Agents that interfere with IL-1β receptor signaling

Abstract

The present disclosure relates to antibodies that specifically bind to and neutralize interleukin-1β (IL-1β), and the use of such antibodies for the therapeutic treatment of IL-1β-mediated diseases and disorders.

Description

Technical Field

[0001] Priority This application claims the benefit of U.S. Provisional Patent Application No. 62 / 963,654, filed on January 21, 2020, the entire content of which is incorporated herein by reference.

[0002] Sequence Listing This application includes a sequence listing that has been electronically submitted in ASCII format, the entire content of which is incorporated herein by reference. The ASCII copy was created on July 8, 2020, named 15271_0001-00304_SL.txt, and is 54,286 bytes in size.

Background Art

[0003] Background Human interleukin-1β (IL-1β) is a pro-inflammatory cytokine that acts as a mediator of the peripheral immune response 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 various cell types.

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

[0005] Canakinumab (Ilaris, ACZ885) is a human monoclonal antibody developed by Novartis that targets interleukin 1β. The mode of action of canakinumab is based on the neutralization of IL-1β signaling (Alten, Gram et al., 2008). Canakinumab was approved for the treatment of cryopyrin-associated periodic syndromes (CAPS) in 2009 and subsequently approved for three additional rare and severe autoinflammatory diseases in 2016 (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] In recent years, IL-1β has been found to be associated with several steps in the development of atherosclerotic plaques, similar to other cardiovascular disease modifiers (McCarty and Frishman 2014). This hypothesis is that these inflammatory chemicals may prevent the heart from healing from damage caused by previous heart attacks. In 2017, a phase III clinical trial using canakinumab revealed a 15% reduction in the total of heart attacks, strokes, and deaths due to cardiovascular disease. In this trial, it was also clarified that the incidence and mortality of lung cancer were significantly reduced.

[0008] Considering the effectiveness of anti-IL-1β antibodies in autoinflammatory diseases, heart attacks, and lung cancer, there is a need to develop further anti-IL-1β antibodies with improved effectiveness and pharmacokinetic profiles.

Summary of the Invention

[0009] Summary The present disclosure provides 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. The 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 mouse monoclonal antibody against human IL-1β named TAVO304, which 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.

[0011] The present disclosure provides the heavy chain variable region of TAVO304, which comprises three complementarity-determining regions (CDRs) named HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown as SEQ ID NOs: 3, 4, and 5, respectively.

[0012] The present disclosure provides the light chain variable region of TAVO304, which comprises three CDRs named LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown as SEQ ID NOs: 6, 7, and 8, respectively.

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

[0014] The present disclosure provides four humanized light chain variable regions of TAVO304 named 304VL1, 304VL2, 304VL3, and 304VL4 having the amino acid sequences shown as SEQ ID NOs: 10, 11, 12, and 13, respectively.

[0015] The present disclosure provides a first humanized antibody against TAVO304, named TAVO7376, comprising a humanized heavy chain variable region 304VH1 and a humanized light chain variable region 304VL1, including 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, named TAVO7377, comprising a humanized heavy chain variable region 304VH1 and a humanized light chain variable region 304VL2, including 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, named TAVO7378, comprising a humanized heavy chain variable region 304VH1 and a humanized light chain variable region 304VL3, including 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, named TAVO7379, comprising a humanized heavy chain variable region 304VH1 and a humanized light chain variable region 304VL4, including 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 contain only the antigen-binding portion including Fab, F(ab’)2, or scFv fragments. The antibody backbone may be modified to affect functionality, for example, to eliminate residual effector functions.

[0020] The present disclosure also provides an isolated engineered anti-IL-1β IgG antibody having an extended half-life when compared to the parental wild-type antibody. The extended half-life is achieved by using any one set of mutations selected from M252Y / S254T / T256E, M428L / N434S, T250Q / M428L, N434A, and T307A / E380A / N434A (residue numbering follows the EU index) when compared to the parental wild-type antibody, and the C H2 and C H3 domains of the antibody are engineered.

[0021] The present disclosure also provides an isolated engineered anti-IL-1β IgG antibody having enhanced resistance to proteolysis by a protease that cleaves the wild-type antibody at or between residues 222-237 (EU numbering). The resistance to proteolysis can be achieved by engineering the E233P / L234V / L235A mutations (residue numbering follows the EU index) in the hinge region having a G236 deletion when compared to the parental wild-type antibody.

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

[0023] The present disclosure also provides a vector comprising the polynucleotide disclosed herein.

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

[0025] The present disclosure also provides a method for producing the anti-IL-1β monoclonal antibody disclosed herein, the method comprising culturing the 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 pharmaceutically acceptable carrier.

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

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

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

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

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

[0032] The present disclosure also provides a method for 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 for treating a 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 for 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 one section of the specification that describes specific aspects of the disclosure, and those described only in the examples or drawings, can be combined with any one or more other embodiments, unless explicitly disclaimed or inappropriate. Brief Description of the Drawings

Brief Description of the Drawings

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

[0037] Figure 2 Response of reporter gene expression to stimulation by human and cynomolgus monkey IL-1β in the HEK-Blue IL-1β reporter assay.

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

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

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

[0041] Figure 6 Binding of four humanized anti-IL-1β IgG1 antibodies to (A) human and (B) cynomolgus monkey IL-1β.

[0042] Figure 7 Neutralization of (A) human and (B) cynomolgus monkey IL-1β-driven reporter gene activation by four humanized anti-IL-1β IgG1 antibodies in the 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 the MRC-5 cytokine release assay.

Mode for Carrying Out the Invention

[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 defined otherwise, 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] Any methods and materials similar or equivalent to those described herein may be used in the practice of the disclosure related hereto, but 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 context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells and the like.

[0048] "Antibody" is construed broadly and includes 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 that includes an antigen-binding site of the required specificity, including murine, human, humanized and chimeric monoclonal antibodies.

[0049] "Full-length antibody molecule" is composed of two heavy chains (HCs) and two light chains (LCs) interconnected 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 the 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 hypervariable regions called complementarity-determining regions (CDRs) separated by framework regions (FRs). Each VH and VL is composed of three CDRs and four FR segments, respectively, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0050] "Complementary determining region (CDR)" is the "antigen-binding site" in an antibody. CDRs can be defined using various terms: (i) Complementary 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), refers to the regions of antibody variable domains that are hypervariable in the structures 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 definitions of antigen-binding sites. The correspondence of CDR, HV, and IMGT descriptions is 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 by Kabat, Chothia, or IMGT above, unless explicitly stated otherwise in the specification.

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

[0052] "Antibody fragment" refers to a portion of an immunoglobulin molecule that retains a heavy chain and / or light chain antigen-binding site, such as 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), such as those consisting of one VH domain. The VH and VL domains may be linked via a synthetic linker to form various types of single-chain antibody designs, where the VH / VL domains may pair intramolecularly, or intermolecularly if the VH and VL domains are expressed by separate single-chain antibody constructs, to form a monovalent antigen-binding site, such as a single-chain Fv (scFv), or a bispecific antibody. For example, as described in International Publication Nos. WO1998 / 44001, WO1988 / 01649, WO1994 / 13804, and WO1992 / 01047.

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

[0054] "Isolated antibody" refers to an antibody or antibody fragment that substantially does not contain other antibodies having different antigenic properties. "Isolated antibody" includes antibodies isolated to sufficient purity, for example, antibodies having a purity of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[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. A humanized antibody may contain substitutions within the framework, and the framework need not be an exact copy of the expressed human immunoglobulin or human immunoglobulin germline gene sequence.

[0056] "Human antibody" refers to an antibody having heavy and light chain variable regions that are both derived from human-origin sequences and 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 the constant region, that constant region is also derived from a human-origin sequence.

[0057] Throughout this specification, the numbering of amino acid residues in the antibody constant region follows the EU index described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), unless otherwise explicitly stated.

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

Table 1

[0059] The constant region sequence of mammalian IgG heavy chain is C H1 -hinge-C H2 -C H3 and is designated in the sequence as such. The "hinge", "hinge region" or "hinge domain" of IgG is generally defined as including Glu216 of human IgG1 according to the EU index and ending at Pro230. Functionally, however, the flexible part of the chain may be considered to include additional residues such as the upper and lower hinge regions, such as from Glu216 to Gly237. The lower hinge refers to residues 233-239 of the Fc region to which FcγR binding is generally ascribed. The 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 bond. Since numbering is according to the EU index, the boundaries may vary slightly, but the C H1 domain is adjacent to the V H domain, is on the amino-terminal side of the hinge region of the immunoglobulin heavy chain molecule, and includes, for example, from EU positions approximately 118-215, the first (most amino-terminal) constant region of the immunoglobulin heavy chain. The Fc domain extends from amino acid 231 to amino acid 447; the C H2 domain extends from around Ala231 to Lys340 or Gly341, and the C H3 extends from Gly341 or Gln342 to Lys447. The residues of the IgG heavy chain constant region in the C H1 region end with Lys. Fc domain-containing molecules include at least the C H2 and C H3 domains of the antibody constant region, and thus include at least the region from around Ala231 to Lys447 of the IgG heavy chain constant region. Fc domain-containing molecules may optionally include at least a portion of the hinge region.

[0060] "Epitope" refers to the part of an antigen to which an antibody specifically binds. An epitope typically consists of the active (such as polar, nonpolar, or hydrophobic) surface groupings of components such as amino acids or polysaccharide side chains, and may have specific three-dimensional structural features, as well as specific charge characteristics. An epitope may be composed of contiguous and / or discontinuous amino acids that form a conformational spatial unit. For discontinuous epitopes, amino acids from different parts of the linear sequence of the antigen are brought into proximity in three-dimensional space through the folding of the antigen molecule. The antibody "epitope" depends on the methodology used to identify the epitope.

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

[0062] The terms "peptide", "polypeptide", and "protein" are used interchangeably herein and 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 can also include polypeptides having post-translational modifications such as cleavage of signal peptides simultaneous with translation of the polypeptide 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 that includes modifications such as deletions, additions, and substitutions (such as of conservative nature known to those of skill in the art) to the native sequence, as long as the protein maintains the desired activity. These modifications can be intentional, such as through site-directed mutagenesis, or can be accidental, such as through mutations in the host producing the protein or errors in PCR amplification or other recombinant DNA methods.

[0064] As used herein with respect to a nucleic acid molecule, the term "recombinant" means a polynucleotide of genomic, cDNA, viral, semi-synthetic, and / or synthetic origin, which by virtue of its origin or manipulation, does not accompany all or part of the polynucleotide sequence with which it is originally associated. As used herein with respect to a protein or polypeptide, the term "recombinant" refers to a polypeptide produced by expression from a recombinant polynucleotide. As used herein with respect to a host cell or virus, the term "recombinant" refers to a host cell or virus into which a recombinant polynucleotide has been introduced. Recombinant is also used herein with respect to a substance (e.g., a cell, nucleic acid, protein, or vector) to indicate that the substance has been modified by the introduction of a heterogeneous substance (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 that are ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule.

[0066] "Vector" refers to a polynucleotide that can be replicated within a biological system or transferred between such systems. A vector polynucleotide typically contains elements such as an origin of replication, a polyadenylation signal, or a selectable marker that function to facilitate the replication or maintenance of these polynucleotides in a biological system, such as a biological system reconstituted using a biological component capable of replicating cells, viruses, animals, plants, and vectors. The vector polynucleotide may be a single-stranded (ss) or double-stranded DNA or RNA molecule, cDNA, or a hybrid thereof.

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

[0068] "Valency" refers to the presence of a specific number of binding sites specific for a particular antigen within a molecule. Thus, the terms "monovalent", "divalent", "tetravalent" and "hexavalent" refer to the presence of 1, 2, 4 and 6 binding sites specific for a particular antigen within the 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 occur naturally in a cell from which the heterologous nucleic acid sequence, protein or polypeptide is 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. Heterologous nucleic acids may be derived from different organisms or animal species, while such nucleic acids need not be derived from a different species to be heterologous. For example, in some cases, a synthetic nucleic acid sequence or the polypeptide encoded therein can be heterologous to the cell into which it is introduced in that the cell did not previously contain that synthetic nucleic acid. Thus, for example, a synthetic nucleic acid sequence or one or more components of the polypeptide encoded therein can be considered heterologous to a human cell even if one or more of the components were originally derived from a human cell.

[0070] As used herein, "host cell" refers to a eukaryotic cell in vivo or in vitro, or a cell (e.g., a cell line) derived from a multicellular organism cultured as a unicellular entity, which eukaryotic cell can be or has been used as a recipient of a nucleic acid (e.g., an expression vector containing a nucleotide sequence encoding a multimeric polypeptide of the present disclosure), and includes progeny of the original cell that have been genetically modified by that nucleic acid. It is understood that progeny of a single cell need not necessarily be identical in form, or genomic or total DNA complementarity, to the original parent due to natural, accidental or intentional 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 introduction 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, into a suitable eukaryotic host cell.

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

[0072] As used herein, the terms "treatment", "treating", etc. refer to obtaining the desired pharmaceutical and / or physiological effect. This effect may be prophylactic in the sense of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in the sense of partial or complete cure of the disease and / or the adverse effects attributable to the disease. "Treatment" as used herein encompasses any treatment of a disease in a mammal, such as a human, and includes (a) preventing the occurrence of a disease in a subject who may have a predisposition to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., stopping the progression of the disease; and (c) alleviating the disease, i.e., causing regression of the disease.

[0073] The terms "individual", "subject", "host", and "patient", which are used interchangeably herein, refer to mammals and include, but are not limited to, rodents (e.g., rats, mice), rabbits (e.g., rabbits), non-human primates, humans, dogs, cats, artiodactyls (e.g., horses, cows, sheep, pigs, goats), etc.

[0074] "Therapeutically effective amount" or "effective amount" refers to the amount of an agent or the total amount of two agents that is sufficient to effect such treatment of a disease when administered to a mammal or other subject for the treatment of that disease. The "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 should be understood that the present disclosure is, of course, subject to variations and is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0076] As used herein, monoclonal antibodies and antigen-binding fragments thereof are described that specifically bind to, neutralize, inhibit, block, suppress, reduce, or interfere with the activity of at least one IL-1β, such as the functional activity of IL-1β against its receptor IL-1RI. These anti-IL-1β antibodies and antigen-binding fragments can be therapeutically administered to a subject for treating IL-1β-mediated diseases. <Composition of Anti-IL-1β Antibody and Antigen-Binding Fragment>

[0077] The present disclosure provides a mouse monoclonal antibody named TAVO304 that is identified from mouse hybridoma screening and specifically binds to neutralize, inhibit, block, suppress, reduce, or interfere with at least one activity of human interleukin 1β (IL-1β). TAVO304 includes 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 includes three complementarity-determining regions named HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown as SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively. The light-chain variable region of TAVO304 includes three complementarity-determining regions named LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown 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]

Chemical Structure

[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] [Chemical formula]

[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] [Chemical formula]

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

[0087] [Chemical formula]

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

[0089] [Chemical formula]

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

[0091] [Chemical formula]

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

[0093] [Chemical formula]

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

[0095]

Chemical formula

[0096] Mouse anti-human IL-1β antibody TAVO304 can be humanized by grafting mouse CDRs onto a human germline scaffold. A small number of key mouse residues are conserved by back-mutations to achieve higher stability and better expression while minimizing immunogenicity. For TAVO304, one humanized VH variant (304VH1) is designed based on IGHV3-48 * 01, and four humanized VL variants are designed using a set of back-mutations using 304VL1 and 304VL2 based on IGKV1-39 * 01 and 304VL3 and 304VL4 based on IGKV2-40 * 01.

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

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

[0099]

Chemical formula

[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 named 304VL1, 304VL2, 304VL3, and 304VL4, which have amino acid sequences shown as SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively.

[0102] Sequence of humanized light chain variable region 304VL1 (SEQ ID NO: 10)

[0103]

Chemical formula

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

[0105] Sequence of humanized light chain variable region 304VL2 (SEQ ID NO: 11)

[0106]

Chemical formula

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

[0108] Sequence of humanized light chain variable region 304VL3 (SEQ ID NO: 12)

[0109]

Chemical formula

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

[0111] Sequence of humanized light chain variable region 304VL4 (SEQ ID NO: 13)

[0112]

Chemical formula

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

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

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

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

[0117] The present disclosure provides a fourth humanized IgG1 antibody against TAVO304, which comprises a humanized heavy chain variable region 304VH1 and a humanized light chain variable region 304VL4, named TAVO7379, and having a heavy chain sequence shown as SEQ ID NO: 14 and a 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]

Chemical formula

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

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

[0122]

Chemical formula

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

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

[0125]

Chemical formula

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

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

[0128]

Chemical formula

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

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

[0131]

Chemical formula

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

[0133] The present disclosure also provides the preparation of bispecific or multispecific antibodies capable of engaging 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 named 304VH1 having the amino acid sequence shown as SEQ ID NO: 9 and one of four humanized light chain variable regions of TAVO304 named 304VL1, 304VL2, 304VL3, and 304VL4 having the amino acid sequences shown as SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively. For example, this can be a bispecific antibody having a Fab or scFv domain having 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 two, three, or four epitopes.

[0134] The IL-1β binding antibodies and fragments of the present disclosure include antigen-binding fragments that retain sufficient ability to specifically bind to IL-1β. As used herein, an IL-1β binding fragment may include any three or more contiguous amino acids of an antibody (e.g., four or more, five or more, six or more, eight or more, or ten or more contiguous 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 the intact antibody, are cleared more rapidly from circulation, and can have less non-specific tissue binding than the intact antibody. These fragments can be produced from intact antibodies by proteolytic cleavage using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab’)2 fragments) using well-known methods.

[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 to create two antigen-binding sites.

[0136] The IL-1β binding antibodies and fragments of the present disclosure may also include single-chain antibody fragments (scFvs) that bind to IL-1β. The scFv comprises an antibody light chain variable region (V L ) operably linked to an antibody heavy chain variable region (V H ) that together or individually form a binding site that binds to IL-1β. Such IL-1β binding fragments can be prepared by methods known in the art, such as 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 cell.

[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 include an IgG Fc domain, such as at least one isotype of 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 engineered into an IgG4 antibody to enhance IgG4 stability.

[0143] IgG1 Fc (SEQ ID NO: 19):

[0144]

Chemical Structure

[0145] IgG2 Fc (SEQ ID NO: 20):

[0146]

Chemical Structure

[0147] IgG3 Fc (SEQ ID NO: 21):

[0148]

Chem.

[0149] IgG4 Fc (SEQ ID NO: 22)

[0150]

Chem.

[0151] The anti-IL-1β antibody may comprise a modified Fc region, wherein the modified Fc region contains at least one amino acid modification with respect to the wild-type Fc region. In some embodiments, the anti-IL-1β antibody is provided with a modified Fc region, wherein the native Fc region is modified such that the half-life of the antibody is extended when compared to the parental wild-type antibody in a biological environment, such as the serum half-life or the half-life measured by an in vitro assay.

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

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

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

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

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

[0157] In certain embodiments, the half-life extension can also be achieved by engineering the 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 evaluated in PK studies in mice for antibodies with native IgG Fc.

[0159] IgG1 Fc with M252Y / S254T / T256E mutations (SEQ ID NO: 23)

[0160]

Chemical Structure

[0161] IgG1 Fc with M428L / N434S mutations (SEQ ID NO: 24)

[0162]

Chemical Structure

[0163] IgG1 Fc with T250Q / M428L mutations (SEQ ID NO: 25)

[0164]

Chemical Structure

[0165] IgG1 Fc with N434A mutation (SEQ ID NO: 26)

[0166]

Chem.

[0167] IgG1 Fc with T307A / E380A / N434A mutations (SEQ ID NO: 27)

[0168]

Chem.

[0169] In some embodiments, the anti-IL-1β antibody is provided with a modified Fc region that has been modified to enhance antibody resistance 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 the E233P / L234V / L235A mutations (residue numbering according to the EU index) in the hinge region where G236 is deleted, compared to the parental wild-type antibody of SEQ ID NO: 28 (Kinder, Greenplate et al., 2013).

[0171] IgG1 Fc with E233P / L234V / L235A mutations and deletion of G236 (SEQ ID NO: 28)

[0172]

Chem.

[0173] When the functionality of the effector is not desired, the antibodies of the present disclosure may be further engineered to introduce at least one mutation in the antibody Fc that reduces the binding of the antibody to activating Fcγ receptors (FcγR) and / or reduces Fc effector functions 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 the binding of the antibody to activating FcγR 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 that may be made singly or in combination are the K214T, E233P, L234V, L234A, deletion of G236, 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 combinatorial mutations that may be made to reduce ADCC are 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 / A327G / P331A / D365E / L358M on IgG1, 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. Hybrid IgGs such as Fc having 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 antibody is provided with a modified Fc region, wherein the native Fc region is modified to promote the generation of bispecific antibodies by Fc heterodimerization.

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

[0179] In certain embodiments, Fc heterodimerization can also be achieved by Fc mutations to promote a Knob-in-Hole strategy (see, e.g., International Publication WO2006 / 028936). Amino acids with small side chains (holes) are introduced into one Fc domain, and amino acids with large side chains (knobs) are introduced into the other Fc domain. After co-expression of these two heavy chains, a heterodimer is formed as a result of the preferential interaction between the heavy chain with a "hole" and the heavy chain with a "knob" (Ridgway, Presta et al., 1996).

[0180] Exemplary Fc mutation pairs that form knobs and holes are as follows: 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 an electrostatically-matched interaction strategy (Gunasekaran, Pentony et al., 2010). The mutations can be engineered such that positively charged residues are generated in one Fc domain and negatively charged residues are generated in the other Fc domain, as described in U.S. Patent Publication US2010 / 0015133; U.S. Patent Publication US2009 / 0182127; U.S. Patent Publication US2010 / 028637 or U.S. Patent Publication US2011 / 0123532. Heavy chain heterodimerization can be formed by the electrostatically-matched interaction 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 properties of the antibody containing the amino acid sequence. Conservative modifications include amino acid substitutions, additions, and deletions. Conservative substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are well defined and include amino acids having 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), β-branched side chains (e.g., threonine, valine, isoleucine), and sulfur-containing side chains (cysteine, methionine). Further, as previously described for alanine scanning mutagenesis, any native residue within a polypeptide may be substituted with alanine ((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. Patent No. 4,683,195). Alternatively, libraries of variants may be generated using, for example, random (NNK) or non-random codons, such as the DVK codon encoding 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The resulting antibody variants 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 can occur in vivo or in vitro. For example, the antibodies of the present disclosure may be conjugated with polyethylene glycol (PEGylated) to improve their pharmacokinetic profile. The conjugation may be carried out by techniques known to those skilled 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 they are within the scope of the present disclosure. Antibody stability is affected by a number of factors, including (1) the core packing of individual domains that affects intrinsic stability, (2) protein / protein interface interactions that impact HC and LC pairing, (3) the burial of polar and charged residues, (4) the hydrogen bond network for polar and charged residues, and (5) the distribution of surface charges and polar residues in other intramolecular and intermolecular forces (Worn and Pluckthun 2001). Potential structures that destabilize residues may be identified based on the crystal structure of the antibody or, in some cases, by molecular modeling, and the effect of residues on antibody stability may be tested by generating mutants with mutations in the identified residues and evaluating them. One way to increase antibody stability is to raise the thermal transition midpoint (Tm) measured by differential scanning calorimetry (DSC). In general, the protein Tm correlates with its stability and is inversely correlated with the degradation process that depends on the tendency of the protein to unfold and its susceptibility to unfolding and denaturation in solution (Remmele and Gombotz 2000). A number of studies have found a correlation between the ranking of the physical stability of formulations measured as thermal stability by DSC and the 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 have suggested that the Tm of the 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 production and drug stability. One example for IgG1 is H224S (or H224Q) in hinge 221-DKTHTC-226 (SEQ ID NO: 33) (EU numbering) that 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 present disclosure can be encoded by a single nucleic acid (e.g., a single nucleic acid comprising a nucleotide sequence encoding the 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 a non-limiting example, the present disclosure provides a nucleic acid sequence of SEQ ID NO: 29 encoding the IgG1 heavy chain sequence of TAVO7378 of SEQ ID NO: 14, and a nucleic acid sequence of SEQ ID NO: 30 encoding the light chain sequence of TAVO7378 of SEQ ID NO: 17.

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

[0190]

Chemical formula

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

[0192]

Chemical formula

[0193] The nucleic acids described herein can be inserted into vectors, such as nucleic acid expression vectors and / or targeting vectors. Such vectors can be used in a variety of ways, for example, for the expression of an IL-1β binding antibody or antibody fragment in a cell or transgenic animal. The vector is typically selected to be functional in the host cell in which the vector is to be used. Nucleic acid molecules encoding an IL-1β binding antibody or fragment may be amplified / expressed in prokaryotic, yeast, insect (baculovirus system), 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 containing donor and acceptor splicing sites, a leader sequence for secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.

[0194] In most cases, the 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 the host cell results in the 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 that can affect glycosylation may be added.

[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 the nucleic acid or vector of the present disclosure such 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, the 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 methods for culturing and selecting transformed host cells in vitro are known in the art and include calcium chloride-mediated transformation, transduction, conjugation, triparental mating (triparental conjugation), DEAE, dextran-mediated transfection, infection, membrane fusion using liposomes, high-velocity bombardment using DNA-coated microparticles, direct microinjection into single cells, and the use of electroporation.

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

[0198] In certain embodiments, two or more vectors encoding an IL-1β binding antibody or antigen-binding fragment thereof together 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 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 the selectively binding agent 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 includes anti-IL-1β antibodies and fragments that selectively bind to IL-1β in binding to IL-1β with a higher affinity than to other antigens. These anti-IL-1β antibodies and fragments can selectively bind to human IL-1β, but can also bind detectably to non-human IL-1β. Alternatively or additionally, these antibodies or fragments can bind to human IL-1β and to IL-1β of other mammals. For example, these antibodies or fragments can 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 against 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 interleukin-1 receptor type I (IL-1R1). For example, the binding of IL-1β to IL-1R1 can be determined by immobilizing an IL-1β-binding antibody, using this immobilized antibody to capture IL-1β to determine whether IL-1β has bound to the antibody, and contacting the bound IL-1β / antibody complex with soluble IL-1RI to determine whether soluble IL-1RI has bound to the complex. The protocol may also include contacting soluble IL-1RI with the immobilized antibody prior to contacting with IL-1β to confirm that soluble IL-1RI does not bind to the immobilized antibody. This protocol can be carried out using a Biacore® instrument for kinetic analysis of the binding interaction. Such a protocol can also be used to determine whether an antibody or other molecule permits or blocks the binding of IL-1β to IL-1 receptor type I.

[0203] For other IL-1β / IL-1RI binding assays, the permissive or blocking of IL-1β binding to 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 its IL-1β-binding fragment. Blocking is identified in the assay readout information as a specified reduction in the binding of IL-1β to IL-1 receptor type I in the presence of an anti-IL-1β antibody or its IL-1β-binding fragment, compared to a control sample containing the corresponding buffer or diluent but no IL-1β antibody or its IL-1β-binding fragment. The assay readout information may be viewed qualitatively as indicating the presence or absence of blocking, or quantitatively as indicating the percent or fold reduction in binding due to the presence of the antibody or antibody fragment. When an IL-1β-binding antibody or IL-1β-binding fragment substantially blocks the binding of IL-1β to IL-1RI, the binding of IL-1β 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 10000-fold, or more, compared to the 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 a PepSpot™ peptide array (JPT Peptide Technologies, Berlin, Germany), in which 12-amino acid peptides that overlap the previous peptide by 11 amino acids are synthesized directly on a membrane across the entire IL-1β amino acid sequence. Alternatively or additionally, antibody competition experiments may be performed, such assays being well known in the art.

[0205] IL-1β antibodies or antibody fragments that are preferred for use according to the present disclosure generally bind to human IL-1β with high affinity (e.g., as determined using BIACORE), and have an equilibrium binding dissociation constant (KD) for IL-1β of, for example, 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 present 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 present disclosure do not cross-react with any other target other than IL-1β. For example, the antibodies and fragments of the present 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 at least about 250-fold) higher selectivity for the binding of IL-1β over the binding of IL-1α.

[0208] The present disclosure also encompasses a neutralizing antibody or a neutralizing fragment thereof that binds to IL-1β so as to neutralize the biological activity of IL-1β. Neutralization of the biological activity of IL-1β can be evaluated by assay for one or more indicators of IL-1β biological activity, such as reporter gene expression by IL-1β stimulation in a reporter assay, IL-6 release by IL-1β stimulation from human fibroblasts or other cells, or proliferation by IL-1β induction of T helper cells. Neutralization of the biological activity of IL-1β can also be evaluated 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 interleukin-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, suppress, 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, suppress, 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 antibody or fragment binding thereto can sterically or conformationally inhibit, block, suppress, reduce or interfere with the target activity of IL-1β. In yet another embodiment, the antibody or fragment thereof of the present disclosure binds 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 antibody or fragment binding thereto results in enhanced clearance of IL-1β.

[0210] Generally, 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 the binding of IL-1β to IL-1 receptor type I is blocked. More preferably, an IL-1β-binding antibody or IL-1β-binding fragment neutralizes the biological activity of IL-1β by binding to IL-1β without substantially preventing the binding of the bound IL-1β to IL-1 receptor type I. A potential advantage of such antibodies and fragments is that they can still allow IL-1β to bind to IL-1RI while binding and neutralizing IL-1β. This can result in a more effective reduction of IL-1α biological activity as well as IL-1β biological activity because there are fewer unbound IL-1RI sites available for IL-1α to bind. Thus, the IL-1β-binding antibodies and fragments of the present disclosure are useful in a desirable manner for neutralizing the in vitro and in vivo biological activity of IL-1.

[0211] The antibody or fragment of the invention may be a neutralizing antibody or fragment that specifically binds to an IL-1β epitope that affects the biological activity of IL-1β. The antibody or fragment 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 the loss of the biological activity of IL-1β that contains 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 contain a therapeutically or prophylactically effective amount of an IL-1β-binding antibody or antibody fragment of the present disclosure in a suitable carrier, such as a mixture with a pharmaceutically acceptable agent. Typically, the IL-1β-binding antibodies and antibody fragments of the present disclosure are sufficiently purified for administration to animals prior to formulation into a pharmaceutical composition.

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

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

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

[0216] The pharmaceutical compositions described herein can be formulated for controlled or sustained delivery, extended release, and / or increased stability or half-life in a particular local environment in a manner that provides a local concentration of the product (e.g., bolus, depot effect). <Method of 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, allergic and allergic-like conditions, hypersensitivity reactions, autoimmune diseases, severe infections, and organ or tissue transplant rejection reactions.

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

[0219] Specific autoimmune diseases for which the antibodies of the present disclosure may be used include autoimmune blood diseases (including, for example, hemolytic anemia, aplastic anemia, polycythemia vera, and idiopathic thrombocytopenia), systemic lupus erythematosus, polychondritis, sclerodema, Wegener's granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, Stevens-Johnson syndrome, idiopathic sprue, autoimmune inflammatory bowel diseases (including, for example, ulcerative colitis, Crohn's disease, and irritable bowel syndrome), endocrine ophthalmopathy, Graves' disease, sarcoidosis, multiple sclerosis, primary biliary cirrhosis, juvenile diabetes (type I diabetes), uveitis (anterior and posterior segments), dry keratoconjunctivitis and vernal keratoconjunctivitis, interstitial pulmonary fibrosis, psoriatic arthritis, and glomerulonephritis (including, regardless of the presence or absence of nephrotic syndrome, for example, idiopathic nephrotic syndrome or minimal change nephropathy).

[0220] The antibodies of the present disclosure are also useful for the treatment, prevention, or improvement of asthma, bronchitis, emphysema, and other obstructive or inflammatory diseases of the airway.

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

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

[0223] The antibodies of the present disclosure are also useful for the reduction, prevention, or treatment of cardiovascular events and / or cardiovascular diseases, including myocardial infarction, stroke, cardiovascular death, congestive heart failure, cardiac arrest, acute coronary syndrome, angina pectoris, or revascularization procedures.

[0224] The antibodies of the present disclosure are also useful for the prevention or treatment of cancers 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 the 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 states characterized by insulin resistance. Such methods may be used to treat mammalian subjects (e.g., humans) suffering from type 2 diabetes, type 1 diabetes, obesity, hyperglycemia, hyperinsulinemia, insulin resistance, and conditions and states characterized by insulin resistance, or to prevent their onset 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 use, the antibodies and fragments of the invention may be used in diagnostic methods for detecting 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] A method for detecting IL-1β in a biological sample can include the steps of contacting the biological sample with one or more antibodies or fragments of the present invention, and detecting either the antibody or fragment bound to IL-1β or the unbound antibody or fragment, whereby IL-1β in the biological sample can be detected. 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, avidin family, fluorescent materials, luminescent materials, and radioactive materials.

Example

[0228] Examples The following examples are provided to explain the present disclosure in more detail. These are intended to illustrate the present disclosure and not to limit it. <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. ELISA-based binding assays were 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 ELISA plates. Increasing concentrations of the TAVO304 antibody were applied to the plates, and its binding to recombinant human IL-1β was detected with an HRP-conjugated anti-mouse secondary antibody. TAVO304 was observed to bind to 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 monkey IL-1β was also evaluated in a similar ELISA assay by coating plates with mouse and rhesus monkey IL-1β, respectively. TAVO304 did not show significant binding affinity to mouse IL-1β (Figure 1). In contrast, it showed good binding affinity to rhesus monkey IL-1β and had an EC50 of 12.2 ng / mL. <Example 2: In Vitro Assay for IL-1β Neutralization by TAVO304>

[0231] To evaluate the functional activity of IL-1β, a HEK-Blue IL-1β reporter assay was developed. In this assay, HEK-Blue IL-1β cells (Invivogen) express a secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of the IFN-β minimal promoter fused to the IL-1β receptor IL-1R and five NF-κB and five AP-1 binding sites. Binding of IL-1β to the receptor IL-1R on the surface of HEK-Blue IL-1β cells triggers a signaling cascade, leading to 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 evaluated using this assay. It was observed that both human IL-1β and rhesus monkey IL-1β were able to induce reporter gene expression in a dose-dependent manner and had EC50 values 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 the blockade of human IL-1β-driven reporter gene expression. Increasing concentrations of the antibody were applied to HEK-Blue IL-1β reporter cells together with 10 ng / mL of human IL-1β. After overnight incubation, SEAP reporter gene expression was quantified. TAVO304 neutralized IL-1β-mediated reporter gene expression in a dose-dependent manner, with an IC50 of 0.12 μg / mL (Figure 3), and was observed to be 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 the blockade of cynomolgus IL-1β-driven reporter gene expression. Increasing amounts of the antibody were applied to HEK-Blue IL-1β reporter cells together with 0.25 ng / mL of cynomolgus IL-1β. TAVO304 was observed to neutralize cynomolgus IL-1β-mediated reporter gene expression in a dose-dependent manner, with an IC50 of 3.6 ng / mL (Figure 3). <Example 3: Humanization of the Mouse Anti-Human IL-1β Antibody TAVO304>

[0235] The mouse anti-human IL-1β antibody TAVO304 was humanized by transplanting mouse CDRs onto a human germline scaffold. A few key mouse residues were conserved by backmutation to achieve higher stability and better expression while minimizing immunogenicity. For TAVO304, one humanized VH variant (304VH1) was designed based on IGHV3-48 * 01, and four humanized VL variants were 304VL1 and 304VL2 using IGKV1-39 * 01, and IGKV2-40 *Designed using 304VL3 and 304VL4 based on 01, it had two reverse mutations (Figure 4). Four humanized TAVO304 antibodies with IgG1 Fc, named TAVO7376, TAVO7377, TAVO7378, and TAVO7379, were generated using the 304VH1 variant that pairs with 304VL1, 304VL2, 304VL3, and 304VL4 respectively, from the combination of a humanized VH variant and four humanized VL variants (Figure 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 (Thermo Scientific) according to the instructions of the transfection kit. The cells were centrifuged and sedimented 5 days after transfection, and the supernatant was passed through a 0.2 μm filter. Purification of the expressed antibody in the supernatant was performed by affinity chromatography on a Protein A agarose column (GE Healthcare Life Sciences). The purified antibody was buffer-exchanged to DPBS, pH 7.2 by dialysis, and the protein concentration was determined by UV absorbance at 280 nm.

[0237] The 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 near 150 kDa. <Example 5: Binding of Humanized TAVO304 to IL-1β>

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

[0239] The binding of the four humanized antibodies to cynomolgus IL-1β was also evaluated in a similar ELISA assay by coating cynomolgus IL-1β onto the plate. All four humanized antibodies, TAVO7376, TAVO7377, TAVO7378, and TAVO7379, were observed to bind to recombinant cynomolgus IL-1β in a dose-dependent manner with similar potency (Figure 6).

[0240] To evaluate binding specificity, cytokines homologous to IL-1β, such as IL-1α, were evaluated 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, did not show significant binding to human IL-1α at 1 μg / mL in the ELISA-based binding assay, indicating specificity for IL-1β binding for these humanized antibodies.

[0241] To further measure the binding of the humanized antibodies to immobilized recombinant IL-1β, a surface plasmon resonance (SPR) binding assay was performed using a Biacore. This assay can measure not only binding affinity but also kinetic rate constants and the thermodynamics of binding. <Example 6: In Vitro Reporter Assay for IL-1β Neutralization by Humanized TAVO304>

[0242] The TAVO7376, TAVO7377, TAVO7378 and TAVO7379 were evaluated for the blockade of human IL-1β-driven reporter gene expression using the HEK-Blue IL-1β reporter assay. Increasing amounts of antibody were applied to HEK-Blue IL-1β reporter cells together with 10 ng / mL of human IL-1β. After overnight incubation, SEAP reporter gene expression was quantified. It was observed that all four humanized antibodies dose-dependently neutralized IL-1β-mediated reporter gene expression with similar potency (IC50 around 0.09 μg / mL, Figure 7). These potencies in the neutralization of human IL-1β were similar to those of TAVO304 and were much more potent than the reference antibody refAb-1.

[0243] Using the same assay, the four humanized antibodies were evaluated for the blockade of cynomolgus IL-1β-driven reporter gene expression. Increasing amounts of antibody were applied to HEK-Blue IL-1β reporter cells together with 0.25 ng / mL of cynomolgus IL-1β. It was observed that the TAVO7376, TAVO7377, TAVO7378 and TAVO7379 antibodies dose-dependently neutralized cynomolgus IL-1β-mediated reporter gene expression with similar potency (IC50 around 0.018 μg / mL, Figure 7). <Example 7: In vitro MRC-5 cell-based cytokine release assay for IL-1β neutralization by humanized TAVO304>

[0244] IL-1β can drive the activation of human lung fibroblast cell line MRC-5 and stimulate IL-6 release. To measure the humanized TAVO304 antibody in the neutralization of IL-1β-mediated MRC-5 cell activation, an in vitro assay was prepared. Increasing amounts of antibody were applied to MRC-5 cells (ATCC) with 1 ng / mL of human IL-1β for 24 hours, and the induced IL-6 release from the cells could be quantified by an IL-6 assay kit (R&D systems). The 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, Figure 8) and were observed to be 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 the antibody half-life. Specifically, the M428L / N434S mutation has been shown to extend the antibody half-life by increasing the FcRn binding affinity (Booth, Ramakrishnan et al., 2018). Furthermore, the L234A / L235A Fc mutation can abolish the ADCC and CDC effector functions of IgG1 antibodies (Hezareh, Hessell et al., 2001). Therefore, a 304VH1-based humanized IgG1 heavy chain with the L234A / L235A / M428L / N434S (AALS) mutation was generated, which contains 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 an extended half-life and reduced effector function was generated and named TAVO11878.

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

[0247] [Chemical]

[0248] The sequence of the variable domain of the heavy chain is underlined. The AALS mutations are shown in bold.

[0249] To investigate whether the 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. Recombinant mouse FcRn (R&D systems) at 1 μg / mL is coated onto ELISA plates. Increasing concentrations of the TAVO11878 and TAVO7378 antibodies are applied to these plates, and their binding to recombinant FcRn at pH 6.0 is detected with an HRP-conjugated anti-mouse secondary antibody.

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

[0251] To improve the in vivo stability of a humanized antibody, additional Fc mutations can be introduced into an IgG1 antibody to enhance 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 the E233P mutation with a G236 deletion (residue numbering follows the EU index). Accordingly, a humanized IgG1 heavy chain based on 304VH1 with the E233P, L234A, L235A, M428L, N434S mutations with a G236 deletion is generated, which contains 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, an extended half-life, and reduced effector function is generated and named TAVO18378.

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

[0253]

Chemical formula

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

[0255] To study whether the engineered anti-IL-1β antibodies having these Fc mutations have improved resistance to proteolysis, TAVO18378 is subjected to digestion with recombinant IgG protease IdeZ (New England Biolabs) at 37°C for 30 minutes, followed by SDS-PAGE analysis under reducing conditions to evaluate 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) at 37°C for 24 hours, followed by SDS-PAGE under reducing conditions to evaluate the integrity of its heavy chain. The above-described Fc mutations below the hinge region are expected to be able to promote the resistance of the Fc-engineered anti-IL-1β antibody 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 the Fc-engineered anti-IL-1β antibody, TAVO18378 is evaluated in a mouse arthritis model. The mouse IL-1 receptor can be activated by human IL-1β, and thus the mouse arthritis model can be used to evaluate the efficacy of an antibody 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-expression cell clones are selected. The mouse arthritis 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 against joint swelling, proteoglycan synthesis, and joint histopathology is evaluated.

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[0278] Ridgway, J. B., L. G. Presta and P. Carter (1996). "'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization." Protein Eng 9(7): 617-621.

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[0281] Vafa, O., G. L. Gilliland, R. J. Brezski, B. Strake, T. Wilkinson, E. R. Lacy, B. Scallon, A. Teplyakov, T. J. Malia and W. R. Strohl (2014). "An engineered Fc variant of an IgG eliminates all immune effector functions via structural perturbations." Methods 65(1): 114-126. Worn, A. and A. Pluckthun (2001). "Stability engineering of antibody single-chain Fv fragments." J Mol Biol305(5): 989-1010.

[0282] Xu, D., M. L. Alegre, S. S. Varga, A. L. Rothermel, A. M. Collins, V. L. Pulito, L. S. Hanna, K. P. Dolan, P. W. Parren, J. A. Bluestone, L. K. Jolliffe and R. A. Zivin (2000). "In vitro characterization of five humanized OKT3 effector function variant antibodies." Cell Immunol 200(1): 16-26.

[0283] Yang, K., A. Basu, M. Wang, R. Chintala, M. C. Hsieh, S. Liu, J. Hua, Z. Zhang, J. Zhou, M. Li, H. Phyu, G. Petti, M. Mendez, H. Janjua, P. Peng, C. Longley, V. Borowski, M. Mehlig and D. Filpula (2003). "Tailoring structure-function and pharmacokinetic properties of single-chain Fv proteins by site-specific PEGylation." Protein Eng 16(10): 761-770. Yates, Z., K. Gunasekaran, H. Zhou, Z. Hu, Z. Liu, R. R. Ketchem and B. Yan (2010). "Histidine residue mediates radical-induced hinge cleavage of human IgG1." J Biol Chem 285(24): 18662-18671.

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

Claims

**Claim 1** An isolated antibody or antigen-binding fragment thereof that specifically binds to IL-1β, comprising: (a) a heavy-chain variable region comprising a heavy-chain complementarity-determining region 1 (HCDR1) having the amino acid sequence represented by SEQ ID NO: 3, a heavy-chain complementarity-determining region 2 (HCDR2) having the amino acid sequence represented by SEQ ID NO: 4, and a heavy-chain complementarity-determining region 3 (HCDR3) having the amino acid sequence represented by SEQ ID NO: 5, and (b) a light-chain variable region comprising a light-chain complementarity-determining region 1 (LCDR1) having the amino acid sequence represented by SEQ ID NO: 6, a light-chain complementarity-determining region 2 (LCDR2) having the amino acid sequence represented by SEQ ID NO: 7, and a light-chain complementarity-determining region 3 (LCDR3) having the amino acid sequence represented by SEQ ID NO: 8 An isolated antibody or antigen-binding fragment thereof. **Claim 2** The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is a mouse monoclonal antibody or antibody fragment having a heavy-chain variable region represented by SEQ ID NO: 1 and a light-chain variable region represented by SEQ ID NO:

2. **Claim 3** The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is a humanized antibody or antibody fragment. **Claim 4** The antibody or antigen-binding fragment thereof according to claim 3, wherein the humanized antibody or fragment comprises a heavy-chain variable region represented by SEQ ID NO:

9. **Claim 5** The antibody or antigen-binding fragment thereof according to claim 3, wherein the humanized antibody or fragment comprises a light-chain variable region represented by SEQ ID NO:

10. **Claim 6** The antibody or antigen-binding fragment thereof according to claim 3, wherein the humanized antibody or fragment comprises a light-chain variable region represented by SEQ ID NO:

11. **Claim 7** The antibody or antigen-binding fragment thereof according to claim 3, wherein the humanized antibody or fragment comprises a light-chain variable region represented by SEQ ID NO:

12. **Claim 8** The antibody or antigen-binding fragment thereof according to claim 3, wherein the humanized antibody or fragment comprises a light-chain variable region represented by SEQ ID NO:

13. **Claim 9** The antibody or antigen-binding fragment thereof according to claim 3, wherein the humanized antibody or fragment comprises a human IgG1 heavy-chain sequence represented by SEQ ID NO:

14. **Claim 10** The antibody or antigen-binding fragment thereof according to claim 3, wherein the humanized antibody or fragment comprises the human light chain sequence shown as SEQ ID NO:

15.

11. The antibody or antigen-binding fragment thereof according to claim 3, wherein the humanized antibody or fragment comprises the human light chain sequence shown as SEQ ID NO:

16.

12. The antibody or antigen-binding fragment thereof according to claim 3, wherein the humanized antibody or fragment comprises the human light chain sequence shown as SEQ ID NO:

17.

13. The antibody or antigen-binding fragment thereof according to claim 3, wherein the humanized antibody or fragment comprises the human light chain sequence shown as SEQ ID NO:

18.

14. The antibody is IgG １ , IgG ２ , IgG ３ or IgG ４ is an isotype, the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and 10 to 13.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, wherein the antibody has one or more Fc mutations that extend the half-life of the engineered antibody as compared to the parental wild-type antibody.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the antibody has one or more Fc mutations that enhance the resistance of the engineered antibody to proteolysis by proteases as compared to the parental wild-type antibody.

17. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, wherein the antibody has one or more Fc mutations that reduce or eliminate the effector function of the engineered antibody as compared to the parental wild-type antibody.

18. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, 10 to 13, and 15 to 17, wherein the antibody has Fc mutations of L234A, L235A, M428L, and N434S according to residue numbering according to the EU index that extend the half-life of the engineered antibody and reduce the effector function as compared to the parental wild-type antibody.

19. The antibody or antigen-binding fragment thereof according to claim 3, wherein the humanized antibody or fragment comprises a human IgG1 heavy chain having Fc mutations of L234A, L235A, M428L, and N434S shown as SEQ ID NO:

31.

20. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, 10 to 13, and 15 to 18, wherein the antibody has an extended half-life, reduced effector function, and resistance to proteolysis compared to the parental wild-type antibody, and has Fc mutations of E233P, L234A, L235A, M428L, and N434S and a deletion of G236 with residue numbering according to the EU index.

21. The antibody or antigen-binding fragment thereof according to claim 3, wherein the humanized antibody or fragment comprises a human IgG1 heavy chain having Fc mutations of E233P, L234A, L235A, M428L, and N434S and a deletion of G236 as shown in SEQ ID NO:

32.

22. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, wherein the antibody or fragment blocks the binding of human IL-1β to its receptor IL-1RI.

23. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, wherein the antibody or fragment blocks the binding of cynomolgus IL-1β to its receptor IL-1RI.

24. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, wherein the antibody or fragment neutralizes, reduces, or interferes with the functional activity of IL-1β with respect to its receptor IL-1RI.

25. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, wherein the antibody or fragment neutralizes IL-1β-driven reporter gene activation in a HEK-Blue IL-1β reporter assay.

26. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, wherein the antibody or fragment inhibits IL-1β-driven IL-6 release from human fibroblast MRC-5 in a cytokine release assay.

27. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, wherein the antibody or fragment inhibits IL-1β-driven knee joint arthritis in a murine arthritis model.

28. An isolated polynucleotide encoding an anti-IL-1β IgG antibody or antigen-binding fragment thereof according to any one of claims 1 to 21.

29. A vector comprising the polynucleotide according to claim 28.

30. The vector according to claim 29, which is an expression vector.

31. A host cell comprising the vector according to claim 29 or 30.

32. A method for producing an anti-IL-1β IgG antibody or an antigen-binding fragment thereof according to any one of claims 1 to 21, comprising culturing the host cell according to claim 31 under conditions in which the anti-IL-1β IgG antibody or an antigen-binding fragment thereof is expressed, and isolating the anti-IL-1β IgG antibody or an antigen-binding fragment thereof.

33. An in vitro method for measuring the half-life of an anti-IL-1β IgG antibody or an antigen-binding fragment thereof according to any one of claims 1 to 21, comprising measuring the half-life of the antibody or an antigen-binding fragment thereof in an in vitro assay.

34. An in vitro method for measuring the resistance of an anti-IL-1β IgG antibody or an antigen-binding fragment thereof according to any one of claims 1 to 21 to proteolysis, (a) digesting the antibody or an antigen-binding fragment thereof with a protease, and (b) measuring the degree of digestion of the antibody or an antigen-binding fragment thereof A method comprising the steps of.

35. Use for a method of treating an IL-1β-mediated disease or disorder in a subject in need of treatment, the method comprising administering to the subject an effective amount of an anti-IL-1β IgG antibody or an antigen-binding fragment thereof, an anti-IL-1β IgG antibody or an antigen-binding fragment thereof according to any one of claims 1 to 21.

36. The anti-IL-1β IgG antibody or an antigen-binding fragment thereof according to claim 35, wherein the IL-1β-mediated disease or disorder is an inflammatory disease.

37. The anti-IL-1β IgG antibody or an antigen-binding fragment thereof according to claim 35, wherein the IL-1β-mediated disease or disorder is a cardiovascular disease.

38. The anti-IL-1β IgG antibody or an antigen-binding fragment thereof according to claim 35, wherein the IL-1β-mediated disease or disorder is lung cancer.

39. The anti-IL-1β IgG antibody or an antigen-binding fragment thereof according to claim 35, wherein the IL-1β-mediated disease or disorder is type 2 diabetes.

40. The anti-IL-1β IgG antibody or an antigen-binding fragment thereof according to claim 35, wherein the IL-1β-mediated disease or disorder is breast cancer.

41. The anti-IL-1β IgG antibody or antigen-binding fragment thereof according to claim 35, wherein the IL-1β-mediated disease or disorder is pancreatic cancer.

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