Agents that interfere with il-1β receptor signalling

Abstract

To provide antibodies that specifically bind interleukin-1β (IL-1β).SOLUTION: The present invention provides an isolated humanized antibody or an antigen-binding fragment thereof that specifically binds to human IL-1β with potency equivalent to that of a mouse monoclonal antibody having a heavy chain variable region and a light chain variable region with a specific sequence. The present invention also provides the use of such an antibody for the therapeutic treatment of IL-1β-mediated diseases and disorders.SELECTED DRAWING: None

Description

[Technical field]

[0001] Priority This application claims priority to U.S. Provisional Patent Application No. 62 / 963,654, filed January 21, 2020, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on July 8, 2020, is named 15271_0001-00304_SL.txt, and is 54,286 bytes in size. [Background technology]

[0003] background Human interleukin 1β (IL-1β) is a proinflammatory cytokine that acts as a mediator of peripheral immune responses during infection and inflammation. IL-1β is initially synthesized in the form of a precursor peptide (pro-IL-1β), which is cleaved by caspase-1 in the inflammasome complex and secreted extracellularly. IL-1β can be released by a variety of cell types.

[0004] There are two IL-1 receptors, IL-1RI and IL-1RII. IL-1β exerts its effects on target cells through the receptor IL-1RI. Dysregulated IL-1β activity is characteristic of autoimmune diseases and can occur either due to abnormally increased levels of cytokines or due to a qualitative or quantitative defect in the IL-1RI endogenous antagonist. IL-1β has been specifically implicated in several autoinflammatory diseases.

[0005] Canakinumab (Ilaris, ACZ885) is a human monoclonal antibody developed by Novartis that targets interleukin-1β. Canakinumab's mode of action is based on neutralization of IL-1β signaling (Alten, Gram et al. 2008). Canakinumab was approved in 2009 for the treatment of cryopyrin-associated periodic syndromes (CAPS), followed by approval in 2016 for three additional rare and severe autoinflammatory diseases (Gram 2016).

[0006] Gevokizumab (XOMA052) is another monoclonal antibody targeting IL-1β developed by XOMA. Gevokizumab is claimed to be a regulatory therapeutic antibody that modulates IL-1β bioactivity by reducing its affinity for the IL-1RI:IL-1RAcP signaling complex (Owyang, Issafras et al. 2011) (Issafras, Corbin et al. 2013).

[0007] Recently, IL-1β, as well as other cardiovascular disease modifiers, have been found to be involved in several steps in the development of atherosclerotic plaques (McCarty and Frishman 2014). The hypothesis is that these inflammatory chemicals may prevent the heart from healing from damage caused by a previous heart attack. In 2017, a Phase III clinical trial with canakinumab revealed a combined 15% reduction in heart attacks, strokes, and deaths from cardiovascular disease. The trial also revealed a significant reduction in lung cancer incidence and mortality.

[0008] Given the efficacy of anti-IL-1β antibodies in autoinflammatory diseases, heart attack and lung cancer, there is a need for the development of additional anti-IL-1β antibodies with improved efficacy and pharmacokinetic profiles. Summary of the Invention

[0009] overview The present disclosure provides monoclonal antibodies and antigen-binding fragments thereof that specifically bind to and neutralize, inhibit, block, suppress, reduce, or interfere with at least one activity of IL-1β. Activities of IL-1β that can be neutralized, inhibited, blocked, suppressed, reduced, or interfered with by the antibodies or fragments thereof disclosed herein include, but are not limited to, neutralization of IL-1β activation of its receptor, IL-1RI.

[0010] The present disclosure provides a murine monoclonal antibody against human IL-1β, designated TAVO304, comprising a heavy chain variable region sequence having the amino acid sequence of SEQ ID NO:1 and a light chain variable region sequence having the amino acid sequence of SEQ ID NO:2.

[0011] The present disclosure provides a heavy chain variable region of TAVO304, which comprises three complementarity determining regions (CDRs) designated HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth as SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively.

[0012] The present disclosure provides the light chain variable region of TAVO304, which comprises three CDRs designated LCDR1, LCDR2 and LCDR3, having the amino acid sequences set forth as SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0013] The present disclosure provides one humanized heavy chain variable region of TAVO304, designated 304VH1, having the amino acid sequence set forth as SEQ ID NO:9.

[0014] The present disclosure provides four humanized light chain variable regions of TAVO304, designated 304VL1, 304VL2, 304VL3 and 304VL4, having the amino acid sequences set forth as SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, respectively.

[0015] The present disclosure provides a first humanized antibody against TAVO304, designated TAVO7376, which comprises a humanized heavy chain variable region 304VH1 and a humanized light chain variable region 304VL1, comprising the heavy chain sequence shown as SEQ ID NO:14 and the light chain sequence shown as SEQ ID NO:15.

[0016] The present disclosure provides a second humanized antibody against TAVO304, designated TAVO7377, which comprises a humanized heavy chain variable region 304VH1 and a humanized light chain variable region 304VL2, comprising the heavy chain sequence shown as SEQ ID NO:14 and the light chain sequence shown as SEQ ID NO:16.

[0017] The present disclosure provides a third humanized antibody against TAVO304, designated TAVO7378, which comprises a humanized heavy chain variable region 304VH1 and a humanized light chain variable region 304VL3, comprising the heavy chain sequence shown as SEQ ID NO:14 and the light chain sequence shown as SEQ ID NO:17.

[0018] The present disclosure provides a fourth humanized antibody against TAVO304, designated TAVO7379, which comprises a humanized heavy chain variable region 304VH1 and a humanized light chain variable region 304VL4, comprising the heavy chain sequence shown as SEQ ID NO:14 and the light chain sequence shown as SEQ ID NO:18.

[0019] The antibodies disclosed herein can be full-length IgG1, IgG2, IgG3, IgG4 antibodies or may comprise only the antigen-binding portion, including Fab, F(ab')2, or scFv fragments. The antibody scaffold may be modified to affect functionality, for example to eliminate residual effector functions.

[0020] The present disclosure also provides isolated engineered anti-IL-1β IgG antibodies having an extended half-life when compared to the parent wild-type antibody. The extended half-life can be achieved by increasing the C value of the antibody with any one set of mutations selected from M252Y / S254T / T256E, M428L / N434S, T250Q / M428L, N434A and T307A / E380A / N434A (residue numbering according to the EU index) when compared to the parent wild-type antibody. H2 and C H3 This can be achieved by manipulating the domain.

[0021] The disclosure also provides isolated engineered anti-IL-1β IgG antibodies that have enhanced resistance to proteolysis by proteases that cleave the wild-type antibody at or between residues 222-237 (EU numbering). Resistance to proteolysis can be achieved by engineering E233P / L234V / L235A mutations (residue numbering is according to the EU index) into the hinge region that has a G236 deletion when compared to the parent wild-type antibody.

[0022] The present disclosure also provides polynucleotides comprising polynucleotide sequences that encode the polypeptide sequences of the anti-IL-1β monoclonal antibodies and antigen-binding fragments disclosed herein.

[0023] The present disclosure also provides vectors comprising the polynucleotides disclosed herein.

[0024] The present disclosure also provides a host cell comprising the vectors disclosed herein.

[0025] The present disclosure also provides a method of producing an anti-IL-1β monoclonal antibody disclosed herein, comprising culturing a host cell disclosed herein under conditions in which the antibody is expressed, and purifying the antibody.

[0026] The present disclosure also provides a pharmaceutical composition comprising an anti-IL-1β monoclonal antibody disclosed herein and a pharma- ceutically acceptable carrier.

[0027] The present disclosure also provides a method for detecting binding of an anti-IL-1β monoclonal antibody to IL-1β.

[0028] The present disclosure also provides a method of blocking the binding of IL-1β to its receptor, IL-1RI, by an anti-IL-1β monoclonal antibody to IL-1β.

[0029] The present disclosure also provides a method of neutralizing the functional activity of IL-1β against its receptor, IL-1RI, by an anti-IL-1β monoclonal antibody against IL-1β.

[0030] The present disclosure also provides methods for determining the half-life of an engineered anti-IL-1β monoclonal antibody.

[0031] The present disclosure also provides methods for determining the resistance of an engineered anti-IL-1β monoclonal antibody to proteolysis.

[0032] The present disclosure also provides a method of treating an anti-inflammatory disease in a subject comprising administering a therapeutically effective amount of an anti-IL-1β monoclonal antibody.

[0033] The present disclosure also provides a method of treating cardiovascular disease in a subject, comprising administering a therapeutically effective amount of an anti-IL-1β monoclonal antibody.

[0034] The present disclosure also provides a method of treating lung cancer in a subject, comprising administering a therapeutically effective amount of an anti-IL-1β monoclonal antibody.

[0035] Any embodiment of the disclosure described herein, including those described only in a section of the specification describing specific aspects of the disclosure and those described only in the examples or drawings, may be combined with any other one or more embodiments, unless expressly disclaimed or inappropriate. BRIEF DESCRIPTION OF THE DRAWINGS [Brief description of the drawings]

[0036] [Figure 1] Binding to (A) human, (B) rhesus and (C) mouse IL-1β by mouse monoclonal anti-human IL-1β antibody TAVO304.

[0037] [Diagram 2] Response of reporter gene expression to stimulation with human and rhesus IL-1β in the HEK-Blue IL-1β reporter assay.

[0038] [Diagram 3] Neutralization of (A) human or (B) rhesus IL-1β-driven reporter gene activation by TAVO304 or reference antibody refAb-1 (canakinumab) in a HEK-Blue reporter assay.

[0039] [Figure 4] Sequence alignment of TAVO304 heavy and light chain variable regions with humanized VH and VL variants. Four humanized antibodies can be formed by pairing the humanized VH variant (304VH1) with the four humanized VL variants (304VL1, 304VL2, 304VL3, and 304VL4). Figure 4 discloses, in order of appearance, SEQ ID NOs: 2, 10-13, 1, and 9, respectively.

[0040] [Diagram 5] SDS-PAGE analysis of four humanized anti-IL-1β IgG1 antibodies under (A) non-reducing and (B) reducing conditions.

[0041] [Figure 6] Binding to (A) human and (B) rhesus IL-1β by four humanized anti-IL-1b IgG1 antibodies.

[0042] [Figure 7] Neutralization of (A) human and (B) rhesus IL-1β-driven reporter gene activation by four humanized anti-IL-1β IgG1 antibodies in a HEK-Blue reporter assay.

[0043] [Figure 8] Neutralization of human IL-1β-driven IL-6 release from human lung fibroblast MRC-5 cells by four humanized anti-IL-1β IgG1 antibodies or the reference antibody refAb-1 (canakinumab) in an MRC-5 cytokine release assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] Detailed Description of the Disclosure <Definition> All publications cited herein, including but not limited to patents and patent applications, are hereby incorporated by reference as if fully set forth.

[0045] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. 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 disclosure belongs.

[0046] Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure, exemplary materials and methods are described herein.

[0047] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and so forth.

[0048] "Antibody" is interpreted broadly and includes immunoglobulin molecules, including monoclonal antibodies, including murine, human, humanized and chimeric monoclonal antibodies, antibody fragments, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies, and any other modified configuration of an immunoglobulin molecule which contains an antigen binding site of the required specificity.

[0049] A "full-length antibody molecule" is composed of two heavy chains (HC) and two light chains (LC) inter-connected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (composed of CH1, hinge, CH2, and CH3 domains). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions may be further divided into regions of high variability called complementarity determining regions (CDRs) separated by framework regions (FRs). Each VH and VL is composed of three CDR and four FR segments, respectively, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0050] The "complementarity determining region (CDR)" is the "antigen binding site" in an antibody. CDRs can be defined using various terms: (i) complementarity determining regions (CDRs), three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3), are based on sequence variability (Wu et al. (1970), J Exp Med 132: 211-50 (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). (ii) "hypervariable region", "HVR", or "HV", three in VH (H1, H2, H3) and three in VL (L1, L2, L3), refer to the regions of an antibody variable domain that are hypervariable in the structure defined by Chothia and Lesk (Chothia et al. (1987), J Mol Biol 196: 901-17). The International ImMunoGeneTics (IMGT) database (http: / / www_imgt_org) provides standardized numbering and definition of antigen binding sites. The correspondence of CDRs, HVs, and IMGT delineations are described in Lefranc et al. (2003), Dev Comp Immunol 27: 55-77. The terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2" and "LCDR3" as used herein include CDRs defined by any of the methods described in Kabat, Chothia or IMGT, above, unless expressly stated otherwise in the specification.

[0051] Immunoglobulins can be assigned to five major classes, IgA, IgD, IgE, IgG, and IgM, depending on their heavy chain constant region amino acid sequences. IgA and IgG are further subclassified as IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4 isotypes. Antibody light chains of any vertebrate species can be assigned to one of two types, kappa (κ) and lambda (λ), based on the amino acid sequences of their constant regions.

[0052] "Antibody fragment" refers to a portion of an immunoglobulin molecule that retains the heavy and / or light chain antigen-binding sites, e.g., heavy chain complementarity determining regions (HCDR) 1, 2 and 3, light chain complementarity determining regions (LCDR) 1, 2 and 3, heavy chain variable region (VH), or light chain variable region (VL). Antibody fragments include Fab, F(ab')2, Fd and Fv fragments, as well as domain antibodies (dAbs), e.g., consisting of one VH domain. The VH and VL domains may be linked via synthetic linkers to form various types of single chain antibody designs, where the VH / VL domains may be paired intramolecularly or intermolecularly, such as when the VH and VL domains are expressed by separate single chain antibody constructs, to form monovalent antigen-binding sites, such as single chain Fvs (scFvs), or bispecific antibodies. For example, they are described in International Publication Nos. WO1998 / 44001, WO1988 / 01649, WO1994 / 13804 and WO1992 / 01047.

[0053] "Monoclonal antibody" refers to an antibody population having a single amino acid composition in each heavy and light chain, except for known possible modifications such as removal of the C-terminal lysine from the antibody heavy chain. A monoclonal antibody typically binds to one antigenic epitope, except that a bispecific monoclonal antibody binds to two distinct antigenic epitopes. A monoclonal antibody may have heterogeneous glycosylation within the antibody population. A monoclonal antibody may be monospecific or polyspecific, or may be monovalent, bivalent or multivalent. Bispecific antibodies are included in the term monoclonal antibody.

[0054] An "isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies having distinct antigenic specificities. An "isolated antibody" encompasses antibodies that have been isolated to a sufficient degree of purity, e.g., antibodies that are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure.

[0055] "Humanized antibody" refers to an antibody in which the antigen binding site is derived from a non-human species and the variable region framework is derived from a human immunoglobulin sequence. Humanized antibodies may contain substitutions within the framework, and the framework may not be an exact copy of an expressed human immunoglobulin or human immunoglobulin germline gene sequence.

[0056] "Human antibody" refers to an antibody having heavy and light chain variable regions in which both the framework and antigen binding sites are derived from sequences of human origin and which are optimized to have a minimal immune response when administered to a human subject. If the antibody contains a constant region or a portion of a constant region, the constant region also is derived from sequences of human origin.

[0057] Throughout this specification, the numbering of amino acid residues in antibody constant regions is according to the EU index as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), unless expressly stated otherwise.

[0058] In this specification, conventional one-letter and three-letter amino acid codes are used, as shown in Table 1. [Table 1]

[0059] The constant region sequence of the mammalian IgG heavy chain is C H1 - Hinge-C H2 -C H3 The "hinge", "hinge region" or "hinge domain" of IgG is generally defined as including Glu216 and ending at Pro230 of human IgG1 according to the EU index, although functionally the flexible portion of the chain may be considered to include additional residues designated the upper and lower hinge regions, such as from Glu216 to Gly237, with the lower hinge referring to residues 233-239 of the Fc region to which FcγR binding is generally ascribed. Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy chain S-S bonds. Numbering is according to the EU index, so the boundaries may vary slightly, but the C H1 The domain is V H Adjacent to the Fc domain and amino-terminal to the hinge region of the immunoglobulin heavy chain molecule, e.g., from about EU positions 118 to 215, is the first (most amino-terminal) constant region of the immunoglobulin heavy chain. The Fc domain extends from amino acid 231 to amino acid 447; H2 The domain is from Ala231 to Lys340 or Gly341, C H3 is from Gly341 or Gln342 to Lys447. H1 The IgG heavy chain constant region residues in the region end with Lys. Fc domain-containing molecules comprise at least C of an antibody constant region. H2 and C H3 domain, and thus comprises at least the region of the IgG heavy chain constant region from about Ala231 to Lys447. The Fc domain-containing molecule may optionally also include at least a portion of the hinge region.

[0060] "Epitope" refers to the portion of an antigen to which an antibody specifically binds. An epitope typically consists of an active (such as polar, non-polar or hydrophobic) surface grouping of moieties such as amino acids or polysaccharide side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be composed of contiguous and / or discontinuous amino acids that form a conformational space unit. For discontinuous epitopes, amino acids from different parts of the antigen's linear sequence are in close proximity in three-dimensional space through folding of the antigen molecule. Antibody "epitopes" depend on the methodology used to identify the epitope.

[0061] As used herein, a "leader sequence" includes any signal peptide that can be processed by a mammalian cell, including the human B2M leader. Such sequences are well known in the art.

[0062] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. These terms also include polypeptides having co-translational modifications of the polypeptide (e.g., signal peptide cleavage) and post-translational modifications, such as disulfide bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage, etc.

[0063] Furthermore, as used herein, "polypeptide" refers to a protein containing modifications such as deletions, additions, and substitutions (e.g., of a conservative nature as would be known to one of skill in the art) to the native sequence, so long as the protein maintains a desired activity. These modifications can be deliberate, such as through site-directed mutagenesis, or can be accidental, such as through mutations of hosts that produce the protein or errors in PCR amplification or other recombinant DNA techniques.

[0064] The term "recombinant" as used herein with respect to a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, and / or synthetic origin that is not associated, by reason of its origin or manipulation, with all or part of the polynucleotide sequence with which it is naturally associated. The term "recombinant" as used herein with respect to a protein or polypeptide refers to a polypeptide produced by expression from a recombinant polynucleotide. The term "recombinant" as used with respect to a host cell or virus refers to a host cell or virus into which a recombinant polynucleotide has been introduced. Recombinant is also used herein with respect to an entity (e.g., a cell, nucleic acid, protein, or vector) to refer to that entity having been altered by the introduction of a heterologous entity (e.g., a cell, nucleic acid, protein, or vector).

[0065] The terms "polynucleotide," "oligonucleotide," "nucleic acid," and "nucleic acid molecule" are used interchangeably herein and include polymeric forms of nucleotides, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule.

[0066] "Vector" refers to a polynucleotide that can be replicated in a biological system or can be transferred between such systems. Vector polynucleotides typically contain elements such as origins of replication, polyadenylation signals, or selection markers that function to facilitate the replication or maintenance of these polynucleotides in biological systems such as cells, viruses, animals, plants, and biological systems reconstituted with biological components capable of replicating the vector. Vector polynucleotides may be single-stranded (single-stranded) or double-stranded, DNA or RNA molecules, cDNA, or hybrids thereof.

[0067] An "expression vector" refers to a vector that can be utilized to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector in a biological system or in a reconstituted biological system.

[0068] "Valency" refers to the presence of a particular number of specific binding sites for a given antigen within a molecule. Thus, the terms "monovalent," "bivalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six specific binding sites for a given antigen within a molecule, respectively.

[0069] As used herein, the term "heterologous" when used with respect to a nucleic acid sequence, protein or polypeptide means that these molecules do not naturally occur in the cell from which the heterologous nucleic acid sequence, protein or polypeptide was derived. For example, a nucleic acid sequence encoding a human polypeptide inserted into a cell that is not a human cell is a heterologous nucleic acid sequence in that particular context. While a heterologous nucleic acid may be derived from a different organism or animal species, such a nucleic acid need not be derived from another organism species to be heterologous. For example, in some cases, a synthetic nucleic acid sequence or a polypeptide encoded therein can be heterologous to a cell into which it is introduced, in that the cell did not previously contain the synthetic nucleic acid. Thus, for example, a synthetic nucleic acid sequence or a polypeptide encoded therein can be considered heterologous to a human cell, even if one or more components of the synthetic nucleic acid sequence or the polypeptide encoded therein were originally derived from a human cell.

[0070] As used herein, a "host cell" refers to an in vivo or in vitro eukaryotic cell, or a cell derived from a multicellular organism cultured as a unicellular organism (e.g., a cell line), which can be or has been used as a recipient of a nucleic acid (e.g., an expression vector comprising a nucleotide sequence encoding a multimeric polypeptide of the present disclosure), including the progeny of the original cell that has been genetically modified by the nucleic acid. It is understood that the progeny of a single cell need not necessarily be completely identical in morphology or in genome or total DNA complement to the original parent due to natural, accidental or deliberate mutations. A "recombinant host cell" (also referred to as a "genetically modified host cell") is a host cell into which a heterologous nucleic acid, e.g., an expression vector, has been introduced. For example, a genetically modified eukaryotic host cell has been genetically modified by the introduction into a suitable eukaryotic host cell of a heterologous nucleic acid, e.g., a foreign nucleic acid that is foreign to the eukaryotic host cell or a recombinant nucleic acid not normally found in the eukaryotic host cell.

[0071] "Specific binding" or "specifically binds" or "binds" refers to an antibody binding to a specific antigen with higher affinity than to other antigens. Typically, an antibody has an equilibrium dissociation constant (KD) for that binding of about 1×10 -8 M or less, for example, about 1×10 -9 M or less, approximately 1×10 -10 M or less, approximately 1×10 -11 M or less, or about 1 x 10 -12 An antibody "specifically binds" when it has a KD that is equal to or less than M, typically at least 100-fold lower than the KD for binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using standard procedures.

[0072] As used herein, the terms "treatment", "treating" and the like refer to obtaining a desired pharmaceutical and / or physiological effect. The effect may be prophylactic, meaning a complete or partial prevention of a disease or its symptoms, and / or therapeutic, meaning a partial or complete cure of a disease and / or adverse effects attributable to the disease. As used herein, "treatment" encompasses any treatment of disease in a mammal, e.g., a human, including (a) preventing the onset of disease in a subject who may be predisposed to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., halting the progression of the disease; and (c) relieving the disease, i.e., causing regression of the disease.

[0073] The terms "individual," "subject," "host," and "patient," as used interchangeably herein, refer to mammals, including, but not limited to, murines (e.g., rats, mice), lagomorphs (e.g., rabbits), non-human primates, humans, canines, felines, cloven-hoofed animals (e.g., horses, cattle, sheep, pigs, goats), and the like.

[0074] A "therapeutically effective amount" or "effective amount" refers to the amount of an agent, or the combined amount of two agents, that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. A "therapeutically effective amount" will vary depending on the agent(s), the disease and its severity, and the age, weight, etc., of the subject to be treated.

[0075] It is to be understood that this disclosure is not limited to particular embodiments described, as these may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0076] Described herein are monoclonal antibodies and antigen-binding fragments thereof that specifically bind and neutralize, inhibit, block, suppress, reduce, or interfere with at least one activity of IL-1β, such as the functional activity of IL-1β on its receptor IL-1RI. These anti-IL-1β antibodies and antigen-binding fragments can be administered therapeutically to a subject to treat an IL-1β mediated disease. <Composition of anti-IL-1β antibodies and antigen-binding fragments>

[0077] The present disclosure provides a murine monoclonal antibody, designated TAVO304, identified from a murine hybridoma screen that specifically binds to and neutralizes, inhibits, blocks, suppresses, reduces, or interferes with at least one activity of human interleukin 1 beta (IL-1β). TAVO304 comprises a heavy chain variable region sequence having the amino acid sequence of SEQ ID NO:1, and a light chain variable region sequence having the amino acid sequence of SEQ ID NO:2. The heavy chain variable region of TAVO304 comprises three complementarity determining regions, designated HCDR1, HCDR2, and HCDR3, having the amino acid sequences set forth as SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively. The light chain variable region of TAVO304 comprises three complementarity determining regions, designated LCDR1, LCDR2, and LCDR3, having the amino acid sequences set forth as SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively.

[0078] Heavy chain variable region sequence of anti-human IL-1β mouse monoclonal antibody TAVO304 (SEQ ID NO:1)

[0079] [ka]

[0080] The sequences of the three CDRs of the heavy chain are underlined.

[0081] Light chain variable region sequence of anti-human IL-1β mouse monoclonal antibody TAVO304 (SEQ ID NO:2)

[0082] [ka]

[0083] The sequences of the three CDRs of the light chain are underlined.

[0084] HCDR1 sequence of anti-human IL-1β mouse monoclonal antibody TAVO304 (SEQ ID NO:3)

[0085] [ka]

[0086] HCDR2 sequence of anti-human IL-1β mouse monoclonal antibody TAVO304 (SEQ ID NO:4)

[0087] [ka]

[0088] HCDR3 sequence of anti-human IL-1β mouse monoclonal antibody TAVO304 (SEQ ID NO:5)

[0089] [ka]

[0090] LCDR1 sequence of anti-human IL-1β mouse monoclonal antibody TAVO304 (SEQ ID NO:6)

[0091] [ka]

[0092] LCDR2 sequence of anti-human IL-1β mouse monoclonal antibody TAVO304 (SEQ ID NO:7)

[0093] [ka]

[0094] LCDR3 sequence of anti-human IL-1β mouse monoclonal antibody TAVO304 (SEQ ID NO:8)

[0095] [ka]

[0096] The murine anti-human IL-1β antibody TAVO304 can be humanized by grafting murine CDRs onto a human germline scaffold. A few key murine residues are preserved by backmutation to achieve higher stability and better expression while minimizing immunogenicity. For TAVO304, one humanized VH variant (304VH1) was engineered to target IGHV3-48. * Four humanized VL variants were designed based on IGKV1-39. * IGKV2-40 using 304VL1 and 304VL2 based on 01 * 01 based on 304VL3 and 304VL4, with a set of back mutations designed.

[0097] Based on the above, the present disclosure provides one humanized heavy chain variable region of TAVO304, designated 304VH1, having the amino acid sequence set forth as SEQ ID NO:9.

[0098] Humanized heavy chain variable region 304 VH1 sequence (SEQ ID NO:9)

[0099] [ka]

[0100] The sequences of the three CDRs of the humanized heavy chain are underlined.

[0101] The present disclosure provides four humanized light chain variable regions of TAVO304, designated 304VL1, 304VL2, 304VL3 and 304VL4, having the amino acid sequences set forth as SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, respectively.

[0102] Humanized light chain variable region 304VL1 sequence (SEQ ID NO:10)

[0103] [ka]

[0104] The sequences of the three CDRs of the humanized light chain are underlined.

[0105] Humanized light chain variable region 304VL2 sequence (SEQ ID NO:11)

[0106] [ka]

[0107] The sequences of the three CDRs of the humanized light chain are underlined.

[0108] Humanized light chain variable region 304VL3 sequence (SEQ ID NO:12)

[0109] [ka]

[0110] The sequences of the three CDRs of the humanized light chain are underlined.

[0111] Humanized light chain variable region 304VL4 sequence (SEQ ID NO:13)

[0112] [ka]

[0113] The sequences of the three CDRs of the humanized light chain are underlined.

[0114] Four humanized antibodies against TAVO304 can be generated by pairing the humanized 304VH1 heavy chain with four humanized light chains. The present disclosure provides one humanized IgG1 antibody against TAVO304, designated TAVO7376, comprising a humanized heavy chain variable region 304VH1 and a humanized light chain variable region 304VL1, comprising the heavy chain sequence shown as SEQ ID NO:14 and the light chain sequence shown as SEQ ID NO:15.

[0115] The present disclosure provides a second humanized IgG1 antibody against TAVO304, designated TAVO7377, comprising a humanized heavy chain variable region 304VH1 and a humanized light chain variable region 304VL2, the heavy chain sequence of which is shown as SEQ ID NO:14 and the light chain sequence of which is shown as SEQ ID NO:16.

[0116] The present disclosure provides a third humanized IgG1 antibody against TAVO304, designated TAVO7378, comprising a humanized heavy chain variable region 304VH1 and a humanized light chain variable region 304VL3, the humanized heavy chain sequence being shown as SEQ ID NO:14 and the light chain sequence being shown as SEQ ID NO:17.

[0117] The present disclosure provides a fourth humanized IgG1 antibody against TAVO304, designated TAVO7379, comprising a humanized heavy chain variable region 304VH1 and a humanized light chain variable region 304VL4, comprising the heavy chain sequence shown as SEQ ID NO:14 and the light chain sequence shown as SEQ ID NO:18.

[0118] Anti-human IL-1β humanized IgG1 antibody heavy chain based on 304VH1 (SEQ ID NO: 14)

[0119] [ka]

[0120] The sequence of the heavy chain variable domain is underlined, and the K409R mutation is shown in bold.

[0121] Anti-human IL-1β humanized IgG1 antibody light chain based on 304VL1 (SEQ ID NO: 15)

[0122] [ka]

[0123] The light chain variable domain sequence is underlined.

[0124] Anti-human IL-1β humanized IgG1 antibody light chain based on 304VL2 (SEQ ID NO: 16)

[0125] [ka]

[0126] The light chain variable domain sequence is underlined.

[0127] Anti-human IL-1β humanized IgG1 antibody light chain based on 304VL3 (SEQ ID NO: 17)

[0128] [ka]

[0129] The light chain variable domain sequence is underlined.

[0130] Anti-human IL-1β humanized IgG1 antibody light chain based on 304VL4 (SEQ ID NO: 18)

[0131] [ka]

[0132] The light chain variable domain sequence is underlined.

[0133] The present disclosure also provides for the preparation of bispecific or multispecific antibodies that can include engagement of any two, three, or four IL-1β epitopes by having a Fab or scFv domain that includes one humanized heavy chain variable region of TAVO304, designated 304VH1, having the amino acid sequence set forth as SEQ ID NO:9, together with one of four humanized light chain variable regions of TAVO304, designated 304VL1, 304VL2, 304VL3, and 304VL4, having the amino acid sequences set forth as SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively. For example, it can be a bispecific antibody having Fab or scFv domains with 304VH1 and 304VL1 and 304VH1 and 304VL2; 304VH1 and 304VL1 and 304VH1 and 304VL3; 304VH1 and 304VL1 and 304VH1 and 304VL4; or any other permutation of domains that can engage 2, 3, or 4 epitopes.

[0134] The IL-1β binding antibodies and fragments of the present disclosure include antigen-binding fragments that retain the full ability to specifically bind to IL-1β. As used herein, an IL-1β binding fragment may contain any three or more consecutive amino acids of an antibody (e.g., four or more, five or more, six or more, eight or more, or even ten or more consecutive amino acids), and includes Fab, Fab', F(ab')2, and F(v) fragments, or individual light or heavy chain variable regions or portions thereof. These fragments lack the Fc fragment of intact antibodies, may be cleared more rapidly from the circulation, and may have less nonspecific tissue binding than intact antibodies. These fragments can be produced from intact antibodies using well-known methods, for example, by proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).

[0135] The IL-1β binding antibodies and fragments of the present disclosure may also include bispecific antibodies, which are bivalent antibodies in which the VH and VL domains are expressed on a single polypeptide chain but use a short linker that does not allow pairing between the two domains on the same chain, thereby allowing the domains to pair with complementary domains on another chain and create two antigen-binding sites.

[0136] The IL-1β binding antibodies and fragments of the present disclosure may also include single chain antibody fragments (scFv) that bind IL-1β. An scFv is an antibody light chain variable region (VLCV) in which the heavy chain variable region and the light chain variable region together or individually form a binding site that binds IL-1β. L operably linked to an antibody heavy chain variable region (V H Such IL-1β binding fragments can be prepared by methods known in the art, such as, for example, synthesis or PCR-mediated amplification of the variable portions of the heavy and light chains of an antibody molecule and a flexible protein linker composed of the amino acids Gly and Ser. The resulting DNA fragment is cloned into E. coli or mammalian cells for expression. The expressed IL-1β binding fragment is purified from the host cells.

[0137] The IL-1β binding antibodies and fragments of the present disclosure include full-length antibodies comprising two heavy chains and two light chains. Exemplary human or humanized antibodies include IgG, IgM, IgE, IgA, and IgD antibodies. The antibodies can be of any class (IgG, IgM, IgE, IgGA, IgD, etc.) or isotype. For example, a human antibody can comprise an IgG Fc domain, such as at least one of the following isotypes: IgG1, IgG2, IgG3, or IgG4.

[0138] In some cases, the IgG Fc domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the IgG1 Fc sequence of SEQ ID NO:19.

[0139] In some cases, the IgG Fc domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the IgG2 Fc sequence of SEQ ID NO:20.

[0140] In some cases, the IgG Fc domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the IgG3 Fc sequence of SEQ ID NO:21.

[0141] In some cases, the IgG Fc domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the IgG4 Fc sequence of SEQ ID NO:22.

[0142] The S228P mutation may be made in an IgG4 antibody to enhance IgG4 stability.

[0143] IgG1Fc (SEQ ID NO: 19):

[0144] [ka]

[0145] IgG2Fc (SEQ ID NO: 20):

[0146] [ka]

[0147] IgG3Fc (SEQ ID NO:21):

[0148] [ka]

[0149] IgG4Fc (SEQ ID NO:22)

[0150] [ka]

[0151] The anti-IL-1β antibodies may comprise an altered Fc region, where the altered Fc region comprises at least one amino acid alteration relative to the wild-type Fc region. In some embodiments, the anti-IL-1β antibodies are provided with an altered Fc region, where the native Fc region has been altered such that the half-life of the antibody is extended when compared to the parent wild-type antibody in a biological environment, e.g., serum half-life or half-life as measured by an in vitro assay.

[0152] Exemplary mutations which may be made singly or in combination are T250Q, M252Y, I253A, S254T, T256E, P257I, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A and H435R mutations.

[0153] In certain embodiments, increased half-life can be achieved by engineering M252Y / S254T / T256E mutations (residue numbering according to the EU index) in the IgG1 Fc of SEQ ID NO: 23 (Dall'Acqua, Kiener et al. 2006).

[0154] In certain embodiments, increased half-life can also be achieved by engineering M428L / N434S mutations in the IgG1 Fc of SEQ ID NO:24 (Zalevsky, Chamberlain et al. 2010).

[0155] In certain embodiments, increased half-life can also be achieved by engineering T250Q / M428L mutations in the IgG1 Fc of SEQ ID NO:25 (Hinton, Xiong et al. 2006).

[0156] In certain embodiments, increased half-life can also be achieved by engineering an N434A mutation in the IgG1 Fc of SEQ ID NO:26 (Shields, Namenuk et al. 2001).

[0157] In certain embodiments, increased half-life can also be achieved by engineering T307A / E380A / N434A mutations in the IgG1 Fc of SEQ ID NO:27 (Petkova, Akilesh et al. 2006).

[0158] The effect of Fc engineering on half-life extension can be assessed in PK studies in mice for antibodies with native IgG Fc.

[0159] IgG1Fc with M252Y / S254T / T256E mutations (SEQ ID NO: 23)

[0160] [ka]

[0161] IgG1Fc with M428L / N434S mutations (SEQ ID NO:24)

[0162] [ka]

[0163] IgG1Fc with T250Q / M428L mutations (SEQ ID NO:25)

[0164] [ka]

[0165] IgG1Fc with N434A mutation (SEQ ID NO:26)

[0166] [ka]

[0167] IgG1Fc with T307A / E380A / N434A mutations (SEQ ID NO:27)

[0168] [ka]

[0169] In some embodiments, the anti-IL-1β antibodies are provided with an altered Fc region, in which the native Fc region has been altered to enhance resistance of the antibody to proteolysis by proteases that cleave the wild-type antibody at or between residues 222-237 (EU numbering).

[0170] In certain embodiments, resistance to proteolysis can be achieved by engineering E233P / L234V / L235A mutations (residue numbering according to the EU index) in the hinge region that deletes G236 compared to the parent wild-type antibody of SEQ ID NO:28 (Kinder, Greenplate et al. 2013).

[0171] IgG1Fc with E233P / L234V / L235A mutations and deletion of G236 (SEQ ID NO:28)

[0172] [ka]

[0173] If effector functionality is undesirable, the antibodies of the disclosure may be further engineered to introduce at least one mutation in the antibody Fc that reduces binding of the antibody to activating Fcγ receptors (FcγR) and / or reduces an Fc effector function such as C1q binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) or phagocytosis (ADCP).

[0174] Fc positions that may be mutated to reduce binding of the antibody to activating FcγRs and subsequently reduce effector function are described, for example, in (Xu, Alegre et al. 2000) (Vafa, Gilliland et al. 2014) (Bolt, Routledge et al. 1993) (Chu, Vostiar et al. 2008) (Shields, Namenuk et al. 2001).

[0175] Exemplary mutations which may be made singly or in combination are K214T, E233P, L234V, L234A, G236 deletion, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, A330S and P331S mutations on IgG1, IgG2, IgG3 or IgG4.

[0176] Exemplary combination mutations that may be made to reduce ADCC include L234A / L235A on IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S on IgG2, F234A / L235A on IgG4, S228P / F234A / L235A on IgG4, N297A on IgG1, IgG2, IgG3 or IgG4, V234A / G237A on IgG2, K214T / E233P / L234V / L235A / G236 deletion / A331S on IgG1, and K214T / E233P / L234V / L235A / G236 deletion / A331S on IgG2. 27G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S on IgG2, S267E / L328F on IgG1, L234F / L235E / D265A on IgG1, L234A / L235A / G237A / P238S / H268A / A330S / P331S on IgG1, S228P / F234A / L235A / G237A / P238S on IgG4, and S228P / F234A / L235A / G236 deletion / G237A / P238S on IgG4. Fc-like hybrid IgG with residues 117-260 from IgG2 and residues 261-447 from IgG4. 2 / 4 The Fc domain may also be used.

[0177] In some embodiments, the anti-IL-1β antibodies are provided with an altered Fc region, in which the native Fc region has been altered to promote the generation of bispecific antibodies by Fc heterodimerization.

[0178] In certain embodiments, Fc heterodimerization can be achieved by engineering F405L and K409R mutations in two parent antibodies and generating bispecific antibodies in a process known as Fab arm exchange (Labrijn, Meesters et al. 2014).

[0179] In certain embodiments, Fc heterodimerization can also be achieved by Fc mutations to facilitate a knob-in-hole strategy (see, for example, International Publication WO 2006 / 028936). An amino acid with a small side chain (hole) is introduced into one Fc domain, and an amino acid with a large side chain (knob) is introduced into the other Fc domain. After co-expression of the two heavy chains, a heterodimer forms as a result of preferential interaction between the heavy chain with the "hole" and the heavy chain with the "knob" (Ridgway, Presta et al. 1996).

[0180] Exemplary Fc mutation pairs that form knobs and holes are: T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S and T366W / T366S / L368A / Y407V.

[0181] In certain embodiments, Fc heterodimerization can also be achieved by Fc mutations to promote electrostatically-matched interaction strategies (Gunasekaran, Pentony et al., 2010). Mutations can be engineered to generate positively charged residues in one Fc domain and negatively charged residues in the other Fc domain, as described in US Patent Publication US2010 / 0015133; US Patent Publication US2009 / 0182127; US Patent Publication US2010 / 028637 or US Patent Publication US2011 / 0123532. Heavy chain heterodimerization can be formed by electrostatically-matched interactions between two mutated Fcs.

[0182] Antibodies of the present disclosure that further include conservative modifications are within the scope of the present disclosure.

[0183] "Conservative modifications" refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing that amino acid sequence. Conservative modifications include amino acid substitutions, additions, and deletions. A conservative substitution is one in which an amino acid is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been well defined and include amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine) and sulfur-containing side chains (cysteine, methionine). Additionally, any native residue in the polypeptide may be substituted with alanine, as previously described for alanine scanning mutagenesis ((MacLennan, Rice et al. 1998); (Sasaki and Sutoh 1998)). Amino acid substitutions to the antibodies of the present disclosure may be made by known methods, such as, for example, PCR mutagenesis (U.S. Pat. No. 4,683,195). Alternatively, libraries of mutants may be generated, for example, using random (NNK) or non-random codons, such as the DVK codons that code for 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The resulting antibody mutants may be tested for their properties using the assays described herein.

[0184] The antibodies of the present disclosure may be post-translationally modified by processes such as glycosylation, isomerization, deglycosylation, or non-natural covalent modifications such as the addition of polyethylene glycol moieties (PEGylation) and lipidation. Such modifications may occur in vivo or in vitro. For example, the antibodies of the present disclosure may be conjugated with polyethylene glycol (PEGylation) to improve their pharmacokinetic profile. Conjugation may be performed by techniques known to those of skill in the art. Conjugation of therapeutic antibodies with PEG has been shown to enhance pharmacodynamics while not interfering with function (Leong, DeForge et al. 2001, Yang, Basu et al. 2003, Knight, Jordan et al. 2004).

[0185] The antibodies of the present disclosure may be modified to improve stability, selectivity, cross-reactivity, affinity, immunogenicity, or other desired biological or biophysical properties, and are within the scope of the present disclosure. Antibody stability is influenced by a number of factors, including (1) the core packing of individual domains, which affects intrinsic stability; (2) protein / protein interface interactions, which impact HC and LC pairing; (3) burial of polar and charged residues; (4) hydrogen bond networks for polar and charged residues; and (5) distribution of surface charge and polar residues among other intra- and intermolecular forces (Worn and Pluckthun 2001). Potential structure destabilizing residues may be identified based on the crystal structure of the antibody or in some cases by molecular modeling, and the effect of the residues on antibody stability may be tested by generating and evaluating mutants with mutations at the identified residues. One method for increasing antibody stability is to increase the thermal transition midpoint (Tm) as measured by differential scanning calorimetry (DSC). In general, protein Tm correlates with its stability and inversely correlates with the susceptibility to unfolding and denaturation in solution as well as degradation processes that depend on the tendency of the protein to unfold (Remmele and Gombotz 2000). Numerous studies have found correlations between the ranking of formulation physical stability as measured by thermal stability by DSC and physical stability measured by other methods (Maa and Hsu 1996; Remmele, Nightlinger et al. 1997; Gupta and Kaisheva 2003; Bedu-Addo, Johnson et al. 2004; Zhang, Roy et al. 2004). Pharmaceutical studies suggest that the Tm of a Fab is related to the long-term physical stability of the corresponding mAb.

[0186] The antibodies of the present disclosure may have amino acid substitutions in the Fc region that improve manufacturing and drug stability. For IgG1, one example is H224S (or H224Q) in the hinge 221-DKTHTC-226 (SEQ ID NO: 33) (EU numbering), which blocks radical-induced cleavage (Yates, Gunasekaran et al. 2010); for IgG4, the S228P mutation blocks half-antibody exchange (Angal, King et al. 1993; Labrijn, Buijsse et al. 2009). <Expression and purification of anti-IL-1β antibodies and fragments>

[0187] The anti-IL-1β antibodies and fragments of the disclosure can be encoded by a single nucleic acid (e.g., a single nucleic acid comprising nucleotide sequences encoding light and heavy chain polypeptides of the antibody) or by two or more separate nucleic acids, each encoding a different portion of the antibody or antibody fragment.

[0188] As non-limiting examples, the disclosure provides the nucleic acid sequence of SEQ ID NO:29, which encodes the IgG1 heavy chain sequence of TAVO7378 of SEQ ID NO:14, and the nucleic acid sequence of SEQ ID NO:30, which encodes the light chain sequence of TAVO7378 of SEQ ID NO:17.

[0189] TAVO7378 heavy chain nucleotide sequence (SEQ ID NO:29)

[0190] [ka]

[0191] TAVO7378 light chain nucleotide sequence (SEQ ID NO:30)

[0192] [ka]

[0193] The nucleic acids described herein can be inserted into a vector, e.g., a nucleic acid expression vector and / or a targeting vector. Such vectors can be used in a variety of ways, e.g., for expression of an IL-1β binding antibody or antibody fragment in a cell or a transgenic animal. The vector is typically selected to be functional in the host cell in which it will be used. The nucleic acid molecule encoding the IL-1β binding antibody or fragment may be amplified / expressed in prokaryotic, yeast, insect (baculovirus systems) and / or eukaryotic host cells. The choice of host cell will depend in part on whether the IL-1β binding antibody or fragment is to be post-translationally modified (e.g., glycosylated and / or phosphorylated). If so, yeast, insect, or mammalian host cells are preferred. Expression vectors typically contain one or more of the following components: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence including donor and acceptor splicing sites, a leader sequence for secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element.

[0194] In most cases, a leader or signal sequence is engineered at the N-terminus of the anti-IL-1β antibody or fragment to guide its secretion. Secretion of the anti-IL-1β antibody or fragment from a host cell results in removal of the signal peptide from the antibody or fragment. Thus, the mature antibody or fragment will lack any leader or signal sequence. In some cases, such as when glycosylation is desired in a eukaryotic host cell expression system, various presequences may be engineered to improve glycosylation or yield. For example, the peptidase cleavage site of the signal peptide may be altered or a prosequence may be added that may affect glycosylation.

[0195] The present disclosure further provides a cell (e.g., an isolated or purified cell) comprising a nucleic acid or vector of the present disclosure. The cell can be any type of cell that can be transformed with a nucleic acid or vector of the present disclosure so that a polypeptide encoded by the nucleic acid or vector of the present disclosure is produced. To express an IL-1β binding antibody or fragment, DNA encoding the partial or full-length light and heavy chains obtained as described herein is inserted into an expression vector such that the genes are operably linked to transcriptional and translational control sequences.

[0196] Methods for introducing nucleic acids and vectors into isolated cells and for culturing and selecting transformed host cells in vitro are known in the art and include the use of calcium chloride-mediated transformation, transduction, conjugation, triparental mating, DEAE, dextran-mediated transfection, infection, membrane fusion using liposomes, high velocity bombardment using DNA-coated particle guns, direct microinjection into single cells, and electroporation.

[0197] After introducing a nucleic acid or vector of this disclosure into a cell, the cell is cultured under conditions suitable for expression of the encoded sequence. The antibody, antigen-binding fragment, or portion of the antibody can then be isolated from the cell.

[0198] In certain embodiments, two or more vectors that together encode an IL-1β binding antibody or antigen-binding fragment thereof can be introduced into a cell.

[0199] Purification of the IL-1β-binding antibody or fragment secreted in the cell culture medium can be achieved using a variety of techniques, including affinity, immunoaffinity or ion-exchange chromatography, molecular sieve chromatography, preparative gel electrophoresis or isoelectric focusing electrophoresis, chromatofocusing, and high-performance liquid chromatography. For example, an antibody containing an Fc region may be purified by affinity chromatography using protein A that selectively binds to the Fc region.

[0200] Modified forms of the antibody or antigen-binding fragment can be prepared by using a hexahistidine (SEQ ID NO: 34), which is an affinity tag, or other small peptides such as FLAG (Eastman Kodak Co., New Haven, Conn.) or myc (Invitrogen) at its carboxyl or amino terminus, and may be purified by a one-step affinity column. For example, since polyhistidine binds to nickel with high affinity and specificity, a nickel affinity column (e.g., Qiagen® nickel column) can be used for the purification of a selectively binding agent attached with a polyhistidine tag. In some cases, two or more purification steps may be used. <Binding and Activity of Anti-IL-1β Antibodies and Fragments Against IL-1β>

[0201] The present disclosure encompasses anti-IL-1β antibodies and fragments that selectively bind to IL-1β in that they bind to IL-1β with higher affinity than to other antigens. These anti-IL-1β antibodies and fragments may selectively bind to human IL-1β, but may also detectably bind to non-human IL-1β. Alternatively or additionally, these antibodies or fragments may bind to human IL-1β and to IL-1β of other mammals. For example, these antibodies or fragments may bind to one or more of rodent IL-1β, primate IL-1β, canine IL-1β, and rabbit IL-1β, or guinea pig IL-1β. Alternatively or additionally, these IL-1β binding antibodies or IL-1β binding fragments may have the same or substantially the same potency for recombinant human IL-1β and endogenous human IL-1β.

[0202] In vitro and cell-based assays are well described in the art for use in determining the binding of IL-1β to the IL-1 receptor type I (IL-1R1). For example, the binding of IL-1β to the IL-1 receptor type I may be determined by immobilizing an IL-1β-binding antibody, capturing IL-1β with the immobilized antibody to determine whether IL-1β is bound to the antibody, and contacting the bound IL-1β / antibody complex with a soluble form of IL-1RI to determine whether soluble IL-1RI is bound to the complex. The protocol may also include contacting the soluble IL-1RI with the immobilized antibody before contacting with IL-1β to ensure that the soluble IL-1RI does not bind to the immobilized antibody. This protocol may be performed using a Biacore® instrument for kinetic analysis of the binding interaction. Such a protocol may also be used to determine whether an antibody or other molecule permits or blocks the binding of IL-1β to the IL-1 receptor type I.

[0203] For other IL-1β / IL-1RI binding assays, the permissiveness or blocking of IL-1β binding to the IL-1 receptor type I may be determined by comparing the binding of IL-1β to IL-1RI in the presence or absence of an IL-1β antibody or IL-1β binding fragment thereof. Blocking is identified in the assay readout as a specified reduction in IL-1β binding to the IL-1 receptor type I in the presence of an anti-IL-1β antibody or IL-1β binding fragment thereof, as compared to a control sample containing the corresponding buffer or diluent but no IL-1β antibody or IL-1β binding fragment thereof. The assay readout may be viewed qualitatively as indicating the presence or absence of blocking, or may be viewed quantitatively as indicating the percent or fold reduction in binding due to the presence of the antibody or antibody fragment. If an IL-1β binding antibody or IL-1β binding fragment substantially blocks IL-1β binding to IL-1RI, IL-1β binding to IL-1RI is reduced by at least 10-fold, alternatively at least about 20-fold, alternatively at least about 50-fold, alternatively at least about 100-fold, alternatively at least about 1000-fold, alternatively at least about 10,000-fold, or more, compared to binding of the same concentrations of IL-1β and IL-1RI in the absence of the antibody or fragment.

[0204] The key amino acid residues (epitopes) bound by the IL-1β binding antibodies or fragments described in this disclosure may be determined using a peptide array, such as, for example, a PepSpot™ peptide array (JPT Peptide Technologies, Berlin, Germany), where a scan of 12 amino acid peptides spanning the entire IL-1β amino acid sequence is synthesized directly onto a membrane, each peptide overlapping the previous peptide by 11 amino acids. Alternatively or additionally, antibody competition experiments may be performed and such assays are well known in the art.

[0205] Preferred IL-1β antibodies or antibody fragments for use in accordance with the present disclosure generally bind to human IL-1β with high affinity (e.g., as determined using BIACORE), e.g., having an equilibrium binding dissociation constant (KD) for IL-1β of about 10 nM or less, about 5 nM or less, about 1 nM or less, about 500 pM or less, or more preferably about 250 pM or less, about 100 pM or less, about 50 pM or less, about 25 pM or less, about 10 pM or less, about 5 pM or less, about 3 pM or less, about 1 pM or less, about 0.75 pM or less, about 0.5 pM or less, or about 0.3 pM or less.

[0206] The antibodies or fragments of the disclosure may bind to IL-1β with an EC50 of, for example, about 10 nM or less, about 5 nM or less, about 2 nM or less, about 1 nM or less, about 0.75 nM or less, about 0.5 nM or less, about 0.4 nM or less, about 0.3 nM or less, or about 0.2 nM or less, as determined by enzyme-linked immunosorbent assay (ELISA).

[0207] Preferably, the antibodies or antibody fragments of the disclosure do not cross-react with any other targets other than IL-1β. For example, the antibodies and fragments of the invention may bind to IL-1β but do not detectably bind to IL-1α or have at least about 100-fold (e.g., at least about 150-fold, at least about 200-fold, or even at least about 250-fold) greater selectivity for their binding of IL-1β over their binding of IL-1α.

[0208] The present disclosure also encompasses neutralizing antibodies or neutralizing fragments thereof that bind IL-1β in a manner that neutralizes the biological activity of IL-1β. Neutralization of IL-1β biological activity can be assessed by assaying for one or more indicators of IL-1β biological activity, such as IL-1β-stimulated reporter gene expression in a reporter assay, IL-1β-stimulated IL-6 release from human fibroblasts or other cells, IL-1β, or IL-1β-induced proliferation of T helper cells. Neutralization of IL-1β biological activity can also be assessed in vivo by a mouse arthritis model. Preferably, the IL-1β binding antibodies and fragments of the present disclosure neutralize the biological activity of IL-1β associated with the signaling function of the IL-1 receptor type I (IL-1RI) bound by IL-1β.

[0209] In one embodiment, the antibody or fragment thereof of the present disclosure can neutralize, inhibit, block, inhibit, reduce or interfere with the activity of IL-1β by binding to an epitope of IL-1β that is directly involved in the target activity of IL-1β. In another embodiment, the antibody or fragment thereof of the present disclosure can neutralize, inhibit, block, inhibit, reduce or interfere with the activity of IL-1β by binding to an epitope of IL-1β that is not directly involved in the target activity of IL-1β, but the antibody or fragment binding thereto can sterically or conformationally inhibit, block, inhibit, reduce or interfere with the target activity of IL-1β. In yet another embodiment, the antibody or fragment thereof of the present disclosure can bind to an epitope of IL-1β that is not directly involved in the target activity of IL-1β (i.e., a non-blocking antibody), but the antibody or fragment binding thereto results in enhanced clearance of IL-1β.

[0210] In general, the antibodies and fragments of the present disclosure can neutralize, inhibit, block, suppress, reduce or interfere with the biological activity of IL-1β, regardless of whether or not the binding of IL-1β to the IL1 receptor type I is blocked. More preferably, the IL-1β binding antibodies or IL-1β binding fragments neutralize the biological activity of IL-1β by binding to IL-1β without substantially preventing the binding of its bound IL-1β to the IL-1 receptor type I. A potential advantage of such antibodies and fragments is that they can bind and neutralize IL-1β while still allowing IL-1β to bind to IL-1RI. This can result in an effective reduction of IL-1α biological activity as well as IL-1β biological activity since there are fewer unbound IL-1RI sites for IL-1α to bind. Thus, the IL-1β binding antibodies and fragments of the present disclosure are useful in methods in which it is desirable to neutralize the in vitro and in vivo biological activity of IL-1.

[0211] The antibodies or fragments of the invention may be neutralizing antibodies or fragments that specifically bind to an IL-1β epitope that affects the biological activity of IL-1β. The antibodies or fragments of the invention can bind to a neutralization-sensitive epitope of IL-1β. When a neutralization-sensitive epitope of IL-1β is bound by one of the antibodies or fragments of the invention, the result is a loss of the biological activity of IL-1β that includes that epitope. <Pharmaceutical Composition>

[0212] The IL-1β binding antibodies and antibody fragments for use according to the present disclosure can be formulated into compositions, particularly pharmaceutical compositions, for use in the methods herein. Such compositions comprise a therapeutically or prophylactically effective amount of the IL-1β binding antibodies or antibody fragments of the present disclosure in admixture with a suitable carrier, e.g., a pharma- ceutically acceptable agent. Typically, the IL-1β binding antibodies and antibody fragments of the present disclosure are sufficiently purified for administration to an animal prior to formulation into a pharmaceutical composition.

[0213] Pharmaceutically acceptable agents include carriers, excipients, diluents, antioxidants, preservatives, colorants, flavorings and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, isotonicity agents, co-solvents, wetting agents, complexing agents, buffering agents, antibacterial agents, and surface active agents.

[0214] The compositions may be in liquid form or in lyophilized or freeze-dried form and may contain one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives and / or bulking agents.

[0215] The composition can be suitable for parenteral administration. Exemplary compositions are suitable for injection or infusion into animals by any route available to those of skill in the art, such as intra-articular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intracemall), intraventricular, intramuscular, intraocular, intraarterial, intralesional, intrarectal, transdermal, oral and inhalation routes.

[0216] The pharmaceutical compositions described herein can be formulated for controlled or sustained delivery in a manner that provides local concentrations of product (e.g., bolus, depo effect), sustained release, and / or enhanced stability or half-life in a particular local environment. <How to use>

[0217] The antibodies and fragments of the present invention are useful for the prevention and treatment of IL-1β mediated diseases or medical conditions, such as inflammatory conditions, allergies and allergic conditions, hypersensitivity reactions, autoimmune diseases, severe infections, and organ or tissue transplant rejection.

[0218] The antibodies of the disclosure are particularly useful for the treatment, prevention or amelioration of autoimmune diseases and inflammatory conditions, particularly inflammatory conditions with an etiology involving an autoimmune component, such as inflammatory conditions associated with bone loss and rheumatic diseases, such as arthritis (e.g., rheumatoid arthritis, chronic arthritis, and osteoarthritis) and rheumatic diseases, inflammatory pain, hypersensitivity (including both airway hypersensitivity and skin hypersensitivity) and allergies.

[0219] Specific autoimmune diseases in which the antibodies of the present disclosure may be used include autoimmune blood diseases (including, for example, hemolytic anemia, aplastic anemia, true erythrocytic anemia, and idiopathic thrombocytopenia), systemic lupus erythematosus, polychondritis, edema or scleroderma, Wegener's granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, Stevens-Johnson syndrome, idiopathic sprue, autoimmune inflammatory bowel disease, and the like. These conditions include, but are not limited to, chronic obstructive pulmonary disease (COD), chronic obstructive pulmonary disease (COPD), chronic obstructive pulmonary disease (COPD), chronic myocardial infarction (COPD), chronic obstructive pulmonary disease (COPD), chronic myocardial infarction (CPO), chronic obstructive pulmonary disease (COPD), chronic obstructive pulmonary disease (COPD), chronic myocardial infarction (CPO), chronic obstructive pulmonary disease (COPD), chronic obstructive pulmonary disease (COPD), chronic obstructive pulmonary disease (COPD), chronic obstructive pulmonary disease (COPD), chronic obstructive pulmonary disease (COPD), chronic obstructive pulmonary disease (COPD), chronic obstructive pulmonary disease (COPD), chronic obstructive pulmonary disease (COPD), chronic obstructive pulmonary disease (COPD), chronic obstructive pulmonary disease (OP ...

[0220] The antibodies of the disclosure are also useful for treating, preventing, or ameliorating asthma, bronchitis, pneumoconiosis, emphysema, and other obstructive or inflammatory diseases of the airways.

[0221] The antibodies and fragments of the invention are also contemplated for use in treating recipients of heart, lung, combined heart-lung, liver, kidney, pancreas, skin or cornea transplants, including graft rejection and xenograft rejection, or for the prevention of graft-versus-host disease, such as after bone marrow transplantation, or atherosclerosis associated with organ transplantation.

[0222] The antibodies of the present disclosure are particularly useful for the treatment of diseases of bone metabolism, including osteoarthritis, osteoporosis and other inflammatory arthritis, and osteopenia in general, including age-related osteopenia, and periodontal disease in particular.

[0223] The antibodies of the disclosure are also useful for reducing, preventing or treating cardiovascular events and / or cardiovascular disease, including myocardial infarction, stroke, cardiovascular death, congestive heart failure, cardiac arrest, acute coronary syndrome, angina, or revascularization procedures.

[0224] The antibodies of the present disclosure are also useful for the prevention or treatment of cancer, including lung cancer, pancreatic cancer and breast cancer. In one embodiment, the anti-IL-1β antibody or fragment thereof disclosed herein may be used in an effective amount for the treatment and / or prevention of lung cancer. In another embodiment, the anti-IL-1β antibody or fragment thereof disclosed herein may be used in an effective amount for the treatment and / or prevention of pancreatic cancer. In another embodiment, the anti-IL-1β antibody or fragment thereof disclosed herein may be used in an effective amount for the treatment and / or prevention of breast cancer.

[0225] An effective amount of an anti-IL-1β antibody may be used in the present disclosure for the treatment and / or prevention of type 1 diabetes, type 2 diabetes, obesity, hyperglycemia, hyperinsulinemia, insulin resistance, and conditions and conditions characterized by insulin resistance. Such methods may be used to treat mammalian subjects (e.g., humans) suffering from, or to prevent the onset of, type 2 diabetes, type 1 diabetes, obesity, hyperglycemia, hyperinsulinemia, insulin resistance, and conditions and conditions characterized by insulin resistance in at-risk subjects. In one embodiment, the anti-IL-1β antibody or fragment thereof disclosed herein may be used in an effective amount for the treatment and / or prevention of type 2 diabetes.

[0226] In addition to therapeutic uses, the antibodies and fragments of the present invention may be used in diagnostic methods to detect IL-1β (e.g., in biological samples such as serum or plasma) using immunoassays such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry.

[0227] Methods for detecting IL-1β in a biological sample can include contacting the biological sample with one or more of the antibodies or fragments of the invention and detecting either the antibody or fragment bound to IL-1β or the unbound antibody or fragment, thereby detecting IL-1β in the biological sample. The antibody or fragment can be directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. EXAMPLES

[0228] Working Example The following examples are provided to more fully illustrate the present disclosure and are intended to illustrate, but not limit, the present disclosure. Example 1: IL-1β binding affinity for mouse anti-IL-1β antibody TAVO304

[0229] TAVO304 was identified as a mouse anti-human IL-1β antibody by hybridoma screening. An ELISA-based binding assay was used to evaluate TAVO304 binding to recombinant human IL-1β. In this assay, 1 μg / ml recombinant human IL-1β (R&D systems) was coated onto an ELISA plate. Increasing concentrations of TAVO304 antibody were applied to the plate, and its binding to recombinant human IL-1β was detected by an HRP-conjugated anti-mouse secondary antibody. TAVO304 was observed to bind recombinant human IL-1β in a dose-dependent manner, with an EC50 of 3.6 ng / mL (Figure 1).

[0230] The binding of TAVO304 to mouse and rhesus IL-1β was also assessed in a similar ELISA assay by coating plates with mouse and rhesus IL-1β, respectively. TAVO304 showed no significant binding affinity to mouse IL-1β (Figure 1). In contrast, it showed good binding affinity to rhesus IL-1β, with an EC50 of 12.2ng / mL. Example 2: In vitro assay for IL-1β neutralization by TAVO304

[0231] To assess the functional activity of IL-1β, we developed the HEK-Blue IL-1β reporter assay. In this assay, HEK-Blue IL-1β cells (Invivogen) express the IL-1β receptor IL-1R and the secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and five AP-1 binding sites. Binding of IL-1β to its receptor IL-1R on the surface of HEK-Blue IL-1β cells triggers a signaling cascade leading to the activation of NF-κB and subsequent production of SEAP (Figure 2).

[0232] The response of the HEK-Blue IL-1β reporter cell line to IL-1β was assessed using this assay, and it was observed that both human IL-1β and rhesus IL-1β were able to induce reporter gene expression in a dose-dependent manner, with EC50s of 1.14 ng / mL and 0.16 ng / mL, respectively (Figure 2).

[0233] The HEK-Blue IL-1β reporter assay was then used to evaluate TAVO304 in blocking human IL-1β-driven reporter gene expression. Increasing concentrations of antibody were applied to HEK-Blue IL-1β reporter cells along with 10 ng / mL human IL-1β. After overnight incubation, SEAP reporter gene expression was quantified. TAVO304 was observed to neutralize IL-1β-mediated reporter gene expression in a dose-dependent manner with an IC50 of 0.12 μg / mL (Figure 3), being much more potent (>6-fold) than the reference antibody refAb-1 in IL-1β inhibition.

[0234] The same assay was also used to evaluate TAVO304 in blocking rhesus IL-1β-driven reporter gene expression. Increasing amounts of antibody were applied to HEK-Blue IL-1β reporter cells along with 0.25 ng / mL rhesus IL-1β. TAVO304 was observed to dose-dependently neutralize rhesus IL-1β-mediated reporter gene expression with an IC50 of 3.6 ng / mL (Figure 3). Example 3: Humanization of mouse anti-human IL-1β antibody TAVO304

[0235] The murine anti-human IL-1β antibody TAVO304 was humanized by grafting murine CDRs onto a human germline scaffold. A few key murine residues were preserved by backmutation to achieve higher stability and better expression while minimizing immunogenicity. For TAVO304, one humanized VH variant (304VH1) was found to be specifically targeted to IGHV3-48. * 01, and four humanized VL variants were designed based on IGKV1-39 * Based on 01, using 304VL1 and 304VL2, and IGKV2-40 *Based on TAVO304 antibody TAVO7376, TAVO7377, TAVO7378, and TAVO7379, four humanized TAVO304 antibodies with IgG1 Fc were generated by combining the humanized VH variants with the four humanized VL variants, with the 304VH1 variants paired with 304VL1, 304VL2, 304VL3, and 304VL4, respectively (FIG. 4). Example 4: Expression and purification of humanized TAVO304

[0236] Plasmids encoding the heavy and light chains of TAVO7376, TAVO7377, TAVO7378, and TAVO7379 were co-transfected into Expi293F cells according to the transfection kit instructions (Thermo Scientific). Cells were spun down 5 days after transfection and the supernatant was passed through a 0.2 μm filter. Purification of expressed antibodies in the supernatant was performed by affinity chromatography on a Protein A agarose column (GE Healthcare Life Sciences). Purified antibodies were buffer exchanged into DPBS, pH 7.2 by dialysis and protein concentration was determined by UV absorbance at 280 nm.

[0237] Purified TAVO7376, TAVO7377, TAVO7378 and TAVO7379 were subjected to SDS-PAGE analysis (Figure 5). Under reducing conditions, all four antibodies had heavy and light chains with the expected molecular weights. Under non-reducing conditions, all four antibodies migrated as major protein bands with molecular weights around 150 kDa. Example 5: Binding of humanized TAVO304 to IL-1β

[0238] An ELISA-based binding assay was used to evaluate humanized TAVO304 binding to recombinant human IL-1β. In this assay, 1 μg / ml of recombinant human IL-1β (R&D systems) was coated onto an ELISA plate. Increasing concentrations of humanized TAVO304 antibody were applied to the plate and its binding to recombinant human IL-1β was detected by an HRP-conjugated anti-mouse secondary antibody. All four humanized antibodies TAVO7376, TAVO7377, TAVO7378 and TAVO7379 were observed to bind recombinant human IL-1β in a dose-dependent manner with similar potency and comparable to the mouse antibody TAVO304 (Figure 6).

[0239] The binding of the four humanized antibodies to rhesus IL-1β was also assessed in a similar ELISA assay by coating rhesus IL-1β to plates. All four humanized antibodies, TAVO7376, TAVO7377, TAVO7378, and TAVO7379, bound to recombinant rhesus IL-1β in a dose-dependent manner with similar potency (Figure 6).

[0240] To assess binding specificity, cytokines homologous to IL-1β, such as IL-1α, were assessed for binding to the humanized antibodies in an ELISA-based binding assay using purified recombinant proteins. All four humanized antibodies, TAVO7376, TAVO7377, TAVO7378, and TAVO7379, showed no significant binding to human IL-1α at 1 μg / mL in an ELISA-based binding assay, demonstrating the specificity of IL-1β binding for these humanized antibodies.

[0241] To further measure the binding of the humanized antibodies to immobilized recombinant IL-1β, surface plasmon resonance (SPR) binding assays are performed using Biacore, which can measure not only the binding affinity but also the kinetic rate constants and thermodynamics of binding. Example 6: In vitro reporter assay for IL-1β neutralization by humanized TAVO304

[0242] TAVO7376, TAVO7377, TAVO7378 and TAVO7379 were evaluated in blocking human IL-1β-driven reporter gene expression using the HEK-Blue IL-1β reporter assay. Increasing amounts of antibodies were applied to HEK-Blue IL-1β reporter cells along with 10 ng / mL human IL-1β. After overnight incubation, SEAP reporter gene expression was quantified. All four humanized antibodies were observed to dose-dependently neutralize IL-1β-mediated reporter gene expression with similar potency (IC50 near 0.09 μg / mL, FIG. 7). Their potency in neutralizing human IL-1β was similar to that of TAVO304 and much more potent than the reference antibody refAb-1.

[0243] Using the same assay, four humanized antibodies were evaluated in blocking rhesus IL-1β-driven reporter gene expression. Increasing amounts of antibody were applied to HEK-Blue IL-1β reporter cells along with 0.25 ng / mL rhesus IL-1β. TAVO7376, TAVO7377, TAVO7378 and TAVO7379 antibodies were observed to dose-dependently neutralize rhesus IL-1β-mediated reporter gene expression with similar potency (IC50 near 0.018 μg / mL, FIG. 7). Example 7: In vitro MRC-5 cell-based cytokine release assay for IL-1β neutralization by humanized TAVO304

[0244] IL-1β can drive activation of human lung fibroblast cell line MRC-5 and stimulate IL-6 release. An in vitro assay was set up to measure the humanized TAVO304 antibody in neutralizing IL-1β-mediated MRC-5 cell activation. Increasing amounts of the antibody were applied to MRC-5 cells (ATCC) with 1 ng / mL human IL-1β for 24 hours, and the induced IL-6 release from the cells can be quantified by an IL-6 assay kit (R&D systems). It was observed that TAVO7376, TAVO7377, TAVO7378 and TAVO7379 antibodies dose-dependently blocked human IL-1β-mediated IL-6 release from MRC-5 cells with similar potency (IC50 around 50 ng / mL, FIG. 8) and were more potent than the reference antibody refAb-1 in IL-1β inhibition. Example 8: Fc engineering of humanized antibodies for extended half-life, reduced effector function and proteolytic resistance

[0245] To improve the PK profile of humanized antibodies, Fc mutations can be introduced into IgG1 antibodies to extend antibody half-life. Specifically, M428L / N434S mutations have been shown to extend antibody half-life by increasing FcRn binding affinity (Booth, Ramakrishnan et al., 2018). Furthermore, L234A / L235A Fc mutations can abolish ADCC and CDC effector functions of IgG1 antibodies (Hezareh, Hessell et al., 2001). Thus, a 304VH1-based humanized IgG1 heavy chain with L234A / L235A / M428L / N434S (AALS) mutations was generated, which comprises the sequence shown as SEQ ID NO:31. By pairing this Fc-engineered 304VH1 heavy chain with the humanized 304VL3 light chain shown as SEQ ID NO:17, a humanized TAVO304 antibody with extended half-life and reduced effector function was generated and named TAVO11878.

[0246] 304 VH1 heavy chain with AALS mutation (SEQ ID NO:31)

[0247] [ka]

[0248] The heavy chain variable domain sequence is underlined, and the AALS mutation is shown in bold.

[0249] To study whether Fc-engineered antibodies have improved FcRn binding affinity, the binding of TAVO11878 and TAVO7378 to mouse FcRn is evaluated in an ELISA-based binding assay. 1 μg / mL of recombinant mouse FcRn (R&D systems) is coated onto an ELISA plate. Increasing concentrations of TAVO11878 and TAVO7378 antibodies are applied to the plate, and their binding to recombinant FcRn at pH 6.0 is detected by HRP-conjugated anti-mouse secondary antibody.

[0250] To study the PK profile of engineered anti-IL-1β antibodies, TAVO11878 and TAVO7378 are subjected to a cynomolgus monkey PK model to evaluate any effect of Fc engineering on improved PK profile relative to antibodies with native IgG1 Fc.

[0251] To improve the in vivo stability of humanized antibodies, additional Fc mutations can be introduced into the IgG1 antibody to enhance the antibody resistance to proteolysis. Many proteases can cleave wild-type IgG antibodies at or between residues 222-237 (EU numbering). Resistance to proteolysis can be achieved by engineering an E233P mutation (residue numbering according to the EU index) with a deletion of G236. Thus, a humanized IgG1 heavy chain based on 304VH1 with E233P, L234A, L235A, M428L, N434S mutations with a G236 deletion was generated, which comprises the sequence shown as SEQ ID NO: 32. By pairing this Fc-engineered 304VH1 heavy chain with the humanized 304VL3 light chain shown as SEQ ID NO: 17, a humanized TAVO304 antibody with resistance to proteolysis, extended half-life and reduced effector function was generated and named TAVO18378.

[0252] 304 VH1 heavy chain with E233P, L234A, L235A, M428L, N434S mutations with G236 deletion (SEQ ID NO: 32)

[0253] [ka]

[0254] The heavy chain variable domain sequence is underlined, and the E233P, L234A, L235A, M428L, N434S mutations are shown in bold.

[0255] To study whether engineered anti-IL-1β antibodies with these Fc mutations have improved resistance to proteolysis, TAVO18378 is subjected to digestion with recombinant IgG protease IdeZ (New England Biolabs) for 30 minutes at 37° C., followed by SDS-PAGE analysis under reducing conditions to assess the integrity of its heavy chain. In addition to IgG protease IdeZ, TAVO18378 is also subjected to digestion with recombinant matrix metalloproteinase 3, MMP3 (Enzo Life Sciences) for 24 hours at 37° C., followed by SDS-PAGE under reducing conditions to assess the integrity of its heavy chain. It is expected that the above-mentioned Fc mutations below the hinge region can promote the resistance of Fc-engineered anti-IL-1β antibodies to degradation by proteases including IdeZ and MMP3. Example 9: In vivo efficacy of humanized anti-IL-1β antibody against arthritis

[0256] To study the in vivo efficacy of Fc-engineered anti-IL-1β antibodies, TAVO18378 is evaluated in a mouse arthritis model. Mouse IL-1 receptors can be activated by human IL-1β, and therefore a mouse arthritis model can be used to evaluate the efficacy of antibodies in blocking human IL-1β in vivo (Alten, Gram et al. 2008).

[0257] An NIH3T3 cell line stably expressing high levels of human IL-1β is established by transfecting NIH3T3 cells with a human IL-1β expression construct, and high-level expressing cell clones are selected. A mouse joint inflammation model is prepared by intra-articular injection of NIH3T3:hIL-1β cells into the right knee joint of DBA-1 mice. After dosing, the efficacy of the antibody on joint swelling, proteoglycan synthesis, and joint histopathology is evaluated.

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[0286] All documents cited herein (including the entire disclosures of those documents / publications), and all patents, patent applications, publications and books mentioned herein are hereby incorporated by reference in their entirety.

Claims

[Claim 1] An isolated humanized antibody or antigen-binding fragment thereof that specifically binds to human IL-1β with a potency comparable to that of a murine monoclonal antibody having a heavy chain variable region shown as SEQ ID NO:1 and a light chain variable region shown as SEQ ID NO:2.

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