Materials and methods for IL-1β binding protein

Abstract

In certain aspects, provided herein are antibodies that bind to IL-1β, and compositions comprising the antibodies. Methods of making and using the antibodies are also provided.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 297,436, filed January 7, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] (Sequence Listing) This application contains a computer readable sequence listing submitted herewith in XML file format, the entire contents of which are incorporated herein by reference in their entirety. The sequence listing XML file submitted herewith is entitled "14620-563-228_SEQ_LISTING.xml", was created on December 1, 2022, and is 123,349 bytes in size.

[0003] FIELD OF THEINVENTION This application relates to anti-IL-1β antibodies, nucleic acids and expression vectors encoding the antibodies, recombinant cells containing the vectors, and compositions comprising the antibodies. Methods of making the antibodies and methods of using the antibodies to treat diseases, including cancer, are also provided. [Background technology]

[0004] IL-1β is a pleiotropic cytokine with multiple roles in both physiological and pathological conditions. In cancer, IL-1β promotes a tumor-supportive microenvironment through various mechanisms. For example, it has been suggested that IL-1β promotes the production of mutagenic reactive oxygen species that may lead to tumorigenesis (Taniguchi K et al, Nat Rev Immunol. 2018; 18(5): 309-324). Furthermore, the IL-1 pathway promotes the expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), two important proangiogenic factors that lead to the formation of capillaries, a hallmark of tumor progression and essential for tumor invasiveness and metastasis (Voronov E et al, Proc Natl Acad Sci US A. 2003; 100(5): 2645-2650, Voronov E et al, Front Physiol. 2014; 5: 114). IL-1β also promotes epithelial-mesenchymal transition (EMT) in vitro, a key step in the early stages of the metastatic cascade (Li R et al, Sci Rep. 2020; 10(1): 377). Within the TME, IL-1β can recruit and reprogram multiple cell types; for example, IL-1β has been shown to promote macrophage and neutrophil infiltration, recruit immunosuppressive myeloid populations (e.g., MDSCs, TAMs, and TANs), and attenuate antitumor T cell infiltration and activation (Bunt SK et al, J Immunol. 2006; 176(1): 284-290).

[0005] Furthermore, clinical data has provided evidence for the important role of the IL-1 pathway in cancer (Ridker PM et al, Lancet. 2017;390(10105):1833-1842). Therefore, there is a need in the art for high affinity and potent anti-IL-1β antibody molecules that can neutralize the IL-1β signaling pathway for cancer therapy. Summary of the Invention [Means for solving the problem]

[0006] In one embodiment, an antibody that binds to IL-1β, (1) (i) a VH having the amino acid sequence of SEQ ID NO: 7, comprising a VH CDR1, a VH CDR2, and a VH CDR3, each having the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3, respectively; and (ii) a VL having the amino acid sequence of SEQ ID NO: 8, comprising a VL CDR1, a VL CDR2, and a VL CDR3, each having the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3, respectively; (2) (i) a VH having the amino acid sequence of SEQ ID NO: 9, comprising a VH CDR1, a VH CDR2, and a VH CDR3, each having the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3, respectively; and (ii) a VL having the amino acid sequence of SEQ ID NO: 10, comprising a VL CDR1, a VL CDR2, and a VL CDR3, each having the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3, respectively; or (3) Provided herein is an antibody comprising: (i) a VH having the amino acid sequence of SEQ ID NO: 11, including a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3, respectively; and (ii) a VL having the amino acid sequence of SEQ ID NO: 12, including a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3, respectively.

[0007] In some embodiments, (i) the amino acid sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are numbered according to the Kabat numbering system; (ii) the amino acid sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are numbered according to the Chothia numbering system; (iii) the amino acid sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are numbered according to the AbM numbering system; (iv) the amino acid sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are numbered according to the Contact numbering system; and / or (v) the amino acid sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are numbered according to the Contact numbering system. The amino acid sequences of CDR2 and VL CDR3 follow the IMGT numbering system.

[0008] In another embodiment, an antibody that binds to IL-1β, (1)(i) a VH comprising a VH CDR1 having an amino acid sequence selected from SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO: 49, SEQ ID NO: 67, and SEQ ID NO: 85, a VH CDR2 having an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 32, SEQ ID NO: 50, SEQ ID NO: 68, and SEQ ID NO: 86, and a VH CDR3 having an amino acid sequence selected from SEQ ID NO: 15, SEQ ID NO: 33, SEQ ID NO: 51, SEQ ID NO: 69, and SEQ ID NO: 87; and (ii) a VL comprising a VL CDR1 having an amino acid sequence selected from SEQ ID NO: 16, SEQ ID NO: 34, SEQ ID NO: 52, SEQ ID NO: 70, and SEQ ID NO: 88, a VL CDR2 having an amino acid sequence selected from SEQ ID NO: 17, SEQ ID NO: 35, SEQ ID NO: 53, SEQ ID NO: 71, and SEQ ID NO: 89, and a VL CDR3 having an amino acid sequence selected from SEQ ID NO: 18, SEQ ID NO: 36, SEQ ID NO: 54, SEQ ID NO: 72, and SEQ ID NO: 90; (2)(i) a VH comprising a VH CDR1 having an amino acid sequence selected from SEQ ID NO:19, SEQ ID NO:37, SEQ ID NO:55, SEQ ID NO:73, and SEQ ID NO:91, a VH CDR2 having an amino acid sequence selected from SEQ ID NO:20, SEQ ID NO:38, SEQ ID NO:56, SEQ ID NO:74, and SEQ ID NO:92, and a VH CDR3 having an amino acid sequence selected from SEQ ID NO:21, SEQ ID NO:39, SEQ ID NO:57, SEQ ID NO:75, and SEQ ID NO:93; and (ii) a VL comprising a VL CDR1 having an amino acid sequence selected from SEQ ID NO:22, SEQ ID NO:40, SEQ ID NO:58, SEQ ID NO:76, and SEQ ID NO:94, a VL CDR2 having an amino acid sequence selected from SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:59, SEQ ID NO:77, and SEQ ID NO:95, and a VL CDR3 having an amino acid sequence selected from SEQ ID NO:24, SEQ ID NO:42, SEQ ID NO:60, SEQ ID NO:78, and SEQ ID NO:96, or (3)(i) a VH comprising a VH CDR1 having an amino acid sequence selected from SEQ ID NO:25, SEQ ID NO:43, SEQ ID NO:61, SEQ ID NO:79, and SEQ ID NO:97, a VH CDR2 having an amino acid sequence selected from SEQ ID NO:26, SEQ ID NO:44, SEQ ID NO:62, SEQ ID NO:80, and SEQ ID NO:98, and a VH CDR3 having an amino acid sequence selected from SEQ ID NO:27, SEQ ID NO:45, SEQ ID NO:63, SEQ ID NO:81, and SEQ ID NO:99; and (ii) Provided herein is an antibody comprising a VL comprising a VL CDR1 having an amino acid sequence selected from SEQ ID NO:28, SEQ ID NO:46, SEQ ID NO:64, SEQ ID NO:82, and SEQ ID NO:100, a VL CDR2 having an amino acid sequence selected from SEQ ID NO:29, SEQ ID NO:47, SEQ ID NO:65, SEQ ID NO:83, and SEQ ID NO:101, and a VL CDR3 having an amino acid sequence selected from SEQ ID NO:30, SEQ ID NO:48, SEQ ID NO:66, SEQ ID NO:84, and SEQ ID NO:102.

[0009] In another embodiment, an antibody that binds to IL-1β, (1) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 13, a VH CDR2 having the amino acid sequence of SEQ ID NO: 14, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 15; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 16, a VL CDR2 having the amino acid sequence of SEQ ID NO: 17, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 18; (2) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 19, a VH CDR2 having the amino acid sequence of SEQ ID NO: 20, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 21; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 22, a VL CDR2 having the amino acid sequence of SEQ ID NO: 23, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 24; (3) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 25, a VH CDR2 having the amino acid sequence of SEQ ID NO: 26, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 27; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 28, a VL CDR2 having the amino acid sequence of SEQ ID NO: 29, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 30; (4) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 31, a VH CDR2 having the amino acid sequence of SEQ ID NO: 32, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 33; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 34, a VL CDR2 having the amino acid sequence of SEQ ID NO: 35, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 36; (5) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 37, a VH CDR2 having the amino acid sequence of SEQ ID NO: 38, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 39; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 40, a VL CDR2 having the amino acid sequence of SEQ ID NO: 41, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 42; (6) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 43, a VH CDR2 having the amino acid sequence of SEQ ID NO: 44, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 45; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 46, a VL CDR2 having the amino acid sequence of SEQ ID NO: 47, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 48; (7) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 49, a VH CDR2 having the amino acid sequence of SEQ ID NO: 50, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 51; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 52, a VL CDR2 having the amino acid sequence of SEQ ID NO: 53, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 54; (8) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 55, a VH CDR2 having the amino acid sequence of SEQ ID NO: 56, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 57; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 58, a VL CDR2 having the amino acid sequence of SEQ ID NO: 59, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 60; (9) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 61, a VH CDR2 having the amino acid sequence of SEQ ID NO: 62, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 63; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 64, a VL CDR2 having the amino acid sequence of SEQ ID NO: 65, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 66; (10) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 67, a VH CDR2 having the amino acid sequence of SEQ ID NO: 68, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 69; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 70, a VL CDR2 having the amino acid sequence of SEQ ID NO: 71, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 72; (11) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 73, a VH CDR2 having the amino acid sequence of SEQ ID NO: 74, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 75; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 76, a VL CDR2 having the amino acid sequence of SEQ ID NO: 77, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 78; (12) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 79, a VH CDR2 having the amino acid sequence of SEQ ID NO: 80, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 81; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 82, a VL CDR2 having the amino acid sequence of SEQ ID NO: 83, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 84; (13) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 85, a VH CDR2 having the amino acid sequence of SEQ ID NO: 86, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 87; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 88, a VL CDR2 having the amino acid sequence of SEQ ID NO: 89, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 90; (14) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 91, a VH CDR2 having the amino acid sequence of SEQ ID NO: 92, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 93; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 94, a VL CDR2 having the amino acid sequence of SEQ ID NO: 95, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 96; or (15) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 97, a VH CDR2 having the amino acid sequence of SEQ ID NO: 98, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 99; and (ii) An antibody is provided herein, comprising a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 100, a VL CDR2 having the amino acid sequence of SEQ ID NO: 101, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 102.

[0010] In some aspects, the antibodies provided herein further comprise one or more framework regions set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and / or SEQ ID NO:12.

[0011] In some embodiments, (i) the antibody comprises a VH having the amino acid sequence of SEQ ID NO:7, and a VL having the amino acid sequence of SEQ ID NO:8; (ii) the antibody comprises a VH having the amino acid sequence of SEQ ID NO:9, and a VL having the amino acid sequence of SEQ ID NO:10; or (iii) the antibody comprises a VH having the amino acid sequence of SEQ ID NO:11, and a VL having the amino acid sequence of SEQ ID NO:12.

[0012] In some embodiments, the antibodies provided herein are humanized antibodies.

[0013] In some embodiments, the antibodies provided herein are IgG antibodies. In some embodiments, the IgG antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.

[0014] In some embodiments, the antibodies provided herein comprise a κ light chain.

[0015] In some embodiments, the antibodies provided herein comprise a λ light chain.

[0016] In some embodiments, the antibodies provided herein comprise a mutated Fc region. In some embodiments, the mutated Fc region comprises M252Y / S254T / T256E (YTE) mutations.

[0017] In some embodiments, the antibodies provided herein are monoclonal antibodies.

[0018] In some embodiments, the antibodies provided herein bind to an IL-1β antigen.

[0019] In some embodiments, the antibodies provided herein bind to an IL-1β epitope.

[0020] In some embodiments, the antibodies provided herein specifically bind to IL-1β.

[0021] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 form a binding site for an antigen of IL-1β.

[0022] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 form a binding site for an epitope of IL-1β.

[0023] In some embodiments, the antibodies provided herein are multispecific. In some embodiments, the antibodies provided herein are capable of binding to at least two antigens. In some embodiments, the antibodies provided herein are capable of binding to at least three antigens. In some embodiments, the antibodies provided herein are capable of binding to at least four antigens. In some embodiments, the antibodies provided herein are capable of binding to at least five antigens.

[0024] In another aspect, provided herein is a binding molecule comprising an antibody disclosed herein. In some embodiments, the antibody is genetically fused or chemically conjugated to an agent.

[0025] In another aspect, provided herein are nucleic acids encoding the antibodies provided herein.

[0026] In another aspect, provided herein is a vector comprising a nucleic acid disclosed herein.

[0027] In one aspect, provided herein is a host cell comprising a vector provided herein.

[0028] In another aspect, provided herein is a kit comprising a vector provided herein and a package therefor.

[0029] In another aspect, provided herein is a kit comprising an antibody provided herein and packaging therefor.

[0030] In another aspect, provided herein is a pharmaceutical composition comprising an antibody provided herein and one or more pharma- ceutically acceptable excipients.

[0031] In one aspect, provided herein is a method of producing a pharmaceutical composition, the method comprising combining an antibody with one or more pharma- ceutically acceptable excipients to obtain a pharmaceutical composition.

[0032] In another aspect, provided herein is a method of inhibiting IL-1β, or IL-1β-mediated signaling in a cell, comprising contacting the cell with an antibody provided herein.

[0033] In another aspect, provided herein is a method of inhibiting IL-1β-induced production of IL-6, ENA-78 (CXCL5), and / or G-CSF in a cell, comprising contacting the cell with an antibody provided herein.

[0034] In another aspect, provided herein is a method of reducing production of IL-6, ENA-78 (CXCL5), and / or G-CSF in a cell, the method comprising contacting the cell with an antibody provided herein.

[0035] In another aspect, provided herein is a method of inhibiting the growth or proliferation of IL-1β expressing cells, comprising contacting the cells with an antibody combination provided herein.

[0036] In some embodiments, the cell or cells are present in a subject having a disease or disorder.

[0037] In yet another aspect, provided herein is a method of inhibiting IL-1β in a subject, the method comprising administering to the subject an antibody provided herein.

[0038] In yet another aspect, provided herein is a method of treating a disease or disorder in a subject, comprising administering to the subject an antibody provided herein.

[0039] In some embodiments, the disease or disorder is an IL-1β related disease or disorder. In some embodiments, the IL-1β related disease or disorder is an inflammatory disease or disorder. In some embodiments, the IL-1β related disease or disorder is cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the non-small cell lung cancer has reached stage 0, stage 1, stage 2, stage 3, or stage 4. In some embodiments, the cancer is renal cancer. In some embodiments, the cancer is renal cancer. In some embodiments, the renal cell carcinoma has reached stage 1, stage 2, or stage 3. [Brief description of the drawings]

[0040] [Figure 1]Figure 1 shows epitope mapping of selected antibodies on IL-1β using hydrogen-deuterium exchange based LC-MS. At the top, the sequence shown is the fragment of SEQ ID NO: 109, residues 117-269, which corresponds to the sequence of the mature IL-1β protein (SEQ ID NO: 110). Double underlining indicates strong epitopes (ΔΔG on binding ≦-2 kcal / mol) and single underlining indicates weak epitopes (-2<ΔΔG on binding ≦-1 kcal / mol). At the bottom, the epitopes are superimposed on the X-ray crystal structure of IL-1β (PDB ID 1I1B). Black indicates strong epitopes and grey indicates weak epitopes. [Diagram 2] Potency of 05H21A, 08F17A, and 15N14A (all with the YTE mutation in the Fc region) in the NF-kB / AP-1 reporter system. The neutralizing activity of the lead anti-IL-1β antibody panel was assessed in HEK-Blue reporter cells. Dose response curves and IC50 values ​​of the panel of lead anti-IL-1β mAbs are shown. [Diagram 3] The inhibitory activity of a panel of anti-IL-1β lead antibodies was evaluated in MRC5 human lung fibroblasts. The figure shows dose-response curves and corresponding IC50 determinations of the lead anti-IL-1β mAb panel. [Figure 4A] Potency of 05H21A, 08F17A, and 15N14A in human lung fibroblasts. The neutralizing activity of a panel of lead anti-IL-1β antibodies was evaluated in normal human lung fibroblasts (NHLF donors 34325 and 35234). The figure shows dose-response curves from donor 34325 based on IL-6 (A) and CXCL5 (B) release measurements, and the corresponding IC50 determinations are reported. [Figure 4B] Potency of 05H21A, 08F17A, and 15N14A in human lung fibroblasts. The neutralizing activity of a panel of lead anti-IL-1β antibodies was evaluated in normal human lung fibroblasts (NHLF donors 34325 and 35234). The figure shows dose-response curves from donor 34325 based on IL-6 (A) and CXCL5 (B) release measurements, and the corresponding IC50 determinations are reported. [Figure 5A]Potency of 05H21A, 08F17A, and 15N14A in human donor PBMC samples. The neutralizing activity of the lead anti-IL-1β antibody panel was evaluated in one healthy human donor PBMC (donor TS235). (A) and (B) Dose-response curves measuring IL-6 release and calculated IC50 values ​​of the lead panel. [Figure 5B] Potency of 05H21A, 08F17A, and 15N14A in human donor PBMC samples. The neutralizing activity of the lead anti-IL-1β antibody panel was evaluated in one healthy human donor PBMC (donor TS235). (A) and (B) Dose-response curves measuring IL-6 release and calculated IC50 values ​​of the lead panel. [Figure 6] Potency of 05H21A, 08F17A, and 15N14A in human blood assays. Neutralizing activity of the lead anti-IL-1β antibody panel was evaluated in human whole blood samples (donors tested: CC00448, M3767, M5988, M7286, and M7370). IC50 values ​​(nM) based on IL-6, CXCL-5, and G-CSF release measurements by MSD are plotted. [Figure 7A] Potency of 05H21A, 08F17A, and 15N14A in cynomolgus monkey fibroblast samples. Neutralizing activity of the lead anti-IL-1β antibody panel was evaluated in primary cynomolgus monkey skin and lung fibroblasts (CDF and CLF, respectively). (A) and (B) Dose-response curves measuring IL-6 release of the lead panel in CDF and CLF, respectively. Calculated IC50 values ​​for the anti-IL-1β antibody panel are shown. [Figure 7B] Potency of 05H21A, 08F17A, and 15N14A in cynomolgus monkey fibroblast samples. Neutralizing activity of the lead anti-IL-1β antibody panel was evaluated in primary cynomolgus monkey skin and lung fibroblasts (CDF and CLF, respectively). (A) and (B) Dose-response curves measuring IL-6 release of the lead panel in CDF and CLF, respectively. Calculated IC50 values ​​for the anti-IL-1β antibody panel are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] The present disclosure is based in part on novel antibodies that bind to IL-1β, and their superior properties.

[0042] 5.1.Definition The techniques and procedures described or referenced herein include those generally well understood and / or commonly employed by those skilled in the art using conventional techniques, such as the widely used techniques described in Sambrook et al: A Laboratory Manual (3rd ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009), Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); and Antibody Engineering Vols 1 and 2 (Kontermann and Dubel eds., 2d ed. 2010). Unless otherwise defined herein, technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. For the purposes of interpreting this specification, the following explanations of terms apply, and where appropriate, terms used in the singular also include the plural and vice versa. In the event that any explanation of a term provided herein conflicts with any document incorporated by reference, the explanation of the term provided below shall prevail.

[0043] The terms "antibody," "immunoglobulin," or "Ig" are used interchangeably herein and are used in the broadest sense, specifically encompassing monoclonal antibodies (including agonist, antagonist, neutralizing, full-length or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), single-chain antibodies, and fragments thereof, e.g., as described below. Antibodies may be human, humanized, chimeric, and / or affinity matured, and may also be antibodies from other species, e.g., mouse, rabbit, llama, etc. The term "antibody" is intended to include polypeptide products of B cells within the immunoglobulin class of polypeptides capable of binding to a specific molecular antigen and composed of two identical paired polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), the respective amino-terminal portions of each chain containing a variable region of about 100 to about 130 or more amino acids, and the respective carboxy-terminal portions of each chain containing a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2d ed. 1995); and Kuby, Immunology (3d ed. 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, antibodies derived from Camelidae species (e.g., llamas and alpacas), or humanized variants thereof, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above, which refer to portions of antibody heavy or light chain polypeptides that retain some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fvs (scFv) (including, e.g., monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies.In particular, the antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as antigen-binding domains or molecules that contain an antigen-binding site (e.g., one or more CDRs of an antibody) that binds to an antigen. Such antibody fragments can be found, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989), Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995), Huston et al., 1993, Cell Biophysics 22:189-224, Pluckthun and Skerra, 1989, Meth. Enzymol. 178:497-515, and Day, Advanced Immunochemistry (2d ed. 1990). The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule. The antibody can be an agonist antibody or an antagonist antibody. The antibody may be neither an agonist nor an antagonist.

[0044] An "antigen" is a structure to which an antibody can selectively bind. A target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide. In certain embodiments, the antigen is associated with a cell, e.g., present on or within a cell.

[0045] An "intact" antibody is one that contains an antigen-binding site as well as a CL and at least a heavy chain constant region, CH1, CH2, CH3. The constant region may include a human constant region or an amino acid sequence variant thereof. In certain embodiments, an intact antibody has one or more effector functions.

[0046] The term "binding" or "binding" refers to interactions between molecules, including, for example, forming a complex. The interactions can be non-covalent interactions, including, for example, hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interactions between a single antigen-binding site of an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment for that epitope. The dissociation rate (k off ) and association rate (k on ) and the ratio (k off / k on ) is the dissociation constant K D and is inversely related to affinity. D The lower the value, the higher the affinity of the antibody. D The value of k varies for different complexes of antibody and antigen. on and k off The dissociation constant K of the antibodies provided herein depends on both D can be determined using any method provided herein or any other method known to those skilled in the art. The affinity at one binding site does not necessarily reflect the true strength of the interaction between the antibody and the antigen. When a complex antigen containing multiple repeating antigenic determinants, such as a multivalent antigen, contacts an antibody containing multiple binding sites, the interaction of the antibody with the antigen at one site will increase the probability of reaction at a second site. The strength of multiple interactions between such a multivalent antibody and an antigen is called avidity.

[0047] In the context of the binding molecules described herein, terms such as "binds to," "specifically binds to," and similar terms are also used interchangeably herein to refer to binding molecules of an antigen-binding domain that specifically binds to an antigen, such as a polypeptide. Binding molecules or antigen-binding domains that bind to or specifically bind to an antigen can be identified, for example, by immunoassay, Octet®, Biacore®, or other techniques known to those of skill in the art. In some embodiments, a binding molecule or antigen-binding domain binds to or specifically binds to an antigen if it binds to the antigen with a higher affinity than any cross-reactive antigens as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, a specific or selective response is at least twice the signal or noise of background, and may be more than 10 times the background. For a discussion of binding specificity, see, for example, Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989). In certain embodiments, the extent of binding of a binding molecule or antigen-binding domain to a "non-target" protein is less than about 10% of the binding of the binding molecule or antigen-binding domain to its particular target antigen, as determined, for example, by fluorescence activated cell sorting (FACS) analysis or RIA. Binding molecules or antigen-binding domains that bind to an antigen include those that are capable of binding an antigen with sufficient affinity such that the binding molecule is useful, for example, as an antigen-targeted therapeutic and / or diagnostic agent. In certain embodiments, a binding molecule or antigen-binding domain that binds to an antigen has a dissociation constant (K D In certain embodiments, the binding molecule or antigen binding domain binds to an epitope of an antigen that is conserved among antigens of different species.

[0048] In certain embodiments, binding molecules or antigen-binding domains may include "chimeric" sequences in which a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567, and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-55). Chimeric sequences may include humanized sequences.

[0049] In certain embodiments, a binding molecule or antigen-binding domain may comprise a portion of a "humanized" form of a non-human (e.g., camelid, murine, non-human primate) antibody that comprises sequences from a human immunoglobulin (e.g., recipient antibody) in which native CDR residues are replaced by residues from a corresponding CDR of a non-human species (e.g., donor antibody) such as camel, mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some cases, one or more FR region residues of the human immunoglobulin sequence 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 improve antibody performance. The heavy or light chain of a humanized antibody can comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In certain embodiments, a humanized antibody comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Further details are described in Jones et al., Nature 321:522-25 (1986); Riechmann et al., Nature 332:323-29 (1988); Presta, Curr. Op. Struct. Biol., 2:593-96 (1992); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-89 (1992); U.S. Patent Nos. 6,800,738, 6,719,971, 6,639,055, 6,407,213, and 6,054,297.

[0050] In certain embodiments, the binding molecule or antigen-binding domain may comprise a "fully human antibody" or a portion of a "human antibody," which terms are used interchangeably herein and refer to an antibody that comprises a human variable region and, for example, a human constant region. The binding molecule may comprise an antibody sequence. In specific embodiments, these terms refer to an antibody that comprises a variable region and a constant region of human origin. A "fully human" antibody may also encompass, in certain embodiments, an antibody that binds a polypeptide and is encoded by a nucleic acid sequence that is a naturally occurring somatic variant of a human germline immunoglobulin nucleic acid sequence. The term "fully human antibody" includes antibodies that have variable and constant regions that correspond to human germline immunoglobulin sequences as described by Kabat et al. (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242). A "human antibody" is one that has an amino acid sequence that corresponds to that of an antibody produced by a human and / or that has been produced using any of the techniques for producing human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)) and yeast display libraries (Chao et al., Nature Protocols 1:755-68 (2006)).In addition, human monoclonal antibodies can be prepared using the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy 77 (1985); Boerner et al., J. Immunol. 147(1):86-95 (1991), and van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., mice, which have been engineered to produce such antibodies in response to antigen challenge, but whose endogenous loci are disabled (see, e.g., Jakobovits, Curr. Opin. Biotechnol. 6(5):561-66 (1995); Bruggemann and Taussing, Curr. Opin. Biotechnol. 8(4):455-58 (1997); and U.S. Pat. Nos. 6,075,181 and 6,150,584, regarding XENO Mouse™ technology). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA 103:3557-62 (2006) (regarding human antibodies generated with human B cell hybridoma technology).

[0051] In certain embodiments, a binding molecule or antigen-binding domain may comprise a portion of a "recombinant human antibody," which term includes human antibodies prepared, expressed, created, or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse or a cow) that is transgenic and / or transchromosomal for human immunoglobulin genes (see, e.g., Taylor, LD, et al., Nucl. Acids Res. 20:6287-6295 (1992)), or antibodies prepared, expressed, created, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242). However, in certain embodiments, such recombinant human antibodies have been subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0052] In certain embodiments, the binding molecule or antigen-binding domain may comprise a portion of a "monoclonal antibody," a term used herein to refer to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for naturally occurring mutations that may be present in minor amounts, and well-known post-translational modifications such as isomerization and deamidation of amino acids, oxidation of methionine, and deamidation of asparagine and glutamine, and each monoclonal antibody typically recognizes a single epitope on an antigen. In specific embodiments, a "monoclonal antibody," as used herein, is an antibody produced by a single hybridoma or other cell. The term "monoclonal" is not limited to a particular method for making the antibody. For example, monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made using recombinant DNA methods in bacterial, or eukaryotic animal, or plant cells (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 352:624-28 (1991), and Marks et al., J. Mol. Biol. 222:581-97 (1991). Other methods for preparing clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art. See, for example, Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002).

[0053] A typical four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). For IgG, the four-chain unit is generally about 150,000 daltons. Each L chain is linked by a H chain with one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus followed by three constant domains (CH) for each of the α and γ chains and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus followed by a constant domain (CL) at the other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CH1) of the heavy chain. Certain amino acid residues are believed to form an interface between the light and heavy chain variable domains. The pairing of VH and VL together forms a single antigen-binding site. The structure and properties of different classes of antibodies are described, for example, in Basic and Clinical Immunology 71 (Stites et al. eds., 8th ed. 1994), and Immunobiology (Janeway et al. eds., 5th ed. 1995). th (ed. 2001).

[0054] The term "Fab" or "Fab region" refers to the antibody region that binds to an antigen. A conventional IgG usually contains two Fab regions, each in one of the two arms of the Y-shaped IgG structure. Each Fab region is typically composed of one variable region and one constant region of each of the heavy and light chains. More specifically, the variable and constant regions of the heavy chain in the Fab region are the VH and CH1 regions, and the variable and constant regions of the light chain in the Fab region are the VL and CL regions. The VH, CH1, VL, and CL in the Fab region can be arranged in various ways to confer antigen-binding capability in accordance with the present disclosure. For example, the VH and CH1 regions can be on one polypeptide, while the VL and CL regions can be on separate polypeptides, as in the Fab region of a conventional IgG. Alternatively, the VH, CH1, VL, and CL regions can all be on the same polypeptide and oriented in a different order, as described in more detail in the following sections.

[0055] The terms "variable region," "variable domain," "V region," or "V domain" refer to a portion of an antibody's light or heavy chain that is located generally at the amino terminus of the light or heavy chain, has a length of about 120-130 amino acids for heavy chains and about 100-110 amino acids for light chains, and is used in the binding and specificity of each particular antibody to its particular antigen. The variable region of the heavy chain may be referred to as "VH." The variable region of the light chain may be referred to as "VL." The term "variable" refers to certain segments of the variable region that vary widely in sequence between antibodies. The V region mediates antigen binding and determines the specificity of a particular antibody for a particular antigen. However, that variability is not uniform across the 110 amino acid span of the variable region. Instead, the V region consists of less variable (e.g., relatively invariant) stretches called framework regions (FR) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called "hypervariable regions," each about 9-12 amino acids in length. The variable regions of the heavy and light chains each contain four FRs, mostly in a β-sheet structure, connected by three hypervariable regions, which form loops connecting the β-sheet structure and sometimes form part of the β-sheet structure. The hypervariable regions in each chain are held in close proximity together by the FRs, and together with the hypervariable regions of the other chains, contribute to the formation of the antigen-binding site of the antibody (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991)). The constant regions are not directly involved in binding the antibody to the antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable regions vary widely in sequence between different antibodies. In a specific embodiment, the variable regions are human variable regions.

[0056] The term "variable region residue numbering according to Kabat" or "amino acid position numbering as in Kabat", and variations thereof, refers to the numbering system used for the heavy or light chain variable regions 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 shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 (residue 52a according to Kabat) and three inserted residues after residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat). The Kabat numbering of residues can be determined for a given antibody by aligning the sequence of that antibody with the "standard" Kabat numbering sequence at the regions of homology. The Kabat numbering system is generally used when referring to residues in the variable domain (residues 1-107 for the light chain, and 1-113 for the heavy chain) (e.g., Kabat et al., supra). The "EU numbering system," or "EU index," is generally used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index as reported in Kabat et al., supra). "EU index as in Kabat" refers to the residue numbering of the human IgG 1 EU antibody. Other numbering systems have been described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

[0057] The term "heavy chain" when used with respect to an antibody refers to a polypeptide chain of about 50-70 kDa, the amino-terminal portion of which contains a variable region of about 120-130 or more amino acids, and the carboxy-terminal portion of which contains a constant region. The constant region can be one of five distinct types (e.g., isotypes) designated alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains vary in size, with α, δ, and γ containing about 450 amino acids, while μ and ε contain about 550 amino acids. When combined with light chains, these distinct types of heavy chains give rise to five well-known classes (e.g., isotypes) of antibodies, IgA, IgD, IgE, IgG, and IgM (which also includes the four subclasses of IgG, i.e., IgG1, IgG2, IgG3, and IgG4).

[0058] The term "light chain" when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, the amino-terminal portion of which contains a variable region of about 100 to about 110 or more amino acids, and the carboxy-terminal portion of which contains a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two different types, called kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain.

[0059] As used herein, the terms "hypervariable region", "hypervariable region, HVR", "complementarity determining region" and "Complementarity Determining Region, CDR" are used interchangeably. "CDR" refers to one of the three hypervariable regions (H1, H2, or H3) in the non-framework region of an immunoglobulin (Ig or antibody) VH β-sheet framework or one of the three hypervariable regions (L1, L2, or L3) in the non-framework region of an antibody VL β-sheet framework. In a VH domain, CDR1, CDR2, and CDR3 are also referred to as HCDR1, HCDR2, and HCDR3, respectively. In a VL domain, CDR1, CDR2, and CDR3 are also referred to as LCDR1, LCDR2, and LCDR3, respectively. Thus, CDRs are variable region sequences interspersed within framework region sequences.

[0060] CDR regions are well known to those skilled in the art and are defined by well-known numbering systems. For example, Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (see, for example, Kabat et al., (above); Nick Deschacht et al., J Immunol 2010;184:5696-5704). Chothia instead refers to the position of structural loops (see, for example, Chothia and Lesk, J.Mol.Biol.196:901-17(1987)). The ends of the Chothia CDR-H1 loops when numbered using the Kabat numbering convention vary from H32 to H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32, if only 35A is present, the loop ends at 33, and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (see, for example, Antibody Engineering Vol. 2 (Kontermann and Dubel, eds., 2d ed. 2010)). The "contact" hypervariable regions are based on an analysis of available complex crystal structures. Another universal numbering system that has been developed and widely adopted is the ImMunoGeneTics (IMGT) Information System® (see Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)). IMGT is an integrated information system dedicated to immunoglobulins (IG), T cell receptors (TCR), and major histocompatibility complexes (MHC) of humans and other vertebrates. As used herein, CDRs are referred to in terms of both amino acid sequence and position within the light or heavy chain.The "locations" of the CDRs within the structure of immunoglobulin variable domains are conserved among species and reside in structures called loops, so that the CDR and framework residues are easily identified by using a numbering system that aligns the variable domain sequences according to structural features. This information can be used to graft and replace CDR residues from one species of immunoglobulin into an acceptor framework, typically from a human antibody. A further numbering system (AHon) has been developed by Honegger and Pluckthun, J. Mol. Biol. 309:657-70 (2001). The correspondence between numbering systems, including, for example, Kaba numbering and the IMGT specific numbering system, is well known to those skilled in the art (see, for example, Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra). The residues of each of these hypervariable regions or CDRs are illustrated in Table 1 below.

[0061] [Table 1]

[0062] The boundaries of a given CDR may vary depending on the scheme used for identification. Thus, unless otherwise stated, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof, such as a variable region, and the individual CDRs of an antibody or region thereof (e.g., CDR-H1, CDR-H2) should be understood to encompass the complementarity determining regions defined by any of the known schemes described herein above. In some cases, a scheme for the identification of a particular CDR or CDRs is specified, such as the CDRs defined by the IMGT, Kabat, Chothia, or Contact method. In other cases, the specific amino acid sequence of the CDR is included. It should be noted that a CDR region may also be defined by a combination of various numbering systems, such as a combination of the Kabat numbering system and the Chothia numbering system, or a combination of the Kabat numbering system and the IMGT numbering system. Thus, a term such as "CDRs shown in a particular VH" includes any CDR1 defined, but not limited to, by the exemplary CDR numbering systems described above. Given a variable region (eg, VH or VL), one of skill in the art will understand that the CDRs within that region may be defined by different numbering systems, or a combination thereof.

[0063] The hypervariable regions may include "extended hypervariable regions" such as: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH.

[0064] The term "constant region" or "constant domain" refers to the carboxy-terminal portions of the light and heavy chains that are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as interaction with Fc receptors. The term refers to the portion of the immunoglobulin molecule that has a more conserved amino acid sequence compared to the other portion of the immunoglobulin, the variable region, which contains the antigen-binding site. The constant region may include the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0065] The term "framework" or "FR" refers to variable region residues that flank the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain, diabodies, linear, and bispecific antibodies. FR residues are variable domain residues other than the hypervariable region or CDR residues.

[0066] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of an immunoglobulin heavy chain Fc region can vary, the Fc region of a human IgG heavy chain is often defined as extending from the amino acid residue at position Cys226, or from the amino acid residue at position Pro230, to the carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies can include an antibody population in which all K447 residues have been removed, an antibody population in which the K447 residue has not been removed, and an antibody population having a mixture of antibodies with and without the K447 residue. A "functional Fc region" has the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding, CDC, Fc receptor binding, ADCC, phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions generally require that the Fc region be combined with a binding region or domain (e.g., an antibody variable region or domain) and can be assessed using a variety of assays known to those of skill in the art. A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by at least one amino acid modification (e.g., substitution, addition, or deletion). In certain embodiments, a variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or the Fc region of a parent polypeptide, e.g., about 1 to about 10 amino acid substitutions, or about 1 to about 5 amino acid substitutions in the native sequence Fc region or the Fc region of a parent polypeptide. The variant Fc region herein may have at least about 80% homology to a native sequence Fc region and / or the Fc region of a parent polypeptide, or at least about 90% homology thereto, for example at least about 95% homology thereto.

[0067] As used herein, "epitope" is a term of the art and refers to a localized region of an antigen to which a binding molecule (e.g., an antibody) can specifically bind. An epitope can be a linear or conformational epitope, a non-linear epitope, or a discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be consecutive amino acids of a polypeptide (a "linear" epitope), or an epitope can include amino acids from two or more non-contiguous regions of a polypeptide (a "conformational", "non-linear", or "discontinuous" epitope). In general, it will be understood by those skilled in the art that a linear epitope may or may not depend on secondary, tertiary, or quaternary structure. For example, in some embodiments, a binding molecule binds to a group of amino acids, regardless of whether the amino acids are folded into a native three-dimensional protein structure. In other embodiments, the binding molecule requires that the amino acid residues that make up the epitope exhibit a particular conformation (eg, a bend, twist, turn, or fold) in order to recognize and bind to the epitope.

[0068] "Percent (%) amino acid sequence identity" and "homology" for a peptide, polypeptide, or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or 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. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0069] The term "specificity" refers to the selective recognition of an antigen-binding protein of a particular epitope of an antigen. Natural antibodies, for example, are monospecific. The term "multispecificity" as used herein refers to an antigen-binding protein having two or more antigen-binding sites, at least two of which bind to different antigens. "Bispecificity" as used herein refers to an antigen-binding protein having two different antigen-binding specificities. The term "monospecificity" as used herein means that a binding protein has one or more antigen-binding sites, each of which binds to the same antigen.

[0070] The term "valency" as used herein refers to the presence of a certain number of binding sites in an antigen-binding protein. For example, a natural antibody or a full-length antibody has two binding sites and is bivalent. Thus, the terms "trivalent", "tetravalent", "pentavalent" and "hexavalent" refer to the presence of two binding sites, three binding sites, four binding sites, five binding sites and six binding sites in an antigen-binding protein, respectively.

[0071] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also refers to amino acid polymers that are naturally modified or modified by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Also included in the definition are polypeptides that contain one or more analogs of an amino acid, including, but not limited to, unnatural amino acids, as well as other modifications known in the art. Because the polypeptides of the present disclosure may be based on antibodies or other members of the immunoglobulin superfamily, it is understood that in certain embodiments, a "polypeptide" can occur as a single chain or as two or more related chains.

[0072] "Polynucleotide" or "nucleic acid", as used interchangeably herein, refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. "Oligonucleotides", as used herein, refer to short, generally single-stranded, synthetic polynucleotides, generally, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is fully applicable to oligonucleotides as well. Cells producing the binding molecules of the present disclosure can include parent hybridoma cells, as well as bacterial and eukaryotic host cells into which nucleic acid encoding an antibody has been introduced. Unless otherwise specified, the left end of any single-stranded polynucleotide sequence disclosed herein is the 5' end. The leftward direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of the nascent RNA transcript is referred to as the transcription direction. The region of sequence on the DNA strand that has the same sequence as the RNA transcript at the 5' to 5' end of the RNA transcript is referred to as the "upstream sequence" and the region of sequence on the DNA strand that has the same sequence as the RNA transcript at the 3' to 3' end of the RNA transcript is referred to as the "downstream sequence."

[0073] An "isolated nucleic acid" is a nucleic acid, e.g., RNA, DNA, or mixed nucleic acid, that is substantially separated from other genomic DNA sequences, and from proteins or complexes, such as ribosomes and polymerases, that are naturally associated with the natural sequence. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. Additionally, an "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. In certain embodiments, one or more nucleic acid molecules encoding an antibody described herein are isolated or purified. This term encompasses a nucleic acid sequence that has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates, and chemically synthesized analogs, or biologically synthesized analogs in heterologous systems. A substantially pure molecule may include isolated forms of the molecule. Specifically, an "isolated" nucleic acid molecule encoding an antibody described herein is a nucleic acid molecule that has been identified and isolated from at least one contaminating nucleic acid molecule with which it is normally associated in the production environment.

[0074] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes nucleotide sequences that are degenerate versions of each other and all nucleotide sequences that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns to the extent that in some cases the nucleotide sequence encoding the protein may contain introns.

[0075] The term "control sequences" 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.

[0076] As used herein, the term "operably linked" and similar phrases (e.g., genetically fused), when used with respect to nucleic acids or amino acids, refers to an operable linkage in which nucleic acid or amino acid sequences, respectively, are placed in a functional relationship with each other. For example, operably linked promoters, enhancer elements, open reading frames, 5' and 3' UTRs, and terminator sequences result in the correct production of a nucleic acid molecule (e.g., RNA). In some embodiments, operably linked nucleic acid elements result in the transcription of the open reading frame and ultimately the production of a polypeptide (i.e., expression of the open reading frame). As another example, an operably linked peptide refers to one in which functional regions are positioned at an appropriate distance from each other to confer the intended function of each region.

[0077] The term "vector" refers to a material used to carry or contain a nucleic acid sequence, including, for example, a nucleic acid sequence encoding a binding molecule (e.g., an antibody) described herein, to introduce a nucleic acid sequence into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may contain an operable selection sequence or marker that can be stably integrated into a host cell chromosome. In addition, the vector may contain one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that may be included are, for example, those that provide resistance to antibiotics or toxins, complement deficiencies in auxiliary auxotrophies, or supply critical nutrients that are absent in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more nucleic acid molecules are to be co-expressed (e.g., both heavy and light chains of an antibody, or antibody VH and VL), both nucleic acid molecules may, for example, be inserted into a single expression vector or into separate expression vectors. In the case of expression in a single vector, the encoding nucleic acids may be operably linked to one common expression control sequence or to different expression control sequences, such as one inducible promoter and one constitutive promoter. Introduction of the nucleic acid molecule into the host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blot or amplification of mRNA by polymerase chain reaction (PCR), immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will understand that the nucleic acid molecule will be expressed in sufficient amounts to produce the desired product, and will further understand that expression levels can be optimized to obtain sufficient expression using methods well known to those skilled in the art.

[0078] As used herein, the term "host" refers to an animal, such as a mammal (eg, a human).

[0079] The term "host cell," as used herein, refers to a particular subject cell into which a nucleic acid molecule can be transfected, and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur during the subsequent generation or integration of the nucleic acid molecule into the host cell genome.

[0080] The terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. Such cells include the primary subject cell and its progeny.

[0081] As used herein, the term "pharmaceutical acceptable" means approved by a federal or state regulatory agency for use in animals, and more particularly for use in humans, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias.

[0082] "Excipient" refers to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, encapsulating material, etc. Excipients include, for example, encapsulating materials or additives such as absorption enhancers, antioxidants, binders, buffers, carriers, coating agents, colorants, diluents, disintegrants, emulsifiers, expanders, fillers, flavorings, wetting agents, lubricants, flavorings, preservatives, propellants, release agents, bactericides, sweeteners, solubilizers, wetting agents, and mixtures thereof. The term "excipient" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete) or vehicle.

[0083] In some embodiments, the excipient is a pharma- ceutically acceptable excipient. Examples of pharma-ceutically acceptable excipients include buffers such as phosphates, citrates, and other organic acids, antioxidants; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides, proteins; hydrophilic polymers, amino acids, monosaccharides, disaccharides, and other carbohydrates; chelating agents; sugar alcohols, salt-forming counterions, and / or non-ionic surfactants. Other examples of pharma-ceutically acceptable excipients are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th ed. 1990).

[0084] In one embodiment, each component is "pharmaceutical acceptable" in the sense of being compatible with the other components of the pharmaceutical formulation and suitable for use in contact with human and animal tissues or organs without undue toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio. See, e.g., Lippincott WILliams & WILkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed., Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed., Ash and Ash Eds., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed., Gibson Ed., CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, a pharmaceutical acceptable excipient is non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed. In some embodiments, the pharma- ceutically acceptable excipient is an aqueous pH buffered solution.

[0085] In some embodiments, the excipient is a sterile liquid, such as water and oils (including those of petroleum, animal, vegetable, or synthetic origin). Water is an exemplary excipient when the composition (e.g., pharmaceutical composition) is administered intravenously. Saline and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, particularly for injectable solutions. If necessary, the composition can further contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, or the like.

[0086] Compositions, including pharmaceutical compositions, can contain, for example, a binding molecule (eg, an antibody described herein) in isolated or purified form, together with a suitable amount of excipients.

[0087] The term "effective amount" or "therapeutically effective amount" as used herein refers to the amount of an antibody or therapeutic molecule contained in a drug and antibody, or pharmaceutical composition provided herein, that is sufficient to bring about the desired result.

[0088] The terms "subject" and "patient" may be used interchangeably herein. As used herein, in certain embodiments, a subject is a mammal, either non-primate or primate (such as a human). In specific embodiments, a subject is a human. In one embodiment, a subject is a mammal, e.g., a human, that has been diagnosed with a disease or disorder. In another embodiment, a subject is a mammal, e.g., a human, that is at risk of developing a disease or disorder.

[0089] "Administering" or "administration" refers to the act of injecting or otherwise physically delivering a substance present outside the body to a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art.

[0090] As used herein, the terms "treatment" and "treatment / treating" refer to the reduction or amelioration of the progression, severity, and / or duration of a disease or condition resulting from the administration of one or more therapeutics. Treatment may be determined by assessing whether there has been a reduction, alleviation, and / or remission of one or more symptoms associated with the underlying disease, such that an improvement is observed in the patient, even though the patient may still be suffering from the underlying disease. The term "treating" includes both management and amelioration of disease. The terms "manage", "managing", and "management" refer to the beneficial effects that a subject derives from a treatment, and do not necessarily result in a cure of the disease.

[0091] The terms "prevent," "preventing," and "prevention" refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom (e.g., diabetes or cancer).

[0092] The terms "intercept," "intercepting," and "interception" refer to providing early treatment of a disease process (e.g., a precursor and / or pre-malignant disease) to prevent, inhibit, or slow disease progression (e.g., to prevent, inhibit, or slow progression to a later stage cancer, such as a malignant cancer).

[0093] As used herein, "delaying" the onset of cancer means to postpone, prevent, slow, delay, stabilize, and / or postpone the onset of the disease. This delay can be of various durations, depending on the disease history and / or the individual receiving the treatment. As will be apparent to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. A method of "delaying" the onset of cancer is one that reduces the probability of disease onset in a given time frame and / or reduces the extent of disease in a given time frame, compared to not using the method. Such comparisons are typically based on clinical studies using a statistically significant number of individuals. The onset of cancer can be detected using standard methods, including, but not limited to, computed tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography, or biopsy. Onset can also refer to the progression of cancer, which may be initially undetectable, including onset, recurrence, and onset.

[0094] As used herein, "IL-1β-related disease or disorder" refers to a disease or disorder involving a tissue or cell in which IL-1β is expressed or overexpressed. In some embodiments, an IL-1β-related disease or disorder involves a cell in which IL-1β is aberrantly expressed. In other embodiments, an IL-1β-related disease or disorder involves a cell in which IL-1β is deficient in at least one of its activities.

[0095] The terms "about" and "approximately" refer to within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0096] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0097] Whenever an embodiment is described herein with the term "comprising," it is understood that other similar embodiments that would otherwise be described in terms of "consisting of" and / or "consisting essentially of" are also provided. Whenever an embodiment is described herein with the phrase "consisting essentially of," it is also understood that similar embodiments that would otherwise be described in terms of "consisting of" are also provided.

[0098] The term "between" when used in phrases such as "between A and B" or "between A and B" refers to a range that includes both A and B.

[0099] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B, A or B, A (alone), and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A (alone), B (alone), and C (alone).

[0100] 5.2.IL-1β binding molecule 5.2.1. Antibodies that bind to IL-1β In one aspect, an antibody capable of binding to IL-1β is provided herein. IL-1β is a pleiotropic cytokine with multiple roles in both physiological and pathological conditions. In cancer, IL-1β promotes a tumor-supportive microenvironment through a variety of mechanisms. In addition, the IL-1 pathway promotes the expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), two important pro-angiogenic factors that lead to the formation of capillary blood vessels, a hallmark of tumor progression and essential for tumor invasiveness and metastasis. IL-1β also promotes epithelial-mesenchymal transition (EMT) in vitro, which is a key step in the early stages of the metastatic cascade. IL-1β can recruit and reprogram multiple cell types. For example, IL-1β has been shown to promote macrophage and neutrophil infiltration, recruit immunosuppressive myeloid populations (e.g., MDSC), enhance neutrophil infiltration, and attenuate T cell infiltration and activation. The nucleic acid and amino acid sequences of IL-1β are known (see GCID:GC02M112829, HGNC:5992, NCBI Entrez Gene:3553, Ensembl:ENSG00000125538, OMIM®:147720, and UniProtKB / Swiss-Prot:P01584). In some embodiments, the antibodies provided herein bind to human IL-1β. In some embodiments, the anti-IL-1β antibodies provided herein modulate one or more IL-1β activities. In some embodiments, the anti-IL-1β antibodies provided herein are antagonistic antibodies.

[0101] In one embodiment, the antibody according to the present disclosure is an IL-1β antagonist. In another embodiment, the antibody or functional fragment comprising the antigen-binding portion binds to the target protein IL-1β and reduces the binding of IL-1β to the interleukin type 1 receptor (IL-1RI) to basal levels. In one aspect of this embodiment, the antibody or functional fragment reduces the amount of IL-1β that binds to IL-1RI. In a further aspect of this embodiment, the antibody or functional fragment completely prevents IL-1β from binding to IL-1RI. In a further embodiment, the antibody or functional fragment inhibits IL-1 signaling activation. It is understood that an antibody that inhibits one or more of these IL-1β functional properties (e.g., biochemical activity, immunochemical activity, cellular activity, physiological activity, or other biological activity, etc.) as determined according to methodologies known in the art and described herein, is associated with a statistically significant decrease in the particular activity compared to the activity seen in the absence of the antibody (e.g., or in the presence of a control antibody of irrelevant specificity). In some embodiments, antibodies that inhibit IL-1β activity result in a statistically significant decrease in a measured parameter of at least 10%, at least 50%, 80%, or 90%, and in certain embodiments, antibodies of the disclosure may inhibit greater than 95%, 98%, or 99% of IL-1β functional activity.

[0102] In some embodiments, the anti-IL-1β antibodies provided herein have a concentration of ≦1 μM, ≦200 nM, ≦100 nM, ≦50 nM, ≦20 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.05 nM, ≦0.02 nM, ≦0.01 nM, ≦0.001 nM (e.g., 20 pM, 22 pM, 64 pM, 70 pM, 180 pM, or 1.7 nM, e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 M, for example, 10 -10 M~10 -13It binds to IL-1β (e.g., human IL-1β) with a dissociation constant (KD) of 1 M. Various methods of measuring binding affinity are known in the art and for purposes of the present disclosure, any can be used including, for example, by RIA (Chen et al., 1999, J. Mol Biol 293:865-81) performed using a Fab version of the antibody of interest and its antigen, or by biolayer interferometry (BLI), or surface plasmon resonance (SPR) assays, e.g., by Octet®, using the Octet® Red96 system, or by Biacore®, e.g., using a Biacore® TM-2000 or Biacore® TM-3000. Additionally, the "on rate" or "rate of association" or "association rate" or "kon" can be measured by the same techniques, biolayer interferometry (BLI) or surface plasmon resonance (SPR), described above, using, for example, an Octet® Red96, Biacore® TM-2000, or Biacore® TM-3000 system.

[0103] In some embodiments, the anti-IL-1β antibodies provided herein are those described in Section 7 below. Thus, in some embodiments, the antibodies provided herein comprise one or more CDR sequences of any one of SEQ ID NOs: 13-102. The sequences of the CDRs can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to the IMGT numbering. In some embodiments, the CDRs are according to the Kabat numbering. In some embodiments, the CDRs are according to the AbM numbering. In other embodiments, the CDRs are according to the Chothia numbering. In other embodiments, the CDRs are according to the Contact numbering. In some embodiments, the anti-IL-1β antibodies are humanized. In some embodiments, the anti-IL-1β antibodies comprise an acceptor human framework, e.g., a human immunoglobulin framework, or a human consensus framework.

[0104] In some embodiments, the anti-IL-1β antibodies provided herein comprise HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO:7. In some embodiments, the anti-IL-1β antibodies provided herein comprise HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO:9. In some embodiments, the anti-IL-1β antibodies provided herein comprise HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO:11. The CDR sequences can be determined according to any well-known numbering system, or combination thereof. In some embodiments, the CDRs are according to the IMGT numbering. In some embodiments, the CDRs are according to the Kabat numbering. In some embodiments, the CDRs are according to the AbM numbering. In other embodiments, the CDRs are according to the Chothia numbering. In other embodiments, the CDRs are according to the Contact numbering.

[0105] In some embodiments, the anti-IL-1β antibodies provided herein comprise LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 8. In some embodiments, the anti-IL-1β antibodies provided herein comprise LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 10. In some embodiments, the anti-IL-1β antibodies provided herein comprise LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 12. The CDR sequences can be determined according to any well-known numbering system, or combination thereof. In some embodiments, the CDRs are according to the IMGT numbering. In some embodiments, the CDRs are according to the Kabat numbering. In some embodiments, the CDRs are according to the AbM numbering. In other embodiments, the CDRs are according to the Chothia numbering. In other embodiments, the CDRs are according to the Contact numbering.

[0106] In some embodiments, the antibodies or antigen-binding fragments provided herein comprise HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO:7, and LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NO:8. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO:9, and LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NO:10. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO:11, and LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NO:12. The CDR sequences can be determined according to any well-known numbering system, or combination thereof. In some embodiments, the CDRs are according to the IMGT numbering. In some embodiments, the CDRs are according to the Kabat numbering. In some embodiments, the CDRs are according to the AbM numbering. In other embodiments, the CDRs are according to the Chothia numbering. In other embodiments, the CDRs are according to the Contact numbering.

[0107] In another embodiment, an antibody that binds IL-1β, comprising: (i) an HCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, and 97; and (ii) an HCDR2 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, and 98. (iii) an HCDR2 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93 and 99; (iv) an LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOs: 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94 and 100; (v) an LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOs: 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95 and 101; and / or (vi) an LCDR2 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, and 102.Provided herein are antibodies comprising an LCDR3 comprising the amino acid sequence: In some embodiments, the anti-IL-1β antibody is humanized. In some embodiments, the anti-IL-1β antibody comprises an acceptor human framework, such as a human immunoglobulin framework, or a human consensus framework.

[0108] In some particular embodiments, in the antibodies or antigen-binding fragments provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 13, HCDR2 comprises the amino acid sequence of SEQ ID NO: 14, HCDR3 comprises the amino acid sequence of SEQ ID NO: 15, LCDR1 comprises the amino acid sequence of SEQ ID NO: 16, LCDR2 comprises the amino acid sequence of SEQ ID NO: 17, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 18. In some particular embodiments, in the antibodies or antigen-binding fragments provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 31, HCDR2 comprises the amino acid sequence of SEQ ID NO: 32, HCDR3 comprises the amino acid sequence of SEQ ID NO: 33, LCDR1 comprises the amino acid sequence of SEQ ID NO: 34, LCDR2 comprises the amino acid sequence of SEQ ID NO: 35, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 36. In some particular embodiments, in the antibodies or antigen-binding fragments provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 49, HCDR2 comprises the amino acid sequence of SEQ ID NO: 50, HCDR3 comprises the amino acid sequence of SEQ ID NO: 51, LCDR1 comprises the amino acid sequence of SEQ ID NO: 52, LCDR2 comprises the amino acid sequence of SEQ ID NO: 53, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 54. In some particular embodiments, in the antibodies or antigen-binding fragments provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 67, HCDR2 comprises the amino acid sequence of SEQ ID NO: 68, HCDR3 comprises the amino acid sequence of SEQ ID NO: 69, LCDR1 comprises the amino acid sequence of SEQ ID NO: 70, LCDR2 comprises the amino acid sequence of SEQ ID NO: 71, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 72. In some specific embodiments, in the antibodies or antigen-binding fragments provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 85, HCDR2 comprises the amino acid sequence of SEQ ID NO: 86, HCDR3 comprises the amino acid sequence of SEQ ID NO: 87, LCDR1 comprises the amino acid sequence of SEQ ID NO: 88, LCDR2 comprises the amino acid sequence of SEQ ID NO: 89, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 90.

[0109] In some particular embodiments, in the antibodies or antigen-binding fragments provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 19, HCDR2 comprises the amino acid sequence of SEQ ID NO: 20, HCDR3 comprises the amino acid sequence of SEQ ID NO: 21, LCDR1 comprises the amino acid sequence of SEQ ID NO: 22, LCDR2 comprises the amino acid sequence of SEQ ID NO: 23, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 24. In some particular embodiments, in the antibodies or antigen-binding fragments provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 37, HCDR2 comprises the amino acid sequence of SEQ ID NO: 38, HCDR3 comprises the amino acid sequence of SEQ ID NO: 39, LCDR1 comprises the amino acid sequence of SEQ ID NO: 40, LCDR2 comprises the amino acid sequence of SEQ ID NO: 41, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 42. In some particular embodiments, in the antibodies or antigen-binding fragments provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 55, HCDR2 comprises the amino acid sequence of SEQ ID NO: 56, HCDR3 comprises the amino acid sequence of SEQ ID NO: 57, LCDR1 comprises the amino acid sequence of SEQ ID NO: 58, LCDR2 comprises the amino acid sequence of SEQ ID NO: 59, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 60. In some particular embodiments, in the antibodies or antigen-binding fragments provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 73, HCDR2 comprises the amino acid sequence of SEQ ID NO: 74, HCDR3 comprises the amino acid sequence of SEQ ID NO: 75, LCDR1 comprises the amino acid sequence of SEQ ID NO: 76, LCDR2 comprises the amino acid sequence of SEQ ID NO: 77, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 78. In some specific embodiments, in the antibodies or antigen-binding fragments provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 91, HCDR2 comprises the amino acid sequence of SEQ ID NO: 92, HCDR3 comprises the amino acid sequence of SEQ ID NO: 93, LCDR1 comprises the amino acid sequence of SEQ ID NO: 94, LCDR2 comprises the amino acid sequence of SEQ ID NO: 95, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 96.

[0110] In some specific embodiments, in the antibodies or antigen-binding fragments provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 25, HCDR2 comprises the amino acid sequence of SEQ ID NO: 26, HCDR3 comprises the amino acid sequence of SEQ ID NO: 27, LCDR1 comprises the amino acid sequence of SEQ ID NO: 28, LCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 30. In some specific embodiments, in the antibodies or antigen-binding fragments provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 43, HCDR2 comprises the amino acid sequence of SEQ ID NO: 44, HCDR3 comprises the amino acid sequence of SEQ ID NO: 45, LCDR1 comprises the amino acid sequence of SEQ ID NO: 46, LCDR2 comprises the amino acid sequence of SEQ ID NO: 47, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 48. In some particular embodiments, in the antibodies or antigen-binding fragments provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 61, HCDR2 comprises the amino acid sequence of SEQ ID NO: 62, HCDR3 comprises the amino acid sequence of SEQ ID NO: 63, LCDR1 comprises the amino acid sequence of SEQ ID NO: 64, LCDR2 comprises the amino acid sequence of SEQ ID NO: 65, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 66. In some particular embodiments, in the antibodies or antigen-binding fragments provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 79, HCDR2 comprises the amino acid sequence of SEQ ID NO: 80, HCDR3 comprises the amino acid sequence of SEQ ID NO: 81, LCDR1 comprises the amino acid sequence of SEQ ID NO: 82, LCDR2 comprises the amino acid sequence of SEQ ID NO: 83, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 84. In some specific embodiments, in the antibodies or antigen-binding fragments provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 97, HCDR2 comprises the amino acid sequence of SEQ ID NO: 98, HCDR3 comprises the amino acid sequence of SEQ ID NO: 99, LCDR1 comprises the amino acid sequence of SEQ ID NO: 100, LCDR2 comprises the amino acid sequence of SEQ ID NO: 101, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 102.

[0111] In some embodiments, the antibody further comprises one or more framework regions of SEQ ID NOs: 13-102. In some embodiments, the antibody provided herein is a humanized antibody. The framework regions described herein are determined based on the boundaries of the CDR numbering system. In other words, when the CDRs are determined by, for example, Kabat, IMGT, or Chothia, the framework regions are the amino acid residues surrounding the CDRs of the variable region from the N-terminus to the C-terminus, i.e., in the format FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the amino acid residues N-terminal to the CDR1 amino acid residues, e.g., as defined by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; FR2 is defined as the amino acid residues between the CDR1 and CDR2 amino acid residues, e.g., as defined by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; FR3 is defined as the amino acid residues between the CDR2 and CDR3 amino acid residues, e.g., as defined by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; and FR4 is defined as the amino acid residues C-terminal to the CDR3 amino acid residues, e.g., as defined by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system.

[0112] In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH comprising the amino acid sequence of SEQ ID NO: 7, and a VL comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH comprising the amino acid sequence of SEQ ID NO: 9, and a VL comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH comprising the amino acid sequence of SEQ ID NO: 11, and a VL comprising the amino acid sequence of SEQ ID NO: 12.

[0113] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise an amino acid sequence that has a certain percentage of identity compared to any of the antibodies provided herein, such as those described in Section 6 below.

[0114] The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm that can be used to compare two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264 2268 (1990), modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873 5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215:403 (1990). BLAST nucleotide searches can be performed with the parameters of the NBLAST nucleotide program set to, for example, score=100 and word length=12 to obtain nucleotide sequences that are homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program parameters set, for example, to score 50 and word length = 3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described by Altschul et al. (Nucleic Acids Res. 25:3389 3402 (1997). Alternatively, PSI BLAST can be used to perform an iterated search that detects distant relationships between molecules (ibid.). When using BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm used to compare sequences is the algorithm of Myers and Miller, CABIOS 4:11-17 (1998).Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating the percent identity, typically only exact matches are counted.

[0115] In some embodiments, the antibodies provided herein contain substitutions (e.g., conservative substitutions), insertions, or deletions compared to a reference sequence, but an anti-IL-1β antibody comprising the sequence retains the ability to bind to IL-1β. In some embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted compared to the reference amino acid sequence. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., within the FRs). In some embodiments, the anti-IL-1β antibodies provided herein include post-translational modifications of the reference sequence.

[0116] In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:7, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:8. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:9, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:10.In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 11, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 12. In all of the above embodiments, the antibodies bind IL-1β.

[0117] In some embodiments, functional epitopes can be mapped, for example by combinatorial alanine scanning, to identify amino acids in the IL-1β protein required for interaction with the anti-IL-1β antibodies provided herein. In some aspects, the three-dimensional and crystal structures of the anti-IL-1β antibodies bound to IL-1β can be used to identify the epitopes. In some embodiments, the present disclosure provides antibodies that specifically bind to the same epitope as any of the anti-IL-1β antibodies provided herein. For example, in some embodiments, the antibodies or antigen-binding fragments provided herein bind to the same epitope as an anti-IL-1β antibody comprising a VH comprising the amino acid sequence of SEQ ID NO:7 and a VL comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, the antibodies or antigen-binding fragments provided herein bind to the same epitope as an anti-IL-1β antibody comprising a VH comprising the amino acid sequence of SEQ ID NO:9 and a VL comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, the antibody or antigen-binding fragment provided herein binds to the same epitope as an anti-IL-1β antibody comprising a VH comprising the amino acid sequence of SEQ ID NO:11, and a VL comprising the amino acid sequence of SEQ ID NO:12.

[0118] In some embodiments, provided herein is an anti-IL-1β antibody or antigen-binding fragment thereof that specifically binds to IL-1β competitively with any one of the anti-IL-1β antibodies described herein. In some embodiments, the antibody or antigen-binding fragment provided herein specifically binds to IL-1β competitively with an anti-IL-1β antibody comprising a VH comprising the amino acid sequence of SEQ ID NO:7 and a VL comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, the antibody or antigen-binding fragment provided herein specifically binds to IL-1β competitively with an anti-IL-1β antibody comprising a VH comprising the amino acid sequence of SEQ ID NO:9 and a VL comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, the antibody or antigen-binding fragment provided herein specifically binds to IL-1β competitively with an anti-IL-1β antibody comprising a VH comprising the amino acid sequence of SEQ ID NO:11 and a VL comprising the amino acid sequence of SEQ ID NO:12.

[0119] In some embodiments, provided herein is an IL-1β binding protein comprising any one of the anti-IL-1β antibodies described above. In some embodiments, the IL-1β binding protein is a monoclonal antibody, including a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the anti-IL-1β antibody is an antibody fragment, e.g., an scFv. In some embodiments, the IL-1β binding protein is a fusion protein comprising an anti-IL-1β antibody provided herein. In other embodiments, the IL-1β binding protein is a multispecific antibody comprising an anti-IL-1β antibody provided herein. Other exemplary IL-1β binding molecules are described in more detail in the following sections.

[0120] In some embodiments, an anti-IL-1β antibody or antigen binding protein according to any of the above embodiments may incorporate any of the features described in Sections 5.2.2-5.2.7 below, either alone or in combination.

[0121] 5.2.2. Antibody fragments As used herein, the term "antibody" also includes various antibody fragments thereof. Antibodies provided herein include, but are not limited to, immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. Immunoglobulin molecules provided herein can be any class of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, and IgA), or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). In some embodiments, the antibody is an IgG antibody. In some embodiments, the IgG antibody is an IgG1 antibody. In some embodiments, the IgG antibody is an IgG2, IgG3, or IgG4 antibody.

[0122] Antibody variants and derivatives include antibody functional fragments that retain the ability to bind antigen. Exemplary functional fragments include Fab fragments (e.g., an antibody fragment containing an antigen-binding domain and comprising a light chain and a portion of a heavy chain bridged by a disulfide bond), Fab' (e.g., an antibody fragment containing a single antigen-binding domain comprising a Fab and an additional portion of a heavy chain up to the hinge region), F(ab')2 (e.g., two Fab' molecules linked by an interchain disulfide bond at the hinge region of the heavy chain, which Fab' molecules can be directed against the same or different epitopes), bispecific Fab (e.g., two Fab molecules having two antigen-binding domains, each of which may be directed against a different epitope), single chains containing variable regions also known as scFvs (e.g. the variable antigen-binding determining regions of a single light and heavy chain of an antibody bound together by a chain of 10-25 amino acids), disulfide-linked Fvs, or dsFvs (e.g. the variable antigen-binding determining regions of a single light and heavy chain of an antibody bound together by disulfide bonds), camelized VHs (e.g. some amino acids in the VH interface are not found in the heavy chain of a native camel antibody), and a single heavy chain variable antigen-binding determining region of an antibody, which is a single heavy chain variable antigen-binding determining region of an antibody, a bispecific scFv (e.g., an scFv or dsFv molecule having two antigen-binding domains, each of which can be directed to a different epitope), a diabody (e.g., a dimerized scFv formed when the VH domain of a first scFv associates with the VL domain of a second scFv and the VL domain of the first scFv associates with the VH domain of the second scFv, wherein the two antigen-binding regions of the diabody can be directed to the same or different epitopes). Examples of such bodies include scFvs, which are formed in a similar manner to diabodies, but where three antigen binding domains are generated in a single complex, and where the three antigen binding domains can be directed against the same or different epitopes), triabodies (e.g., trimerizing scFvs, which are formed in a similar manner to diabodies, but where four antigen binding domains are generated in a single complex, and where the four antigen binding domains can be directed against the same or different epitopes), and tetrabodies (e.g., tetramerizing scFvs, which are formed in a similar manner to diabodies, but where four antigen binding domains are generated in a single complex, and where the four antigen binding domains can be directed against the same or different epitopes).

[0123] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., 1992, J. Biochem. Biophys. Methods 24:107-17, and Brennan et al., 1985, Science 229:81-83). However, these fragments can now be produced directly by recombinant host cells. For example, Fab, Fv, and ScFv antibody fragments can all be expressed and secreted in E. coli or yeast cells, allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries described above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., 1992, Bio / Technology 10:163-67). By another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Fab and F(ab')2 fragments with increased in vivo half-life containing salvage receptor binding epitope residues are described, for example, in U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. In certain embodiments, the antibody is a single chain Fv fragment (scFv) (see, for example, WO 93 / 16185, U.S. Pat. Nos. 5,571,894, and 5,587,458). Fvs and scFvs have intact binding sites that are devoid of constant regions, and therefore, they may be suitable for reduced non-specific binding during in vivo use. scFv fusion proteins can be constructed to provide fusion of an effector protein at either the amino or carboxy terminus of the scFv (see, for example, Borrebaeck ed., supra). An antibody fragment may also be a "linear antibody" as described, for example, in the references cited above. Such linear antibodies may be monospecific or multispecific, such as bispecific.

[0124] Humanized Antibodies The antibodies described herein include humanized antibodies. Humanized antibodies, such as the humanized antibodies disclosed herein, can be prepared by any of a variety of techniques, including CDR grafting (EP 239,400, WO 91 / 09967, U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP 592,106, 519,596, Padlan, Molecular Immunology 28(4 / 5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814(1994); and Roguska et al., 1994, PNAS 91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and, for example, U.S. Pat. al.,Protein Eng.13(5):353-60(2000),Morea et al.,Methods 20(3):267 79(2000),Baca et al.,J.Biol.Chem.272(16):10678-84(1997),Roguska et al.,Protein Eng.9(10):895 904(1996),Couto et al.,Cancer Res. 55(23 Supp):5973s-5977s (1995), Couto et al., Cancer Res. 55(8):1717-22 (1995), Sandhu JS, Gene 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol. 235(3):959-73 (1994). See also U.S. Patent Application Publication No. 2005 / 0042664(A1) (February 24, 2005), each of which is incorporated herein by reference in its entirety.

[0125] In some embodiments, the antibodies provided herein can be humanized antibodies that bind IL-1β, including human IL-1β. For example, the humanized antibodies of the present disclosure can include one or more CDRs set forth in SEQ ID NOs: 13-102. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues and are typically taken from an "import" variable domain. Humanization can be performed, for example, by substituting hypervariable region sequences for the corresponding sequences of a human antibody according to the methods of Jones et al., Nature 321:522-25 (1986), Riechmann et al., Nature 332:323-27 (1988), and Verhoeyen et al., Science 239:1534-36 (1988). In a specific embodiment, humanization of the antibodies provided herein is performed as described in Section 6 below.

[0126] In some cases, humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the CDRs of a parent non-human antibody are grafted onto a human antibody framework. For example, Padlan et al. determined that only about one-third of the residues in the CDRs actually contact the antigen, and called these "specificity determining residues" or SDRs (Padlan et al., FASEB J. 9:133-39 (1995)). In the technique of SDR grafting, only the SDR residues are grafted onto a human antibody framework (see, e.g., Kashmiri et al., Methods 36:25-34 (2005)).

[0127] The choice of human variable domains used in making humanized antibodies can be important to reduce antigenicity. For example, according to the so-called "best-fit" method, the sequence of the variable domain of a non-human antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to the human sequence of the non-human antibody can be selected as the human framework of the humanized antibody (Sims et al., J. Immunol. 151:2296-308 (1993), and Chothia et al., J. Mol. Biol. 196:901-17 (1987)). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-89 (1992); and Presta et al., J. Immunol. 151:2623-32 (1993)). In some cases, the framework is selected from the most abundant human subclass, V L 6 Subgroup I(V L 6I), and VH subgroup III (V H III) are derived from the consensus sequence. Alternatively, human germline genes are used as the source of the framework regions.

[0128] In an alternative paradigm based on CDR comparison, called superhumanization, the homology of FRs is irrelevant. This method consists of comparing non-human sequences and functional human germline gene repertoires. Then, genes encoding the same or closely related canonical structures as the mouse sequences are selected. Next, among the genes sharing the canonical structures with non-human antibodies, the genes with the highest homology within the CDRs are selected as FR donors. Finally, non-human CDRs are grafted onto these FRs (see, for example, Tan et al., J. Immunol. 169:1119-25 (2002)).

[0129] Furthermore, it is generally desirable for antibodies to be humanized with retention of affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformations for selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, Protein Eng. 13:819-24 (2002)), Modeller (Sali and Blundell, J. Mol. Biol. 234:779-815 (1993)), and Swiss PDB Viewer (Guex and Peitsch, Electrophoresis 18:2714-23 (1997)). Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, for example, the analysis of residues that influence the ability of the candidate immunoglobulin to bind an antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen, is achieved. In general, the hypervariable region residues are directly and most substantially involved in antigen binding.

[0130] Another method of antibody humanization is based on a metric of antibody humanness called Human String Content (HSC). This method compares the mouse sequence with a repertoire of human germline genes and scores the differences as HSC. The target sequence is then humanized by maximizing its HSC, rather than using a global identity measure to generate diverse humanized variants (Lazar et al., Mol. Immunol. 44:1986-98 (2007)).

[0131] In addition to the above methods, empirical methods can be used to generate and select humanized antibodies. These methods include those based on generating large libraries of humanized variants and selecting the best clones using enrichment or high-throughput screening techniques. Antibody variants can be isolated from phage, ribosomal, and yeast display libraries, as well as by bacterial colony screening (see, e.g., Hoogenboom, Nat. Biotechnol. 23:1105-16 (2005); Dufner et al., Trends Biotechnol. 24:523-29 (2006); Feldhaus et al., Nat. Biotechnol. 21:163-70 (2003); and Schlapschy et al., Protein Eng. Des. Sel. 17:847-60 (2004)).

[0132] In the FR library approach, multiple residue variants are introduced at specific positions in the FR, and the library is then screened to select the FR that best supports the grafted CDR. The substituted residues may include some or all of the "vernier" residues identified as potentially contributing to the CDR structure (see, e.g., Foote and Winter, J. Mol. Biol. 224:487-99 (1992)), or from a more limited set of target residues identified by Baca et al. J. Biol. Chem. 272:10678-84 (1997).

[0133] In FR shuffling, entire FRs are combined with non-human CDRs rather than creating a combinatorial library of selected residue variants (see, e.g., Dall'Acqua et al., Methods 36:43-60 (2005)). A one-step FR shuffling process can be used. Such a process has been shown to be efficient because the resulting antibodies exhibit improved biochemical and physicochemical properties, including enhanced expression, increased affinity, and thermal stability (see, e.g., Damschroder et al., Mol. Immunol. 44:3049-60 (2007)).

[0134] The "humaneering" method is based on the experimental identification of essential minimum specificity determinants (MSDs) and the sequential substitution of non-human fragments into a human FR library and evaluation of binding. This approach typically identifies antibodies of multiple subclasses that retain the epitope and have distinct CDRs in the human V segments.

[0135] "Human engineering" methods involve modifying non-human antibodies or antibody fragments by making specific changes to the amino acid sequence of the antibody to produce modified antibodies that have reduced immunogenicity in humans, yet retain the desired binding properties of the original non-human antibody. In general, this technique involves classifying amino acid residues of the non-human antibody as "low risk", "medium risk", or "high risk" residues. The classification is performed using a global risk / benefit calculation that evaluates the predicted benefit of making a particular substitution (e.g., for immunogenicity in humans) against the risk that the substitution will affect the folding of the resulting antibody. Specific human amino acid residues (e.g., low or medium risk) to be substituted at a given position of the non-human antibody sequence can be selected by aligning amino acid sequences from the variable regions of the non-human antibody with the corresponding regions of specific or consensus human antibody sequences. Amino acid residues at low or medium risk positions in the non-human sequence can be substituted with the corresponding residues in the human antibody sequence depending on the alignment. Techniques for making human engineered proteins are described in detail in Studnicka et al., Protein Engineering 7:805-14 (1994), U.S. Patent Nos. 5,766,886, 5,770,196, 5,821,123, and 5,869,619, and WO 93 / 11794.

[0136] Composite human antibodies can be generated, for example, using Composite Human Antibody™ technology (Antitope Ltd., Cambridge, United Kingdom). To generate composite human antibodies, fragments of multiple human antibody variable region sequences are engineered in such a way that they avoid T-cell epitopes, thereby minimizing the immunogenicity of the resulting antibody.

[0137] A deimmunized antibody is an antibody from which T cell epitopes have been removed. Methods for making deimmunized antibodies have been described. See, for example, Jones et al., Methods Mol Biol. 525:405-23 (2009), xiv, and De Groot et al., Cell. Immunol. 244:148-153 (2006). Deimmunized antibodies include a T cell epitope-depleted variable region and a human constant region. Briefly, the variable region of an antibody is cloned, and then T cell epitopes are identified by testing overlapping peptides derived from the variable region of the antibody in a T cell proliferation assay. The T cell epitopes are identified by an in silico method to identify peptides that bind to human MHC class II. Mutations are introduced into the variable region to abolish binding to human MHC class II. The mutated variable region is then utilized to generate the deimmunized antibody.

[0138] 5.2.4. Antibody Variants In some embodiments, amino acid sequence modifications of the antibodies that bind IL-1β described herein are contemplated. For example, it may be desirable to optimize the binding affinity and / or other biological properties of the antibody, including but not limited to specificity, thermostability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility. Thus, in addition to the antibodies that bind IL-1β described herein, it is contemplated that mutants of the antibodies that bind IL-1β described herein can be prepared. For example, antibody mutants can be prepared by introducing appropriate nucleotide changes into the encoding DNA and / or by synthesizing the desired antibody or polypeptide. Those skilled in the art who understand that amino acids are changed can modify post-translational processes of the antibody.

[0139] chemical modification In some embodiments, the antibodies provided herein are chemically modified, for example, by covalently attaching any type of molecule to the antibody. Antibody derivatives may include antibodies that have been chemically modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, binding to cellular ligands or other proteins, or conjugation to one or more immunoglobulin domains (e.g., Fc or portions of Fc). Any of a number of chemical modifications may be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, and the like. Additionally, the antibody may include one or more non-classical amino acids.

[0140] In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may conveniently be accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0141] When the antibodies provided herein are fused to an Fc region, the carbohydrate attached thereto may be altered. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally attached by N-linkage to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides may include various carbohydrates, such as 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 binding molecules provided herein may be made to generate variants with specific improved properties.

[0142] In other embodiments, when an antibody provided herein is fused to an Fc region, the antibody variants provided herein can have a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody can be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at 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 about position 297 (EU numbering of Fc region residues) of the Fc region. However, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Application Publication Nos. 2003 / 0157108 and 2004 / 0093621. Exemplary publications relating to "defucosylated" or "fucose-deficient" antibody variants include U.S. Patent Application Publication Nos. 2003 / 0157108, WO 2000 / 61739, 2001 / 29246, U.S. Patent Application Publication Nos. 2003 / 0115614, 2002 / 0164328, 2004 / 0093621, 2004 / 0093622, and 2004 / 0093623. No. 132140, 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 See 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; and, for example, α-1,6-fucosyltransferase gene, FUT8 knockout CHO cells (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107).

[0143] The binding molecules, including antibodies, provided herein further provide bisected oligosaccharides, e.g., where the biantennary oligosaccharide attached to the Fc region is bisected by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function. Examples of such variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.), U.S. Pat. No. 6,602,684 (Umana et al.), and U.S. Pat. App. Pub. No. 2005 / 0123546 (Umana et al.). Variants are also provided that have at least one galactose residue in the oligosaccharide attached to the Fc region. Such variants may have improved CDC function. Such variants are described, for example, in WO 1997 / 30087, WO 1998 / 58964, and WO 1999 / 22764.

[0144] In the antibodies and molecules comprising an Fc region of the invention, one or more amino acid modifications may be introduced into the Fc region, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid alteration (e.g., substitution) at one or more amino acid positions.

[0145] In certain embodiments, the invention contemplates antibody variants that possess some, but not all, effector functions that make them desirable candidates for applications in which the half-life of the binding molecule in vivo is important, but in which certain effector functions (such as complement and ADCC) are unnecessary or deleterious. 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 FγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest include those described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986); and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ non-radioactive cytotoxicity assay for flow cytometry (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 (see, e.g., animal models such as those disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998)). C1q binding assays can also be performed to confirm that the antibody is unable to bind C1q and thus lacks CDC activity. (See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402).To assess complement activation, CDC assays can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life measurements can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0146] Antibodies with reduced effector function include those with substitutions at one or more of 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 the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0147] Certain variants have been described that have 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)).

[0148] In certain embodiments, the variants include an Fc region having one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues). In some embodiments, changes may be 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).

[0149] Binding molecules with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which account 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(A1) (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having one or more substitutions of Fc region residues: 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 of Fc region residue 434 (U.S. Patent No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent Nos. 5,648,260, 5,624,821, and WO 94 / 29351 for other examples of Fc region variants.

[0150] In certain embodiments, it may be desirable to create a cysteine ​​engineered antibody in which one or more residues of an antibody are replaced with a cysteine ​​residue. In certain embodiments, the replaced residues are located at accessible sites of the antibody. As further described herein, by replacing those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker drug moieties, to create an immunoconjugate.

[0151] Substitutions, deletions, or insertions Mutations may be substitutions, deletions, or insertions of one or more codons encoding the antibody or polypeptide, resulting in a change in the amino acid sequence compared to the native sequence antibody or polypeptide. Target sites for substitutional mutagenesis include the CDRs and FRs.

[0152] Amino acid substitutions can be the result of replacing one amino acid with another amino acid with similar structural and / or chemical properties, such as, for example, a conservative amino acid substitution, such as a replacement of a leucine with a serine. Standard techniques known to those of skill in the art can be used to introduce mutations into the nucleotide sequences encoding the molecules provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions. Insertions or deletions can optionally range from about 1 to 5 amino acids. In certain embodiments, the substitutions, deletions, or insertions include fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions compared to the original molecule. In certain embodiments, the substitutions are conservative amino acid substitutions made at one or more predicted non-essential amino acid residues. Permitted variations can be determined by systematically making insertions, deletions, or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the parent antibody.

[0153] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing multiple residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue.

[0154] Antibodies generated by conservative amino acid substitutions are included in the present disclosure. In conservative amino acid substitutions, an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. These families include amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed and the activity of the protein determined. Conservative (e.g., within a group of amino acids with similar properties and / or side chains) substitutions can be made to maintain properties or not significantly change properties. Exemplary substitutions are shown in Table 2 below.

[0155] [Table 2]

[0156] Amino acids can be grouped according to similarities in the properties of their side chains (see, e.g., Lehninger, Biochemistry 73-75 (2d ed. 1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H)). Alternatively, naturally occurring residues may be divided into groups based on 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 influence chain orientation: Gly, Pro, (6) aromatic: Trp, Tyr, Phe. For example, any cysteine ​​residue that is not involved in maintaining the proper conformation of the antibody may be substituted with another amino acid, e.g., alanine or serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Non-conservative substitutions would involve exchanging a member of one of these classes for a member of another class.

[0157] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further study will have a modification (e.g., an improvement) in a particular biological property (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody and / or will have substantially retained a particular biological property of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which can be conveniently generated (e.g., using phage display-based affinity maturation techniques such as those described herein). Briefly, one or more CDR residues are mutated and the mutated antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0158] Changes (e.g., substitutions) can be made in the CDRs, for example to improve antibody affinity. Such changes can be made in CDR "hot spots," i.e., residues encoded by codons that undergo high frequency of mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in the SDRs (a-CDRs), and the resulting mutated antibodies, or fragments thereof, are tested for binding affinity. Affinity maturation by constructing and reselecting from a secondary library 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 created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves a CDR-directed approach, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. A more detailed description of affinity maturation is provided in the sections below.

[0159] In some embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, so long as such changes do not significantly reduce the ability of the antibody to bind antigen. For example, conservative changes that do not significantly reduce binding affinity (e.g., conservative substitutions as provided herein) may be made in a CDR. In some embodiments of the mutated antibodies provided herein, each CDR is either unchanged or contains no more than one, two, or three amino acid substitutions.

[0160] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described in Cunningham and Wells, Science, 244:1081-1085 (1989). In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact and adjacent residues can be targeted or eliminated as candidates for substitution. Mutants can be screened to determine whether they contain the desired properties.

[0161] 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 is 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., ADEPT) or a polypeptide which extends the serum half-life of the antibody.

[0162] Modifications can be made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, PCR mutagenesis, etc. Site-directed mutagenesis (see, e.g., Carter, Biochem J. 237:1-7 (1986); and Zoller et al., Nucl. Acids Res. 10:6487-500 (1982)), cassette mutagenesis (see, e.g., Wells et al., Gene 34:315-23 (1985)), or other known techniques can be performed on the cloned DNA to produce the antibody mutant DNA.

[0163] Fc mutations To facilitate the formation of heterodimers between two heavy chains (e.g., a heavy chain with and without a fusion of an anti-IL-1β antibody or antigen-binding fragment thereof, or a heavy chain with an Fc of an anti-IL-1β arm and a heavy chain with an Fc of a tissue-targeting arm), heterodimer mutations are introduced into the Fc of the two heavy chains. Examples of such Fc mutations include, but are not limited to, Zymework mutations (see, e.g., U.S. Pat. No. 10,457,742) and "knobs-in-holes" mutations (see, e.g., Ridgway et al., Protein Eng., 9(7):617-621, 1996). Other heterodimer mutations can also be used in the present disclosure. In some embodiments, a modified CH3 as described herein is used to facilitate the formation of heterodimers between two heavy chains.

[0164] In certain embodiments, each of the two heavy chains of the antibody comprises one or more heterodimerization mutations or one or more knob and hole mutations. In certain aspects, the one or more heterodimerization mutations are in the CH3 domain.

[0165] In certain embodiments, the Fc region of the antibody or antigen-binding fragment thereof contains a substitution that alters binding of the antibody or antigen-binding fragment thereof to the neonatal Fc receptor (FcRn). In certain embodiments, the Fc region of the antibody or antigen-binding fragment thereof contains a substitution that enhances binding of the antibody or antigen-binding fragment thereof to the neonatal Fc receptor (FcRn). In certain embodiments, the Fc region of the antibody or antigen-binding fragment thereof contains a substitution that enhances binding of the antibody or antigen-binding fragment thereof to the neonatal Fc receptor (FcRn) at a pH of about 6. In certain embodiments, the Fc region of the antibody or antigen-binding fragment thereof contains a substitution that enhances binding of the antibody or antigen-binding fragment thereof to the neonatal Fc receptor (FcRn) at a pH of about 6, thereby enhancing FcRn-mediated endosomal recycling. In certain embodiments, the Fc region of the antibody or antigen-binding fragment thereof contains substitutions that enhance binding of the antibody or antigen-binding fragment thereof to the neonatal Fc receptor (FcRn) at a pH of about 6, thereby resulting in longer serum exposure. In certain embodiments, the Fc region of the antibody or antigen-binding fragment thereof contains substitutions that enhance binding at acidic pH. In certain embodiments, the Fc region of the antibody or antigen-binding fragment thereof has M252Y / S254T / T256E (YTE) mutations, where the numbering of amino acid residues is according to the EU index.

[0166] 5.2.5. In Vitro Affinity Maturation In some embodiments, antibody variants with improved properties, such as affinity, stability, or expression level, compared to the parent antibody can be prepared by in vitro affinity maturation. Similar to natural prototypes, in vitro affinity maturation is based on the principle of mutation and selection. Libraries of antibodies are displayed on the surface of an organism (e.g., phage, bacteria, yeast, or mammalian cells) or in association with the encoding mRNA or DNA (e.g., covalently or non-covalently). Affinity selection of the displayed antibodies allows the isolation of organisms or complexes that carry the genetic information encoding the antibody. After two to three rounds of mutation and selection using display methods such as phage display, antibody fragments with affinities in the low nanomolar range are typically obtained. Affinity matured antibodies can have nanomolar or picomolar affinities for the target antigen.

[0167] Phage display is a widespread method for the display and selection of antibodies. Antibodies are displayed on the surface of Fd or M13 bacteriophage as fusions to bacteriophage coat proteins. Selection involves exposure to antigen to allow the phage-displayed antibodies to bind to the target, a process called "panning." Phage that bind antigen are recovered and used to infect bacteria to generate phage for further rounds of selection. For reviews, see, e.g., Hoogenboom, Methods. Mol. Biol. 178:1-37 (2002), and Bradbury and Marks, J. Immunol. Methods 290:29-49 (2004).

[0168] In yeast display systems (see, e.g., Boder et al., Nat. Biotech. 15:553-57 (1997), and Chao et al., Nat. Protocols 1:755-68 (2006)), antibodies can be fused to the adhesive subunit of the yeast agglutinin protein Aga2p, which binds to the yeast cell wall via a disulfide bond to Aga1p. Display of the protein via Aga2p keeps the protein away from the cell surface, minimizing potential interactions with other molecules on the yeast cell wall. The library is screened using magnetic separation and flow cytometry to select for antibodies with improved affinity or stability. Binding to the soluble antigen of interest is determined by labeling the yeast with biotinylated antigen and a secondary reagent such as streptavidin conjugated to a fluorophore. Variation in surface expression of the antibody can be measured by immunofluorescence labeling of either hemagglutinin or c-Myc epitope tags flanking the single-chain antibody (e.g., scFv). Expression has been shown to correlate with stability of the displayed protein, so antibodies can be selected for improved stability and affinity (see, e.g., Shusta et al., J. Mol. Biol. 292:949-56 (1999)). An added advantage of yeast display is that the displayed protein folds in the endoplasmic reticulum of the eukaryotic yeast cell, taking advantage of endoplasmic reticulum chaperones and quality control machinery. Once maturation is complete, antibody affinity can be conveniently "titrated" while displayed on the yeast surface, obviating the need for expression and purification of each clone. A theoretical limitation of yeast surface display is the potentially small size of functional libraries compared to other display methods. However, recent approaches have utilized yeast cell mating systems to allow for the generation of 10 14 They have generated combinatorial diversity estimated to be of size (see, e.g., U.S. Patent Application Publication No. 2003 / 0186374, and Blaise et al., Gene 342:211-18 (2004)).

[0169] In ribosome display, antibody-ribosome-mRNA (ARM) complexes are generated and selected in a cell-free system. A DNA library encoding a specific antibody library is genetically fused to a spacer sequence lacking a stop codon. This spacer sequence remains attached to the peptidyl-tRNA upon translation and occupies the ribosomal tunnel, allowing the protein of interest to protrude from the ribosome and fold. The resulting mRNA, ribosome, and protein complex binds to a surface-bound ligand, allowing the antibody and its encoding mRNA to be simultaneously isolated by affinity capture with the ligand. The ribosome-bound mRNA can then be reverse transcribed back to cDNA, which can then be subjected to mutagenesis and used for the next round of selection (see, e.g., Fukuda et al., Nucleic Acids Res. 34:e127 (2006)). In mRNA display, puromycin is used as an adapter molecule to form a covalent link between the antibody and the mRNA (see Wilson et al., Proc. Natl. Acad. Sci. USA 98:3750-55 (2001)).

[0170] These methods are performed entirely in vitro and therefore offer two main advantages over other selection techniques: First, the diversity of the library is not limited by the transformation efficiency of bacterial cells, but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, random mutations can be easily introduced after each round of selection, for example by non-proofreading polymerases, since there is no need to transform the library after the diversity step.

[0171] In some embodiments, a mammalian display system can be used.

[0172] Diversity can also be introduced into the CDRs of an antibody library in a targeted manner or through random introduction. The former approach includes sequentially targeting all CDRs of an antibody through high- or low-level mutagenesis, or targeting isolated hotspots of somatic hypermutation (see, e.g., Ho et al., J. Biol. Chem. 280:607-17 (2005)), or residues suspected to affect affinity on experimental or structural grounds. Diversity can also be introduced by replacing naturally diverse regions through DNA shuffling or similar techniques (see, e.g., Lu et al., J. Biol. Chem. 278:43496-507 (2003); U.S. Patent Nos. 5,565,332 and 6,989,250). Alternative techniques target hypervariable loops extending into framework region residues (see, e.g., Bond et al., J. Mol. Biol. 348:699-709 (2005)), utilize loop deletions and insertions in the CDRs, or use hybridization-based diversification (see, e.g., U.S. Patent Publication No. 2004 / 0005709). Additional methods for generating diversity in CDRs are disclosed, for example, in U.S. Patent No. 7,985,840. Additional methods that can be used for the generation of antibody libraries and / or antibody affinity maturation are disclosed in, e.g., U.S. Pat. Nos. 8,685,897 and 8,603,930, and U.S. Patent Application Publication Nos. 2014 / 0170705, 2014 / 0094392, 2012 / 0028301, 2011 / 0183855, and 2009 / 0075378, each of which is incorporated herein by reference.

[0173] Screening of libraries can be accomplished by a variety of techniques known in the art, for example, antibodies can be immobilized on solid supports, columns, pins, or cellulose / poly(vinylidene fluoride) membranes / other filters, expressed in host cells immobilized on adsorption plates or used in cell sorting, or conjugated to biotin for capture on streptavidin-coated beads, or used in any other manner to pan the display library.

[0174] For reviews of in vitro affinity maturation methods, see, e.g., Hoogenboom, Nature Biotechnology 23:1105-16 (2005); Quiroz and Sinclair, Revista Ingeneria Biomedia 4:39-51 (2010), and references therein.

[0175] 5.2.6. Antibody Modifications Covalent modifications of single domain antibodies are included within the scope of this disclosure. Covalent modifications include reacting targeted amino acid residues of the antibody with organic derivatizing agents capable of reacting with selected side chains or the N- or C-terminal residues of the antibody. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine and histidine side chains (see, e.g., Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)), acetylation of the N-terminal amine, and amidation of the C-terminal carboxyl group.

[0176] Other types of covalent modifications of antibodies within the scope of the disclosure include altering the native glycosylation pattern of the antibody or polypeptide, as described above (see, e.g., Beck et al., Curr. Pharm. Biotechnol. 9:482-501 (2008), and Walsh, Drug Discov. Today 15:773-80 (2010)), and linking the antibody to one of a variety of nonproteinaceous polymers, such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, e.g., by methods described in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337. The antibodies that bind IL-1β of the present disclosure may also be genetically fused or conjugated to one or more immunoglobulin constant regions or portions thereof (e.g., Fc) to extend half-life and / or improve known Fc-mediated effector functions.

[0177] The antibodies that bind IL-1β of the present disclosure can also be modified to form chimeric molecules that include the antibody that binds IL-1β fused to another heterologous polypeptide or amino acid sequence, such as an epitope tag (see, e.g., Terpe, Appl. Microbiol. Biotechnol. 60:523-33 (2003)), or the Fc region of an IgG molecule (see, e.g., Aruffo, Antibody Fusion Proteins 221-42 (Chamow and Ashkenazi eds., 1999)).

[0178] Also provided herein are fusion proteins comprising an antibody that binds IL-1β of the present disclosure and a heterologous polypeptide, in some embodiments, the heterologous polypeptide to which the antibody is genetically fused or chemically conjugated is useful for targeting the antibody to cells that have IL-1β expressed on the cell surface.

[0179] Also provided herein are panels of antibodies that bind to the IL-1β antigen. In certain embodiments, the panel of antibodies has different association rates, different dissociation rates, different affinities, and / or different specificities for the IL-1β antigen. In some aspects, the panel comprises or consists of about 10 to about 1000 or more antibodies. The panel of antibodies can be used in assays such as ELISA, for example in 96-well or 384-well plates.

[0180] 5.2.7. Other Binding Molecules, Including Antibodies In another aspect, provided herein are binding molecules comprising the anti-IL-1β antibodies provided herein. In some embodiments, the antibodies against IL-1β provided herein are part of other binding molecules. Exemplary binding molecules of the present disclosure are described herein.

[0181] Fusion proteins In various embodiments, the antibodies provided herein may be genetically fused or chemically conjugated to other agents, such as protein-based entities. The antibodies may be chemically conjugated to the agents or otherwise non-covalently conjugated to the agents. The agents may be peptides or antibodies (or fragments thereof).

[0182] Thus, in some embodiments, provided herein are antibodies that are recombinantly fused or chemically conjugated (covalently or non-covalently conjugated) to a heterologous protein or polypeptide (or a fragment thereof, e.g., about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500 amino acids, or more than 500 amino acids) to generate a fusion protein, and uses thereof. In particular, provided herein are fusion proteins comprising an antigen-binding fragment (e.g., CDR1, CDR2, and / or CDR3) of an antibody provided herein and a heterologous protein, polypeptide, or peptide.

[0183] Additionally, the antibodies provided herein can be fused to marker or "tag" sequences, such as peptides, to facilitate purification. In certain embodiments, the marker or tag amino acid sequences are a hexahistidine peptide, a hemagglutinin ("HA") tag, and a "FLAG" tag.

[0184] Methods of fusing or conjugating moieties, including polypeptides, to antibodies are known (see, e.g., Arnon et al., Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy, in Monoclonal Antibodies and Cancer Therapy 243-56 (Reisfeld et al. eds., 1985); Hellstrom et al., Antibodies for Drug Delivery, in Controlled Drug Delivery 623-53 (Robinson et al. eds., 2d ed. 1987); Thorpe, Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review, in Monoclonal Antibodies: Biological and Clinical Applications 475-506 (Pinchera et al. eds., 1985); Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy, in Monoclonal Antibodies for Cancer Detection and Therapy 2001 eds., 1987). 303-16 (Baldwin et al. eds., 1985); Thorpe et al. al., Immunol. Rev. 62:119-58 (1982), U.S. Patent No. 5,336,603, U.S. Patent No. 5,622,929, U.S. Patent No. 5,359, No. 046, No. 5,349,053, No. 5,447,851, No. 5,723,125, No. 5,783,181, No. 5,908,626 , European Patent No. 5,844,095, and European Patent No. 5,112,946, European Patent No. 307,434, European Patent No. 367,166, European Patent No. 394,827, International Publication Nos. 91 / 06570, 96 / 04388, 96 / 22024, 97 / 34631 and 99 / 04813, Ashkenazi et al.,Proc.Natl.Acad.Sci.USA, 88:10535-39 (1991); Traunecker et al., Nature, 331:84-86 (1988); Zheng et al., J. Immunol. 154:5590-600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-41 (1992).

[0185] Fusion proteins can be generated, for example, by techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to alter the activities of the antibodies provided herein, including, for example, antibodies with higher affinities and lower dissociation rates (see, e.g., U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998)). Antibodies, or the encoded antibodies, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. Polynucleotides encoding the antibodies provided herein may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc., of one or more heterologous molecules.

[0186] In some embodiments, an antibody provided herein is conjugated to a second antibody to form an antibody heteroconjugate.

[0187] In various embodiments, the antibody is genetically fused to the agent. Genetic fusion can be achieved by placing a linker (e.g., a polypeptide) between the antibody and the agent. The linker can be a flexible linker.

[0188] In various embodiments, the antibody is genetically conjugated to the therapeutic molecule, and the hinge region connects the antibody to the therapeutic molecule.

[0189] Also provided herein are methods for producing the various fusion proteins provided herein. The various methods described in Section 5.4 can also be utilized to produce the fusion proteins provided herein.

[0190] In a specific embodiment, the fusion proteins provided herein are recombinantly expressed. Recombinant expression of the fusion proteins provided herein may require the construction of an expression vector comprising a polynucleotide encoding the protein or a fragment thereof. Once a polynucleotide encoding the protein or a fragment thereof provided herein is obtained, a vector for producing the molecule can be produced by recombinant DNA technology using techniques well known in the art. Thus, described herein are methods for expressing a polynucleotide comprising a coding nucleotide sequence to prepare a protein. Methods well known to those skilled in the art can be used to construct an expression vector comprising a coding sequence and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA technology, synthetic technology, and in vivo genetic recombination. Also provided are replicable vectors comprising a nucleotide sequence encoding the fusion proteins provided herein, or fragments thereof, or CDRs, operably linked to a promoter.

[0191] The expression vector can be transfected into a host cell by conventional techniques and the transfected cells can then be cultured by conventional techniques to produce the fusion proteins provided herein. Accordingly, also provided herein is a host cell comprising a polynucleotide encoding a fusion protein provided herein, or a fragment thereof, operably linked to a heterologous promoter.

[0192] A variety of host-expression vector systems can be utilized to express the fusion proteins provided herein. Such host expression systems represent vehicles in which a coding sequence of interest may be produced and subsequently purified, but also cells which, when transformed or transfected with the appropriate nucleotide coding sequence, may express in situ the antibody molecules provided herein. These host expression systems include microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors comprising the antibody coding sequence, yeast (e.g., Saccharomyces cerevisiae) transformed with recombinant yeast expression vectors containing the antibody coding sequence, and yeast (e.g., Saccharomyces cerevisiae) transformed with recombinant yeast expression vectors containing the antibody coding sequence. Examples of suitable expression vectors include, but are not limited to, insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the coding sequence, plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody coding sequence, or mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) harboring recombinant expression constructs containing promoters from the genome of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter). In particular for expression of whole recombinant antibody molecules, bacterial cells such as E. coli, or eukaryotic cells can be used for expression of recombinant fusion proteins. For example, mammalian cells such as Chinese hamster ovary cells (CHO) provide an effective expression system for antibodies or variants thereof when conjugated with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus. In specific embodiments, expression of a nucleotide sequence encoding a fusion protein provided herein is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0193] In bacterial systems, many expression vectors can be advantageously selected depending on the intended use of the fusion protein to be expressed. For example, when producing large quantities of such fusion proteins, a vector that directs the expression of a high level of fusion protein product that is easily purified to produce a pharmaceutical composition of the fusion protein may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO 12:1791 (1983)), pIN vector (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)), and the like, in which the coding sequence can be ligated into the vector in frame with the lac Z coding region separately to produce the fusion protein. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0194] In mammalian host cells, many virus-based expression systems can be utilized. When adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated with the adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome (e.g., regions El or E3) can result in recombinant viruses that are viable and capable of expressing the fusion protein in infected hosts (see, e.g., Logan & Shenk, Proc. Natl. Acad. Sci. USA 8 1:355-359 (1984)). Specific initiation signals may also be required for efficient translation of the inserted coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., Methods in Enzymol. 153:51-544 (1987)).

[0195] Additionally, a host cell line can be selected which modulates expression of the inserted sequences or modifies and processes the gene product in the specific manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. By selecting the appropriate cell line or host system, the expressed foreign protein can be correctly modified and processed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O and HsS78Bst cells.

[0196] Stable expression can be utilized to produce recombinant proteins in long-term, high yields. For example, cell lines can be engineered that stably express the fusion protein. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers. After introduction of the foreign DNA, engineered cells can be grown in enriched medium for 1-2 days and then switched to selective medium. The selectable marker on the recombinant plasmid confers resistance to selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow to form foci that can then be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express the fusion protein. Such engineered cell lines can be particularly useful for screening and evaluating compositions that interact directly or indirectly with the binding molecule.

[0197] A number of selection systems may be used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:8-17 (1980)), genes may be utilized in tk-, hgprt-, or aprt- cells, respectively. Antimetabolite resistance may also be used as a selection criterion for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)), gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)), and neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993), and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993), May, TIB TECH 11(5):155-215 (1993)), and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods generally known in the art in recombinant DNA technology can be routinely applied to select the desired recombinant clones, as described, for example, in Ausubel, et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Chapters 12 and 13, Dracopoli, et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated herein by reference in their entireties.

[0198] The expression level of the fusion protein can be increased by vector amplification (for review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)). If the vector-based marker that expresses the fusion protein is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the fusion protein gene, production of the fusion protein will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

[0199] A host cell may be co-transfected with multiple expression vectors provided herein. The vectors may contain identical selectable markers that allow for equal expression of each encoded polypeptide. Alternatively, a single vector may be used that encodes and is capable of expressing multiple polypeptides. The coding sequences may comprise cDNA or genomic DNA.

[0200] Once the fusion proteins provided herein are produced by recombinant expression, they can be purified by any method known in the art for purification of polypeptides (e.g., immunoglobulin molecules), such as, for example, chromatography (e.g., ion exchange, affinity, particularly affinity for specific antigens following Protein A, sizing column chromatography, and kappa selection affinity chromatography), centrifugation, differential lysis, or by any other standard technique for purification of proteins. Additionally, the fusion protein molecules provided herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0201] Immunoconjugates In some embodiments, the disclosure also provides immunoconjugates comprising any of the anti-IL-1β antibodies described herein conjugated to one or more cytotoxic agents, e.g., chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.

[0202] In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC) conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent No. 0 425 235). B1), auristatins such as the monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see, e.g., U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298), dolastatins, calicheamicins or derivatives thereof (see, e.g., U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. Res. 58:2925-2928 (1998)), anthracyclines such as daunomycin or doxorubicin (see, e.g., Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., al., J. Med. Chem. 45:4336-4343 (2002), and U.S. Patent No. 6,630,579), methotrexate; vindesine; taxanes, such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel, trichothecenes, and CC1065.

[0203] In some embodiments, the immunoconjugate comprises an antibody of the invention as described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, PAP-S), momordica charantia inhibitor, curtin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, trichothecenes.

[0204] In some embodiments, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes can be used to produce the radioconjugate. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 Radioactive isotopes include, for example, tc99m, or I123 for scintigraphic examinations, or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as, for example, iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0205] Conjugates of antibodies and cytotoxic agents can be made using a variety of bifunctional protein coupling agents, such as, for example, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl substrates), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. See WO 94 / 11026.

[0206] The linker may be a "cleavable linker" that facilitates the release of the conjugated drug in cells, although non-cleavable linkers are also contemplated herein.The linkers used in the conjugates of the present disclosure include, but are not limited to, acid-labile linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers that contain amino acids such as valine and / or citrulline, e.g., citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linkers, thioether linkers, or hydrophilic linkers designed to avoid multidrug transporter-mediated resistance.

[0207] The immunoconjugates or ADCs herein can be prepared with crosslinkers such as, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA).

[0208] In other embodiments, the antibodies provided herein are conjugated or recombinantly fused to, for example, a diagnostic molecule. Such diagnosis and detection can be accomplished, for example, by coupling the antibody to a detectable substance, which includes various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidin / biotin and avidin / biotin; fluorescent substances, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent substances, such as, but not limited to, luminol; bioluminescent substances, such as, but not limited to, luciferase, luciferin, or aequorin; chemiluminescent substances, such as gamma-, Auger-, beta-, alpha-, or positron-emitting radioisotopes of 225Ac.

[0209] Polynucleotides In certain embodiments, the present disclosure provides polynucleotides encoding the antibodies herein that bind IL-1β, and fusion proteins comprising the antibodies that bind IL-1β described herein. The polynucleotides of the present disclosure may be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, which may be double-stranded or single-stranded, and if single-stranded, may be the coding strand or the non-coding (antisense) strand. In some embodiments, the polynucleotide is in the form of cDNA. In some embodiments, the polynucleotide is a synthetic polynucleotide.

[0210] The present disclosure further relates to variants of the polynucleotides described herein, e.g., variants encoding fragments, analogs, and / or derivatives of the disclosed antibodies that bind IL-1β. In certain embodiments, the present disclosure provides polynucleotides, including polynucleotides having a nucleotide sequence at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in some embodiments at least about 96%, 97%, 98%, or 99% identical to a polynucleotide encoding an antibody that binds IL-1β of the present disclosure. As used herein, the phrase "a polynucleotide having a nucleotide sequence at least, e.g., 95% "identical" to a reference nucleotide sequence" is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to 5 mutations for every 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides of the reference sequence may be deleted or replaced with another nucleotide, or up to 5% of the total number of nucleotides of the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence, or anywhere between these terminal positions, and may be interspersed individually among the nucleotides of the reference sequence or in one or more contiguous groups within the reference sequence.

[0211] Polynucleotide variants may contain alterations in coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain alterations that result in silent substitutions, additions, or deletions, but do not change the properties or activities of the encoded polypeptide. In some embodiments, polynucleotide variants contain silent substitutions that do not result in changes to the amino acid sequence of a polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants can be generated for a variety of reasons, for example, to optimize codon expression for a particular host (i.e., changing the codons of human mRNA to those preferred by a bacterial host, such as E. coli). In some embodiments, polynucleotide variants contain at least one silent mutation in a non-coding or coding region of the sequence.

[0212] In some embodiments, polynucleotide variants are produced to modulate or alter expression (or expression levels) of an encoded polypeptide. In some embodiments, polynucleotide variants are produced to increase expression of an encoded polypeptide. In some embodiments, polynucleotide variants are produced to decrease expression of an encoded polypeptide. In some embodiments, polynucleotide variants increase expression of an encoded polypeptide compared to the parent polynucleotide sequence. In some embodiments, polynucleotide variants decrease expression of an encoded polypeptide compared to the parent polynucleotide sequence.

[0213] Also provided are vectors comprising the nucleic acid molecules described herein. In one embodiment, the nucleic acid molecules can be incorporated into a recombinant expression vector. The present disclosure provides recombinant expression vectors comprising any of the nucleic acids of the present disclosure. As used herein, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that allows expression of an mRNA, protein, polypeptide, or peptide by a host cell, where the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with a cell under conditions sufficient to express the mRNA, protein, polypeptide, or peptide in the cell. The vectors described herein are not naturally occurring in their entirety. However, portions of the vectors may be naturally occurring. The recombinant expression vectors described may comprise any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially derived from natural sources, and may contain natural, non-natural, or modified nucleotides. The recombinant expression vectors may comprise naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. Non-naturally occurring or altered nucleotides or internucleotide linkages do not inhibit the transcription or replication of the vector.

[0214] In one embodiment, the recombinant expression vector of the present disclosure may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host. Suitable vectors include vectors designed for propagation and propagation, or for expression, or both, such as plasmids and viruses. The vector may be selected from the group consisting of pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors such as λGT10, λGT11, λEMBL4, and λNM1149, λZapII (Stratagene) may be used. Examples of plant expression vectors include pBI01, pBI01.2, pBI121, pBI101.3, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The recombinant expression vector can be a viral vector, such as a retroviral vector, for example a gamma retroviral vector.

[0215] In one embodiment, recombinant expression vectors are prepared using standard recombinant DNA techniques, for example, as described in Sambrook et al., supra, and Ausubel et al., supra. Expression vector constructs, either circular or linear, can be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. Replication systems can be derived, for example, from ColE1, SV40, 2μ plasmid, λ, bovine papilloma virus, and the like.

[0216] The recombinant expression vector may include regulatory sequences, such as transcription and translation initiation and termination codons, that are specific to the type of host (e.g., bacterial, plant, fungal, or animal) into which the vector will be introduced, as appropriate, considering whether the vector is DNA- or RNA-based.

[0217] The recombinant expression vector may contain one or more marker genes that allow for the selection of transformed or transfected hosts. Marker genes include biocide resistance (e.g., resistance to antibiotics, heavy metals, etc.), supplementation to provide prototrophy in auxotrophic hosts, etc. Suitable marker genes for the described expression vectors include, for example, the neomycin / G418 resistance gene, the histidinol x resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0218] The recombinant expression vector may comprise a native promoter or a canonical promoter operably linked to the nucleotide sequence of the present disclosure. The selection of a promoter, for example, strong, weak, tissue-specific, inducible, and development-specific, is within the skill of a person skilled in the art. Similarly, it is also within the skill of a person skilled in the art to combine a nucleotide sequence with a promoter. The promoter may be a non-viral promoter or a viral promoter, for example, a cytomegalovirus (CMV) promoter, a RSV promoter, a SV40 promoter, or a promoter found in the long terminal repeat of murine stem cell virus.

[0219] Recombinant expression vectors can be designed for either transient expression, stable expression, or both, and can be made for constitutive or inducible expression.

[0220] Additionally, the recombinant expression vector can be made to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the death of a cell expressing the suicide gene. A suicide gene can be a gene that confers sensitivity to an agent (e.g., a drug) on ​​a cell expressing the gene, causing the cell to die when the cell is contacted or exposed to the agent. Suicide genes are known in the art and include, for example, Herpes Simplex Virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0221] In certain embodiments, the polynucleotide is isolated. In certain embodiments, the polynucleotide is substantially pure.

[0222] Also provided are host cells comprising the nucleic acid molecules described herein. A host cell can be any cell that contains a heterologous nucleic acid. The heterologous nucleic acid can be a vector (e.g., an expression vector). For example, a host cell can be a cell from any organism that is selected, modified, transformed, grown, used, or engineered in any way for the production of a substance by the cell, such as the expression by the cell of a gene, DNA or RNA sequence, protein, or enzyme. An appropriate host can be determined. For example, a host cell can be selected based on the vector backbone and the desired outcome. By way of example, a plasmid or cosmid can be introduced into a prokaryotic host cell to replicate some types of vectors. Bacterial cells, such as, but not limited to, DH5α, JM109, and KCB, SURE® competent cells, and SOLOPACK Gold cells can be used as host cells for vector replication and / or expression. Additionally, bacterial cells, such as E. coli LE392, can be used as host cells for phage viruses. Eukaryotic cells that can be used as host cells include, but are not limited to, yeast (e.g., YPH499, YPH500, and YPH501), insect, and mammalian. Examples of mammalian eukaryotic host cells for replicating and / or expressing vectors include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, Saos, PC12, SP2 / 0 (American Type Culture Collection (ATCC), Manassas, VA, CRL-1581), NS0 (European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC number 85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) mouse cell lines. An exemplary human myeloma cell line is U266 (ATCC CRL-TIB-196).Other useful cell lines include those derived from Chinese Hamster Ovary (CHO) cells, such as CHO-K1SV (Lonza Biologics, Walkersville, MD), CHO-K1 (ATCC CRL-61), or DG44.

[0223] 5.4. Antibody Preparation and Production Method Methods for preparing antibodies are described. See, for example, Els Pardon et al, Nature Protocol, 9(3):674 (2014). Antibodies (e.g., scFv fragments) can be obtained using methods known in the art, for example, by immunizing camelid species (e.g., camels or llamas) and obtaining hybridomas therefrom, or by cloning a library of antibodies using molecular biology techniques known in the art and then selecting by ELISA using individual clones of the unselected library, or by using phage display.

[0224] The antibodies provided herein can be produced by culturing cells transformed or transfected with a vector containing nucleic acid encoding the antibody. Polynucleotide sequences encoding the polypeptide components of the antibodies of the present disclosure can be obtained using standard recombinant techniques. The desired polynucleotide sequence can be isolated and sequenced from antibody producing cells, such as hybridoma cells or B cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesis agents or PCR techniques. Once obtained, the polypeptide encoding sequence is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a host cell. Many vectors available and known in the art can be used for the purposes of the present disclosure. The selection of an appropriate vector depends primarily on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed with the vector. Suitable host cells for expressing the antibodies of the present disclosure include prokaryotes such as archaea and eubacteria, including gram-negative or gram-positive organisms, eukaryotic microbes such as filamentous fungi and yeast, invertebrate cells such as insect cells and plant cells, and vertebrate cells such as mammalian host cell lines. Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. Antibodies produced by the host cells are purified using standard protein purification methods known in the art.

[0225] Methods for antibody production, including vector construction, expression, and purification, are further described in Pluckthun et al., Antibody Engineering: Producing antibodies in Escherichia coli: From PCR to fermentation 203-52 (McCafferty et al. eds., 1996), Kwong and Rader, E. coli Expression and Purification of Fab Antibody Fragments, in Current Protocols in Protein Science (2009), Tachibana and Takekoshi, Production of Antibody Fab Fragments in Escherichia coli, in Antibody Expression and Production (Al-Rubeai ed., 2011), and Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed., 2009).

[0226] Of course, it is contemplated that alternative methods well known in the art may be employed to prepare anti-IL-1β antibodies. For example, the appropriate amino acid sequence or portions thereof may be produced by direct peptide synthesis using solid-phase techniques (see, e.g., Stewart et al., Solid-Phase Peptide Synthesis (1969), and Merrifield, J. Am. Chem. Soc. 85:2149-54 (1963)). In vitro protein synthesis may be performed manually or by automation. Various portions of the anti-IL-1β antibody may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired anti-IL-1β antibody. Alternatively, the antibodies may be purified from cells or body fluids (such as milk) of transgenic animals genetically engineered to express the antibodies, as disclosed, for example, in U.S. Pat. Nos. 5,545,807 and 5,827,690.

[0227] Polyclonal antibodies Polyclonal antibodies are generally raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. The relevant antigen is conjugated to a protein that is immunogenic in the species being immunized, such as keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, via a bifunctional or derivatizing agent, such as maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine ​​residues), N-hydroxysuccinimide (conjugation via lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R. 1 N=C=NR, where R and R 1 are independently lower alkyl groups). Examples of adjuvants that can be used include Freund's complete adjuvant, and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization protocol can be selected by one of skill in the art without undue experimentation.

[0228] For example, animals are immunized against the antigen, immunogenic conjugate, or derivative by combining, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted by subcutaneous injection at multiple sites with 1 / 5 to 1 / 10 of the original amount of peptide or conjugate in Freund's complete adjuvant. Seven to 14 days later, the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer reaches a plateau. Conjugates can also be made in recombinant cell culture as protein fusions. Agglutinating agents such as alum are also suitable for enhancing the immune response.

[0229] Monoclonal antibodies Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

[0230] Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (e.g., U.S. Pat. No. 4,816,567).

[0231] In the hybridoma method, a suitable host animal is immunized to elicit lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0232] The immunizing agent generally comprises an antigen protein, or a fusion variant thereof. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp.59-103. The immortalized cell line is usually a transformed mammalian cell. The hybridoma cells thus prepared are seeded and grown in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of the unfused parent myeloma cells. Preferred immortalized cell lines are those that fuse efficiently, support stable high-level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.

[0233] The culture medium in which the hybridoma cells grow is assayed for the production of monoclonal antibodies against the antigen. The medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the desired antigen. Such methods and assays are well known in the art. For example, binding affinity can be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0234] Once hybridoma cells are identified that produce antibodies with the desired specificity, affinity, and / or activity, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM medium or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as tumors within a mammal.

[0235] The monoclonal antibodies secreted by the subclones can be suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0236] Monoclonal antibodies can also be produced by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567 or as described above. DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector, which can then be transfected into host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, that do not normally produce immunoglobulin proteins, and the monoclonal antibodies can be synthesized in such recombinant host cells. For review articles on recombinant expression in bacteria of DNA encoding the antibody, see Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pliickthun, Immunol. Revs. 130:151-188 (1992).

[0237] In further embodiments, antibodies can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990), Clackson et al., Nature, 352:624-628 (1991), and Marks et al., J. Mol. Biol., 222:581-597 (1991). Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993)). Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies.

[0238] The DNA can also be modified, for example, by substituting the coding sequence (U.S. Pat. No. 4,816,567; Morrison, et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)) or by covalently linking to the coding sequence all or part of the coding sequence of a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted to create a chimeric, bivalent antibody containing one antigen binding site with specificity for an antigen and another antigen binding site with specificity for a different antigen.

[0239] Chimeric or hybrid antibodies can also be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0240] Recombinant production in prokaryotic cells Polynucleic acid sequences encoding the antibodies of the present disclosure can be obtained using standard recombinant techniques. The desired polynucleotide sequences can be isolated and sequenced from antibody producing cells, such as hybridoma cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesis agents or PCR techniques. Once obtained, the sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic host cells. Many vectors available and known in the art can be used for the purposes of the present disclosure. The selection of an appropriate vector depends mainly on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains various components, depending on its function (amplification or expression of heterologous polynucleotides, or both) and its compatibility with the particular host cell in which it resides. For example, vector components may generally include, but are not limited to, an origin of replication, a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, a heterologous nucleic acid insert, and a transcription termination sequence.

[0241] Generally, plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used in connection with these hosts. The vector usually carries a replication site, as well as marking sequences that can provide phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species. Examples of pBR322 derivatives used to express specific antibodies are described in detail in Carter et al., U.S. Patent No. 5,648,237.

[0242] Additionally, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, bacteriophages such as GEM™-11 can be utilized in generating recombinant vectors that can be used to transform susceptible host cells such as E. coli LE392.

[0243] The expression vector of the present application may contain two or more promoter-cistron pairs, one encoding each of the polypeptide components. A promoter is a non-translated regulatory sequence located upstream (5') of a cistron that regulates its expression. Prokaryotic promoters are typically divided into two classes: inducible and constitutive. An inducible promoter is a promoter that initiates an increase in the transcription level of the cistron under its control in response to a change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature.

[0244] A large number of promoters recognized by various potential host cells are well known. The selected promoter can be operably linked to the cistron DNA encoding the antibody of the present invention by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present application. Both native promoter sequences and many heterologous promoters can be used to direct the amplification and / or expression of the target gene. In some embodiments, heterologous promoters are utilized because they generally allow greater transcription and higher yields of the expressed target gene compared to the native target polypeptide promoter.

[0245] Promoters suitable for use in prokaryotic host cells include the PhoA promoter, the -galactamase and lactose promoter systems, the tryptophan (trp) promoter system, and hybrid promoters such as the tac or trc promoter. However, other promoters that are functional in bacteria (e.g., other known bacterial promoters or phage promoters) are suitable as well. Their nucleic acid sequences have been published, allowing one skilled in the art to operably ligate them to the cistron encoding the target peptide (Siebenlist et al. Cell 20:269 (1980)), and to provide any required restriction sites using linkers or adapters.

[0246] In one embodiment, each cistron in the recombinant vector contains a secretory signal sequence component that directs translocation of the expressed polypeptide across a membrane. In general, the signal sequence may be a component of the vector or may be part of the target polypeptide DNA that is inserted into the vector. The signal sequence selected for the purposes of the present invention should be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the signal sequence native to the heterologous polypeptide, the signal sequence may be substituted by a prokaryotic signal sequence selected from the group consisting of, for example, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, PelB, OMPa, and MBP.

[0247] In some embodiments, production of antibodies according to the present disclosure can occur in the cytoplasm of the host cell and therefore does not require the presence of secretion signal sequences within each cistron. - The recombinant β-actin (strain) provides favorable cytoplasmic conditions for disulfide bond formation, thereby allowing proper folding and assembly of the expressed protein subunits.

[0248] Suitable prokaryotic host cells for expressing the antibodies of the present disclosure include Archaea and Eubacteria, such as Gram-negative or Gram-positive bacteria. Examples of useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B. subtilis), Enterobacteriaceae, Pseudomonas species (e.g., Pseudomonas aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In some embodiments, Gram-negative cells are used. In one embodiment, E. coli cells are used as hosts. An example of an E. coli strain is the W3110 strain (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219, genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompT A (nmpc-fepE) degP41 kan R(ATCC Accession No. 27325, including strain 33D3 having the gene encoding E. coli 1776 (ATCC 31,537), and E. coli RV308 (ATCC 31,608) are also suitable. These examples are illustrative and not limiting. Methods for constructing derivatives of any of the above-mentioned bacteria with defined genotypes are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). In general, it is necessary to select a suitable bacterium taking into account the replicative ability of the replicon in the bacterial cell. For example, E. coli, Serratia, or Salmonella species can be suitably used as hosts when well-known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon.

[0249] Typically, the host cell should secrete minimal amounts of proteolytic enzymes, and it may be desirable to incorporate additional protease inhibitors into the cell culture.

[0250] Host cells are transformed with the above expression vectors and cultured in conventional nutrient media modified as appropriate to induce promoters, select transformants, or amplify genes encoding the desired sequences. Transformation means introducing DNA into a prokaryotic host so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is performed using standard techniques appropriate for such cells. A calcium treatment using calcium chloride is generally used for bacterial cells that contain substantial cell wall barriers. Another method for transformation uses polyethylene glycol / DMSO. Yet another technique used is electroporation.

[0251] The host cells used to produce the antibody of the present invention can be grown in a medium known in the art and suitable for culturing the selected host cells. An example of a suitable medium includes Luria Broth (LB) + necessary nutritional supplements. In some embodiments, the medium also contains a selection agent selected based on the construction of the expression vector to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium for the growth of cells expressing an ampicillin resistance gene.

[0252] Any necessary supplements other than carbon, nitrogen, and inorganic phosphate sources may also be included at appropriate concentrations, introduced alone or in mixture with other supplements or media, such as complex nitrogen sources. Optionally, the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithioerythritol, and dithiothreitol. Prokaryotic host cells are cultured at an appropriate temperature and pH.

[0253] When an inducible promoter is used in the expression vector of the present disclosure, protein expression is induced under conditions suitable for the activation of the promoter. In one aspect of the present application, the PhoA promoter is used to control the transcription of the polypeptide. Thus, the transformed host cell is cultured in a phosphate-limited medium for induction. Preferably, the phosphate-limited medium is CRAP medium (see, for example, Simmons et al., J.Immunol.Methods 263:133-147 (2002)). As known in the art, various other inducers can be used according to the vector construct used.

[0254] The expressed antibodies of the present disclosure are secreted into the periplasm of the host cells and recovered therefrom. Protein recovery typically involves disrupting the microorganisms, generally by means such as osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris or whole cells may be removed by centrifugation or filtration. The protein may be further purified, for example, by affinity resin chromatography. Alternatively, the protein may be transported into the culture medium and isolated therein. The cells may be removed from the culture and the culture supernatant may be filtered and concentrated for further purification of the produced protein. The expressed polypeptide may be further isolated and characterized using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assays.

[0255] Alternatively, protein production is carried out in large quantities by fermentation processes. A variety of large-scale fed-batch fermentation procedures are available for the production of recombinant proteins. Various fermentation conditions can be modified to improve the production yield and quality of the antibodies of the present disclosure. For example, chaperone proteins have been demonstrated to promote proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. J Bio Chem 274:19601-19605 (1999), U.S. Patent No. 6,083,715, U.S. Patent No. 6,027,888, Bothmann and Pluckthun, J. Biol. See Pluckthun, J. Biol. Chem. 275:17106-17113 (2000), Arie et al., Mol. Microbiol. 39:199-210 (2001).

[0256] To minimize proteolysis of expressed heterologous proteins (especially those that are proteolytically sensitive), certain host strains that are deficient in proteolytic enzymes can be used in the present invention, for example, as described in U.S. Patent Nos. 5,264,365 and 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996). E. coli strains that are deficient in proteolytic enzymes and transformed with plasmids that overexpress one or more chaperone proteins can be used as host cells in the expression system encoding the antibodies of the present application.

[0257] The antibodies produced herein can be further purified to obtain preparations that are substantially homogeneous for further assays and uses. Standard protein purification methods known in the art can be used. The following procedures are examples of suitable purification procedures: fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on cation exchange resins on silica or DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration, for example using Sephadex G-75. For example, Protein A immobilized on a solid phase can be used in some embodiments for immunoaffinity purification of binding molecules of the present disclosure. The solid phase on which Protein A is immobilized is preferably a column comprising a glass or silica surface, more preferably a controlled pore glass column or a silicic acid column. In some embodiments, the column is coated with a reagent such as glycerol to prevent nonspecific attachment of contaminants. The solid phase is then washed to remove contaminants nonspecifically bound to the solid phase. Finally, the antibody of interest is recovered from the solid phase by elution.

[0258] Recombinant production in eukaryotic cells For eukaryotic expression, the vector components generally include one or more of the following, but are not limited to: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0259] Vectors for use in eukaryotic hosts may also contain an insert encoding a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected is preferably one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences and viral secretory leaders (e.g., herpes simplex gD signal) are available. The DNA of such precursor regions can be ligated in reading frame to DNA encoding the antibody of the present application.

[0260] Generally, the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter).

[0261] Expression and cloning vectors can contain a selection gene, also called a selectable marker. Selection genes can encode proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, complement auxotrophic deficiencies, or supply vital nutrients unavailable from complex media.

[0262] One example of a selection scheme utilizes a drug to arrest the growth of the host cell. Cells successfully transformed with a heterologous gene produce a protein that confers drug resistance and therefore survive the selection regime. Examples of such dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.

[0263] Another example of a suitable selection marker for mammalian cells is one that allows the identification of cellular components that can incorporate nucleic acid encoding the antibody of the present application. For example, cells transformed with a DHFR selection gene are first identified by culturing all transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. An exemplary suitable host cell when wild-type DHFR is used is the Chinese Hamster Ovary (CHO) cell line, which is deficient in DHFR activity. Alternatively, host cells transformed or co-transformed with a DNA sequence encoding a polypeptide, a wild-type DHFR protein, and another selection marker, such as aminoglycoside 3'-phosphotransferase (APH), (particularly wild-type hosts containing endogenous DHFR), can be selected by growing the cells in a medium containing a selection agent for the selection marker, such as an aminoglycoside antibiotic.

[0264] Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to a nucleic acid encoding a desired polypeptide sequence. Eukaryotic genes have an AT-rich region located approximately 25-30 bases upstream from the site where transcription begins. Additional sequences found 70-80 bases upstream from the start of transcription of many genes may be included. The 3' end of most eukaryotic genes may be a signal for addition of a polyA tail to the 3' end of the coding sequence. All of these sequences may be inserted into eukaryotic expression vectors.

[0265] Transcription of a polypeptide from a vector in a mammalian host cell can be controlled by promoters derived from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and Simian Virus 40 (SV40), heterologous mammalian promoters such as the actin promoter, or promoters derived from immunoglobulin promoters, heat shock promoters, provided that such promoters are compatible with the host cell system.

[0266] Transcription of DNA encoding the antibody of the present disclosure by higher eukaryotic cells is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing elements for activation of eukaryotic promoters. The enhancer can be spliced ​​into the vector at a 5' or 3' position relative to the polypeptide coding sequence, but is preferably located at a site 5' from the promoter.

[0267] In eukaryotic host cells (nucleated cells from yeast, fungi, insects, plants, animals, humans, or other multicellular organisms), the expression vectors used also contain sequences necessary for the termination of transcription and stabilization of the mRNA. Such sequences are commonly available from the 5' and, occasionally, 3' untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments that are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding a polypeptide. One useful transcription termination component is the bovine growth hormone polyadenylation region.

[0268] Suitable host cells for cloning or expressing the DNA in the vectors herein include the higher eukaryotic cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Other examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651), human embryonic kidney line (subcloned 293 or 293 cells for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TR1 cells (Mather et al., Annals NYAcad. Sci. 383:44-68 (1982)), MRC 5 cells, FS4 cells, and a human hepatoma line (Hep G2).

[0269] Host cells can be transformed with the above-described expression or cloning vectors for producing antibodies and cultured in conventional nutrient media modified as appropriate, e.g., to induce promoters, select transformants, or amplify genes encoding desired sequences.

[0270] The host cells used to produce the antibodies of the present invention can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al. Any of the media described in U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO 90 / 03430, WO 87 / 00195, or U.S. Reissue Patent No. 30,985 can be used as a culture medium for the host cells. Any of these media can be supplemented with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, etc.) as necessary. The culture medium may be supplemented with nutrients such as calcium, calcium, magnesium, and phosphate, buffers (e.g., HEPES), nucleotides (e.g., adenosine, thymidine, and the like), antibiotics (e.g., Gentamicin™ agents), trace elements (defined as inorganic compounds, usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may be included at appropriate concentrations that would be known to one of skill in the art. Culture conditions such as temperature, pH, and the like will be those conventionally used with the host cell selected for expression and will be apparent to one of skill in the art.

[0271] When using recombinant techniques, the antibody may be produced intracellularly, in the periplasmic space, or directly secreted into the medium. As a first step, if the antibody is produced intracellularly, particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. If the antibody is secreted into the medium, the supernatant from such an expression system is generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors, such as PMSF, may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0272] Protein compositions prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. In some embodiments, protein compositions prepared from cells can be purified using the AKTA chromatography system. The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices, such as controlled pore glass or poly(styrene-divinyl)benzene, allow for faster flow rates and shorter processing times than can be achieved using agarose. Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica, chromatography on heparin, SEPHAROSE® chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the antibody to be recovered. Following any preliminary purification step(s), the mixture containing the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography.

[0273] 5.5. Pharmaceutical Compositions In one aspect, the present disclosure further provides a pharmaceutical composition comprising at least one antibody or antigen-binding fragment thereof of the present disclosure. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein and a pharma- ceutical acceptable excipient.

[0274] Pharmaceutical compositions containing an antibody or antigen-binding fragment thereof are prepared by mixing the protein having the desired purity, optionally with a physiologically acceptable excipient (see, e.g., Remington, Remington's Pharmaceutical Sciences (18th ed. 1980)), in the form of an aqueous solution or in lyophilized or other dried form for storage.

[0275] The antibodies or antigen-binding fragments thereof of the disclosure can be formulated in any suitable form for delivery to the target cells / tissues, for example, as microcapsules or macroemulsions (Remington, supra; Park, et al., Molecules, 2005, 10:146-61; Malik et al., 2007, Curr. Drug. Deliv. 4:141-51), sustained release formulations ((Putney and Burke, 1998, Nature Biotechnol. 16:153-57), or in liposomes (Maclean et al., 1997, Int. J. Oncol. 11:325-32; Kontermann, 2006, Curr. Opin. Mol. Ther. 8:39-45).

[0276] The antibodies or antigen-binding fragments thereof provided herein can also be encapsulated within microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, prepared, for example, by coacervation techniques or by interfacial polymerization, respectively, in colloidal drug delivery systems (such as, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed, for example, in Remington, supra.

[0277] A variety of compositions and delivery systems are known and can be used with the antibodies or antigen-binding fragments thereof described herein, including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibodies or antigen-binding fragments thereof, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-32), construction of a nucleic acid as part of a retrovirus or other vector, and the like. In another embodiment, the composition can be provided as a controlled or sustained release system. 262:4429-32), construction of a nucleic acid as part of a retrovirus or other vector, and the like. In another embodiment, the composition can be provided as a controlled or sustained release system. In one embodiment, a pump can be used to achieve controlled or sustained release (e.g., Langer, supra; Sefton, 1987, Crit. Ref. Biomed. Eng. 14:201-40; Buchwald et al., 1980, Surgery 88:507-16; and Saudek et al., 1989, N. Engl. J. Med. 321:569-74).In another embodiment, polymeric materials can be used to achieve controlled or sustained release of a prophylactic or therapeutic agent (e.g., an antibody or antigen-binding fragment thereof described herein), or a composition provided herein (see, e.g., Medical Applications of Controlled Release (Langer and Wise, eds., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball, eds., 1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61-126; Levy et al., Science 228:190-92 (1985); During et al., 1989, Ann. Neurol. 25:351-56; Howard et al., 1990; (see, for example, U.S. Pat. Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463, and 5,128,326; WO 99 / 15154 and WO 99 / 20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymers used in the sustained release formulations are inert, free of leachable impurities, stable on storage, sterile, and biodegradable.

[0278] In yet another embodiment, a controlled or sustained release system can be placed in close proximity to a specific target tissue, such as the nasal cavity or lungs, such that only a fraction of the systemic dose is required (see, e.g., Goodson, Medical Applications of Controlled Release Vol. 2, 115-38 (1984)). Controlled release systems are discussed, for example, by Langer, 1990, Science 249:1527-33. Any technique known to one of skill in the art can be used to prepare sustained release formulations comprising one or more antibodies or antigen-binding fragments thereof described herein (see, e.g., U.S. Pat. No. 4,526,938; WO 91 / 05548 and WO 96 / 20698; Ning, et al., Radiotherapy & Oncology, 39:179-89 (1996); Song et al., 1995, PDA J. of Pharma. Sci. & Tech. 50:372-97; Cleek et al., 1997, Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-54; and Lam et al., 1997, Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-60).

[0279] 5.6 How to use antibodies In one aspect, provided herein is a method of attenuating the activity of IL-1β in a cell, the method comprising exposing the cell to an effective amount of an antibody provided herein.

[0280] In some embodiments, the antibodies provided herein inhibit the IL-1β signaling pathway. In some embodiments, the antibodies provided herein inhibit IL-1β biological activity. In some embodiments, the antibodies provided herein inhibit IL-6 production. In some embodiments, the antibodies provided herein inhibit CXCL5 production. In some embodiments, the antibodies provided herein inhibit G-CSF production. In some embodiments, the antibodies provided herein inhibit IL-6, CXCL5, and G-CSF production. In some embodiments, the antibodies provided herein inhibit IL-1β biological activity in human fibroblasts. In some embodiments, the antibodies provided herein inhibit IL-6, CXCL5, and G-CSF production in human fibroblasts. In some embodiments, the antibodies provided herein inhibit IL-1β biological activity in human fibroblasts. In some embodiments, the antibodies provided herein inhibit IL-1β biological activity in human peripheral blood mononuclear cell (PBMC) samples. In some embodiments, the antibodies provided herein inhibit IL-1β biological activity in human whole blood samples.

[0281] In some embodiments, the antibodies provided herein cross-react with cynomolgus IL-1β. In some embodiments, the antibodies provided herein inhibit IL-1β biological activity in cynomolgus fibroblasts.

[0282] . In some embodiments, the antibodies provided herein attenuate IL-1β activity by at least about 10%. In some embodiments, the antibodies provided herein attenuate IL-1β activity by at least about 20%. In some embodiments, the antibodies provided herein attenuate IL-1β activity by at least about 30%. In some embodiments, the antibodies provided herein attenuate IL-1β activity by at least about 40%. In some embodiments, the antibodies provided herein attenuate IL-1β activity by at least about 50%. In some embodiments, the antibodies provided herein attenuate IL-1β activity by at least about 60%. In some embodiments, the antibodies provided herein attenuate IL-1β activity by at least about 70%. In some embodiments, the antibodies provided herein attenuate IL-1β activity by at least about 80%. In some embodiments, the antibodies provided herein attenuate IL-1β activity by at least about 90%. In some embodiments, the antibodies provided herein attenuate IL-1β activity by at least about 95%. In some embodiments, the antibodies provided herein attenuate IL-1β activity by at least about 98%. In some embodiments, the antibodies provided herein attenuate IL-1β activity by about 100%. In certain embodiments, the antibodies described herein may attenuate (e.g., partially attenuate) IL-1β activity by at least about 15% to about 65%. In certain embodiments, the antibodies described herein may attenuate (e.g., partially attenuate) IL-1β activity by at least about 20% to about 65%. In certain embodiments, the antibodies described herein may attenuate (e.g., partially attenuate) IL-1β activity by at least about 30% to about 65%.

[0283] A non-limiting example of an IL-1β activity is IL-1β-mediated signaling. Thus, in certain embodiments, provided herein is a method of attenuating (e.g., partially attenuating) IL-1β-mediated signaling in a cell, comprising exposing the cell to an effective amount of an antibody or antigen-binding fragment thereof provided herein.

[0284] In some embodiments, the antibodies provided herein attenuate IL-1β mediated signaling by at least about 10%. In some embodiments, the antibodies provided herein attenuate IL-1β mediated signaling by at least about 20%. In some embodiments, the antibodies provided herein attenuate IL-1β mediated signaling by at least about 30%. In some embodiments, the antibodies provided herein attenuate IL-1β mediated signaling by at least about 40%. In some embodiments, the antibodies provided herein attenuate IL-1β mediated signaling by at least about 50%. In some embodiments, the antibodies provided herein attenuate IL-1β mediated signaling by at least about 60%. In some embodiments, the antibodies provided herein attenuate IL-1β mediated signaling by at least about 70%. In some embodiments, the antibodies provided herein attenuate IL-1β mediated signaling by at least about 80%. In some embodiments, the antibodies provided herein attenuate IL-1β mediated signaling by at least about 90%. In some embodiments, the antibodies provided herein attenuate IL-1β mediated signaling by at least about 95%. In some embodiments, the antibodies provided herein attenuate IL-1β mediated signaling by at least about 98%. In some embodiments, the antibodies provided herein attenuate IL-1β mediated signaling by about 100%. In certain embodiments, the antibodies described herein may attenuate (e.g., partially attenuate) IL-1β mediated signaling by at least about 15% to about 65%. In certain embodiments, the antibodies described herein may attenuate (e.g., partially attenuate) IL-1β mediated signaling by at least about 20% to about 65%. In certain embodiments, the antibodies described herein may attenuate (e.g., partially attenuate) IL-1β mediated signaling by at least about 30% to about 65%.

[0285] In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of IL-6 by at least about 10%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of IL-6 by at least about 20%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of IL-6 by at least about 30%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of IL-6 by at least about 40%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of IL-6 by at least about 50%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of IL-6 by at least about 60%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of IL-6 by at least about 70%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of IL-6 by at least about 80%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of IL-6 by at least about 90%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of IL-6 by at least about 95%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of IL-6 by at least about 98%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of IL-6 by about 100%. In certain embodiments, the antibodies described herein may attenuate (e.g., partially attenuate) IL-1β-induced production of IL-6 by at least about 15% to about 65%. In certain embodiments, the antibodies described herein may attenuate (e.g., partially attenuate) IL-1β-induced production of IL-6 by at least about 20% to about 65%. In certain embodiments, the antibodies described herein may attenuate (eg, partially attenuate) IL-1β-induced production of IL-6 by at least about 30% to about 65%.

[0286] In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of CXCL5 by at least about 10%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of CXCL5 by at least about 20%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of CXCL5 by at least about 30%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of CXCL5 by at least about 40%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of CXCL5 by at least about 50%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of CXCL5 by at least about 60%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of CXCL5 by at least about 70%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of CXCL5 by at least about 80%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of CXCL5 by at least about 90%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of CXCL5 by at least about 95%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of CXCL5 by at least about 98%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of CXCL5 by about 100%. In certain embodiments, the antibodies described herein may attenuate (e.g., partially attenuate) IL-1β-induced production of CXCL5 by at least about 15% to about 65%. In certain embodiments, the antibodies described herein may attenuate (e.g., partially attenuate) IL-1β-induced production of CXCL5 by at least about 20% to about 65%. In certain embodiments, the antibodies described herein may attenuate (eg, partially attenuate) IL-1β-induced production of CXCL5 by at least about 30% to about 65%.

[0287] In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of G-CSF by at least about 10%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of G-CSF by at least about 20%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of G-CSF by at least about 30%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of G-CSF by at least about 40%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of G-CSF by at least about 50%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of G-CSF by at least about 60%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of G-CSF by at least about 70%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of G-CSF by at least about 80%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of G-CSF by at least about 90%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of G-CSF by at least about 95%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of G-CSF by at least about 98%. In some embodiments, the antibodies provided herein attenuate IL-1β-induced production of G-CSF by about 100%. In certain embodiments, the antibodies described herein can attenuate (e.g., partially attenuate) IL-1β-induced production of G-CSF by at least about 15% to about 65%. In certain embodiments, the antibodies described herein can attenuate (e.g., partially attenuate) IL-1β-induced production of G-CSF by at least about 20% to about 65%. In certain embodiments, the antibodies described herein may attenuate (eg, partially attenuate) IL-1β induced production of G-CSF by at least about 30% to about 65%.

[0288] In some embodiments, the antibodies provided herein attenuate the binding of IL-1β to at least one of its receptors.

[0289] Another non-limiting example of IL-1β activity is binding to IL-1R1. Thus, in certain embodiments, provided herein is a method of attenuating (e.g., partially attenuating) IL-1β binding to IL-1R1, comprising exposing a cell to an effective amount of an antibody or antigen-binding fragment thereof provided herein.

[0290] In some embodiments, the antibodies provided herein attenuate the binding of IL-1β to IL-1R1 by at least about 10%. In some embodiments, the antibodies provided herein attenuate the binding of IL-1β to IL-1R1 by at least about 20%. In some embodiments, the antibodies provided herein attenuate the binding of IL-1β to IL-1R1 by at least about 30%. In some embodiments, the antibodies provided herein attenuate the binding of IL-1β to IL-1R1 by at least about 40%. In some embodiments, the antibodies provided herein attenuate the binding of IL-1β to IL-1R1 by at least about 50%. In some embodiments, the antibodies provided herein attenuate the binding of IL-1β to IL-1R1 by at least about 60%. In some embodiments, the antibodies provided herein attenuate the binding of IL-1β to IL-1R1 by at least about 70%. In some embodiments, the antibodies provided herein attenuate the binding of IL-1β to IL-1R1 by at least about 80%. In some embodiments, the antibodies provided herein attenuate the binding of IL-1β to IL-1R1 by at least about 90%. In some embodiments, the antibodies provided herein attenuate the binding of IL-1β to IL-1R1 by at least about 95%. In some embodiments, the antibodies provided herein attenuate the binding of IL-1β to IL-1R1 by at least about 98%. In some embodiments, the antibodies provided herein attenuate the binding of IL-1β to IL-1R1 by about 100%. In certain embodiments, the antibodies described herein may attenuate (e.g., partially attenuate) the binding of IL-1β to IL-1R1 by at least about 15% to about 65%. In certain embodiments, the antibodies described herein may attenuate (e.g., partially attenuate) IL-1β binding to IL-1R1 by at least about 20% to about 65%. In certain embodiments, the antibodies described herein may attenuate (e.g., partially attenuate) IL-1β binding to IL-1R1 by at least about 30% to about 65%.

[0291] Yet another non-limiting example of IL-1β activity is signal transduction mediated by IL-1R1. Thus, in certain embodiments, provided herein is a method of attenuating (e.g., partially attenuating) IL-1R1-mediated signal transduction in a cell, comprising exposing the cell to an effective amount of an antibody or antigen-binding fragment thereof provided herein.

[0292] In some embodiments, the antibodies provided herein attenuate IL-1R1 mediated signaling by at least about 10%. In some embodiments, the antibodies provided herein attenuate IL-1R1 mediated signaling by at least about 20%. In some embodiments, the antibodies provided herein attenuate IL-1R1 mediated signaling by at least about 30%. In some embodiments, the antibodies provided herein attenuate IL-1R1 mediated signaling by at least about 40%. In some embodiments, the antibodies provided herein attenuate IL-1R1 mediated signaling by at least about 50%. In some embodiments, the antibodies provided herein attenuate IL-1R1 mediated signaling by at least about 60%. In some embodiments, the antibodies provided herein attenuate IL-1R1 mediated signaling by at least about 70%. In some embodiments, the antibodies provided herein attenuate IL-1R1 mediated signaling by at least about 80%. In some embodiments, the antibodies provided herein attenuate IL-1R1 mediated signaling by at least about 90%. In some embodiments, the antibodies provided herein attenuate IL-1R1-mediated signaling by at least about 95%. In some embodiments, the antibodies provided herein attenuate IL-1R1-mediated signaling by at least about 98%. In some embodiments, the antibodies provided herein attenuate IL-1R1-mediated signaling by about 100%. In certain embodiments, the antibodies described herein may attenuate (e.g., partially attenuate) IL-1R1-mediated signaling by at least about 15% to about 65%. In certain embodiments, the antibodies described herein may attenuate (e.g., partially attenuate) IL-1R1-mediated signaling by at least about 20% to about 65%. In certain embodiments, the antibodies described herein may attenuate (e.g., partially attenuate) IL-1R1-mediated signaling by at least about 30% to about 65%.

[0293] In some embodiments, the antibodies provided herein inhibit the initiation and progression of the IL-1 signaling pathway by at least about 10%. In some embodiments, the antibodies provided herein inhibit the initiation and progression of the IL-1 signaling pathway by at least about 20%. In some embodiments, the antibodies provided herein inhibit the initiation and progression of the IL-1 signaling pathway by at least about 30%. In some embodiments, the antibodies provided herein inhibit the initiation and progression of the IL-1 signaling pathway by at least about 40%. In some embodiments, the antibodies provided herein inhibit the initiation and progression of the IL-1 signaling pathway by at least about 50%. In some embodiments, the antibodies provided herein inhibit the initiation and progression of the IL-1 signaling pathway by at least about 60%. In some embodiments, the antibodies provided herein inhibit the initiation and progression of the IL-1 signaling pathway by at least about 70%. In some embodiments, the antibodies provided herein inhibit the initiation and progression of the IL-1 signaling pathway by at least about 80%. In some embodiments, the antibodies provided herein inhibit the initiation and progression of the IL-1 signaling pathway by at least about 90%. In some embodiments, the antibodies provided herein inhibit the initiation and progression of the IL-1 signaling pathway by at least about 95%. In some embodiments, the antibodies provided herein inhibit the initiation and progression of the IL-1 signaling pathway by at least about 98%. In some embodiments, the antibodies provided herein inhibit the initiation and progression of the IL-1 signaling pathway by about 100%. In certain embodiments, the antibodies described herein may attenuate (e.g., partially attenuate) the initiation and progression of the IL-1 signaling pathway by at least about 15% to about 65%. In certain embodiments, the antibodies described herein may attenuate (e.g., partially attenuate) the initiation and progression of the IL-1 signaling pathway by at least about 20% to about 65%. In certain embodiments, the antibodies described herein may attenuate (e.g., partially attenuate) the initiation and progression of the IL-1 signaling pathway by at least about 30% to about 65%.

[0294] In another aspect, provided herein is a method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof provided herein. In one embodiment, the disease or disorder is an IL-1β mediated disease or disorder. In one embodiment, the disease or disorder is an IL-1R1 mediated disease or disorder. Also provided herein is a method of treating a disease or disorder, wherein the subject is administered one or more therapeutic agents in combination with an antibody provided herein.

[0295] The present disclosure also relates to methods of using the antibodies provided herein to inhibit or antagonize the function of IL-1β to inhibit IL-1 signaling pathway activation, thereby regulating inflammation and providing treatment for pathological disorders such as cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is renal cell carcinoma.

[0296] In some embodiments, provided herein is a method of treating an IL-1β mediated disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of an isolated IL-1β antibody or antigen-binding fragment thereof described herein. In some embodiments, provided herein is a method of treating an inflammatory disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of an isolated IL-1β antibody or antigen-binding fragment thereof described herein. In some embodiments, the IL-1β mediated disease or disorder is cancer, such as lung cancer or kidney cancer.

[0297] In another aspect, provided herein is a method of treating lung cancer in a subject, comprising administering to the subject an effective amount of an isolated IL-1β antibody or antigen-binding fragment thereof described herein. Administration can include, for example, systemic delivery or local delivery.

[0298] In some embodiments, the subject is diagnosed with lung cancer (e.g., non-small cell lung cancer). In some embodiments, the subject is diagnosed with stage 0 non-small cell lung cancer (NSCLC). In some embodiments, the subject is diagnosed with stage 1 NSCLC. In some embodiments, the subject is diagnosed with stage 2 NSCLC. In some embodiments, the subject is diagnosed with stage 2 NSCLC and has undergone surgery. In some embodiments, the subject is diagnosed with stage 3 NSCLC. In some embodiments, the subject is diagnosed with stage 3 NSCLC and has undergone surgery. In some embodiments, the subject is diagnosed with stage 4 NSCLC.

[0299] In another aspect, provided herein is a method of treating renal cancer in a subject, comprising administering to the subject an effective amount of an isolated IL-1β antibody, or antigen-binding fragment thereof, described herein.

[0300] In some embodiments, the subject is diagnosed with kidney cancer, e.g., renal cell carcinoma (RCC). In some embodiments, the subject is diagnosed with stage 1 RCC. In some embodiments, the subject is diagnosed with stage 2 RCC. In some embodiments, the subject is diagnosed with stage 3 RCC.

[0301] In some embodiments, the antibodies provided herein are used to block lung cancer. In some embodiments, the subject is identified as being at risk for developing lung cancer. In another aspect, provided herein is a method of treating lung cancer in a subject, comprising administering to the subject an effective amount of an isolated IL-1β antibody or antigen-binding fragment thereof described herein.

[0302] Subjects at risk of developing lung cancer can be identified by a variety of factors known in the art. In some embodiments, the subject is determined to have one or more pulmonary nodules, such as precancerous pulmonary nodules (e.g., identified by computed tomography imaging). In some embodiments, the one or more pulmonary nodules are precancerous. In some embodiments, the subject is between about 50 and about 80 years old, and / or the subject has a smoking history, e.g., a 20 pack-year smoking history. In some embodiments, the subject has an elevated level of C-reactive protein (CRP).

[0303] According to further embodiments, a subject at risk of developing lung cancer is identified according to the methods described in WO 2021 / 146516 ("SYSTEM AND METHOD FOR PREDICTING THE RISK OF FUTURE LUNG CANCER"), which is incorporated by reference herein, and the at-risk subject is administered an effective amount of an isolated IL-1β antibody or antigen-binding fragment thereof described herein. For example, the method may include identifying a patient at risk for developing lung cancer by obtaining one or more images (e.g., CT scans) captured from the patient; extracting features from the one or more obtained images (e.g., the extracted features include at least non-nodule specific features, where the non-nodule specific features include one or both of lung parenchymal features or body composition features); predicting one or more future risks of the subject developing lung cancer by analyzing the features extracted from the one or more images by applying one or more trained risk prediction models; and if the patient is identified as at risk for developing lung cancer (e.g., at risk for developing lung cancer within 1 year, 3 years, 5 years, or 10 years), administering to the patient an effective amount of an isolated IL-1β antibody or antigen-binding fragment thereof, as described herein.

[0304] In some embodiments, the antibodies provided herein are used for the prevention of lung cancer. The prevention may be complete, e.g., the IL-1β-associated condition or disorder may be completely absent. The prevention may also be partial, such that the likelihood of the development of an IL-1β-associated condition or metabolic disorder in a subject is less likely than in a subject not administered the antibody of the present disclosure.

[0305] Methods of administration and dosing are described in more detail below in Section 5.7.

[0306] In another aspect, provided herein is the use of an antibody, or antigen-binding fragment thereof, provided herein in the manufacture of a medicament for treating a disease or disorder in a subject.

[0307] In another aspect, provided herein is the use of a pharmaceutical composition provided herein in the manufacture of a medicament for treating a disease or disorder in a subject.

[0308] In another aspect, provided herein is the use of an antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament, wherein the medicament is for use in a method for detecting the presence of IL-1β in a biological sample, the method comprising contacting the biological sample with the antibody under conditions permitting binding of the antibody to the IL-1β protein, and detecting whether a complex is formed between the antibody and the IL-1β protein.

[0309] In other aspects, the antibodies and fragments thereof of the present disclosure are useful for detecting the presence of IL-1β in a biological sample. The term "detect" as used herein encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises a bodily fluid, cell, or tissue. Diagnostic assays and methods are described in more detail in Section 5.9 below.

[0310] 5.7 Methods of Use, Administration and Dosage In certain embodiments, provided herein is a composition for use in the prevention and / or treatment of a disease or condition comprising an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a composition for use in the prevention of a disease or condition, the composition comprising an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a composition for use in the treatment of a disease or condition, the composition comprising an antibody or antigen-binding fragment thereof provided herein. In some embodiments, the disease or condition is an IL-1β mediated disease. In some embodiments, the disease or condition is an IL-1R1 mediated disease. In some embodiments, the disease or disorder is IL-1β associated. In some embodiments, the disease or disorder is IL-1β associated. In some embodiments, the disease or disorder is an inflammatory disease or disorder. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the lung cancer is stage 0 non-small cell lung cancer. In some embodiments, the lung cancer is stage 1 non-small cell lung cancer. In some embodiments, the lung cancer is stage 2 non-small cell lung cancer. In some embodiments, the lung cancer is stage 3 non-small cell lung cancer. In some embodiments, the lung cancer is stage 4 non-small cell lung cancer. In some embodiments, the cancer is renal cancer. In some embodiments, the cancer is renal cell carcinoma. In some embodiments, the renal cell carcinoma is stage 1 renal cell carcinoma. In some embodiments, the renal cell carcinoma is stage 2 renal cell carcinoma. In some embodiments, the renal cell carcinoma is stage 3 renal cell carcinoma. In some embodiments, the subject is a subject in need of treatment thereof. In some embodiments, the subject has a disease or condition. In other embodiments, the subject is at risk of having a disease or condition. In some embodiments, the administration results in the prevention, management, treatment, or amelioration of the disease or condition.

[0311] In one embodiment, provided herein is a composition for use in the prevention and / or treatment of a symptom of a disease or condition, the composition comprising an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a composition for use in the prevention of a symptom of a disease or condition, the composition comprising an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a composition for use in the treatment of a symptom of a disease or condition, the composition comprising an antibody or antigen-binding fragment thereof provided herein. In some embodiments, the disease or condition is an IL-1β mediated disease. In some embodiments, the disease or condition is an IL-1R1 mediated disease. In some embodiments, the disease or disorder is IL-1β associated. In some embodiments, the disease or disorder is IL-1β associated. In some embodiments, the disease or disorder is an inflammatory disease or disorder. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the lung cancer is stage 0 non-small cell lung cancer. In some embodiments, the lung cancer is stage 1 non-small cell lung cancer. In some embodiments, the lung cancer is stage 2 non-small cell lung cancer. In some embodiments, the lung cancer is stage 3 non-small cell lung cancer. In some embodiments, the lung cancer is stage 4 non-small cell lung cancer. In some embodiments, the cancer is renal cancer. In some embodiments, the cancer is renal cell carcinoma. In some embodiments, the renal cell carcinoma is stage 1 renal cell carcinoma. In some embodiments, the renal cell carcinoma is stage 2 renal cell carcinoma. In some embodiments, the renal cell carcinoma is stage 3 renal cell carcinoma. In certain embodiments, the subject is a subject in need of treatment thereof. In some embodiments, the subject has a disease or condition. In other embodiments, the subject is at risk of having a disease or condition. In some embodiments, the administration results in prevention or treatment of a symptom of the disease or condition.

[0312] In another embodiment, provided herein is a method of preventing and / or treating a disease or condition in a subject comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a method of preventing a disease or condition in a subject comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a method of treating a disease or condition in a subject comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In some embodiments, the disease or condition is an IL-1β mediated disease. In some embodiments, the disease or condition is an IL-1R1 mediated disease. In some embodiments, the disease or disorder is IL-1β associated. In some embodiments, the disease or disorder is IL-1β associated. In some embodiments, the disease or disorder is an inflammatory disease or disorder. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the lung cancer is stage 0 non-small cell lung cancer. In some embodiments, the lung cancer is stage 1 non-small cell lung cancer. In some embodiments, the lung cancer is stage 2 non-small cell lung cancer. In some embodiments, the lung cancer is stage 3 non-small cell lung cancer. In some embodiments, the lung cancer is stage 4 non-small cell lung cancer. In some embodiments, the cancer is renal cancer. In some embodiments, the cancer is renal cell carcinoma. In some embodiments, the renal cell carcinoma is stage 1 renal cell carcinoma. In some embodiments, the renal cell carcinoma is stage 2 renal cell carcinoma. In some embodiments, the renal cell carcinoma is stage 3 renal cell carcinoma. In certain embodiments, the subject is a subject in need of such treatment. In some embodiments, the subject has a disease or condition. In other embodiments, the subject is at risk of having a disease or condition. In some embodiments, the administration results in prevention or treatment of a disease or condition.

[0313] In another embodiment, provided herein is a method of preventing and / or treating a symptom of a disease or condition in a subject, comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a method of preventing a symptom of a disease or condition in a subject, comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a method of treating a symptom of a disease or condition in a subject, comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In some embodiments, the disease or condition is an IL-1β mediated disease. In some embodiments, the disease or condition is an IL-1R1 mediated disease. In some embodiments, the disease or disorder is IL-1β associated. In some embodiments, the disease or disorder is IL-1β associated. In some embodiments, the disease or disorder is an inflammatory disease or disorder. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the lung cancer is stage 0 non-small cell lung cancer. In some embodiments, the lung cancer is stage 1 non-small cell lung cancer. In some embodiments, the lung cancer is stage 2 non-small cell lung cancer. In some embodiments, the lung cancer is stage 3 non-small cell lung cancer. In some embodiments, the lung cancer is stage 4 non-small cell lung cancer. In some embodiments, the cancer is renal cancer. In some embodiments, the cancer is renal cell carcinoma. In some embodiments, the renal cell carcinoma is stage 1 renal cell carcinoma. In some embodiments, the renal cell carcinoma is stage 2 renal cell carcinoma. In some embodiments, the renal cell carcinoma is stage 3 renal cell carcinoma. In certain embodiments, the subject is a subject in need of treatment thereof. In some embodiments, the subject has a disease or condition. In other embodiments, the subject is at risk of having a disease or condition. In some embodiments, the administration results in prevention or treatment of a symptom of the disease or condition.

[0314] Also provided herein are methods of preventing and / or treating a disease or condition by administering to a subject an effective amount of an antibody or antigen-binding fragment thereof provided herein or a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof provided herein. In one aspect, the antibody or antigen-binding fragment thereof is substantially purified (i.e., substantially free of substances that limit its effect or cause undesirable side effects). The subject to which the therapy is administered can be a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey, such as macaque (cynomolgus monkey), or human). In one embodiment, the subject is a human. In another embodiment, the subject is a human having a disease or condition.

[0315] A variety of delivery systems are known and can be used to administer a prophylactic or therapeutic agent (e.g., an antibody or antigen-binding fragment thereof provided herein). Methods of administering a prophylactic or therapeutic agent (e.g., an antibody or antigen-binding fragment thereof provided herein), or pharmaceutical composition include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes). In certain embodiments, a prophylactic or therapeutic agent (e.g., an antibody or antigen-binding fragment thereof provided herein), or pharmaceutical composition is administered intranasally, intramuscularly, intravenously, or subcutaneously.

[0316] In certain embodiments, it may be desirable to administer the prophylactic or therapeutic agents or pharmaceutical compositions provided herein locally to the area in need of treatment. This can be accomplished, for example, but not limited to, by localized infusion, by topical administration (e.g., by intranasal spray), by injection, or by implant.

[0317] In certain embodiments, the compositions provided herein comprise one, two or more of the antibodies or antigen-binding fragments thereof provided herein. In another embodiment, the compositions provided herein comprise one, two or more of the antibodies or antigen-binding fragments thereof provided herein and a prophylactic or therapeutic agent other than the antibodies or antigen-binding fragments thereof provided herein. In one embodiment, the agent is known to be useful or is used or is currently used for the prevention, management, treatment and / or amelioration of a disease or condition. In addition to the prophylactic or therapeutic agent, the compositions provided herein may also comprise one or more excipients.

[0318] The compositions provided herein include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., compositions suitable for administration to a subject or patient) that can be used to prepare unit dosage forms. In some embodiments, the compositions provided herein are pharmaceutical compositions. Such compositions include a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic agents (e.g., an antibody or antigen-binding fragment thereof, or other prophylactic or therapeutic agent provided herein) and one or more pharma- ceutically acceptable excipients. The pharmaceutical compositions can be formulated to be suitable for the route of administration to a subject.

[0319] In specific embodiments, the term "excipient" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), or vehicle. Pharmaceutical excipients can be sterile liquids, such as water and oils (including those of petroleum, animal, vegetable, or synthetic origin). Water is a typical excipient when pharmaceutical compositions are administered intravenously. Saline and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, particularly for injectable solutions. If desired, the compositions can further contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Non-limiting examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Such compositions will contain a prophylactically or therapeutically effective amount of the antibodies or antigen-binding fragments thereof provided herein, such as in purified form, together with an appropriate amount of excipient(s) for proper administration to a patient. The formulation should suit the mode of administration.

[0320] In an embodiment, the composition is formulated in accordance with procedures as a pharmaceutical composition adapted for intravenous administration to human beings.

[0321] The components of the compositions provided herein are supplied individually or mixed together in unit dosage form, for example as dry lyophilized powder or water-free concentrate in a sealed container such as an ampoule or sachet indicating the amount of active agent.When the composition is administered by injection, the composition can be dispensed by an injection bottle containing sterile water or saline of pharmaceutical grade.When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0322] The antibodies or antigen-binding fragments thereof provided herein may be packaged in a hermetically sealed container, such as an ampoule or sachet indicating the quantity of antibody. In one embodiment, the antibodies or antigen-binding fragments thereof are supplied as a dry, sterile, lyophilized powder or water-free concentrate in a hermetically sealed container and can be reconstituted, for example, with water or saline solution, to the appropriate concentration for administration to a subject.

[0323] The compositions provided herein can be formulated in a neutral or salt form. Pharmaceutically acceptable salts include those formed with anions and those formed with cations.

[0324] The amount of a prophylactic or therapeutic agent (e.g., an antibody or antigen-binding fragment thereof provided herein), or a composition provided herein, that will be effective in the prevention and / or treatment of a disease or condition can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or condition, and should be decided according to the judgment of the practitioner and each patient's circumstances.

[0325] In certain embodiments, the route of administration of a dose of an antibody or antigen-binding fragment thereof provided herein to a patient is intranasal, intramuscular, intravenous, subcutaneous, or a combination thereof, although other routes described herein are also acceptable. Each dose may or may not be administered by the same route of administration. In some embodiments, an antibody or antigen-binding fragment thereof provided herein may be administered via multiple routes of administration simultaneously with or after other doses of the same or different antibody or antigen-binding fragment thereof provided herein.

[0326] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are administered prophylactically or therapeutically to a subject. The antibodies or antigen-binding fragments thereof provided herein can be administered prophylactically or therapeutically to a subject to prevent, reduce, or ameliorate a disease or condition.

[0327] 5.8 Gene Therapy In certain embodiments, nucleic acids comprising sequences encoding an antibody or functional derivative thereof are administered to a subject for use in the methods provided herein to prevent, manage, treat, and / or ameliorate an IL-1β mediated disease, disorder, or condition, e.g., by gene therapy. Such treatments include treatments performed by administration of an expressed or expressible nucleic acid to a subject. In one embodiment, the nucleic acids produce the antibodies they encode, which mediate a prophylactic or therapeutic effect.

[0328] Any of the methods for recombinant gene expression (or gene therapy) available in the art can be used.

[0329] For general reviews of gene therapy methods, see Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May 1993, TIBTECH 11(5):155-215. Methods generally known in the art of recombinant DNA technology that may be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993), and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

[0330] In certain embodiments, the composition comprises a nucleic acid encoding an antibody provided herein, the nucleic acid being part of an expression vector that expresses the antibody or chimeric protein or its heavy or light chain in a suitable host. In particular, such a nucleic acid has a promoter (e.g., a heterologous promoter) operably linked to the antibody coding region, the promoter being inducible or constitutive, and optionally tissue-specific. In another particular embodiment, a nucleic acid molecule is used in which the antibody coding sequence and any other desired sequences are flanked by regions that promote homologous recombination at the desired site in the genome, thus providing for intrachromosomal expression of the antibody-encoding nucleic acid (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

[0331] Delivery of nucleic acids to a subject can be either direct, where the subject is directly exposed to the nucleic acid or a nucleic acid-carrying vector, or indirect, where cells are first transformed with the nucleic acid in vitro and then transplanted into the subject. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.

[0332] In a specific embodiment, nucleic acid sequences (eg, DNA and mRNA sequences) are directly administered in vivo, where the sequences are expressed to produce the encoded product. This can be accomplished by any of a number of methods known in the art, for example, by constructing them as part of an appropriate nucleic acid expression vector and administering the vector so that the sequences become intracellular, for example, by infection using a defective or attenuated retroviral vector or other viral vector (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA or mRNA, or by use of microparticle bombardment (e.g., gene gun, Biolistic, Dupont), or by coating with lipids or cell surface receptors or transfection agents, encapsulating in liposomes, microparticles, microcapsules, or administering them bound to peptides known to enter the nucleus, by administering them bound to a ligand that undergoes receptor-mediated endocytosis (e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), which can be used to target cell types that specifically express the receptor. In another embodiment, the ligand can include a fusogenic viral peptide to disrupt endosomes, forming a nucleic acid-ligand complex that allows the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell-specific uptake and expression by targeting a specific receptor (see, e.g., WO 92 / 06180, WO 92 / 22635, WO 92 / 20316, WO 93 / 14188, WO 93 / 20221). Alternatively, the nucleic acid can be introduced into the cell by homologous recombination and integrated into the host cell DNA for expression (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935, and Zijlstra et al., 1989, Nature 342:435-438).

[0333] In certain embodiments, viral vectors containing nucleic acid sequences encoding antibodies are used. For example, retroviral vectors can be used (see Miller et al., 1993, Meth. Enzymol. 217:581-599). These retroviral vectors contain the components necessary for correct packaging of viral genome and integration into host cell DNA. The nucleic acid sequences encoding the antibodies used in gene therapy can be cloned into one or more vectors, which facilitates delivery of the gene to a subject. Further details about retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302, which describes the use of retroviral vectors to deliver the MDR1 gene to hematopoietic stem cells to make the stem cells more resistant to chemotherapy. Other references describing the use of retroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin. Invest. 93:644-651; Klein et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.

[0334] Adenoviruses are other viral vectors that can be used in recombinant production of antibodies. Adenoviruses are particularly attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503, present a review of adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy 5:3-10, demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other examples of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., 1991, Science 252:431-434, Rosenfeld et al., 1992, Cell 68:143-155, Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234, WO 94 / 12649, and Wang et al., 1995, Gene Therapy 2:775-783. In some embodiments, adenovirus vectors are used.

[0335] Adeno-associated viruses (AAV) can also be utilized (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300, and U.S. Pat. No. 5,436,146). In certain embodiments, AAV vectors are used to express the anti-IL-1β antibodies provided herein. In certain embodiments, the AAV comprises a nucleic acid encoding a VH domain. In other embodiments, the AAV comprises a nucleic acid encoding a VL domain. In certain embodiments, the AAV comprises a nucleic acid encoding a VH domain and a VL domain. In some embodiments of the methods provided herein, the subject is administered an AAV comprising a nucleic acid encoding a VH domain and an AAV comprising a nucleic acid encoding a VL domain. In other embodiments, the subject is administered an AAV comprising a nucleic acid encoding a VH domain and a VL domain. In certain embodiments, the VH and VL domains are overexpressed.

[0336] Another approach to gene therapy involves transferring a gene into cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate-mediated gene transfer, or viral infection. Usually, the transfer method involves the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. These cells are then delivered to a subject.

[0337] In this embodiment, the nucleic acid is introduced into the cell prior to in vivo administration of the resulting recombinant cell. Such introduction can be by any method known in the art, including, but not limited to, transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequence, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques for the introduction of foreign genes into cells are known in the art (e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644; Clin. Pharma. Ther. 29:69-92 (1985)) and can be used in accordance with the methods provided herein, provided that the necessary development and physiological functions of the recipient cell are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell (eg, heritable and expressible by its cell progeny).

[0338] The resulting recombinant cells can be delivered to a subject by a variety of methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) can be administered intravenously. The amount of cells envisioned for use depends on the desired effect, the patient's condition, etc., and can be determined by one of skill in the art.

[0339] Cells into which nucleic acids may be introduced for purposes of gene therapy encompass any desired available cell type, and include, but are not limited to, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes, blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes, and various stem or progenitor cells, particularly hematopoietic stem or progenitor cells, such as those obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, and the like.

[0340] In certain embodiments, the cells used in gene therapy are autologous to the subject.

[0341] In embodiments in which recombinant cells are used in gene therapy, nucleic acid sequences encoding the antibodies are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In certain embodiments, stem or progenitor cells are used. Any stem and / or progenitor cells that can be isolated and maintained in vitro can potentially be used according to this embodiment of the methods provided herein (see, e.g., WO 94 / 08598; Stemple and Anderson, 1992, Cell 7 1:973-985; Rheinwald, 1980, Meth. Cell Bio. 21A:229; and Pittelkow and Scott, 1986, Mayo Clinic Proc. 61:771).

[0342] In certain embodiments, a nucleic acid to be introduced for purposes of gene therapy contains an inducible promoter operably linked to the coding region, such that expression of the nucleic acid can be controlled by controlling the presence or absence of the appropriate transcription inducer.

[0343] 5.9. Diagnostic Assays and Methods Labeled antibodies that immunospecifically bind to IL-1β antigen, as well as derivatives and analogs thereof, can be used for diagnostic purposes to detect, diagnose, or monitor IL-1β-mediated disease. Thus, provided herein is a method for detecting an IL-1β-mediated disease, comprising: (a) assaying expression of IL-1β antigen in a cell or tissue sample of a subject using one or more antibodies provided herein that immunospecifically bind to IL-1β antigen; and (b) comparing the level of IL-1β antigen to a control level, e.g., the level in a normal tissue sample (e.g., a sample from a patient not having an IL-1β-mediated disease, or a sample from the same patient prior to disease onset), whereby an increase in the assayed level of IL-1β antigen compared to the control level of IL-1β antigen is indicative of an IL-1β-mediated disease.

[0344] Also provided herein is a diagnostic assay for diagnosing an IL-1β mediated disease, comprising: (a) assaying the level of IL-1β antigen in a cell or tissue sample of an individual using one or more antibodies provided herein that immunospecifically bind to the IL-1β antigen; and (b) comparing the level of the IL-1β antigen to a control level, e.g., a level in a normal tissue sample, whereby an increase in the level of the assayed IL-1β antigen compared to the control level of the IL-1β antigen is indicative of an IL-1β mediated disease. In certain embodiments, provided herein is a method of treating an IL-1β mediated disease in a subject, comprising: (a) assaying the level of IL-1β antigen in a cell or tissue sample of the subject using one or more antibodies provided herein that immunospecifically bind to the IL-1β antigen; and (b) comparing the level of the IL-1β antigen to a control level, e.g., a level in a normal tissue sample, whereby an increase in the level of the assayed IL-1β antigen compared to the control level of the IL-1β antigen is indicative of an IL-1β mediated disease. In some aspects, the method further comprises (c) administering to a subject identified as having an IL-1β mediated disease an effective amount of an antibody provided herein. A more definitive diagnosis of an IL-1β mediated disease may enable medical practitioners to employ preventative measures or aggressive treatments earlier, thereby preventing the onset or further progression of an IL-1β mediated disease.

[0345] The antibodies provided herein can be used to assay IL-1β antigen levels in biological samples using classical immunohistological methods as described herein or known to those of skill in the art (see, e.g., Jalkanen et al., 1985, J. Cell. Biol. 101:976-985, and Jalkanen et al., 1987, J. Cell. Biol. 105:3087-3096). Other antibody-based methods useful for detecting protein gene expression include enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs). Suitable antibody assay labels are known in the art and include radioisotopes such as glucose oxidase, iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (121In), technetium (99Tc), luminescent labels such as luminol, and fluorescent labels such as fluorescein and rhodamine, and enzyme labels such as biotin.

[0346] One aspect provided herein is the detection and diagnosis of IL-1β mediated disease in humans. In one embodiment, the diagnosis involves a) administering to a subject (e.g., parenterally, subcutaneously, or intraperitoneally) an effective amount of a labeled antibody that immunospecifically binds to an IL-1β antigen; b) waiting a time interval after administration to allow the labeled antibody to concentrate at sites in the subject where the IL-1β antigen is expressed (and for unbound labeled molecule to be removed to background levels); c) determining the background level; and d) detecting the labeled antibody in the subject, where detection of the labeled antibody above the background level indicates that the subject has an IL-1β mediated disease. The background level can be determined by a variety of methods, including comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.

[0347] It is understood in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety required to produce a diagnostic image. In the case of a radioisotope moiety, for a human subject, the amount of radioactivity injected is usually in the range of about 5-20 millicuries of 99Tc. The labeled antibody then accumulates at the location of cells that contain the specific protein. In vivo tumor imaging is described in Chapter 13 of SW Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments." in Tumor Imaging: The Radiochemical Detection of Cancer, SW Burchiel and BA Rhodes, eds., Masson Publishing Inc. (1982).

[0348] Depending on several variables, including the type of label used and the mode of administration, the time interval after administration to allow the labeled antibody to concentrate at the site of interest and for unbound labeled antibody to be cleared to background levels is 6 to 48 hours, or 6 to 24 hours, or 6 to 12 hours, hi another embodiment, the time interval after administration is 5 to 20 days, or 5 to 10 days.

[0349] In one embodiment, monitoring of the IL-1β mediated disease is performed by repeating the method for diagnosing the IL-1β mediated disease, e.g., one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.

[0350] The presence of the labeled molecule can be detected in the subject using methods known in the art for in vivo scanning.These methods depend on the type of label used.Those skilled in the art can determine the appropriate method for detecting a particular label.The methods and devices that can be used in the diagnostic method provided herein include, but are not limited to, computer tomography (CT), whole body scanning such as positron emission tomography (PET), magnetic resonance imaging (MRI), and ultrasound examination.

[0351] In certain embodiments, the molecule is labeled with a radioisotope and detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Patent No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and detected in the patient using positron emission tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and detected in the patient using magnetic resonance imaging (MRI).

[0352] 5.10 Kits Also provided herein are kits comprising an antibody (e.g., an anti-IL-1β antibody), or composition thereof (e.g., a pharmaceutical composition) provided herein, packaged in suitable packaging material. The kits optionally include a label or package insert containing a description of the components or instructions for in vitro, in vivo, or ex vivo use of the components therein.

[0353] The term "packaging material" refers to a physical structure that contains the components of the kit. The packaging material can maintain the components sterile and can be made of materials commonly used for such purposes (e.g., paper, cardboard fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0354] The kits provided herein can include a label or insert. The label or insert includes "printed matter", such as paper or cardboard, separate from or attached to a component, kit, or packaging material (e.g., box), or attached to, for example, an ampoule, tube, or vial containing a kit component. The label or insert can further include a computer-readable medium, such as a disk (e.g., hard disk, card, memory disk), optical disk such as CD or DVD-ROM / RAM, DVD, MP3, magnetic tape, or electronic storage medium such as RAM and ROM, or hybrids thereof such as magnetic / optical storage medium, flash media, or memory type card. The label or insert can include information identifying manufacturer information, lot number, manufacturer address, and date.

[0355] The kits provided herein can further include other components. Each component of the kit can be enclosed in an individual container, and all of the various containers can be in a single package. The kits can also be designed for refrigeration. The kits can further be designed to include cells that contain an antibody provided herein, or a nucleic acid encoding an antibody provided herein. The cells in the kit can be maintained under appropriate storage conditions until ready for use.

[0356] Also provided herein are panels of antibodies that immunospecifically bind to the IL-1β antigen. In certain embodiments, provided herein are panels of antibodies that have different association rate constants, different dissociation rate constants, different affinities for the IL-1β antigen, and / or different specificities for IL-1β. In certain embodiments, provided herein are panels of about 10, preferably about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, or about 1000 or more antibodies. A panel of antibodies can be used in an assay such as an ELISA in, for example, 96-well or 384-well plates.

[0357] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0358] As used herein, numerical values ​​are often presented in a range format throughout this document. The use of the range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention unless the context clearly indicates otherwise. Thus, the use of a range explicitly includes all possible subranges, all individual numerical values ​​within that range, and all numerical values ​​or numerical ranges, including integers within such ranges and fractions of values ​​or integers within the range, unless the context clearly indicates otherwise. This configuration applies in all contexts throughout this patent document, regardless of the breadth of the range. Thus, for example, a reference to a range of 90-100% includes 91-99%, 92-98%, 93-95%, 91-98%, 91-97%, 91-96%, 91-95%, 91-94%, 91-93%, etc. References to the range of 90-100% include 91%, 92%, 93%, 94%, 95%, 95%, 97% etc, as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5% etc, 92.1%, 92.2%, 92.3%, 92.4%, 92.5% etc.

[0359] Furthermore, references to the ranges 1-3, 3-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-225, and 225-250 include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. In further examples, references to ranges of 25 to 250, 250 to 500, 500 to 1,000, 1,000 to 2,500, 2,500 to 5,000, 5,000 to 25,000, 25,000 to 50,000 include any numerical value or range within or encompassing such values, for example, 25, 26, 27, 28, 29. 250, 251, 252, 253, 254. 500, 501, 502, 503, 504, etc.

[0360] Similarly, as used herein, a series of ranges are disclosed throughout this document. The use of a series of ranges includes the combination of upper and lower ranges to provide another range. This configuration applies in all contexts throughout this patent document, regardless of the breadth of the range. Thus, for example, reference to a series of ranges such as 5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100-150 includes ranges such as 5-20, 5-30, 5-40, 5-50, 5-75, 5-100, 5-150, and 10-30, 10-40, 10-50, 10-75, 10-100, 10-150, and 20-40, 20-50, 20-75, 20-100, 20-150, etc.

[0361] For the sake of brevity, certain abbreviations are used herein. One example is the single letter abbreviations that represent amino acid residues. The amino acids and their corresponding three letter and one letter abbreviations are as follows:

[0362] [Table 3]

[0363] The present invention is generally disclosed herein using positive language to describe a number of embodiments. The present invention also specifically includes embodiments in which certain subject matter, such as substances or materials, method steps and conditions, protocols, procedures, assays or analyses, is entirely or partially excluded. Thus, the present specification generally discloses aspects not expressly included in the present invention, even if the specification is not expressed herein with respect to what the present invention does not include.

[0364] A number of embodiments of the present invention have been described. However, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, the following examples are intended to illustrate, but not limit, the scope of the invention as claimed.

[0365] 6. Embodiment The present invention provides the following non-limiting embodiments.

[0366] In one set of embodiments, the following is provided: 1. An antibody that binds to IL-1β, (1) (i) a VH having the amino acid sequence of SEQ ID NO: 7, comprising a VH CDR1, a VH CDR2, and a VH CDR3, each having the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3, respectively; and (ii) a VL having the amino acid sequence of SEQ ID NO: 8, comprising a VL CDR1, a VL CDR2, and a VL CDR3, each having the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3, respectively; (2) (i) a VH having the amino acid sequence of SEQ ID NO: 9, comprising a VH CDR1, a VH CDR2, and a VH CDR3, each having the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3, respectively; and (ii) a VL having the amino acid sequence of SEQ ID NO: 10, comprising a VL CDR1, a VL CDR2, and a VL CDR3, each having the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3, respectively; or (3) An antibody comprising: (i) a VH having the amino acid sequence of SEQ ID NO: 11, the VH including VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3, respectively; and (ii) a VL having the amino acid sequence of SEQ ID NO: 12, the VL including VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3, respectively. 2. (i) the amino acid sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are numbered according to the Kabat numbering system; (ii) the amino acid sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are numbered according to the Chothia numbering system; (iii) the amino acid sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are numbered according to the AbM numbering system; (iv) the amino acid sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are numbered according to the Contact numbering system; and / or (v) the amino acid sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are numbered according to the Contact numbering system. The antibody of embodiment 1, wherein the amino acid sequences of CDR2 and VL CDR3 are according to the IMGT numbering system. 3. An antibody that binds to IL-1β, (1)(i) a VH comprising a VH CDR1 having an amino acid sequence selected from SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO: 49, SEQ ID NO: 67, and SEQ ID NO: 85, a VH CDR2 having an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 32, SEQ ID NO: 50, SEQ ID NO: 68, and SEQ ID NO: 86, and a VH CDR3 having an amino acid sequence selected from SEQ ID NO: 15, SEQ ID NO: 33, SEQ ID NO: 51, SEQ ID NO: 69, and SEQ ID NO: 87; and (ii) a VL comprising a VL CDR1 having an amino acid sequence selected from SEQ ID NO: 16, SEQ ID NO: 34, SEQ ID NO: 52, SEQ ID NO: 70, and SEQ ID NO: 88, a VL CDR2 having an amino acid sequence selected from SEQ ID NO: 17, SEQ ID NO: 35, SEQ ID NO: 53, SEQ ID NO: 71, and SEQ ID NO: 89, and a VL CDR3 having an amino acid sequence selected from SEQ ID NO: 18, SEQ ID NO: 36, SEQ ID NO: 54, SEQ ID NO: 72, and SEQ ID NO: 90; (2)(i) a VH comprising a VH CDR1 having an amino acid sequence selected from SEQ ID NO:19, SEQ ID NO:37, SEQ ID NO:55, SEQ ID NO:73, and SEQ ID NO:91, a VH CDR2 having an amino acid sequence selected from SEQ ID NO:20, SEQ ID NO:38, SEQ ID NO:56, SEQ ID NO:74, and SEQ ID NO:92, and a VH CDR3 having an amino acid sequence selected from SEQ ID NO:21, SEQ ID NO:39, SEQ ID NO:57, SEQ ID NO:75, and SEQ ID NO:93; and (ii) a VL comprising a VL CDR1 having an amino acid sequence selected from SEQ ID NO:22, SEQ ID NO:40, SEQ ID NO:58, SEQ ID NO:76, and SEQ ID NO:94, a VL CDR2 having an amino acid sequence selected from SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:59, SEQ ID NO:77, and SEQ ID NO:95, and a VL CDR3 having an amino acid sequence selected from SEQ ID NO:24, SEQ ID NO:42, SEQ ID NO:60, SEQ ID NO:78, and SEQ ID NO:96, or (3)(i) a VH comprising a VH CDR1 having an amino acid sequence selected from SEQ ID NO:25, SEQ ID NO:43, SEQ ID NO:61, SEQ ID NO:79, and SEQ ID NO:97, a VH CDR2 having an amino acid sequence selected from SEQ ID NO:26, SEQ ID NO:44, SEQ ID NO:62, SEQ ID NO:80, and SEQ ID NO:98, and a VH CDR3 having an amino acid sequence selected from SEQ ID NO:27, SEQ ID NO:45, SEQ ID NO:63, SEQ ID NO:81, and SEQ ID NO:99; and (ii) an antibody comprising a VL CDR1 having an amino acid sequence selected from SEQ ID NO:28, SEQ ID NO:46, SEQ ID NO:64, SEQ ID NO:82, and SEQ ID NO:100, a VL CDR2 having an amino acid sequence selected from SEQ ID NO:29, SEQ ID NO:47, SEQ ID NO:65, SEQ ID NO:83, and SEQ ID NO:101, and a VL CDR3 having an amino acid sequence selected from SEQ ID NO:30, SEQ ID NO:48, SEQ ID NO:66, SEQ ID NO:84, and SEQ ID NO:102. 4. An antibody that binds to IL-1β, (1) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 13, a VH CDR2 having the amino acid sequence of SEQ ID NO: 14, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 15; and (ii) a VL comprising...

Claims

1. An antibody that binds to IL-1β, (1) (i) a VH having the amino acid sequence of SEQ ID NO: 7, including VH CDR1, VH CDR2, and VH CDR3, each having the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3; and (ii) a VL having the amino acid sequence of SEQ ID NO: 8, including VL CDR1, VL CDR2, and VL CDR3, each having the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3; (2) (i) a VH having the amino acid sequence of SEQ ID NO: 9, comprising VH CDR1, VH CDR2, and VH CDR3, each having the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3; and (ii) a VL having the amino acid sequence of SEQ ID NO: 10, comprising VL CDR1, VL CDR2, and VL CDR3, each having the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3; or (3) An antibody comprising: (i) a VH having the amino acid sequence of SEQ ID NO: 11, including VH CDR1, VH CDR2, and VH CDR3, each having the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3; and (ii) a VL having the amino acid sequence of SEQ ID NO: 12, including VL CDR1, VL CDR2, and VL CDR3, each having the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3.

2. (i) the amino acid sequences of the VH CDR1, the VH CDR2, the VH CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 are numbered according to the Kabat numbering system; (ii) the amino acid sequences of the VH CDR1, the VH CDR2, the VH CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 are numbered according to the Chothia numbering system; (iii) the amino acid sequences of the VH CDR1, the VH CDR2, the VH CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 are numbered according to the AbM numbering system; (iv) the amino acid sequences of the VH CDR1, the VH CDR2, the VH CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 are numbered according to the AbM numbering system; The antibody of claim 1, wherein (v) the amino acid sequences of the VH CDR1, the VH CDR2, the VH CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 are according to the Contact numbering system, and / or (v) the amino acid sequences of the VH CDR1, the VH CDR2, the VH CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 are according to the IMGT numbering system.

3. An antibody that binds to IL-1β, (1) (i) a VH comprising a VH CDR1 having an amino acid sequence selected from SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO: 49, SEQ ID NO: 67, and SEQ ID NO: 85; a VH CDR2 having an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 32, SEQ ID NO: 50, SEQ ID NO: 68, and SEQ ID NO: 86; and a VH CDR3 having an amino acid sequence selected from SEQ ID NO: 15, SEQ ID NO: 33, SEQ ID NO: 51, SEQ ID NO: 69, and SEQ ID NO: 87; and (ii) a VL comprising a VL CDR1 having an amino acid sequence selected from SEQ ID NO: 16, SEQ ID NO: 34, SEQ ID NO: 52, SEQ ID NO: 70, and SEQ ID NO: 88; a VL CDR2 having an amino acid sequence selected from SEQ ID NO: 17, SEQ ID NO: 35, SEQ ID NO: 53, SEQ ID NO: 71, and SEQ ID NO: 89; and a VL CDR3 having an amino acid sequence selected from SEQ ID NO: 18, SEQ ID NO: 36, SEQ ID NO: 54, SEQ ID NO: 72, and SEQ ID NO: 90; (2) (i) a VH comprising a VH CDR1 having an amino acid sequence selected from SEQ ID NO: 19, SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 73, and SEQ ID NO: 91; a VH CDR2 having an amino acid sequence selected from SEQ ID NO: 20, SEQ ID NO: 38, SEQ ID NO: 56, SEQ ID NO: 74, and SEQ ID NO: 92; and a VH CDR3 having an amino acid sequence selected from SEQ ID NO: 21, SEQ ID NO: 39, SEQ ID NO: 57, SEQ ID NO: 75, and SEQ ID NO: 93; and (ii) a VL comprising a VL CDR1 having an amino acid sequence selected from SEQ ID NO:22, SEQ ID NO:40, SEQ ID NO:58, SEQ ID NO:76, and SEQ ID NO:94, a VL CDR2 having an amino acid sequence selected from SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:59, SEQ ID NO:77, and SEQ ID NO:95, and a VL CDR3 having an amino acid sequence selected from SEQ ID NO:24, SEQ ID NO:42, SEQ ID NO:60, SEQ ID NO:78, and SEQ ID NO:96; or (3) (i) a VH comprising a VH CDR1 having an amino acid sequence selected from SEQ ID NO: 25, SEQ ID NO: 43, SEQ ID NO: 61, SEQ ID NO: 79, and SEQ ID NO: 97; a VH CDR2 having an amino acid sequence selected from SEQ ID NO: 26, SEQ ID NO: 44, SEQ ID NO: 62, SEQ ID NO: 80, and SEQ ID NO: 98; and a VH CDR3 having an amino acid sequence selected from SEQ ID NO: 27, SEQ ID NO: 45, SEQ ID NO: 63, SEQ ID NO: 81, and SEQ ID NO: 99; and (ii) an antibody comprising a VL comprising a VL CDR1 having an amino acid sequence selected from SEQ ID NO:28, SEQ ID NO:46, SEQ ID NO:64, SEQ ID NO:82, and SEQ ID NO:100; a VL CDR2 having an amino acid sequence selected from SEQ ID NO:29, SEQ ID NO:47, SEQ ID NO:65, SEQ ID NO:83, and SEQ ID NO:101; and a VL comprising a VL CDR3 having an amino acid sequence selected from SEQ ID NO:30, SEQ ID NO:48, SEQ ID NO:66, SEQ ID NO:84, and SEQ ID NO:

102.

4. An antibody that binds to IL-1β, (1) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 13, a VH CDR2 having the amino acid sequence of SEQ ID NO: 14, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 15; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 16, a VL CDR2 having the amino acid sequence of SEQ ID NO: 17, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 18; (2) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 19, a VH CDR2 having the amino acid sequence of SEQ ID NO: 20, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 21; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 22, a VL CDR2 having the amino acid sequence of SEQ ID NO: 23, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 24; (3) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 25, a VH CDR2 having the amino acid sequence of SEQ ID NO: 26, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 27; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 28, a VL CDR2 having the amino acid sequence of SEQ ID NO: 29, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 30; (4) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 31, a VH CDR2 having the amino acid sequence of SEQ ID NO: 32, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 33; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 34, a VL CDR2 having the amino acid sequence of SEQ ID NO: 35, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 36; (5) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 37, a VH CDR2 having the amino acid sequence of SEQ ID NO: 38, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 39; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 40, a VL CDR2 having the amino acid sequence of SEQ ID NO: 41, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 42; (6) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 43, a VH CDR2 having the amino acid sequence of SEQ ID NO: 44, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 45; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 46, a VL CDR2 having the amino acid sequence of SEQ ID NO: 47, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 48; (7) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 49, a VH CDR2 having the amino acid sequence of SEQ ID NO: 50, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 51; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 52, a VL CDR2 having the amino acid sequence of SEQ ID NO: 53, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 54; (8) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 55, a VH CDR2 having the amino acid sequence of SEQ ID NO: 56, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 57; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 58, a VL CDR2 having the amino acid sequence of SEQ ID NO: 59, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 60; (9) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 61, a VH CDR2 having the amino acid sequence of SEQ ID NO: 62, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 63; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 64, a VL CDR2 having the amino acid sequence of SEQ ID NO: 65, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 66; (10) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 67, a VH CDR2 having the amino acid sequence of SEQ ID NO: 68, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 69; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 70, a VL CDR2 having the amino acid sequence of SEQ ID NO: 71, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 72; (11) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 73, a VH CDR2 having the amino acid sequence of SEQ ID NO: 74, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 75; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 76, a VL CDR2 having the amino acid sequence of SEQ ID NO: 77, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 78; (12) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 79, a VH CDR2 having the amino acid sequence of SEQ ID NO: 80, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 81; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 82, a VL CDR2 having the amino acid sequence of SEQ ID NO: 83, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 84; (13) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 85, a VH CDR2 having the amino acid sequence of SEQ ID NO: 86, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 87; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 88, a VL CDR2 having the amino acid sequence of SEQ ID NO: 89, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 90; (14) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 91, a VH CDR2 having the amino acid sequence of SEQ ID NO: 92, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 93; and (ii) a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 94, a VL CDR2 having the amino acid sequence of SEQ ID NO: 95, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 96; or (15) (i) a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 97, a VH CDR2 having the amino acid sequence of SEQ ID NO: 98, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 99; and (ii) An antibody comprising a VL having a VL CDR1 having the amino acid sequence of SEQ ID NO: 100, a VL CDR2 having the amino acid sequence of SEQ ID NO: 101, and a VL CDR3 having the amino acid sequence of SEQ ID NO:

102.

5. The antibody of claim 1, wherein the antibody further comprises one or more framework regions set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and / or SEQ ID NO:

12.

6. (i) the antibody comprises a VH having the amino acid sequence of SEQ ID NO: 7 and a VL having the amino acid sequence of SEQ ID NO: 8; (ii) the antibody comprises a VH having the amino acid sequence of SEQ ID NO: 9 and a VL having the amino acid sequence of SEQ ID NO: 10; or (iii) the antibody comprises a VH having the amino acid sequence of SEQ ID NO: 11 and a VL having the amino acid sequence of SEQ ID NO: 12; The antibody described in claim 1.

7. A nucleic acid encoding the antibody of any one of claims 1 to 6.

8. A kit comprising the antibody of any one of claims 1 to 6 and a package therefor.

9. A pharmaceutical composition comprising the antibody of any one of claims 1 to 6 and one or more pharmaceutically acceptable excipients.

10. 10. A composition for use in a method of treating an IL-1β mediated disease or disorder in a subject, the composition comprising an antibody of any one of claims 1 to 6, the method comprising administering the composition to the subject.

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