3300B, R&D Systems, Minneapolis, MN, USA), LEGEND MAX TM hIL-33 Kit ELISA (#435907, Biolegend, San Diego, CA, USA), Invitrogen IL-33 Human ELISA Kit (#BMS2048, ThermoFisher Scientific, Carlsbad, CA, USA), U-PLEX® hIL-33 Assay (#K151WFK, MSD, Rockville, MD, USA), Bio-Plex Pro TM Human Th17 Cytokine 15-Plex LUMINEX® Assay (#171AA001M, Bio-Rad, Hercules, CA, USA).

[0421] In addition, two ELISAs were developed using commercially available antibodies (Abs). For the first assay, MaxiSorp® plates (384-well, Nunc, Thermo Fisher Scientific, Rochester, NY, USA) were coated overnight at 4° C. with 2 μg / mL mouse anti-hIL-33 diluted in 0.05 M sodium carbonate buffer, pH 9.6 (#ALX-804-840PF-C100, Enzo, Farmingdale, NY, USA). After washing three times with 0.05% TWEEN® 20 in PBS, pH 7.4, the plates were blocked for 1 hour (h) at room temperature (RT) with 0.5% bovine serum albumin (BSA), 15 ppm PROCLIN® 300 in PBS, pH 7.4. After three washes, titrated hIL-33 was added to the plates and incubated at RT for 2 hours. Plates were washed six times and incubated with biotinylated goat anti-hIL-33 (#BAF3625, R&D Systems, Minneapolis, MN, USA) diluted 1:1000 for 1 h at RT. Bound Ab was detected using streptavidin poly-horseradish peroxidase (HRP) 80 (poly-HRP80) (#65R-5119, Fitzgerald, Acton, MA, USA) and its substrate 3,3',5,5'-tetramethylbenzidine (Moss Inc., Pasadena, MD, USA). The reaction was stopped with 1 M phosphoric acid and absorbance was read at 450 nm.

[0422] For the second ELISA, a similar protocol was followed except that 2 μg / mL rabbit anti-hIL-33 (#500-P261, Peprotech, Minneapolis, MN, USA) was used for plate coating and 50 ng / mL biotinylated mouse anti-hIL-33 clone 6H617 (#NBP2-273338, Novus, Centennial, CO, USA) in combination with streptavidin-HRP (#RPN1231, GE Healthcare Life Sciences, Chicago, IL, USA) was used for detection.

[0423] sensitivity The lower and upper limits of quantification (LLOQ and ULOQ) were defined as the lowest and highest hIL-33 concentrations that could be accurately quantified. These values ​​were determined from a standard curve run in quadruplicate in six independent assays. CV and relative error (RE) were calculated for each point on the standard curve, and those with RE and CV below 20% were included in the reporting range.

[0424] Accuracy (additive recovery) Reduced hIL-33 was spiked (3 to 4 replicates) in serum, plasma, and NF samples from different individuals and in pooled vitreous and aqueous humor samples from two individuals to target high (4.5 pg / mL), medium (1.5 pg / mL), and low (0.5 pg / mL) concentrations. The same high, medium, and low concentrations were spiked in assay buffer as controls (spiked concentrations). Endogenous levels of reduced hIL-33 were determined from unspiked matrix. Spike recovery was calculated using the following formula: % recovery = [measured concentration / expected concentration (endogenous + spiked)] x 100.

[0425] Dilution Linearity Serum, plasma and NF samples from spike recovery experiments were diluted in assay buffer to concentrations of 1:2, 1:4, 1:10 and 1:20, while vitreous and aqueous humor were diluted 1:2, 1:4 and 1:8 (vitreous humor only) due to limited sample volume available. Percent recoveries were calculated from measured concentrations versus expected corrected concentrations.

[0426] The MRD was defined as the lowest dilution required to obtain a reduced hIL-33 concentration of 80-120% of the expected sample recovery. The lower limit of quantification (LLOQ) of the matrix was calculated by multiplying the LLOQ obtained in buffer by the MRD.

[0427] Determination of hsST2 resistance hsST2 resistance was determined by adding various concentrations of hsST2 (0.016 ng / mL to 40 ng / mL) to a hIL-33 standard curve. Values ​​reported are the highest concentrations of hsST2 that yielded acceptable recovery of hIL-33 concentrations. Acceptable recovery was defined as 100% ± 20% of the corresponding hIL-33 concentrations measured in the absence of hsST2.

[0428] Immunodepletion of hIL-33 Immunodepletion was performed by adding buffer, beads conjugated to biotinylated anti-hIL-33, or beads conjugated to anti-gp120 (isotype control) to human vitreous humor samples for 1 h at room temperature (RT). After incubation, the conjugated beads were removed from the matrix. These two steps were repeated and the amount of hIL-33 immunodepleted from the matrix was calculated. Immunodepletion was considered acceptable if the amount of hIL-33 in the sample was reduced by 100% ± 20% SD compared to the value of the control (non-immunodepleted) matrix sample.

[0429] Generation and quality control (QC) of reduced and oxidized hIL-33 Recombinant hIL-33, consisting of the N-terminal sequence MHHHHHHGENLYFQG (His tag and TEV cleavage site; SEQ ID NO: 37) followed by the coding sequence for amino acids 112 to 270 (UniProtKB / Swiss-Prot accession no. 095760), was expressed in E. coli. The protein was purified by Ni-Excel chromatography followed by a SUPERDEX® S75 size-exclusion column pre-equilibrated with PBS (phosphate-buffered saline). Mass spectrometry showed that the purified hIL-33 had the expected molecular weight and amino acid sequence. Examination of sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gels predicted the purity of the material to be greater than 95%. An aliquot of purified hIL-33 was further diluted in PBS buffer to obtain a final concentration of 0.3 mg / mL. Similarly, another aliquot of purified IL-33 was diluted in 60% Iscove's Modified Dulbecco's Medium (IMDM) (Thermo Fischer Scientific, Waltham, MA) to obtain the same final protein concentration. The diluted proteins were then incubated at 37°C for approximately 18 hours. Both diluted proteins were then analyzed on SDS-PAGE gels using mass spectrometry to determine the extent of disulfide bond formation. The oxidized hIL-33 diluted in IMDM medium was then buffer exchanged into the final PBS buffer to remove medium components. The reduced hIL-33 in PBS was supplemented with 0.1 mM tris(2-carboxyethyl)phosphine (TCEP) reducing agent.

[0430] antibody generation Nine Sprague Dawley rats (Charles River, Hollister, CA, USA) were immunized with hIL-33 (Genentech) at 100 μg / animal for the first dose and 50 μg / animal for the remaining boosts, each split between sites: intraperitoneally, sc (subcutaneously) at the base of the tail, sc at the scruff of the neck, and sc at both hind leg joints. A Toll-like receptor cocktail containing monophosphoryl lipid A (MPL) (Sigma-Aldrich, St. Louis, MO, USA), Poly(I:C), R848, and cytosine phosphodiester-linked guanine (CpG) (InvivoGen, San Diego, CA, USA) was used as an adjuvant for the first dose. Antigen-specific hybridomas were generated, sorted, and screened as described (Goldstein et al., Commun Biol, 2019, 2:304). Briefly, immunoglobulin G (IgG) + hIL-33 + Hybridomas were single cell sorted into 96-well plates and cultured for 7 days using hIL-33 conjugated to phycoerythrin (PE) (Novus Biological, Littleton, CO, USA). Supernatants were screened by high-throughput (HTP) ELISA using a robotics platform, and hybridomas showing binding to hIL-33 by ELISA were scaled up in 1 mL cultures. Supernatants were harvested and purified using Protein G affinity chromatography resin (GammaBind Plus, GE Healthcare, Pittsburgh, PA, USA). Anti-hIL-33 Abs were submitted for sequencing and cloned for recombinant production.

[0431] Characterization of anti-hIL-33 Abs by an array-based SPR imaging system An array-based SPR imaging system (CFM / IBIS, CARTERRA® USA) was used to analyze the binding kinetics and epitope-bind a panel of anti-IL-33 MAbs. Purified hybridoma Abs were diluted to 10 μg / ml in 10 mM sodium acetate buffer (pH 4.5). Abs were visualized using amine coupling on a Continuous Flow Microspotter. TM (Carterra, Dublin, CA, USA) was used to directly immobilize Abs onto an SPR sensor prism CMD 200M chip (XanTec Bioanalytics, Germany) to generate arrays of Abs. Kinetics and binning experiments were performed at 25°C in a running buffer consisting of 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA and 0.005% TWEEN® 20 (HBS-TE). Binding of hIL-33 to immobilized Abs was assessed using an IBIS MX96 SPRi (Carterra, Dublin, CA, USA). For kinetic analysis, step concentrations of reduced and oxidized hIL-33 starting from 300 nM were injected for 3 minutes (min) and allowed to dissociate for 10 min. The surface was regenerated with 10 mM glycine (pH 1.7) between cycles. Binding data were processed using Scrubber (BioLogic Software). For Ab binning, reduced and oxidized hIL-33 were injected for 4 min at 100 nM, followed by a second 4 min injection at 100 nM with purified Ab in HBS-TE running buffer. m The cycles were run at 100 µg / mL. Surfaces were regenerated with 10 mM glycine (pH 1.7) between cycles. Binding data were processed using an epitope binning software tool (Carterra, Dublin, Calif., USA).

[0432] Characterization of anti-hIL-33 Ab by surface plasmon resonance (SPR) A Series S Protein A sensor chip (Cytiva, Marlborough, MA, USA) was used to capture a recombinant chimeric human IgG version of anti-hIL-33 Ab using a BIACORE® 8K instrument (Cytiva, Marlborough, MA, USA). Antibody binding to human histidine tagged hIL-33 (Genentech, South San Francisco, CA, USA) was measured using multi-cycle kinetics. Sensorgrams were recorded using a 2 min injection time at a flow rate of 30 μL / min, at 25° C. or 37° C., and with a running buffer of 10 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.4, 150 mM sodium chloride (NaCl), 3 mM ethylenediaminetetraacetic acid (EDTA), and 0.005% TWEEN® 20. After injection, dissociation of IL-33 from the antibody was monitored for 10 min in running buffer. The surface was regenerated between binding cycles with 30 μl injections of 10 mM glycine HCl (pH 1.5). After subtraction of a blank containing only running buffer, the sensorgrams were analyzed using a 1:1 Langmuir binding model with software provided by the manufacturer to calculate kinetic and binding constants.

[0433] Reduced hIL-33 ELISA MAXISORP® plates (384 wells, Nunc, Thermo Fisher Scientific, Rochester, NY, USA) were coated with rat IgG2a anti-human hIL-33 15.21C7 Ab (Genentech, South San Francisco, CA, USA) at 1 μg / mL in PBS, pH 7.4 overnight at 4° C. Plates were then washed with 0.05% TWEEN® 20 in PBS, pH 7.4, and resuspended in 0.5% bovine serum albumin (BSA), 15 ppm ProClin 1000 ng / mL in PBS, pH 7.4. TMThe reduced hIL-33 standards were stored in 1 mM TCEP to keep the protein in its reduced form, and the samples were buffered in ice-cold assay buffer (0.5% BSA, 0.05% TWEEN® 20, 15 ppm ProClin 100 in PBS, pH 7.4) and blocked at 300 for 1 hour (h) at RT. TM The plates were diluted using two half-dilutions in 0.5% BSA, 0.05% TWEEN® 20, 15 ppm ProClin 100 in PBS (pH 7.4), 0.25% CHAPS, 5 mM EDTA, and 0.35 N NaCl, and incubated in the plates for 2 hours at RT. After six washes, the plates were resuspended in 0.5% BSA, 0.05% TWEEN® 20, 15 ppm ProClin 100 in PBS (pH 7.4), 0.25% CHAPS, 5 mM EDTA, and 0.35 N NaCl, and incubated in the plates for 2 hours at RT. TM Biotinylated rat / human chimeric anti-hIL-33 3F10 MAb (Genentech, South San Francisco, CA, USA) diluted at 200 ng / mL was incubated for 1 h at RT. Bound reduced hIL-33 was detected using a 1:10,000 dilution of streptavidin conjugated to horseradish peroxidase (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and its substrate 3,3',5,5'-tetramethylbenzidine (Moss Inc., Pasadena, MD, USA). The reaction was stopped with 1 M phosphoric acid and absorbance was read at 450 nm. Titration curves were fitted using an in-house four-parameter regression program.

[0434] Total hIL-33 ELISA Total hIL-33 ELISA was performed similarly using a 1:1 volume-to-volume (vol / vol) mixture of rat IgG2a anti-hIL-33 15.21C7 (Genentech, South San Francisco, CA, USA) at 0.5 μg / mL and goat anti-hIL-33 Abs (R&D Systems, Minneapolis, MN, USA) at 2 μg / mL for coating, and a 1:1 (vol / vol) mixture of biotinylated goat anti-huST2 (R&D Systems, Minneapolis, MN, USA) at 100 ng / mL and biotinylated goat anti-hIL-33 (R&D Systems, Minneapolis, MN, USA) Ab at 50 ng / mL for detection. Standards consisted of a 1:1 (vol / vol) mixture of oxidized and reduced hIL-33 prepared in ice-cold assay buffer containing 20 ng / mL hsST2.

[0435] Antibody-DNA conjugation 55-mer DNA (SEQ ID NO: 38) 5'-NH2- / 5AmMC6 / TGAAGGTCCTTGGCGATCATTTCGGCGGTGATCGGATGCATTTGGCTACGTCCCT-3' (Integrated DNA Technologies, Coralville, IA) was activated with N-succinimidyl-(N-maleimidomethyl)cyclohexane-1-carboxylate (Pierce, Rockford, IL, USA). Rat IgG2a anti-human IL-33 clone 15.21C7 (Genentech, South San Francisco, CA, USA) was activated with N-succinimidyl-S-acetylthioacetate (Pierce, Rockford, IL, USA) and conjugated to the activated deoxyribonucleic acid (DNA) according to the manufacturer's instructions. Free (e.g., unbound) DNA was removed using a Superose® 12 gel filtration column. Free (eg, unbound) antibody was removed using Vivapure® Q mini columns (Sartorius Stedim Biotech GmbH, Goettingen, Germany).

[0436] A 384-well iPCR method for detecting reduced hIL-33 Biotinylated rat / human chimeric anti-hIL-33 3F10 capture antibody at 0.2 μg / mL, standard or sample, and DNA-labeled rat IgG2a anti-hIL-33 15.21C7 detection antibody at 25 ng / mL were diluted in sample buffer (PBS, pH 7.4, 0.5% BSA, 0.05% TWEEN® 20, 15 ppm ProClin TMThe assay buffer was preincubated in 0.25% 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS), 5 mM EDTA, 0.35 N NaCl, and 250 μg / mL calf thymus DNA for 2 h at room temperature. Calf thymus DNA (Sigma, St. Louis, MO, USA) at 250 μg / mL was heated to 95°C for 10 min and then kept on ice for at least 5 min prior to addition to the assay buffer. Reduced hIL-33 (Genentech, South San Francisco, CA, USA) in 1 mM TCEP was used as a standard and was prepared on ice to reduce oxidation. The concentrations of reduced hIL-33 in human vitreous, aqueous humor, serum, and NF samples were prepared on ice using sample buffer. Streptavidin-coated 384-well real-time PCR plates (Roche Applied Science-Custom Biotech, Penzberg, Germany) were prepared using SuperBlock TM The plates were blocked for 2 h with iPCR wash buffer (ThermoFisher scientific, Waltham, MA, USA) and washed with iPCR wash buffer (0.5% BSA, 0.05% TWEEN® 20, 15 ppm Proclin 100 in PBS, pH 7.4) using a 384-well plate washer (BioTek, Winooski, VT, USA). TM ) and washed 3 times with iPCR wash buffer. The antibody-antigen mix was then added to the plate and incubated for 20 minutes before being washed 13 times with iPCR wash buffer. A 60X PCR mix containing primers (5'-TGAAGGTCCTTGGCGATCA (SEQ ID NO: 39) and 5'-AGGGACGTAGCCAAATGCAT (SEQ ID NO: 40)) and probe (5'-TTCGGCGGTGATCGp-3' (SEQ ID NO: 41)) labeled with fluorescein dye and quencher was diluted to 1X in Universal TaqMan® Master Mix II (Applied Biosystems, Foster City, CA, USA) and PCR grade water before being added to the plate. Real-time PCR was performed using a ViiA TMPCR was performed using a 7 real-time PCR system (Applied Biosystems, Foster City, CA, USA). Thermal cycling conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Standard curves were generated using a four-parameter regression curve-fitting program (Genentech, South San Francisco, CA, USA).

[0437] Human Biological Samples Vitreous and aqueous humor samples from control and acute AMD post-mortem donors were purchased from Lions Eye Institute for Transplant and Research (Tampa, FL, USA) or BioIVT (Westbury, NY, USA). Disease stage (early, intermediate, advanced) or type (wet vs. dry AMD) was not disclosed on the sample sheets provided by the vendors. Serum, NF, and plasma samples from controls and "self-reported" asthma patients were obtained from Genentech Healthy Services (South San Francisco, CA, USA).

[0438] statistical analysis Statistical tests were performed using unpaired two-tailed Student's t-test or Mann-Whitney test. Error bars indicate standard deviation. P values ​​of <0.05 were considered statistically significant.

[0439] Example 2: Development of an ultrasensitive human IL-33 biomarker assay for drug development in age-related macular degeneration and asthma In the past few decades, human interleukin-33 (hIL-33) has emerged as a key contributor to the pathogenesis of numerous inflammatory diseases. Although there are multiple commercially available hIL-33 assays spanning multi-platform technologies, their ability to provide precise hIL-33 concentration measurements in various matrices and to distinguish between active (reduced) and inactive (oxidized) hIL-33 remains unclear. This is especially true for relatively small sample volumes, matrices with low hIL-33 concentrations, and matrices with elevated levels of interleukin-1 receptor-like 1 (sST2), an inactive form of ST2 that competes with membrane-bound ST2 for hIL-33 binding.

[0440] The performance of several commercially available hIL-33 detection assays was tested in various human matrices. Most of the tested commercially available assays were found to lack sensitivity (below to low pg / mL) and / or sufficient target specificity to accurately detect reduced hIL-33 at biologically relevant levels, especially in the presence of human sST2 (hsST2) (e.g., hIL-33 bound to hsST2). To address this, we developed and validated a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) that can detect reduced and total hIL-33 levels even in the presence of high concentrations of sST2 (e.g., hIL-33 bound to hsST2). By incorporating an immuno-polymerase chain reaction (iPCR) platform, the sensitivity of this assay was increased by approximately 52-fold for the reduced form of hIL-33. Using this hIL-33 iPCR assay, hIL-33 was detected in postmortem human vitreous humor samples from donors with age-related macular degeneration (AMD), and hIL-33 levels were found to be significantly elevated when compared to control individuals. No statistically significant differences were observed in aqueous humor from AMD donors and in plasma and nasal fluid (NF) from asthma patients compared to control individuals.

[0441] Unlike existing commercially available hIL-33 assays, the presently disclosed hIL-33 bioassays are extremely sensitive and specific, and can accurately quantify hIL-33 in a variety of human clinical matrices, including those with high levels of hsST2. The present results demonstrate the utility of these assays in clinical trials targeting the hIL-33 / hST2 pathway.

[0442] result Commercially available assays were unable to detect reduced hIL-33 with high sensitivity To identify a sensitive assay capable of detecting reduced hIL-33 at concentrations below 10 pg / mL in biological matrices, we compared various commercially available assay reagents and platforms used in the literature to report hIL-33 concentrations (Table 3). A series of anti-hIL-33 antibodies were prescreened and only those with the highest sensitivity in buffer were selected for testing in the ELISA format. Four selection criteria were established to qualify the assay as a suitable biomarker for human matrices: 1) the limit of quantification of the assay in VH must be <4 pg / mL, since hIL-33 is expected to be absent or present at low levels under non-pathological to moderately pathological conditions; 2) the assay must tolerate at least 10 ng / mL of hsST2, since the mean concentrations of hsST2 in VH and serum samples were 2.05 and 19.8 ng / mL, respectively (Figure 1); 3) the assay must be specific, characterized by its ability to immunodeplete 100% ± 20% of hIL-33 from hIL-33-containing human vitreous humor samples when incubated with anti-hIL-33 Ab; and 4) the maximum sample volume must be 50 μL, since some human ocular matrices are difficult to obtain and high dilutions would compromise the sensitivity of the assay.

[0443] None of the commercially available kits / reagent assays tested met all four criteria (Table 3). Invitrogen's human IL-33 ELISA kit, rabbit (Rb) x hIL-33 (Pepro) / biotin goat x hIL-33 (R&D), U-PLEX human IL-33 MSD kit and Bio-Plex Pro human T helper (Th17) cytokine 15-Plex kit had lower limits of detection in vitreous fluid (Table 3). However, none of them could tolerate the expected average hsST2 concentration in human matrix (Figure 1). The Quanterix Simoa® platform could not improve the quantification limit of the mu anti-hIL-33 (Enzo) / biotin-mu anti-hIL-33 (Novus) pair in buffer, and the required sample volume was not compatible with the assay of ocular samples (Table 3).

[0444] Apart from not being tolerant to hsST2 resistance, the Invitrogen human IL-33 ELISA kit was sensitive and specific (Table 3), fulfilling three of the four criteria. To investigate which form of hIL-33 was specifically recognized by this kit, we compared the hIL-33 provided in the kit with the standard curves generated in-house or using oxidized and reduced hIL-33 (Figure 2). These results indicated that this ELISA kit detects most of the oxidized hIL-33 and has low sensitivity to reduced hIL-33 (Figure 3). In summary, this comparative study suggested that the tested commercial reagents / kits fulfilled the necessary criteria to be qualified as a sensitive reduced hIL-33 detection assay.

[0445] Table 3: Performance of commercially available reagents and platforms for detecting hIL-33 TIFF2024544885000003.tif161170Parameters that did not meet assay criteria selection are highlighted in bold; n / a indicates the parameter was not tested. LLOQ = lower limit of quantification.

[0446] Development of reduced and total hIL-33 ELISA The successful development of two ELISAs capable of quantifying 1) reduced hIL-33 and 2) total hIL-33 (reduced and oxidized) in the vitreous humor was achieved through an extensive antibody generation and screening campaign followed by characterization and validation experiments. A rat immunization campaign resulted in the generation of 3026 antibodies. Using array-based surface plasmon resonance (SPR), purified hybridoma Abs were identified that specifically bound reduced hIL-33 with high affinity and displayed a distinct set of epitopes that did not compete with hsST2 through Ab binning. Eighty-eight pairwise combinations of selected in-house monoclonal Abs and commercially available polyclonal Abs (MAbs or PAbs) were screened for their binding ability to total hIL-33, oxidized hIL-33, or reduced hIL-33 by ELISA. Of the 88 antibody pairs, only two pairs detected reduced hIL-33 with a sensitivity of less than 10 pg / mL in buffer, and only one pair (antibody clones 3F10 and 15.21C7) was hsST2 resistant (up to 40 ng / mL) (Figure 4A). Clones 3F10 and 15.21C7 each showed a constant dissociation (K D ) values ​​of 0.487 and 0.338 nM (Figure 5 and Table 4). The lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) of the reduced hIL-33 ELISA were 4.1 pg / mL and 1000 pg / mL, respectively, with interassay coefficients of variation (CV) of 9.1% and 1.6% (Table 4). The assay was performed in a 384-well plate format with an assay volume of 25 μL.

[0447] Table 4: Rate constants for anti-hIL-33 Abs 15.21C7 and 3F10 binding to reduced hIL-33 at 25°C. TIFF2024544885000004.tif28170

[0448] A total hIL-33 ELISA was implemented in which a combination of reduced (15.21C7) and total hIL-33 specific antibodies was used as coating. Upon addition of saturating concentrations of hsST2, an antibody cocktail consisting of anti-hIL-33 and anti-ST2 monoclonal antibodies was used to detect the total amount of reduced and oxidized hIL-33 present in the mixture. The ratio of each antibody was thoroughly optimized to ensure equal detection of reduced and oxidized hIL-33 (Figure 4B). The LLOQ and ULOQ of the overall ELISA were 4.1 pg / mL and 1000 pg / mL, respectively, with interassay CVs of 10.5% and 0.7% (Table 5).

[0449] Table 5: Standard curve performance of reduced and total hIL-33 ELISA. TIFF2024544885000005.tif102170n=6 independent assay runs LLOQ=4.1pg / mL, ULOQ=1000pg / mL

[0450] Immuno-depletion experiments confirmed that the assay was specific for total hIL-33 in control vitreous humor samples, with 89.1% ± 0.31% of the signal being removed in the presence of beads conjugated to anti-hIL-33 antibodies (Figure 4C). The lowest dilution ratio for serum, plasma, and NF was also established to be 1:2 by testing the linear dilution of total hIL-33 (Table 6). With the LLOQ of the assay being 4.1 pg / mL, the lower limit of detection for serum, plasma, and NF is 8.2 pg / mL.

[0451] In summary, a reduced and total hIL-33 ELISA capable of detecting less than 10 pg / mL hIL-33 in the presence of hsST2 (e.g., hIL-33 bound to hsST2) was successfully implemented and developed.

[0452] Table 6: Dilution linearity of total hIL-33 endogenously expressed in human matrix TIFF2024544885000006.tif80170

[0453] Development and characterization of a reduced hIL-33 iPCR assay Although the sensitivity of the hIL-33 ELISA described above was increased, we still could not measure detectable levels of hIL-33 in the majority of human ocular and serum samples. Therefore, we investigated the iPCR platform, which previously showed great promise in increasing the sensitivity of the detection assay. Similar to the reduced hIL-33 ELISA, clones 3F10 and 15.21C7 were utilized, but it was the latter that was conjugated to the 55-mer DNA amplified during the iPCR reaction, allowing for enhanced signal. The iPCR platform dramatically increased the sensitivity with an LLOQ of 0.078 pg / mL (± 1.1%), representing a 52.6-fold improvement from the ELISA (Table 7) (Figure 6A). Immunodepletion experiments confirmed that the assay was specific for reduced hIL-33 in control vitreous humor samples, with 99.6% ± 0.07% of the signal being eliminated in the presence of beads conjugated to clone 15.21C7 Ab (Figure 6B).

[0454] Table 7: Standard curve performance for reduced hIL-33 iPCR TIFF2024544885000007.tif64170

[0455] n=6 independent assay runs LLOQ=0.078pg / mL, ULOQ=20pg / mL The precision of the assay was assessed by measuring three concentrations of reduced hIL-33 (0.6, 1.8 and 6.5 pg / mL) diluted in assay buffer in four replicates for a total of 12 independently performed runs. All three controls had intra- and interassay CVs below 20% (Table 8), indicating that the results generated were reproducible and precise.

[0456] Table 8: Precision of the reduced hIL-33 iPCR assay TIFF2024544885000008.tif34170 a n = 12 runs;b n=4 replicates

[0457] The linearity of the assay was then evaluated using a set of pooled individual controls and postmortem matrix samples to determine the lowest dilution ratio. Two ocular matrices, serum, plasma, and NF, were tested to implement a biomarker assay that could be used for multiple indications. The range of dilutions tested was narrowed due to the low concentration of endogenous reduced hIL-33 (low pg / mL) and the limited availability of human matrices. The results showed that the recoveries of dilution-adjusted concentrations in ocular, serum, plasma, and NF matrices were within ±20% of the linearity acceptance criteria for all dilutions tested, except for serum at 1:2 (% recovery = 66.3%) (Table 9). Thus, the lowest dilution ratio (MRD) for vitreous, aqueous humor, plasma, and NF was 1:2, and for serum it was 1:4. With an assay LLOQ of 0.078 pg / mL, the lowest quantifiable concentration was 0.156 pg / mL for vitreous, aqueous humor, plasma and NF, and 0.312 pg / mL for serum.

[0458] Table 9: Dilution linearity of reduced hIL-33 endogenously expressed in human matrix TIFF2024544885000009.tif115170

[0459] Additionally, spike recovery experiments were performed to assess accuracy. Low, medium, and high concentrations of reduced hIL-33 were added to these matrices diluted according to their respective MRDs, and percent recovery was determined as the ratio of measured concentrations over expected concentrations (endogenous + spiked reduced hIL-33). All sample recoveries were within the expected range of 80-120%, demonstrating that the iPCR assay can accurately measure reduced hIL-33 in eye, serum, plasma, and NF matrices (Table 10).

[0460] Table 10: Precision test measured by spike recovery of reduced hIL-33 in matrix TIFF2024544885000010.tif112170

[0461] Levels of reduced hIL-33 were significantly higher in vitreous fluid samples from human dry AMD compared to controls.

[0462] Using an iPCR assay, levels of reduced hIL-33 were measured in human vitreous and aqueous humor samples from postmortem normal and AMD donors. Note that no information was available on the severity (early or advanced) or type (wet or dry) of AMD from these donors. In total, 88.7% of vitreous and 57.7% of aqueous humor samples were above the LLOQ. A significant increase in reduced hIL-33 was observed in AMD compared to control samples, consistent with the increased presence of hIL-33-positive cells associated with GA (Xi et al., J Exp Med, 2016, 213:189-207) (Figure 7A). Also, an average of 3.3-fold higher amounts of reduced hIL-33 were detected in postmortem AMD aqueous humor samples compared to control aqueous humor samples, although the difference was not significant (P=0.2773) (Figure 7B). Taken together, these results suggest that the reduced iPCR assay can be used as a predictive and pharmacodynamic biomarker assay.

[0463] Reduced hIL-33 is not significantly upregulated in plasma and NF from patients with self-reported asthma Multiple literature reports have shown that hIL-33 protein levels are upregulated in asthmatic patients (Momen et al., Int J Prev Med, 2017, 8:65; Bahrami Mahneh et al., Int Arch Allergy Immunol, 2015, 168:193-196; Voloshyna et al., Clin Exp Allergy, 2015, 45:1554-1565; Raeiszadeh Jahromi et al., J Asthma, 2014, 51:1004-1013). However, a comprehensive evaluation of commercially available assays available to measure the concentration of hIL-33 revealed that most, if not all, of them are unable to measure active, reduced hIL-33 with the sensitivity required to obtain an informed interpretation. Herein, using the reduced hIL-33 iPCR assay, we determined and compared the levels of reduced hIL-33 in NF and serum from 9 age- and sex-matched controls and asthma patients. Note that both matrices were extracted from the same donor. The results showed that there was no significant difference in reduced hIL-33 concentration between NF and serum (Figures 8A and 8B). In addition, the mean level of reduced hIL-33 in serum was 6.4 pg / mL (Figure 8B). Such a relatively high level could be due to the release of intracellular hIL-33 from platelets during serum preparation. To exclude this hypothesis, similar measurements were performed in matched plasma samples. The mean reduced hIL-33 concentration was similar to serum (6.9 pg / mL), confirming that the concentration of reduced hIL-33 was not an artifact of sample preparation (Figure 8C). The concentration of total hIL-33 (reduced and oxidized) in the same samples was also examined and no significant differences were observed between normal and asthma samples (Figure 9).

[0464] The mean reduced hIL-33 concentration in human serum from asthma patients (n=23 samples) was 6.7 pg / mL compared to 1.06 ng / mL total hIL-33, demonstrating that most hIL-33 is oxidized and inactive (Figure 8D). There was a significant correlation between reduced and total hIL-33 in NF samples, demonstrating that the majority of hIL-33 is reduced and active in this matrix (Figure 8D).

[0465] Taken together, these results do not support upregulation of active hIL-33 in the serum and NF of asthmatic patients.

[0466] Consideration In this study, a thorough evaluation of commercially available assays for measuring hIL-33 in biological matrices was provided. Similar to the observations made by Cohen et al. and Ketelaar et al., most, if not all, commercially available assays did not meet the criteria for sensitive and specific measurement of secreted active hIL-33 (Cohen et al., Nat Commun, 2015, 6:8327; Ketelaar et al., Clin Exp Allergy, 2016, 46:884-887). Such findings suggested that bioactive hIL-33 levels in extracellular matrices reported in the literature must be interpreted with caution and supported the need to implement assays to measure the availability of reduced hIL-33 in healthy and diseased human patients.

[0467] Reported herein is the development of an ultrasensitive iPCR assay, compatible with a 384-well plate format, that accurately measures reduced hIL-33 in ocular matrix, serum, plasma, and NF with detection limits of 0.156, 0.312, 0.156, and 0.156 pg / mL, respectively. The iPCR platform was chosen because it could be successfully used to increase the sensitivity of antigen detection by up to 2 logs compared to standard ELISA. The 384-well plate format was particularly attractive because human ocular samples are extremely difficult to obtain, typically only 100 μL of aqueous humor sample can be obtained per donor. The fully characterized assay has the potential to be utilized as a predictive or pharmacodynamic biomarker assay.

[0468] First, we measured the levels of hIL-33 in human vitreous and aqueous humor samples from healthy subjects and AMD patients. In contrast to rodent eye samples, which contain ng / mL of hIL-33 (data not shown), approximately 1,000-fold less hIL-33 was expected in human eye samples based on a study published by Takeuchi et al. (Takeuchi et al., PLoS One, 2015, 10:e0137358). In this study, the authors reported that hIL-33 in vitreous humor samples from patients with proliferative diabetic retinopathy averaged 17.1 pg / mL, with only 25% of samples above the detection limit. These results indicate that the assay used in the study was suboptimal and could not tolerate ng / mL concentrations of hsST2 (Figure 3). Using the iPCR assay, we were unable to detect hIL-33 in 88.7% of the vitreous humor samples (control and AMD samples) tested. A significant increase in hIL-33 concentrations was observed in postmortem AMD compared to control vitreous humor samples, suggesting that hIL-33 is upregulated in AMD, consistent with previous findings. Elevated levels of hIL-33 were also measured in postmortem aqueous humor samples from donors with AMD compared to control donors, but the difference was not significant (P=0.2773). More aqueous humor samples would be needed to see if statistical significance could be reached. Overall, the concentrations of hIL-33 obtained in vitreous humor samples were higher than those in aqueous humor samples. This is consistent with the predominance of hIL-33 production by cells in the posterior compartment of the eye, which is closer to the vitreous compartment than the aqueous compartment (Xi et al., J Exp Med, 2016, 213:189-207).

[0469] We also analyzed serum, plasma, and NF from healthy controls and asthmatic patients. A strong correlation between reduced and total hIL-33 was observed in NF from asthmatic patients, indicating that most of the total hIL-33 was reduced. This may be explained by the proximity of tissues expressing high levels of hIL-33.

[0470] In contrast, most of the hIL-33 in serum and plasma appears to be oxidized and therefore unable to bind to hsST2. Such observations confirmed the predictions by Cohen et al., who hypothesized that all free (e.g., unbound) hIL-33 reaching plasma is likely to be oxidized within a few hours (Cohen et al., Nat Commun, 2015, 6:8327). Such a control mechanism is essential, since sustained elevation of IL-33 in serum was shown to trigger lethal inflammation in a mouse model in which the IL-33 nuclear localization signal was deleted (Bessa et al., J Autoimmun, 2014, 55:33-41).

[0471] Importantly, the reduced hIL-33 iPCR assay is highly hsST2 resistant, meaning that it cannot distinguish between free (e.g., unbound) reduced hIL-33 and hIL-33 bound to hsST2. This implies that the 6.9 pg / mL (0.38 pM) of hIL-33 measured in plasma may not be fully active because a portion may be bound to its decoy receptor hsST2. The mean concentration of hsST2 in both the control and asthma sample groups was determined to be 14.7 ng / mL (0.4 nM) (Figure 10A). Thus, it is likely that most or all of the reduced hIL-33 was complexed with hsST2 and was unable to signal through its receptor. A similar assumption could not be made for the NF samples, since hsST2 levels were below the LLOQ in approximately 60% of the samples (Figure 10B).

[0472] This is the first study to accurately report reduced and total hIL-33 in human serum and plasma. Other groups have reported serum hIL-33 levels in asthma patients with various severity of disease (Momen et al., Int J Prev Med, 2017, 8:65; Bahrami Mahneh et al., Int Arch Allergy Immunol, 2015, 168:193-196; Voloshyna et al., Clin Exp Allergy, 2015, 45:1554-1565; Raeiszadeh Jahromi et al., J Asthma, 2014, 51:1004-1013). All commercial kits used to generate hIL-33 levels were, after thorough characterization, not suitable to accurately detect reduced hIL-33 for the following reasons: 1) these assays could not tolerate the levels of hsST2 present in serum (~20 ng / mL), 2) their detection limits were not low enough, and / or 3) they specifically detected only the oxidized form of hIL-33. Therefore, caution should be exercised when comparing data generated with these assays.

[0473] These results show no significant difference between reduced hIL-33 levels from healthy subjects and asthmatics. One important point to consider is that the analyzed samples were collected from "self-reported" asthmatics, which likely represent a heterogeneous population across a range of severity. There is no information whether these donors had exacerbated asthma at the time of sample collection, and therefore we cannot exclude the possibility that reduced hIL-33 is indeed significantly higher in such cases. Also, since human lung samples contained high levels of hIL-33, it seemed interesting to test sputum samples from moderate to severe asthmatics, similar to Cohen et al. (Cohen et al., Nat Commun, 2015, 6:8327).

[0474] conclusion In conclusion, commercially available methods for measuring reduced hIL-33 concentrations in multiple human matrices did not meet acceptable standards of sensitivity and specificity. To address this, improved antibodies and a highly sensitive iPCR assay were developed that can specifically and accurately detect reduced hIL-33 across a wide range of matrices. This novel biomarker assay may have broad implications for ongoing and future clinical trials targeting the hIL-33 / hST2 signaling pathway for multiple indications. In a clinical setting, this assay may help identify candidate responders based on basal hIL-33 concentrations and determine target engagement for patients undergoing anti-hIL-33 antibody therapy.

[0475] Example 3: Reduced IL-33 iPCR Assay An iPCR assay using an anti-IL-33 antibody conjugated to DNA was optimized to quantify reduced hIL-33 in aqueous humor samples from patients with geographic atrophy (GA) secondary to age-related macular degeneration (AMD) (dry AMD).

[0476] Figure 11 shows the steps of an exemplary IL-33 iPCR assay. Briefly, streptavidin-coated 384-well PCR plates (MicroCoat, Cat. 840003) were filled with blocking buffer (Thermo Scientific SuperBlock TM Blocking Buffer Cat. 37515) and incubated for 2-3 hours on a shaker at room temperature. Wash the blocked PCR plate with 25 μL / well of wash buffer (1x phosphate-buffered saline (PBS), 0.5% bovine serum albumin (BSA), 15 ppm ProClin TM) and washed with 100 mL of ... TM Each of the standards, samples, and QC controls (40 μL) diluted 1:4 in 100 μg / mL anti-IL-33 antibody (1E1v8, see WO 2021 / 183849 and US 2021 / 0284725, which are incorporated by reference in their entireties) was mixed with 40 μl of the antibody mix in quadruplicate in 96-well minitubes and incubated for 2 hours at room temperature on a shaker set at 400 rpm. The addition of the anti-IL-33 antibody 1E1v8 reduces the oxidation of IL-33 in the samples. The hIL-33-antibody mix was added into the wells of a blocked PCR plate and incubated at room temperature on a shaker set at 400 rpm. After incubation, plates were washed and 10 μL / well of PCR master mix (1x ABI Universal TaqMan 1x) containing forward primer (5'-TGAAGGTCCTTGGCGATCA, SEQ ID NO: 39), reverse primer (5'-AGGGACGTAGCCAAATGCAT, SEQ ID NO: 40), and carboxyfluorescein (FAM) / min or groove binder (MGB)-nonfluorescent quencher (NFQ) tag probe (5'-TTCGGCGGTGATCG, SEQ ID NO: 41)) was added. TM Master Mix II, 1x Custom TaqMan TM Gene Expression Assay Mix (Life Technologies, Assay ID AIT96PT) was added. Real-time quantitative PCR (qPCR) amplification was performed using the ViiA PCR kit using the following cycling conditions: TM 7 instruments were run: 95° C. for 10 min, repeated 40 times (95° C. for 15 sec and 60° C. for 1 min). Data were analyzed using SoftMax Pro, version 6.5.1.

[0477] Standard curve and control accuracy, precision, LLOQ, and ULOQ at the minimum dilution in the sample diluent

[0478] Standard curve reporting ranges were determined and the accuracy and precision of the assays within the established reporting ranges were assessed.

[0479] Accuracy is the ability of an analytical method to obtain test results close to the true value of hIL-33. Precision is the degree of agreement between individual test results when an analytical method is repeated for multiple analyses of a sample. Accuracy and precision are also used to determine the limit of quantification of an assay. The lower limit of quantification (LLOQ) is the lowest concentration of hIL-33 that can be quantified with the applicable precision and accuracy under the experimental conditions stated. The upper limit of quantification (ULOQ) is the highest concentration of hIL-33 that can be quantified for a sample with the applicable precision and accuracy. The reporting range of an assay is the range of concentrations from the LLOQ to the ULOQ within which the interpolated results have an acceptable level of precision and accuracy.

[0480] To determine the accuracy, precision, LLOQ, and ULOQ of the assay, samples were first prepared by spiking exogenous analyte into various concentrations of sample dilutions. Samples used to determine the LLOQ were prepared at well concentrations of 61, 244, and 977 fg / mL. Samples used to determine the ULOQ were prepared at well concentrations of 15,625, 62,500, and 250,000 fg / mL. In addition, low, medium, and high controls spanning the range of the standard curve were prepared at concentrations of 1,000 fg / mL, 8,000 fg / mL, and 800,000 fg / mL, respectively. An endogenous control was obtained by pooling serum samples with detectable levels of hIL-33 and quantified to 4,000 fg / mL. All samples except the LLOQ and ULOQ were then assayed at the minimum dilution in seven consecutive assays over six days.

[0481] The results of accuracy, precision, LLOQ, and ULOQ at the minimum dilution ratio in the sample dilutions are shown in Table 11.

[0482] Table 11: Standard curve, accuracy and precision TIFF2024544885000011.tif82170

[0483] The results for the presence of endogenous hIL-33 in the pooled samples are shown in Table 12.

[0484] Table 12: Endogenous levels of reduced hIL-33 TIFF2024544885000012.tif45170

[0485] The results for accuracy and precision for high, medium, low, and endogenous controls are shown in Table 13.

[0486] Table 13: Accuracy and precision of controls TIFF2024544885000013.tif51170

[0487] Based on the results, the accuracy, intra-assay precision, and total error were acceptable for the endogenous, low, medium, and high controls, and the selected LLOQ and ULOQ. The LLOQ and ULOQ were selected to be 244 and 250,000 fg / mL, respectively (dilution-corrected concentrations: 977 and 1,000,000 fg / mL). The minimum dilution factor (MRD) of the assay was 1 / 4, which was confirmed during accuracy testing by assaying individual serum samples at the lowest dilution factor using specimen diluents.

[0488] The minimum quantifiable concentration is equal to the LLOQ multiplied by the minimum dilution required for accurate quantification of hIL-33 in the sample matrix. Therefore, the minimum quantifiable concentration for this assay is 977 fg / mL.

[0489] The maximum quantifiable concentration is equal to the ULOQ multiplied by the maximum sample dilution that can be accurately measured. The maximum quantifiable concentration for this assay was 1,000,000 fg / mL.

[0490] Parallelism (intrinsic dilution linearity) Parallelism is an important experiment that demonstrates whether the dose response of endogenous hIL-33 in the matrix is ​​sufficiently similar to the dose response of the recombinant standard in the surrogate matrix. This validates the recombinant material as well as the use of the surrogate matrix and the stability of the selected MRD.

[0491] Serum samples from 10 individual patients (1 AMD, 4 asthma, and 5 rheumatoid arthritis) containing measurable amounts of hIL-33 were tested at MRD (1 / 4) followed by one 1 / 2 serial dilution in sample diluent. Parallelism was acceptable based on 9 / 10 (90%) samples passing the %CV≦30% criterion and 9 / 10 (90%) samples passing the %RE for each dilution against the acceptance criterion of MRD<30%. Results are shown in Table 14.

[0492] Table 14: Parallelism of reduced hIL-33 (endogenous dilution linearity) TIFF2024544885000014.tif147170

[0493] Detectability of endogenous levels in human serum and plasma Various aqueous humor, serum, and plasma samples from normal and diseased states were diluted in sample diluent to a minimum dilution of 1 / 4 and tested for endogenous levels. Detectability of IL-33 was acceptable, with 97%-100% of the concentrations in human serum and plasma samples above the LLOQ; however, only 1 / 20 (5%) of the human aqueous humor samples were above the LLOQ. Detectability results are shown in Figure 12.

[0494] specificity Specificity is the ability of the capture antibody to selectively bind to the targeted hIL-33.

[0495] Samples from diseased individuals were incubated with 10 μg / mL of capture antibody for 1 hour at room temperature with shaking. The specificity of the capture antibody was acceptable, with 100% inhibition of analyte concentration in all 10 serum samples. The results are shown in Table 15.

[0496] Table 15: Specificity TIFF2024544885000015.tif108170

[0497] reproducibility Reproducibility is the degree of agreement between individual test results when an analytical method is repeated in an identical manner over time for multiple analyses of samples.

[0498] Twenty sera and plasma samples with detectable levels of hIL-33 were evaluated in duplicate over a minimum of two days. In this experiment, 8 / 20 (40%) serum and plasma samples showed acceptable reproducibility. The poor reproducibility was likely due to expected oxidation of IL-33 in serum and plasma samples.

[0499] The results are shown in Table 16.

[0500] Table 16: Reproducibility TIFF2024544885000016.tif152170

[0501] Interference / Cross-reactivity Interference demonstrates the ability of an assay to accurately quantify an analyte in the presence of compounds known to be in the systemic circulation at the time of sample collection. Interference is observed when analyte recovery is <70% or >130% of the nominal concentration of the analyte in the presence of known concentrations of potentially interfering molecules.

[0502] Cross-reactivity indicates the specificity of the assay in quantifying only the analyte and not unrelated compounds that may be in the systemic circulation at the time of sample collection.

[0503] Each of the high, medium, low, endogenous, and negative (sample dilution) controls was assayed with or without 0, 10, 20, 40, and 80 ng / mL rhST2 (rhST2 / IL-33R Fc Chimera) (R&D Systems Cat.#523-ST-100). No interference with the endogenous controls was observed at 80 ng / mL or less of rhST2; however, interference with the high, medium, and low controls was mixed with biased recoveries at 10 ng / mL rhST2 for the medium control, at 20 ng / mL rhST2 for the high, medium, and low controls, at 40 ng / mL rhST2 for the high and medium controls, and at 80 ng / mL rhST2 for the medium control; no interference was observed at 10 ng / mL rhST2 for the high and low controls, at 40 ng / mL rhST2 for the low control, and at 80 ng / mL rhST2 for the high control. Interference observed in some of the high, medium, and low controls was likely caused by assay variability. No cross-reactivity was observed with rhST2 below 80 ng / mL. Results are shown in Table 17.

[0504] Table 17: Interference / Cross-reactivity TIFF2024544885000017.tif99170

[0505] summary Table 18 below summarizes the reduced IL-33 iPCR assay.

[0506] Table 18: Summary of reduced IL-33 iPCR assays TIFF2024544885000018.tif133170

[0507] Example 4: Total IL-33 iPCR Assay An iPCR assay using anti-IL-33 antibodies conjugated to DNA was optimized to quantify total hIL-33 in aqueous humor samples from patients with geographic atrophy (GA) secondary to age-related macular degeneration (AMD) (dry AMD). The total IL-33 iPCR assay had the same steps and conditions as the reduced IL-33 iPCR assay described in Example 3, except that the samples were treated with 1 mM TCEP for 2 to 4 days at 4° C. prior to mixing with the antibody mixture. The positive control used in the total IL-33 iPCR assay was oxidized IL-33, prepared by diluting IL-33 in Iscove's Modified Dulbecco's Medium (IMDM) in 50 mM Tris, 300 mM NaCl (pH 8.0) and incubating at 37° C. for 1 day.

[0508] The performance of the standard curve and controls for the total IL-33 iPCR assay is shown below in Table 19. The minimum dilution was 1:4, the LLOQ was 977 fg / mL and the ULOQ was 250,000 fg / mL.

[0509] Table 19: Performance of standard curves and controls (N=7) TIFF2024544885000019.tif127170

[0510] Figure 13 shows the detectability of reduced IL-33 in normal and diseased human body fluid, serum, and plasma samples using reduced or total IL-33 iPCR assays. Reduced IL-33 was only detectable in serum samples from AMD, asthma, and RA patients, as well as in pools of normal human serum and normal human plasma.

[0511] Table 20 below shows the reproducibility of the total IL-33 iPCR assay. Eight of eleven (73%) human serum and plasma samples tested showed acceptable reproducibility (i.e., within 30% difference).

[0512] Table 20: Reproducibility TIFF2024544885000020.tif91170

[0513] Other embodiments The foregoing invention has been described in some detail by way of illustration and example for clarity of understanding, but the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein by reference in their entireties.

[0514] array TIFF2024544885000021.tif255170TIFF2024544885000022.tif253170TIFF2024544885000023.tif27170

Claims

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to IL-33, wherein the antibody comprises a binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, and the binding domain comprises the following six complementarity-determining regions (CDRs): (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6 An antibody or antigen-binding fragment thereof comprising:

2. An isolated antibody or antigen-binding fragment thereof that specifically binds to IL-33, wherein the antibody comprises the following six CDRs: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24 1. An antibody or antigen-binding fragment thereof comprising a binding domain comprising:

3. The antibody of claim 2, comprising: (a) a VH domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 25; (b) a VL domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 26; or (c) the VH domain of (a) and the VL domain of (b).

4. The antibody of claim 2, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 25 and the VL domain comprises the amino acid sequence of SEQ ID NO:

26.

5. An antibody described in claim 2, comprising: (a) a HC having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 27; (b) a LC having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 28; or (c) the HC of (a) and the LC of (b).

6. The antibody of claim 2, wherein the HC comprises the amino acid sequence of SEQ ID NO: 27 and the LC comprises the amino acid sequence of SEQ ID NO:

28.

7. The antibody binds to oxidized or reduced IL-33, and / or the antibody binds to IL-33 bound to soluble interleukin-1 receptor-like 1 (sST2) protein and IL-33 not bound to sST2 with a K within 2-fold. Ｄ The antibody of claim 2, which binds to

8. An isolated nucleic acid encoding the antibody of claim 2, or a set of isolated nucleic acids which together encode said antibody.

9. A vector or set of vectors comprising the isolated nucleic acid or set of isolated nucleic acids of claim 8.

10. A host cell comprising the vector or set of vectors according to claim 9.

11. A method for producing an antibody that specifically binds to IL-33, comprising culturing the host cell of claim 10 in a medium.

12. An immunoconjugate comprising the antibody of claim 2, wherein the antibody is linked to a label.

13. 1. A method for detecting the presence or level of IL-33 in a biological sample, comprising: contacting the biological sample with the immunoconjugate of claim 12 and detecting the presence of bound immunoconjugate. A method comprising:

14. 14. The method of claim 13, wherein detecting is by IHC, IF, flow cytometry, ELISA, immunoblotting, or iPCR.

15. 14. The method of claim 13, wherein detecting is by iPCR, the method comprising contacting the biological sample with the immunoconjugate in the presence of a second anti-IL-33 antibody that specifically binds to an epitope on IL-33 that is different from the epitope specifically bound by the anti-IL-33 antibody in the immunoconjugate, and detecting the presence of the bound immunoconjugate.

16. 14. The method of claim 13, wherein the biological sample is a blood sample, an eye sample, a nasal sample, or a cell culture sample.

17. 14. The method of claim 13, wherein the biological sample is derived from a subject having or at risk for an IL-33-mediated disorder.

18. The method of claim 13, wherein the presence or level of IL-33 is the presence or level of total IL-33, reduced IL-33, or IL-33 bound to sST2.

19. 13. An assay for identifying a subject having an IL-33 mediated disorder who is a candidate for treatment comprising an IL-33 axis binding antagonist, the assay comprising determining the presence or level of IL-33 in a biological sample obtained from the subject using the immunoconjugate of claim 12.

20. (a) the antibody of the immunoconjugate of claim 12; and (b) a package insert containing instructions for using the antibody to detect the presence or level of IL-33 in a biological sample. Kit including:

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