Anti-IL-1β Antibody

Abstract

The present invention provides engineered human interleukin-1β antibodies, cells and vectors containing DNA encoding same, and methods for producing said antibodies. Additionally, the present invention provides the use of engineered human interleukin-1β antibodies for treating inflammatory diseases, such as cardiovascular disease and cancer.

Description

[Technical field]

[0001] The present invention relates to the field of medicine. More specifically, the present invention relates to an antibody that binds to human IL-1β (IL-1-beta or IL-1β or interleukin-1β have the same meaning in this specification), which may be useful for treating and / or preventing inflammatory diseases, including but not limited to atherosclerotic cardiovascular disease (ASCVD), heart failure, cancer and rare genetic diseases caused by overproduction of IL-1β. The present invention also relates to a method for treating and / or preventing these inflammatory diseases. [Background technology]

[0002] Cardiovascular disease (CVD) is a category of diseases involving the heart or blood vessels. Common symptoms of CVD include angina, myocardial infarction (MI; commonly known as heart attack), stroke, heart failure, arrhythmia, etc. As CVD is a complex disease, various risk factors have been identified that contribute to the development and progression of CVD. These include dyslipidemia, hypertension, diabetes, smoking, unhealthy diet, physical inactivity, obesity, etc. However, despite efforts to control these traditional risk factors, cardiovascular disease remains the leading cause of death worldwide.

[0003] Research over the past two decades has highlighted the inflammatory process as a key component in the pathogenesis of CVD, particularly ASCVD. Epidemiological data since the mid-1990s have shown that inflammation, measured by either high-sensitivity C-reactive protein (hsCRP) or interleukin-6 (IL-6), is strongly associated with future major adverse cardiovascular events (MACE) in both primary and secondary prevention, independent of traditional risk factors (Ridker et al. (2018) J. Am. Coll. Cardiol. 72: 3320-3331). Preclinical studies have also demonstrated a role for inflammation in the initiation and progression of atherosclerotic plaques (Aday et al. (2019) Front. Cardiovasc. Med. 6: 16 doi: 10.3389 / fcvm.2019. 00016). Importantly, inflammation is also involved in plaque destabilization and rupture, inducing acute cardiovascular events such as MI and stroke.

[0004] The interleukin-1 family is a central component of inflammation and has been well studied as a therapeutic target for various inflammatory diseases (Szekely et al. (2018) Cardiol. Ther: 7:25-44). There are three members of the IL-1 gene family: IL-1α, IL-1β and IL-1 receptor antagonist (IL-1ra). IL-1α and IL-1β are agonists of the IL-1 receptor, whereas IL-1ra is a receptor antagonist specific for the IL-1 receptor and therefore an endogenous competitive inhibitor of IL-1. IL-1β is the major circulating form of IL-1. It is produced as a precursor (pro-IL-1β) and is activated via the NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome under various inflammatory stimuli. Active IL-1β has autocrine, paracrine and endocrine actions and is therefore involved in a wide range of inflammatory diseases.

[0005] Inhibition of IL-1β may also have a role in the treatment of cancers with underlying inflammation. Many malignant tumors arise in areas of chronic inflammation, and insufficient resolution of inflammation may play an important role in tumor invasion, progression and metastasis (Grivennikov et al. (2010) Cell 140: 883-899). Inflammation is pathophysiologically relevant in lung cancer; for example, smoking and other external inhaled toxins induce a persistent inflammatory response. This inflammatory activation is mediated, in part, by activation of the NLRP3 inflammasome, which produces active IL-1β locally. In clinical practice, high baseline concentrations of hsCRP and IL-6 have been found to be associated with later-diagnosed lung cancer. Blockade of IL-1β with canakinumab was associated with reduced total cancer mortality, incident lung cancer and lung cancer mortality (Ridker et al. (2017) Lancet 390:1833-1842).

[0006] There remains a need to provide therapeutic antibodies that bind to human IL-1β. In particular, there is a need to provide IL-1β antibodies with good clinical properties. The present invention encompasses genetically engineered human antibodies against human IL-1β. The antibodies of the present invention exhibit potent IL-1β neutralizing activity and high specificity for IL-1β. Summary of the Invention

[0007] Thus, in some embodiments, the invention provides an antibody that binds to a human IL-1β protein (SEQ ID NO:27) comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3; said HCDR1 comprises AASGFTFSSYSX1S (SEQ ID NO:8), wherein X1 is Phe or Leu; said HCDR2 comprises AISVSGSTYYAX2SVKG (SEQ ID NO: 9), wherein X2 is Pro or Asp; the HCDR3 comprises ARDDLIX3TRGTFYNWFDP (SEQ ID NO: 10), wherein X3 is Thr or Pro; the LCDR1 comprises RASQSISSNLN (SEQ ID NO: 12); the LCDR2 comprises YAASSLQS (SEQ ID NO: 13); and The antibody is provided, wherein the LCDR3 comprises QQSYSIPWT (SEQ ID NO: 14).

[0008] Thus, in some embodiments, the invention provides an antibody that binds human IL-1β protein, comprising: said HCDR1 comprises AASGFTFSSYSX1S (SEQ ID NO:8), wherein X1 is Phe; said HCDR2 comprises AISVSGSTYYAX2SVKG (SEQ ID NO: 9) (wherein X2 is Pro); The antibody is provided wherein the HCDR3 comprises ARDDLIX3TRGTFYNWFDP (SEQ ID NO: 10), where X3 is Thr.

[0009] Thus, in some embodiments, the invention provides an antibody that binds human IL-1β protein, comprising: said HCDR1 comprises AASGFTFSSYSX1S (SEQ ID NO:8), where X1 is Leu; said HCDR2 comprises AISVSGSTYYAX2SVKG (SEQ ID NO: 9) (wherein X2 is Asp); The antibody is provided wherein the HCDR3 comprises ARDDLIX3TRGTFYNWFDP (SEQ ID NO: 10), where X3 is Pro.

[0010] Thus, in some embodiments, the invention provides an antibody that binds human IL-1β protein, comprising: said HCDR1 comprises AASGFTFSSYSX1S (SEQ ID NO:8), where X1 is Leu; said HCDR2 comprises AISVSGSTYYAX2SVKG (SEQ ID NO: 9) (wherein X2 is Asp); The antibody is provided wherein the HCDR3 comprises ARDDLIX3TRGTFYNWFDP (SEQ ID NO: 10), where X3 is Thr.

[0011] Thus, in some embodiments, the invention provides an antibody that binds human IL-1β protein, said HCDR1 comprises AASGFTFSSYSX1S (SEQ ID NO:8), wherein X1 is Phe; said HCDR2 comprises AISVSGSTYYAX2SVKG (SEQ ID NO: 9) (wherein X2 is Asp); The antibody is provided wherein the HCDR3 comprises ARDDLIX3TRGTFYNWFDP (SEQ ID NO: 10), where X3 is Thr.

[0012] In some embodiments, the invention provides an antibody wherein the VH comprises a sequence selected from SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:22, or SEQ ID NO:25, and the VL comprises SEQ ID NO:11. In another embodiment, the invention provides an antibody wherein the VH consists of a sequence selected from SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:22, or SEQ ID NO:25, and the VL consists of SEQ ID NO:11. In some embodiments, the invention provides an antibody wherein the VH comprises SEQ ID NO:7, and the VL comprises SEQ ID NO:11. In some embodiments, the invention provides an antibody wherein the VH comprises SEQ ID NO:17, and the VL comprises SEQ ID NO:11. In some embodiments, the invention provides an antibody wherein the VH comprises SEQ ID NO:22, and the VL comprises SEQ ID NO:11. In some embodiments, the invention provides an antibody wherein the VH comprises SEQ ID NO:25, and the VL comprises SEQ ID NO:11.

[0013] In some embodiments, the invention provides an antibody comprising a heavy chain (HC) comprising a sequence selected from SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:3, SEQ ID NO:20, or SEQ ID NO:23, and a light chain (LC) comprising SEQ ID NO:5. In some embodiments, the invention provides an antibody comprising a HC comprising a sequence selected from SEQ ID NO:1 or SEQ ID NO:3, and a LC comprising SEQ ID NO:5. In some embodiments, the invention provides an antibody comprising a HC comprising a sequence selected from SEQ ID NO:18 or SEQ ID NO:20, and a LC comprising SEQ ID NO:5. In some embodiments, the invention provides an antibody comprising a HC comprising a sequence selected from SEQ ID NO:23, and a LC comprising SEQ ID NO:5.

[0014] In some embodiments, the present invention provides an antibody comprising a heavy chain (HC) comprising amino acids 2-451 selected from SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:3, SEQ ID NO:20, or SEQ ID NO:23, and a light chain (LC) comprising SEQ ID NO:5. In some embodiments, the present invention provides an antibody comprising a HC comprising amino acids 2-451 selected from SEQ ID NO:1 or SEQ ID NO:3, and a LC comprising SEQ ID NO:5. In some embodiments, the present invention provides an antibody comprising a HC comprising amino acids 2-451 of SEQ ID NO:15, and a LC comprising SEQ ID NO:5. In some embodiments, the present invention provides an antibody comprising a HC comprising amino acids 2-451 selected from SEQ ID NO:18 or SEQ ID NO:20, and a LC comprising SEQ ID NO:5. In some embodiments, the present invention provides an antibody comprising a HC comprising amino acids 2-451 of SEQ ID NO:23, and a LC comprising SEQ ID NO:5.

[0015] In some embodiments, the invention provides antibodies comprising a HC consisting of a sequence selected from SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:3, SEQ ID NO:20 or SEQ ID NO:23 and a LC consisting of SEQ ID NO:5. In some embodiments, the invention provides antibodies comprising a HC consisting of a sequence selected from SEQ ID NO:1 or SEQ ID NO:3 and a LC consisting of SEQ ID NO:5. In some embodiments, the invention provides antibodies comprising a HC consisting of a sequence selected from SEQ ID NO:18 or SEQ ID NO:20 and a LC consisting of SEQ ID NO:5. In some embodiments, the invention provides antibodies comprising a HC consisting of a sequence selected from SEQ ID NO:23 and a LC consisting of SEQ ID NO:5.

[0016] In some embodiments, the antibody has an engineered human IgG1 or IgG4 isotype.

[0017] Optionally, certain antibodies of the invention contain an Fc portion derived from human IgG1. IgG1 is well known to bind to proteins of the Fcγ receptor family (FcγR) as well as C1q. Interactions with these receptors may include antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Thus, certain antibodies of the invention, including antibodies IV and V, may introduce specific amino acid substitutions in the IgG1 Fc to inhibit immune effector functions. Mutations in the CH2 region of the anti-IL-1β portion of the antibody may include positions 234, 235 and 265 (EU numbering).

[0018] In some embodiments, the antibody has an engineered human IgG4 isotype. In certain embodiments, the antibody of the present invention is an IgG4 antibody and thus comprises an IgG4 Fc region or an Fc region derived from human IgG4, such as a modified IgG4 Fc region. According to some embodiments, amino acid substitutions are introduced into the IgG4 Fc region. In certain embodiments, the antibody of the present invention is an IgG4 antibody and has a modification in the constant region of both of the two HCs that reduces effector function, such as a modification that includes the amino acid alanine at both residues 239 and 240 (residue numbers are based on the exemplified HC of SEQ ID NO: 18). In certain embodiments, the antibody of the present invention is an IgG4 antibody and has a modification in the constant region of both of the two HCs that reduces effector function, such as a modification that includes the amino acid alanine at both residues 239 and 240, and further has a modification in the constant region of both of the two HCs that improves stability, such as a modification that includes the deletion of the amino acid proline at residue 233 and the amino acid lysine at residue 443 (residue numbers are based on the exemplified HC of SEQ ID NO: 2).

[0019] According to some embodiments, amino acid substitutions are introduced in the IgG4 Fc region. For example, in some embodiments, a serine to proline mutation is introduced at position 228 ("S228P" according to IMGT or EU numbering), a phenylalanine to alanine mutation is introduced at position 234 ("F234A" according to IMGT or EU numbering), and / or a leucine to alanine mutation is introduced at position 235 ("L235A" according to IMGT or EU numbering). According to some embodiments of the antibody of the invention, the Fc region comprises S228P, F234A and L235A (according to EU index numbering). In some embodiments, the invention provides a nucleic acid sequence encoding SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:3, SEQ ID NO:20 or SEQ ID NO:23 and a second nucleic acid encoding SEQ ID NO:5.

[0020] Another embodiment is a vector comprising a first nucleic acid sequence encoding SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:3, SEQ ID NO:20 or SEQ ID NO:23 and a second nucleic acid sequence encoding SEQ ID NO:5. In another embodiment, the nucleic acids are present on separate vectors. Another embodiment is a first vector comprising a nucleic acid sequence encoding SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:3, SEQ ID NO:20 or SEQ ID NO:23 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:5.

[0021] In some embodiments, the present disclosure provides a cell comprising the vector described herein. In one embodiment, the present disclosure provides a cell comprising a first nucleic acid sequence encoding SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:3, SEQ ID NO:20 or SEQ ID NO:23 and a second nucleic acid sequence encoding SEQ ID NO:5. In another embodiment, the present disclosure provides a cell comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:3, SEQ ID NO:20 or SEQ ID NO:23 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:5. In an embodiment, the cell is a mammalian cell.

[0022] In an embodiment, the invention provides an antibody comprising two light chains and two heavy chains, each light chain having the amino acid sequence set forth in SEQ ID NO:5, and each heavy chain having the amino acid sequence set forth in SEQ ID NO:1.

[0023] In one embodiment, the invention provides an antibody comprising two light chains and two heavy chains, each light chain having the amino acid sequence set forth in SEQ ID NO:5, and each heavy chain having the amino acid sequence set forth in SEQ ID NO:15.

[0024] In an embodiment, the invention provides an antibody comprising two light chains and two heavy chains, each light chain having the amino acid sequence set forth in SEQ ID NO:5 and each heavy chain having the amino acid sequence set forth in SEQ ID NO:18. In an embodiment, the invention provides an antibody comprising two light chains and two heavy chains, each light chain having the amino acid sequence set forth in SEQ ID NO:5 and each heavy chain having the amino acid sequence set forth in SEQ ID NO:3. In an embodiment, the invention provides an antibody comprising two light chains and two heavy chains, each light chain having the amino acid sequence set forth in SEQ ID NO:5 and each heavy chain having the amino acid sequence set forth in SEQ ID NO:20. In one embodiment, the invention provides an antibody comprising two light chains and two heavy chains, each light chain having the amino acid sequence set forth in SEQ ID NO:5 and each heavy chain having the amino acid sequence set forth in SEQ ID NO:23.

[0025] In one embodiment, the invention provides a method of producing an antibody comprising culturing cells under conditions in which the antibody is expressed and recovering said expressed antibody from the culture medium. In another embodiment, the invention provides an antibody produced by culturing cells under conditions in which the antibody is expressed and recovering the expressed antibody from the culture medium. Methods for expressing an antibody or culturing cells under conditions in which the antibody is expressed are well known in the art, as are methods for recovering an antibody.

[0026] In an embodiment, the invention provides a pharmaceutical composition comprising an antibody and a pharma- ceutically acceptable excipient, diluent or carrier.

[0027] In an embodiment, the invention provides a method of treating a disease, comprising administering an antibody of the invention and a pharma- ceutically acceptable carrier, diluent or excipient.

[0028] In an embodiment, the invention provides a method for preventing disease, comprising administering an antibody of the invention and a pharma- ceutically acceptable carrier, diluent or excipient.

[0029] In a further embodiment, the present invention provides a method of treating a disease, wherein the disease is an inflammatory disease.

[0030] In a further embodiment, the present invention provides a method of preventing a disease, wherein said disease is an inflammatory disease.

[0031] In an embodiment, the invention provides an antibody of the invention for use in therapy. In one embodiment, the invention provides an antibody of the invention for use in therapy of an inflammatory disease. In a further embodiment, the invention provides an antibody of the invention for use in therapy of an inflammatory disease, said inflammatory disease being a cardiovascular disease. In a further embodiment, the invention provides an antibody of the invention for use in therapy of an inflammatory disease, said inflammatory disease being a cancer.

[0032] In a further embodiment, the invention provides the use of an antibody of the invention in the manufacture of a medicament for the treatment of cardiovascular disease or cancer.In a further embodiment, the invention provides the use of an antibody of the invention that binds to and antagonizes IL-1β protein for use in the treatment of inflammatory disease.

[0033] As used herein, an "antibody" is an immunoglobulin (IgG) molecule that binds to an antigen. Naturally occurring full-length antibodies are IgG molecules that comprise two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. The amino-terminal portion of each chain contains a variable region of about 100 to about 110 amino acids primarily responsible for antigen recognition by the complementarity determining regions (CDRs) contained therein. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.

[0034] The CDRs are interspersed with more highly conserved regions called frame regions (FRs). Each light chain variable region (LCVR, also known as VL) and heavy chain variable region (HCVR, also known as VH) is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are referred to as "LCDR1, LCDR2 and LCDR3" and the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2 and HCDR3". The CDRs contain most of the residues that form specific interactions with the antigen. Numbering and arrangement of the CDR amino acid residues in the VL and VH regions according to the well-known North numbering convention.

[0035] Light chains are classified as kappa or lambda and are characterized by a particular constant region known in the art. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon and define the antibody's isotype as IgG, IgM, IgA, IgD, or IgE, respectively. IgG antibodies can be further divided into subclasses, e.g., IgG1, IgG2, IgG3, IgG4. Each heavy chain type is characterized by a particular constant region with a sequence known in the art.

[0036] In some biological systems and production methods, antibodies may undergo co- and post-translational modifications such as glycosylation, deamidation, acylation, oxidation, cyclization, fucosylation, among other modifications known in the art. Another known modification is the cyclization of glutamine or glutamic acid at the N-terminus of the heavy chain variable region, including the heavy chain, to pyroglutamic acid (often abbreviated as pyrGlu, pyrE, pGlu or pE). Depending on the method and antibody used, the percentage of glutamic acid converted to pyroglutamic acid may vary and may be present as a mixture, encompassing substantially all of the produced antibody or a very low percentage of the antibody.

[0037] As used herein, the term "monoclonal antibody" (mAb) refers to an antibody obtained from a single copy or clone, including, for example, any eukaryotic, prokaryotic, or phage clone, and not the method of producing it. The monoclonal antibodies of the present invention are preferably present in a homogeneous or substantially homogeneous population. An intact mAb contains two heavy chains and two light chains. Monoclonal antibodies can be produced, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic technology, e.g., CDR grafting, or a combination of such technologies or other techniques known in the art.

[0038] "IL-1β" (also known as IL-1 beta or IL-1β or interleukin-1β) refers to the major circulating form of IL-1. It is produced as a precursor (pro-IL-1β or IL-1β proprotein) that is activated via the NLRP3 inflammasome under various inflammatory diseases.

[0039] As used herein, "inflammatory" includes both inflammatory and autoinflammatory diseases. The term "inflammatory disease" includes, but is not limited to, ASCVD, heart failure, cancer, and rare genetic diseases caused by overproduction of IL-1β.

[0040] The term "treatment" (or "treat" or "treating") refers to a process that includes slowing, interrupting, preventing, controlling, halting, reducing or reversing the progression or severity of a symptom, disorder, condition or disease associated with IL-1β activity, but does not necessarily eliminate completely all disease-related symptoms, conditions or disorders associated with IL-1β activity. The term "prevention" (or "preventing") means causing something to not occur, be present or appear and / or preventing or stopping something from being done.

[0041] As used herein, the term "cardiovascular disease" refers to a group of diseases involving the heart or blood vessels. Common symptoms of CVD include, but are not limited to, MI, stroke, ASCVD, heart failure, arrhythmia, etc.

[0042] In addition to the antibodies disclosed herein that exhibit similar functional properties according to the present invention, engineered human antibodies can be generated using several different methods. Certain antibody compounds disclosed herein can be used as templates or parent antibody compounds to produce additional antibody compounds. In one approach, the CDRs of the parent antibody compound are grafted onto a human framework that has high sequence identity with the framework of the parent antibody compound. The sequence identity of the new framework will generally be at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99% identical to the sequence of the corresponding framework of the parent antibody compound. This grafting may result in a decrease in binding affinity compared to the parent antibody. In this case, back mutations can be introduced into the framework at specific positions of the parent framework based on the specific criteria disclosed by Queen et al. Al (1991) Proc. Natl. Acad. Sci US 88:2869. Additional references describing methods useful for humanizing antibodies include U.S. Pat. Nos. 4,816,397, 5,225,539, and 5,693,761; and the computer programs ABMOD and ENCAD described in Levitt (1983) J. Mol. Biol. 168: 595-620; and the methods of Winter and coworkers (Jones et al. (1986) Nature 321:522-525); Riechmann et al. (1988) Nature 332:323-327; and Verhoeyen et al. (1988) Science 239:1534-1536).

[0043] Identification of residues to consider for backmutation can be performed as follows: If an amino acid in a human framework region of the acceptor framework falls into the category of being rare in the human framework at that position, but the corresponding amino acid of the donor immunoglobulin is typical in the human framework at that position, then the framework amino acid of the human germline sequence used (the "acceptor framework") is replaced with a framework amino acid from the framework of the parent antibody compound (the "donor framework").

[0044] When each amino acid in the human framework region of the acceptor framework and the corresponding amino acid in the donor framework is generally uncommon in human frameworks at that position, such an amino acid can be replaced with an amino acid typical of the human framework at that position. This backmutation criterion can restore the activity of the parent antibody compound.

[0045] Another approach involves randomly mutating amino acids in the grafted CDRs without altering the framework, and screening the resulting molecules for binding affinity and other functional properties that are comparable or superior to those of the parent antibody compounds, to generate engineered human antibodies that exhibit similar functional properties to the antibody compounds disclosed herein. A single mutation can also be introduced at each amino acid position in each CDR, and then the effect of said mutations on binding affinity and other functional properties can be evaluated. Single mutations that produce improved properties can be combined to evaluate their effect in combination with each other.

[0046] Moreover, a combination of both of the above approaches is possible. After CDR grafting, in addition to introducing amino acid changes in CDRs, back mutations can be introduced in specific framework regions. This methodology is described in Wu et al., (1999) J. Mol. Biol. 294: 151-162.

[0047] The genetically engineered human antibodies of the present invention can be used as pharmaceuticals in human medicine, administered by a variety of routes. Most preferably, such compositions are for parenteral administration. Such pharmaceutical compositions can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy; 1999). th ed. (1995), A. Gennaro et al., Mack Publishing Co.), comprising the genetically engineered human antibody disclosed herein, and a pharma- ceutically acceptable carrier, diluent or excipient.

[0048] The results of the following assays demonstrate that the exemplified monoclonal antibodies and antigen-binding fragments thereof of the invention bind to and / or neutralize IL-1β and therefore can be used to treat inflammatory diseases (e.g., cardiovascular disease or cancer). EXAMPLES

[0049] Example 1: Antibody Expression and Purification In constructing the IL-1β antibodies of the present invention, significant problems related to chemical and physical stability were encountered, including, inter alia, low binding affinity, deamidation of the variable regions, oxidation, and low potency.

[0050] Therefore, modifications were designed to improve the binding affinity and chemical and physical stability of the antibody. Amino acid modifications were introduced throughout both the heavy and light chains. The antibody of the present application contains multiple residue changes from the original construct and was identified to have high binding affinity and to be chemically and physically stable. None of the modifications that comprise the antibody of the present application were identified in the original construct.

[0051] Exemplary anti-IL-1β antibodies of the present invention are shown in Table 1.

[0052] Antibodies I-VI can be produced and purified as follows: A suitable host cell (e.g., HEK 293 or CHO) is transiently transfected with an expression system for secreting the antibody using an optimal predetermined HC:LC vector ratio encoding the sequences of antibody I_HC, antibody II_HC, antibody III_HC, antibody IV_HC, antibody V_HC, antibody VI_HC and the common LC sequence. The clarified medium into which the antibody is secreted is purified using any of a number of commonly used techniques. For example, the medium can be suitably applied to a Protein A or Protein G column equilibrated with a compatible buffer (e.g., phosphate buffered saline (pH 7.4)). The column is washed to remove non-specifically bound components. The bound antibody is eluted, for example, by a pH gradient (e.g., from 0.1 M sodium phosphate buffer (pH 6.8) to 0.1 M sodium citrate buffer (pH 2.5)). The antibody fraction is pooled after detection by SDS-PAGE or the like. Depending on the intended use, further purification is appropriately selected. The antibody may be concentrated and / or sterile filtered using common techniques. Soluble aggregates and multimers can be effectively removed by common techniques including size exclusion chromatography, hydrophobic interaction chromatography or ion exchange chromatography. The purity of the antibody after these chromatography steps is greater than 99%. The product can be immediately frozen or lyophilized at -70°C, or stored at 4°C for immediate use.

[0053] Example 2: Antibody Discovery and Engineering Four in-house phage libraries were used for three rounds of solution panning against human (hu)IL-1β. Phage plaques were randomly picked and assessed for huIL-1β binding activity. After sequencing and binding confirmation, the panel of anti-huIL-1β phage hits were cloned and expressed as human IgG.

[0054] To increase antibody affinity, mutations are introduced at all individual residues in the heavy chain complementarity determining regions (HCDRs) of the parent IL-1β phage antibody. From the filter lift screening, beneficial mutations are selected to generate a combinatorial library. After filter lift and binding titers are determined, individual combinatorial clones are sequenced to determine binding characteristics. Additionally, framework (FW) substitutions were made in the light chain to revert FW1, FW2, FW3 and FW4 sequences to their germline state to reduce potential immunogenicity and post-translational modifications (PTMs).

[0055] Engineered and / or optimized anti-IL-1β antibodies, designated herein as Antibody I, Antibody II, Antibody III, Antibody IV, Antibody V and Antibody VI, have been obtained, with heavy and light chain variable region amino acid sequences, and the complete heavy and light chain amino acid and encoding nucleotide sequences are listed in the section entitled "Amino Acid and Nucleotide Sequences." Table 1 below provides sequence IDs corresponding to these fragments, as well as the light and heavy chain CDR amino acid sequences. [Table 1]

[0056] Example 3: Neutralization of human or cynomolgus IL-1β in vivo Recombinant human or cynomolgus IL-1β is produced in E. coli as an N-terminal HIS-SUMO fusion protein. The protein is purified using HisPurNi-NTA chromatography followed by removal of endotoxin. The purified fusion protein is then treated with SUMO protease Ulp1 to cleave HIS-SUMO from the fusion protein. The cleaved HIS-SUMO protein is removed from the reaction by HisPurNi-NTA and the untagged IL-1β is further purified to homogeneity using Superdex75 size exclusion chromatography.

[0057] The antibodies of the present application are expected to neutralize IL-1β. Neutralization of IL-1β activity by the antibodies of the present application can be assessed, for example, by one or more IL-1β cell-based activity assays, as described below.

[0058] Screening for neutralizers of IL-1β / IL-1R binding can be initially performed by a high-throughput cell-based assay using HeLa cells expressing the luciferase gene under the control of the NF-κB promoter. This assay uses the NF-κB-luciferase reporter signal as a readout of recombinant IL-1β-induced signaling. Neutralization of IL-1β can then be quantified by measuring the level of reduction in luciferase activity by titration of anti-IL-1β antibodies. Alternatively, another in vitro neutralization assay (e.g., HEK-Blue cell-based assay) is detailed below.

[0059] IL-1β activity was monitored using HEK-Blue™ IL-1β cells (InvivoGen Cat. # hkb-il1b) expressing an NF-κB / AP-1 inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene. Specifically, HEK-Blue™ IL-1β cells are cultured in T-75 flasks in growth medium (DMEM, 4.5 g / l glucose, 2 mM L-glutamine, 10% (v / v) fetal bovine serum, 50 U / mL penicillin, 50 μg / mL streptomycin, 100 μg / mL Normocin™, 100 μg / mL Zeocin™ and 200 μg / mL Hygromycin B Gold) until 90% confluence. Cells are cultured in PBS (Ca ++ and Mg ++The flask was then gently tapped on the side to detach the cells, and the cells were resuspended in 10 mL of test medium (DMEM, 4.5 g / L glucose, 2 mM L-glutamine, 10% (v / v) heat-inactivated FBS (30 min at 56°C), 50 U / mL penicillin, 50 μg / mL streptomycin, 100 μg / mL Normocin™) and counted, with a concentration of 0.33 x 10 in test medium. 6 Dilute to 1000 cells / mL. Recombinant human or cynomolgus IL-1β and test substances are adjusted to the desired concentrations in test medium. In a BioCoat poly-D-lysine plate (Corning 354461), 40 μL of antibody (5× concentration) is mixed with 10 μL of IL-1β (20× concentration, 4 pM final concentration in the assay) and incubated at room temperature for 30 minutes. 150 μL of 0.33×10 6 Cells / mL of the HEK-Blue™ IL-1β cell suspension are dispensed into each well of the poly-D-lysine plate containing the antibody and IL-1β mixture. The plate is incubated overnight at 37° C., 5% CO2, 90% relative humidity. On day 2, 25 μL of medium from the poly-D-lysine plate is transferred to a Costar assay plate (Corning 3695). 75 μL of QUANTI-Blue detection solution (Invivogen Cat. No. rep-qb1, rep-qb2) pre-warmed to 37° C. is added to the assay plate. The assay plate is covered and incubated at 37° C. for 1 hour, after which the OD is measured in a plate reader (SpectraMax Plus, Molecular Device). 650 Read at nm. Data was normalized and expressed as % inhibition of 4 pM IL-1β: 0% inhibition = 4 pM IL-1β, 100% inhibition = 0 pM IL-1β. Neutralizing anti-hIL-1β antibodies block recombinant human IL-1β activity stimulating HEK-Blue™ IL-1β cells. Relative potency of neutralizing antibodies was calculated using a four-parameter logistic fit to obtain IC 50 expressed as a value.

[0060] The human or cynomolgus monkey IL-1β neutralizing activity of the antibodies of the invention (Antibodies I-VI) is summarized in Table 2. Canakinumab is included as a comparison. Antibodies I-VI have comparable or greater relative potency (IC) for neutralizing human IL-1β compared to canakinumab. 50 Antibodies I-VI also exhibit activity in neutralizing cynomolgus IL-1β, whereas canakinumab does not exhibit neutralizing activity against cynomolgus IL-1β in this assay.

[0061] [Table 2]

[0062] Example 4: Neutralization of human IL-1β in vivo Human IL-1β binds to and stimulates the mouse IL-1 receptor, leading to an increase in the mouse cytokine IL-6. An optimized protocol is described below to test the neutralizing activity of the present antibodies in vivo. Specifically, male C57BL / 6 mice obtained from Envigo are used in the study at approximately 9 weeks of age. Mice are fed a normal chow diet (Harlan Teklad diet, 2014) and randomized into treatment groups according to body weight (n=5-8 / group). The present and control antibodies are dissolved in saline and administered subcutaneously at the indicated dose levels. After 24 hours, human IL-1β is dissolved in saline and administered intraperitoneally at a dose level of 1 μg / kg. After 2 hours, blood samples are collected by retro-orbital bleeding and then centrifuged at 2000 g for 3 minutes to isolate serum samples.

[0063] Levels of mouse IL-6 in serum are determined using the V-PLEX Mouse IL-6 Kit (Meso Scale Discovery, Cat# K152QXD-2) according to the manufacturer's instructions. Briefly, MSD plates are washed 3 times with 150 μL of wash buffer. 50 μL of pre-adjusted calibrators (serial dilutions), control and test samples (1:10 dilution) are transferred to appropriate wells on the plate and shaken (500-1000 rpm) at room temperature for 2 hours. The plate is washed 3 times with 150 μL of wash buffer. 25 μL of detection antibody solution is then added to each well, followed by shaking (500-1000 rpm) at room temperature for 2 hours. The plate is washed 3 times with wash buffer. 150 μL of 2× read buffer is added to each well. The plate is read immediately on an MSD SQ120 plate reader. The IL-6 concentrations of the test samples are analyzed from the standard curve using a four parameter logistic fit.

[0064] An isotype-matched control antibody (IgG4-PAA) is used as a negative control in the study. Data are calculated as percent inhibition compared to the mean IL-6 levels of the control group. Statistical significance of mean differences is assessed using Dunnett's post-hoc analysis of one-way ANOVA with JMP11 software. The present antibodies (Antibodies I-III) dose-dependently block the action of human IL-1β to stimulate an increase in mouse IL-6 via the mouse IL-1 receptor in a manner comparable to canakinumab (Tables 3-5). Antibody II shows higher activity compared to canakinumab at the same dose level, consistent with its higher relative potency in neutralizing human IL-1β in vitro. [Table 3] [Table 4] [Table 5]

[0065] Example 5: Binding affinity measurement of Antibody I, Antibody II, and Antibody III by MSD-SET The affinity of Antibody I, Antibody II, Antibody III and Canakinumab for human IL-1β was measured using the MSD (Meso Scale Discovery) electrochemiluminescence assay. First, equilibrated mixtures of antibody and human IL-1β are prepared; the antibody concentrations in the mixtures are kept constant at 1 pM, 10 pM and 100 pM, while the ligand is titrated in the concentration range of 0.9 nM to 0.00004 nM (2.5-fold dilution between concentrations). The equilibrated mixtures are set in sealed non-binding 96-well plates for 72 hours at 37°C.

[0066] Free antibody in the equilibration mixture was detected using MSD Gold streptavidin plates. MSD plates were first blocked with blocking buffer (PBS+1% BSA) for 1 hour on a shaker at 800 rpm, then washed 3 times with wash buffer PBST (PBS+0.05% Tween20). Plates were coated with biotinylated human IL-1β and washed 3 times with PBST. Equilibration mixture was added to the coated plate and incubated with shaking for 2.5 minutes at room temperature, immediately followed by washing 3 times with PBST. Goat anti-human Sulfo-TAG antibody was added to the plate and incubated with shaking for 1 hour at room temperature. After 3 further washes, MSD read buffer diluted 1:2 in MilliQ water was added to the wells. Plates were then read immediately using an MSD Sector Imager SI6000.

[0067] For data evaluation, the MSD instrument readings were imported into a customized Excel or GraphPad Prism 8-based evaluation program, which automatically plotted the titration data and calculated the K D Values ​​and statistical parameters are calculated.

[0068] The present antibodies (Antibodies I-III) demonstrate similar binding affinity to human IL-1β compared to canakinumab (Table 6). [Table 6]

[0069] Amino acid and nucleotide sequences TIFF2024535887000007.tif74164

[0070] TIFF2024535887000008.tif122150

[0071] TIFF2024535887000009.tif72150

[0072] TIFF2024535887000010.tif125164

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[0074] TIFF2024535887000012.tif61150 TIFF2024535887000013.tif30150

[0075] TIFF2024535887000014.tif18164 TIFF2024535887000015.tif19150 TIFF2024535887000016.tif18150 TIFF2024535887000017.tif21150 TIFF2024535887000018.tif12164 TIFF2024535887000019.tif11150

[0076] TIFF2024535887000020.tif11150 TIFF2024535887000021.tif73150 TIFF2024535887000022.tif126150

[0077] TIFF2024535887000023.tif31150

[0078] TIFF2024535887000024.tif73164

[0079] TIFF2024535887000025.tif124164

[0080] TIFF2024535887000026.tif75150

[0081] TIFF2024535887000027.tif124150

[0082] TIFF2024535887000028.tif33164

[0083] TIFF2024535887000029.tif73164

[0084] TIFF2024535887000030.tif123150

[0085] TIFF2024535887000031.tif31150 TIFF2024535887000032.tif41150 TIFF2024535887000033.tif27164 TIFF2024535887000034.tif68164 TIFF2024535887000035.tif34150

Claims

1. An antibody that binds to human IL-1β protein (SEQ ID NO: 27) comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3; The HCDR1 is AASGFTFSSYSX 1 S (SEQ ID NO: 8) (wherein X 1 is Phe or Leu; the HCDR2 is AISVSSGGSTYYAX 2 SVKG (SEQ ID NO: 9) (wherein X 2 is Pro or Asp; the HCDR3 is ARDDLIX 3 TRGTFYNWFDP (SEQ ID NO: 10) (wherein X 3 is Thr or Pro; the LCDR1 comprises RASQSISSNLN (SEQ ID NO: 12); the LCDR2 comprises YAASSLQS (SEQ ID NO: 13); and the LCDR3 comprises QQSYSIPWT (SEQ ID NO: 14); antibody.

2. The HCDR1 is AASGFTFSSYSX 1 S (SEQ ID NO: 8) (wherein X 1 is Phe); the HCDR2 is AISVSSGGSTYYAX 2 SVKG (SEQ ID NO: 9) (wherein X 2 is Pro); and the HCDR3 is ARDDLIX 3 TRGTFYNWFDP (SEQ ID NO: 10) (wherein X 3 The antibody of claim 1, comprising:

3. The HCDR1 is AASGFTFSSYSX 1 S (SEQ ID NO: 8) (wherein X 1 is Leu); the HCDR2 is AISVSSGGSTYYAX 2 SVKG (SEQ ID NO: 9) (wherein X 2 is Asp; and the HCDR3 is ARDDLIX 3 TRGTFYNWFDP (SEQ ID NO: 10) (wherein X 3 The antibody of claim 1, comprising:

4. The HCDR1 is AASGFTFSSYSX 1 S (SEQ ID NO: 8) (wherein X 1 is Leu); the HCDR2 is AISVSSGGSTYYAX 2 SVKG (SEQ ID NO: 9) (wherein X 2 is Asp; and the HCDR3 is ARDDLIX 3 TRGTFYNWFDP (SEQ ID NO: 10) (wherein X 3 The antibody of claim 1, comprising:

5. The HCDR1 is AASGFTFSSYSX 1 S (SEQ ID NO: 8) (wherein X 1 is Phe); the HCDR2 is AISVSSGGSTYYAX 2 SVKG (SEQ ID NO: 9) (wherein X 2 is Asp); the HCDR3 is ARDDLIX 3 TRGTFYNWFDP (SEQ ID NO: 10) (wherein X 3 The antibody of claim 1, comprising:

6. The antibody of claim 1, wherein the VH comprises SEQ ID NO: 7 and the VL comprises SEQ ID NO:

11.

7. The antibody of claim 1, wherein the antibody comprises a heavy chain (HC) comprising a sequence selected from SEQ ID NO: 1 or SEQ ID NO: 3 and a light chain (LC) comprising SEQ ID NO:

5.

8. The antibody of claim 1, wherein the antibody comprises an HC comprising amino acids 2 to 451 selected from SEQ ID NO: 1 or SEQ ID NO: 3 and an LC comprising SEQ ID NO:

5.

9. The antibody of claim 1, wherein the antibody comprises an HC consisting of a sequence selected from SEQ ID NO: 1 or SEQ ID NO: 3 and an LC consisting of SEQ ID NO:

5.

10. The antibody of claim 1, wherein the VH comprises SEQ ID NO: 17 and the VL comprises SEQ ID NO:

11.

11. The antibody of claim 1, wherein the antibody comprises an HC comprising SEQ ID NO: 15 and an LC comprising SEQ ID NO:

5.

12. The antibody of claim 1, wherein the antibody comprises an HC comprising amino acids 2 to 451 of SEQ ID NO: 15 and an LC comprising SEQ ID NO:

5.

13. The antibody of claim 1, wherein the antibody comprises an HC consisting of SEQ ID NO: 15 and an LC consisting of SEQ ID NO:

5.

14. The antibody of claim 1, wherein the VH comprises SEQ ID NO: 22 and the VL comprises SEQ ID NO:

11.

15. The antibody of claim 1, wherein the antibody comprises an HC comprising a sequence selected from SEQ ID NO: 18 or SEQ ID NO: 20 and an LC comprising SEQ ID NO:

5.

16. The antibody of claim 1, wherein the antibody comprises an HC comprising amino acids 2 to 451 selected from SEQ ID NO: 18 or SEQ ID NO: 20 and an LC comprising SEQ ID NO:

5.

17. The antibody of claim 1, wherein the antibody comprises an HC comprising a sequence selected from SEQ ID NO: 18 or SEQ ID NO: 20 and an LC consisting of SEQ ID NO:

5.

18. The antibody of claim 1, wherein the VH comprises SEQ ID NO: 25 and the VL comprises SEQ ID NO:

11.

19. The antibody of claim 1, wherein the antibody comprises an HC comprising SEQ ID NO: 23 and an LC comprising SEQ ID NO:

5.

20. The antibody of claim 1, wherein the antibody comprises an HC comprising amino acids 2 to 451 of SEQ ID NO: 23 and an LC comprising SEQ ID NO:

5.

21. The antibody of claim 1, wherein the antibody comprises an HC consisting of the sequence of SEQ ID NO: 23 and an LC consisting of SEQ ID NO:

5.

22. The antibody of claim 1, wherein the antibody has a human IgG1 or IgG4 isotype.

23. 23. The antibody of claim 22, wherein the antibody has a human IgG4 isotype.

24. 23. The antibody of claim 22, wherein the antibody has a human IgG1 isotype.

25. A nucleic acid comprising a sequence encoding SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:3, SEQ ID NO:20 or SEQ ID NO:23 and a second nucleic acid sequence encoding SEQ ID NO:

5.

26. A composition comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:3, SEQ ID NO:20 or SEQ ID NO:23 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:

5.

27. A cell comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:3, SEQ ID NO:20 or SEQ ID NO:23 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:

5.

28. 28. The cell of claim 27, wherein the cell is a mammalian cell.

29. 29. A method for producing an antibody, comprising culturing the cell of any one of claims 27 to 28 under conditions in which the antibody is expressed, and recovering the expressed antibody from the culture medium.

30. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 24 and a pharmaceutically acceptable excipient, diluent or carrier.

31. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 24 for use in the treatment of an inflammatory disease.

32. 25. Use of an antibody according to any one of claims 1 to 24 in the manufacture of a medicament for the treatment of an inflammatory disease.

33. A pharmaceutical composition comprising an antibody that binds to and antagonizes human IL-1β protein for use in treating inflammatory diseases.