Anti-IL-1β Antibody

Abstract

The present disclosure provides engineered human IL-1β antibodies, cells, and vectors comprising DNA encoding the antibodies, as well as methods for producing the antibodies. In addition, the present disclosure provides uses of engineered human IL-1β antibodies for treating inflammatory diseases such as cardiovascular diseases and cancer.

Description

[0001] The present disclosure relates to the field of medicine. Specifically, the present disclosure relates to antibodies that bind to human IL-1β (IL-1 beta or IL-1β or interleukin-1β have the same meaning herein) and can be used to treat and / or prevent inflammatory diseases, including but not limited to atherosclerotic cardiovascular disease (ASCVD), heart failure, cancer, and rare genetic diseases (such as genetic mutations that lead to excessive production of IL-1β). The present disclosure also relates to methods for treating and / or preventing these inflammatory diseases.

[0002] Cardiovascular disease (CVD) is a class of diseases involving the heart or blood vessels. Common manifestations of CVD include angina, myocardial infarction (MI, commonly known as heart attack), stroke, heart failure, and irregular heartbeat, among others. Due to the complexity of the disease, many risk factors have been identified that contribute to the onset and progression of the disease. These risk factors include dyslipidemia, hypertension, diabetes, tobacco use, unhealthy diet, lack of exercise, and obesity. However, despite much work done to control these traditional risk factors, cardiovascular disease remains the leading cause of death in the United States and worldwide.

[0003] Research over the past two decades has emphasized that the inflammatory process is a key component of the pathogenesis of CVD, especially atherosclerotic cardiovascular disease (ASCVD). Epidemiological data from the mid-1990s showed that inflammation, as determined by high-sensitivity C-reactive protein (hsCRP) or interleukin-6 (IL-6), was strongly associated with future major adverse cardiovascular events (MACE) in 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 the role of 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 can also lead to plaque instability and rupture, triggering acute cardiovascular events such as MI and stroke.

[0004] The interleukin-1 family is a key component of inflammation and has been intensively studied as a therapeutic target for various inflammatory diseases (Szekely et al. (2018) Cardiol. Ther. 7: 25-44). The IL-1 gene family has three members: IL-1, IL-1β, and IL-1 receptor antagonist (IL-1ra). IL-1 and IL-1β are agonists of the IL-1 receptor, while IL-1ra is a specific receptor antagonist and is therefore an endogenous competitive inhibitor of IL-1 (IL-1a or IL-1β). IL-1β is the major circulating form of IL-1. It is produced as a precursor (pro-IL-1β), which is activated by the NLRP3 (NOD, LRR, and pyrin domain-containing protein 3) inflammasome under many inflammatory stimuli. Importantly, it has recently been discovered that multiple factors known to be associated with atherosclerosis can activate the NLRP3 inflammasome. These factors include cholesterol crystals, atherogenic oscillatory flow, hypoxia, and neutrophil extracellular traps, which support a key role for the NLRP3 inflammasome-IL 1β pathway in atherogenesis (Ridker (2016) Circ. Res. 118:145-156).

[0005] The active form of IL-1β has autocrine, paracrine and endocrine effects and is therefore involved in a wide range of inflammatory diseases. Rare genetic diseases such as Muckle Wells Syndrome (MWS), cryopyrin-associated periodic syndrome (CAPS) and neonatal-onset multisystem inflammatory syndrome (NOMIS) are associated with overproduction of IL-1β, among others. Intervention with canakinumab (IL-1β antibody), anakinra (IL-1R antagonist) and rilonocept (IL-1 trap) can all improve the symptoms of these overproduction syndromes (Ridker (2016) Circ. Res. 118: 145-156).

[0006] IL-1β inhibition may also play a role in the treatment of cancers with an inflammatory basis. Many malignancies arise in areas of chronic inflammation, and inadequate resolution of inflammation can play a major role in tumor invasion, progression, and metastasis (Grivennikov et al. (2010) Cell 140: 883-899). Inflammation has pathophysiological significance in lung cancer; for example, smoking and other externally inhaled toxins trigger a persistent inflammatory response. This inflammatory activation is mediated in part by activation of the NLRP3 inflammasome, and active IL-1β is produced locally. In the clinic, high baseline concentrations of hsCRP and IL-6 have been found to be associated with subsequent diagnosis of lung cancer. IL-1β blockade using canakinumab is associated with a reduction in total cancer mortality, lung cancer incidence, and lung cancer mortality (Ridker et al. (2017) Lancet 390: 1833-1842).

[0007] Thus, the present disclosure can be used to treat or prevent many cancers, including but not limited to lung cancer, such as non-small cell lung cancer (NSCLC); breast cancer, such as triple-negative breast cancer (TNBC); prostate cancer, such as metastatic prostate cancer; blood cancers, such as leukemias, lymphomas, myelomas, such as low- or intermediate-risk myelodysplastic leukemia; gastric cancer, including esophageal cancer; ovarian cancer, kidney cancer, liver cancer, such as hepatocellular carcinoma (HCC); skin cancer, such as melanoma; head and neck cancer; brain cancer; colorectal cancer; bladder cancer; pancreatic cancer; and kidney cancer, such as localized renal cancer.

[0008] There remains a need to provide therapeutic antibodies that bind human IL-1 β. In particular, there remains a need to provide IL-1 β antibodies that have favorable clinical properties.

[0009] The present disclosure encompasses engineered human antibodies against human IL-1β. The antibodies disclosed herein have one or more of the following properties: (1) binding to human and cynomolgus macaque IL-1b with desired binding affinity and / or association and dissociation rates; (2) potent IL-1β neutralization activity; (3) high specificity for IL-1β; and (4) low immunogenicity risk.

[0010] The present disclosure provides engineered IL-1β antibodies and vectors comprising DNA encoding the antibodies, as well as methods for producing the antibodies. In addition, the present disclosure provides uses of engineered IL-1β antibodies for treating inflammatory diseases such as cardiovascular disease and cancer, which can benefit from modulation, such as antagonizing IL-1β signaling and / or improving the effects of excessive production of IL-1β.

[0011] Therefore, in some embodiments, the present disclosure provides an antibody that binds to human IL-1β protein, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a heavy chain complementary determining region (HCDR) HCDR1, HCDR2 and HCDR3, and the VL comprises a light chain complementary determining region (LCDR) LCDR1, LCDR2 and LCDR3, wherein

[0012] The HCDR1 comprises AASGFTFSDHYMS (SEQ ID NO: 7),

[0013] The HCDR2 comprises YISSSGSTIYYADSVKG (SEQ ID NO: 8),

[0014] The HCDR3 comprises AREADSSGYYYVGVDV (SEQ ID NO: 9),

[0015] The LCDRI comprises RASQSISSYLN (SEQ ID NO: 11),

[0016] The LCDR2 comprises YGASSDQS (SEQ ID NO: 12), and

[0017] The LCDR3 comprises QQGYYFPPT (SEQ ID NO: 13).

[0018] In some embodiments, the disclosure provides an antibody, wherein the VH comprises SEQ ID NO: 6, and the VL comprises SEQ ID NO: 10. In other embodiments, the disclosure provides an antibody, wherein the VH consists of SEQ ID NO: 6, and the VL consists of SEQ ID NO: 10.

[0019] In some embodiments, the disclosure provides an antibody, wherein the VH comprises SEQ ID NO: 17 or SEQ ID NO: 18, and the VL comprises SEQ ID NO: 10. In other embodiments, the disclosure provides an antibody, wherein the VH consists of SEQ ID NO: 17 or SEQ ID NO: 18, and the VL consists of SEQ ID NO: 10.

[0020] In some embodiments, the disclosure provides an antibody, wherein the antibody comprises a heavy chain (HC) comprising SEQ ID NO: 2 and a light chain (LC) comprising SEQ ID NO: 4. In some embodiments, the antibody comprises a HC consisting of SEQ ID NO: 2 and a LC consisting of SEQ ID NO: 4.

[0021] In some embodiments, the present disclosure provides an antibody, wherein the antibody comprises a heavy chain (HC) comprising SEQ ID NO: 15 or SEQ ID NO: 16 and a light chain (LC) comprising SEQ ID NO: 4. In other embodiments, the present disclosure provides an antibody, wherein the antibody comprises a heavy chain (HC) consisting of SEQ ID NO: 15 or SEQ ID NO: 16 and a light chain (LC) consisting of SEQ ID NO: 4.

[0022] In some embodiments, the disclosure provides an antibody, wherein the antibody comprises a heavy chain (HC) comprising amino acids 2-445 of SEQ ID NO: 2 and a light chain (LC) comprising SEQ ID NO: 4. In other embodiments, the disclosure provides an antibody, wherein the antibody comprises a heavy chain (HC) comprising amino acids 2-445 of SEQ ID NO: 15 or SEQ ID NO: 16 and a light chain (LC) comprising SEQ ID NO: 4.

[0023] In some embodiments, the epitope of an antibody is determined by obtaining an X-ray crystal structure of the antibody:antigen complex and identifying which residues on the antigen are at the same residues on the antibody of interest. In one embodiment, the antibodies of the invention bind to human IL-1β (SEQ ID NO: 1) at an epitope comprising some or all of residues R120, E153, K219, E221, N224, M264, Q265, F266, and S268 of SEQ ID NO: 1.

[0024] In some embodiments, the antibody is of an engineered human IgG1 or IgG4 isotype.

[0025] In a preferred embodiment, the antibody is of an engineered human IgG4 isotype.

[0026] In some embodiments, the disclosure includes a nucleic acid sequence encoding SEQ ID NO: 2 or 4.

[0027] In some embodiments, the disclosure includes a nucleic acid sequence encoding SEQ ID NO: 15 or 4.

[0028] In some embodiments, the disclosure includes a nucleic acid sequence encoding SEQ ID NO: 16 or 4.

[0029] In some embodiments, the present disclosure provides a vector comprising a first nucleic acid sequence encoding SEQ ID NO:2 and a second nucleic acid sequence encoding SEQ ID NO:4.

[0030] In some embodiments, the present disclosure provides a vector comprising a first nucleic acid sequence encoding SEQ ID NO:15 and a second nucleic acid sequence encoding SEQ ID NO:4.

[0031] In some embodiments, the present disclosure provides a vector comprising a first nucleic acid sequence encoding SEQ ID NO:16 and a second nucleic acid sequence encoding SEQ ID NO:4.

[0032] In some embodiments, the present disclosure provides a first vector comprising a nucleic acid sequence encoding SEQ ID NO:2 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:4.

[0033] In some embodiments, the present disclosure provides a first vector comprising a nucleic acid sequence encoding SEQ ID NO:15 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:4.

[0034] In some embodiments, the present disclosure provides a first vector comprising a nucleic acid sequence encoding SEQ ID NO:16 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:4.

[0035] In some embodiments, the present disclosure provides a cell comprising a vector comprising a first nucleic acid sequence encoding SEQ ID NO:2 and a second nucleic acid sequence encoding SEQ ID NO:4.

[0036] In some embodiments, the present disclosure provides a cell comprising a vector comprising a first nucleic acid sequence encoding SEQ ID NO:15 and a second nucleic acid sequence encoding SEQ ID NO:4.

[0037] In some embodiments, the present disclosure provides a cell comprising a vector comprising a first nucleic acid sequence encoding SEQ ID NO:16 and a second nucleic acid sequence encoding SEQ ID NO:4.

[0038] In some embodiments, the present disclosure provides a cell comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO:2 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:4.

[0039] In some embodiments, the present disclosure provides a cell comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 15 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 4.

[0040] In some embodiments, the present disclosure provides a cell comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 16 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 4.

[0041] In one embodiment, the cell is a mammalian cell.

[0042] In one embodiment, the present disclosure provides a method of producing an antibody, the method comprising culturing the cell as described above under conditions such that the antibody is expressed and recovering the expressed antibody from the culture medium.

[0043] In one embodiment, the present disclosure provides an antibody produced by culturing a cell as described above under conditions such that the antibody is expressed and recovering the expressed antibody from the culture medium.

[0044] In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody of the present disclosure and a pharmaceutically acceptable excipient, diluent, or carrier.

[0045] In one embodiment, the disclosure provides an antibody comprising two light chains and two heavy chains, wherein each light chain has the amino acid sequence given in SEQ ID NO:4, and each heavy chain has the amino acid sequence given in SEQ ID NO:2.

[0046] In one embodiment, the present disclosure provides an antibody comprising two light chains and two heavy chains, wherein each light chain has the amino acid sequence given in SEQ ID NO:4, and each heavy chain has the amino acid sequence given in SEQ ID NO:15 or SEQ ID NO:16.

[0047] In one embodiment, the present disclosure provides a method for preventing a disease, the method comprising administering an antibody of the present disclosure and an acceptable carrier, diluent or excipient. In a specific embodiment, the present disclosure provides a method for treating an inflammatory disease, wherein the inflammatory disease is selected from a list including but not limited to the following: cardiovascular disease, cancer, Muir-Wei syndrome (MWS), cryptopyrin-associated periodic syndrome (CAPS), neonatal-onset multisystem inflammatory syndrome (NOMIS), rheumatoid arthritis, systemic juvenile idiopathic arthritis (soJIA), gouty arthritis, chronic obstructive pulmonary disease (COPD), type 1 diabetes, type 2 diabetes, familial cold autoinflammatory syndrome (FCAS) and eye diseases such as age-related macular degeneration.

[0048] In a specific embodiment, the present disclosure provides a method of treating a cardiovascular disease, wherein the cardiovascular disease is selected from the list including but not limited to: atherosclerotic cardiovascular disease (ASCVD) or heart failure.

[0049] In a specific embodiment, the present disclosure provides a method of treating cancer, wherein the type of cancer is selected from a list including but not limited to: lung cancer such as non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), metastatic prostate cancer, low or intermediate risk myelodysplastic leukemia, and localized renal cancer.

[0050] In one embodiment, the present disclosure provides antibodies of the present disclosure for use in therapy. In one embodiment, the present disclosure provides antibodies of the present disclosure for use in treating inflammatory diseases. In one embodiment, the present disclosure provides antibodies of the present disclosure for use in treating cardiovascular diseases. In one embodiment, the present disclosure provides antibodies of the present disclosure for use in treating cancer. In another embodiment, the present disclosure provides antibodies of the present disclosure for use in treating inflammatory diseases, wherein the inflammatory diseases are cardiovascular diseases. In another embodiment, the present disclosure provides antibodies of the present disclosure for use in treating inflammatory diseases, wherein the inflammatory diseases are cancer.

[0051] In another embodiment, the present disclosure provides the use of the antibody of the present disclosure in the manufacture of a medicament for treating an inflammatory disease. In another embodiment, the present disclosure provides the use of the antibody of the present disclosure in the manufacture of a medicament for treating a cardiovascular disease or cancer.

[0052] As used herein, including the appended claims, singular forms of words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0053] As used herein, an "antibody" is an immunoglobulin polypeptide molecule that binds an antigen. A naturally occurring full-length antibody is an immunoglobulin molecule that comprises two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The amino-terminal portion of each chain comprises a variable region of about 100-110 amino acids that is primarily responsible for antigen recognition through the complementary determining regions (CDRs) contained therein. The carboxyl-terminal portion of each chain defines a constant region that is primarily responsible for effector functions.

[0054] CDRs are interspersed with more conserved regions, called framework regions (FRs). Each light chain variable region (LCVR, also called VL) and heavy chain variable region (HCVR, also called VH) consists of 3 CDRs and 4 FRs, arranged from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDRs of the light chain are called "LCDR1, LCDR2 and LCDR3", and the 3 CDRs of the heavy chain are called "HCDR1, HCDR2 and HCDR3". CDRs contain most of the residues that form specific interactions with antigens. The numbering and positioning of CDR amino acid residues in the VL and VH regions are performed according to the well-known Kabat numbering convention.

[0055] The light chain is classified as kappa or lambda and is characterized by a specific constant region known in the art. The heavy chain is classified as gamma, μ, alpha, delta or epsilon and the isotype of the antibody is defined as IgG, IgM, IgA, IgD or IgE, respectively. The IgG antibody can be additionally divided into subclasses, such as IgG1, IgG2, IgG3, IgG4. Every kind of heavy chain type is characterized by a specific constant region with a sequence well known in the art.

[0056] In some biological systems and methods for production, antibodies may undergo co-translational and post-translational modifications, such as glycosylation, deamidation, acylation, oxidation, cyclization, fucosylation, and other modifications well known in the art. Another known modification is the cyclization of glutamine or glutamic acid to pyroglutamic acid (commonly abbreviated as pyrGlu, pyrE, pGlu or pE) at the N-terminus of the heavy chain variable region comprising the heavy chain. Depending on the method and antibody used, the percentage of glutamic acid converted to pyroglutamic acid will vary and may represent a mixture, involving essentially all of the antibodies being produced, or a very low percentage of the antibodies.

[0057] As used herein, the term "monoclonal antibody" (mAb) refers to an antibody derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic or phage clone, rather than the method by which the clone was produced. The mAbs of the present disclosure are preferably present in a homogeneous or substantially homogeneous population. A complete mAb contains 2 heavy chains and 2 light chains. For example, monoclonal antibodies can be produced by hybridoma technology, recombinant technology, phage display technology, synthetic technology (e.g., CDR-grafting), or a combination of such or other technologies known in the art.

[0058] The phrase "engineered human" or "engineered human IL-1β antibody" refers to a monoclonal antibody produced and / or manipulated in this way: having binding and functional properties according to the present invention, binding to human IL-1β and having a framework region engineered to contain a framework sequence substantially similar or identical to a human variant, the framework region surrounding the CDRs derived from a non-human antibody. Human and humanized antibodies are well known in the art. The engineered human antibodies herein may be intentionally modified, for example in the constant region, to alter effector or other biological functional characteristics, or biophysical characteristics (such as stability, developability and / or solubility, etc.) compared to the native sequence. Another embodiment herein includes an engineered human antibody comprising a fully human or substantially fully human heavy chain and / or light chain constant region. Another embodiment herein includes an immunoglobulin molecule comprising 2 HCs and 2 LCs interconnected by disulfide bonds, comprising fully human or substantially fully human HC and LC constant regions.

[0059] Such "antigen-binding fragments" of engineered human antibodies include, for example, Fab fragments, Fab' fragments, F(abz')2 fragments and single-chain Fv fragments.

[0060] "Framework region" or "framework sequence" refers to any one of framework regions 1 to 4. Engineered human antibodies and antigen-binding fragments thereof encompassed by the present disclosure include molecules in which any one or more of framework regions 1 to 4 are substantially or fully human, i.e., in which any of the possible combinations of separate substantially or fully human framework regions 1 to 4 are provided. For example, this includes molecules in which framework region 1 and framework region 2, framework region 1 and framework region 3, framework regions 1, 2, and 3, etc. are substantially or fully human. Substantially human frameworks are those that have at least about 80% sequence identity with known human germline framework sequences. Human framework germline sequences can be obtained from ImMunoGeneTics (IMGT) or from The 20 Immunoglobulin Facts Book, by Marie-Paule Lefranc and Gerard Leffanc, Academic Press, 2001, ISBN012441351. For example, the germline light chain framework can be selected from the group consisting of: A11, A17, A18, A19, A20, A27, A30, L1, L11, L12, L2, L5, L15, L6, L8, O12, O2, and O8, and the germline heavy chain framework region can be selected from the group consisting of: VH2-5, VH2-26, VH2-70, VH 3-20, 25VH3-72, VH1-46, VH3-9, VH3-66, VH3-74, VH4-31, VHI-18, VHI-69, VI-1 3-7, VH3-11, VH3-15, VH3-21, VH3-23, VH3-30, VH3-48, VH4-39, VH4-59 and VH5-51.

[0061] As used herein, "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), which is activated by the NLRP3 inflammasome in many inflammatory diseases. Human pro-IL-1β comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof. The mature human IL-1β protein comprises the amino acid sequence of SEQ ID NO: 14 or a variant thereof.

[0062] As used herein, "inflammatory" includes inflammatory and autoinflammatory diseases. The term "inflammatory disease" includes, but is not limited to, cardiovascular disease, cancer, rare genetic diseases that lead to excessive production of IL-1β, and other diseases that can benefit from regulation, such as antagonizing IL-1b signaling. "Inflammatory disease" may include, but is not limited to, cardiovascular disease, heart failure, cancer, Muir-Wei syndrome (MWS), cryptopyrin-associated periodic syndrome (CAPS), neonatal multisystem inflammatory syndrome (NOMIS), rheumatoid arthritis, systemic juvenile idiopathic arthritis (soJIA), gouty arthritis, chronic obstructive pulmonary disease (COPD), type 1 diabetes, type 2 diabetes, familial cold autoinflammatory syndrome (FCAS) and eye diseases including, for example, age-related macular degeneration.

[0063] The term "cardiovascular disease" herein refers to a class of diseases involving the heart or blood vessels. A non-exhaustive list of CVD manifestations includes, but is not limited to, angina, myocardial infarction (MI, commonly known as heart attack), stroke, heart failure, and arrhythmia.

[0064] The term "cancer" herein refers to a group of diseases involving abnormal cell growth and the potential to invade or spread to other parts of the body. A non-exhaustive list of cancer types includes, but is not limited to, lung cancer such as non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), metastatic prostate cancer, low or moderate risk myelodysplastic leukemia, and localized renal cancer. Thus, cancer can involve cells from solid tissues or organs, such as brain cancer, breast cancer, colorectal cancer, skin cancer, liver cancer, kidney cancer, lung cancer, pancreatic cancer, prostate cancer, head and neck cancer, ovarian cancer, uterine cancer, bladder cancer, gastric cancer (including gastric cancer of the esophagus); connective tissue cancer, such as sarcoma or bone cancer; or blood cancer, such as lymphoma, leukemia, and myeloma. Cancers can also be described by their cell origin, such as cancers originating from epithelial cells in various parts of the body, or adenomas originating from glands.

[0065] When "treatment" is applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids, it refers to the contact of exogenous drugs, therapeutic agents or compositions with animals, humans, subjects, cells, tissues, organs or biological fluids. The term "treatment" (or "treatment" or "medical treatment") applied to humans or research subjects refers to a method that involves slowing down, interrupting, preventing, controlling, stopping, reducing or reversing the progress or severity of symptoms, disorders, conditions or diseases associated with IL-1β activity, but does not necessarily involve the complete elimination of all disease-related symptoms, conditions or disorders associated with IL-1β activity. "Treatment" (or "treatment" or "medical treatment") applied to pharmacokinetics, diagnosis, research and experimental methods covers the contact of reagents with cells, and the contact of reagents with fluids, wherein the fluids are in contact with cells. The term "prevention" (or "prevention") refers to preventing the occurrence, existence or occurrence of something and / or hindering or stopping doing something.

[0066] The term "activation" can refer to cellular activation regulated by internal mechanisms as well as external or environmental factors.

[0067] The "activity" of a molecule can describe or refer to the binding of the molecule to a ligand or receptor, catalytic activity; the ability to stimulate gene expression or cell signaling, differentiation or maturation; antigenic activity, regulation of the activity of other molecules, etc. The "activity" of a molecule can also refer to the activity of regulating or maintaining cell-to-cell interactions such as adhesion, or maintaining the structure of a cell such as a cell membrane or cytoskeleton. "Activity" can also refer to specific activity, such as [catalytic activity] / [mg protein], or [immune activity] / [mg protein], concentration in a biological compartment, etc.

[0068] Engineered human antibodies other than those disclosed herein that exhibit similar functional properties according to the present disclosure can be produced using several different methods. Specific antibody compounds disclosed herein can be used as templates or parent antibody compounds to prepare additional antibody compounds. In one method, the parent antibody compound CDR is grafted to a human framework with high sequence identity to the parent antibody compound framework. The sequence identity of the new framework is generally 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 in the parent antibody compound. Compared to the parent antibody, this grafting can result in a reduction in binding affinity. In this case, according to the specific criteria disclosed in Queen et al. A1 (1991) Proc. Natl. Acad. Sci US 88: 2869, the framework can be backmutated to the parent framework at certain positions. Other references describing methods used in humanizing antibodies include U.S. Pat. Nos. 4,816,397; 5,225,539 and 5,693,761; the computer programs ABMOD and ENCAD described by Levitt (1983) J. Mol. Biol. 168:595-620; and the methods of Winter and colleagues (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).

[0069] The residues to be considered for backmutation can be determined as follows:

[0070] When an amino acid falls into a category where the amino acid in the human framework region of the acceptor framework is unusual for human frameworks at that position, while the corresponding amino acid in the donor immunoglobulin is typical for human frameworks at that position, the framework amino acids of the human germline sequence being used (the "acceptor framework") are replaced with framework amino acids from the framework of the parent antibody compound (the "donor framework").

[0071] When each of the amino acids in the human framework region of the acceptor framework and the corresponding amino acid in the donor framework is usually uncommon for the human framework at that position, such amino acid can be replaced by a typical amino acid of the human framework at that position. This back mutation standard enables one to restore the activity of the parent antibody compound.

[0072] Another method for producing engineered human antibodies that exhibit functional properties similar to those of the antibody compounds disclosed herein involves randomly mutating amino acids within the grafted CDR without changing the framework, and screening the resulting molecules for binding affinity and other functional properties that are as good as or better than the parent antibody compound. Single mutations can also be introduced at each amino acid position within each CDR, and the effects of these mutations on binding affinity and other functional properties are then evaluated. Single mutations that produce improved properties can be combined to evaluate their combined effects with each other.

[0073] Furthermore, a combination of the two aforementioned methods is possible. After CDR grafting, in addition to introducing amino acid changes in the CDRs, specific framework regions can also be backmutated. This method is described in Wu et al., (1999) J. Mol. Biol. 294: 151-162.

[0074] The engineered human antibodies disclosed herein can be used as drugs in human medicine administered by a number of routes. Most preferably, such compositions are for parenteral administration. Such pharmaceutical compositions can be prepared by methods well known in the art (see, e.g., Remington: The Science and Practice of Pharmacy; 19 版 (1995), A. Gennaro et al., Mack Publishing Co.), and comprises an engineered human antibody as disclosed herein, and a pharmaceutically acceptable carrier, diluent or excipient.

[0075] The results of the following assays demonstrate that exemplary monoclonal antibodies and antigen-binding fragments thereof of the present disclosure bind to and / or neutralize IL-1β and thus can be used to treat inflammatory diseases such as cardiovascular disease or cancer.

[0076] Example 1: Antibody expression and purification

[0077] Significant problems of chemical and physical stability were encountered in constructing the anti-IL-1 β antibodies of the present disclosure. For example, problems encountered with the initial constructs included low binding affinity, variable region deamidation, oxidation, and low potency.

[0078] There are significant problems to overcome in terms of binding affinity and chemical and physical stability, and the chemical and physical modifications of the present disclosure unexpectedly overcome these problems. Amino acid modifications are introduced in both the heavy chain and the light chain. The antibodies of the present disclosure include multiple residue changes compared to the original construct and are identified as having high binding affinity and being chemically and physically stable. No modifications were identified in the initial construct that comprise the antibodies of the present disclosure.

[0079] Exemplary antibodies of the present disclosure are provided in Table 1.

[0080] The antibodies disclosed herein can be prepared and purified as described below. Appropriate host cells, such as HEK 293 or CHO, are transiently transfected with an expression system for secreting antibodies using an optimal predetermined HC:LC carrier ratio, and the HC vector and LC vector encode the sequence of antibody I_HC or antibody II_HC, respectively, and the common LC sequence of antibody I and II. The clarified culture medium that has secreted the antibody is purified using any of a number of common techniques. For example, the culture medium can be conveniently applied to a protein A or G chromatographic column that has been balanced with a compatible buffer such as phosphate buffered saline (pH 7.4). The chromatographic column is washed to remove non-specific binding components. For example, the bound antibody is eluted by a pH gradient (such as 0.1M sodium phosphate buffer pH 6.8 to 0.1M sodium citrate buffer pH 2.5). The antibody fractions are detected, such as by SDS-PAGE, and then the antibody fractions are combined. Further purification is optional depending on the intended use. Common techniques can be used to concentrate and / or sterile filter the antibody. Soluble aggregates and multimers can be effectively removed by common techniques, including size exclusion, hydrophobic interaction or ion exchange chromatography. The antibody purity after these chromatography steps is greater than 99%. The product can be immediately frozen at -70°C, or lyophilized or stored at 4°C for immediate use.

[0081] Example 2: Antibody Discovery and Engineering

[0082] A group of human anti-IL-1β antibodies is obtained using a complete human yeast display library, and the group of human anti-IL-1β antibodies is screened to identify agents that may be effective IL-1β neutralizing antibodies. Mutations are systematically introduced into the individual complementary determining regions (CDRs) of each antibody, and the resulting variants are subjected to multiple rounds of selection, and clones with improved affinity are separated by reducing antigen concentration and / or increasing dissociation time. The sequence of a single variant is determined and the sequence is used to construct a combinatorial library, which is subjected to another round of selection with increased stringency to identify addition or synergistic mutation pairing between single CDR regions. Single combinatorial clones are sequenced and binding characteristics are determined. Selected antibodies can also be mutagenized to repair post-translational modifications, such as methionine oxidation, while still maintaining binding affinity to IL-1β. In addition, the antibody is subjected to a framework (FW) replacement to restore these FW1 sequences to their germline state, thereby reducing potential immunogenicity.

[0083] Engineered and / or optimized anti-IL-1β antibodies referred to herein as Antibody I and Antibody II were obtained, which have amino acid sequences of heavy and light chain variable regions, as well as complete heavy and light chain amino acid sequences, and nucleotide sequences encoding the amino acid sequences, as described in the section entitled "Amino Acid and Nucleotide Sequences". The sequence IDs corresponding to these fragments and the light and heavy chain CDR amino acid sequences are shown in Table 1 below.

[0084] Table 1.

[0085] Antibody I (SEQ ID NO) Antibody II (SEQ ID NO) VH 17 18 V L 10 10 HC 15 16 LC 4 4 HC CDR1 7 7 HC CDR2 8 8 HC CDR3 9 9 LC CDR1 11 11 LC CDR2 12 12 LC CDR3 13 13

[0086] Example 3: In vitro neutralization of human or cynomolgus monkey IL-1β

[0087] Recombinant human or cynomolgus macaque IL-1β was produced in E. coli as an N-terminal HIS-SUMO fusion protein. The protein was purified using HisPur Ni-NTA chromatography and then endotoxin was removed. The purified fusion protein was then treated with the SUMO protease Ulp1 to cleave HIS-SUMO from the fusion protein. The cleaved HIS-SUMO protein was then removed from the reaction by HisPur Ni-NTA, and untagged IL-1β was further purified to homogeneity using Superdex 75 size exclusion chromatography.

[0088] It is expected that the antibodies of the present disclosure can neutralize IL-1 β. Neutralization of IL-1 β activity by Antibody I and / or Antibody II can be assessed by one or more IL-1 β cell-based activity assays, for example, as described below.

[0089] Screening for neutralizers of IL-1β / IL-1R binding can initially be accomplished by a high-throughput cell-based assay using HeLa cells expressing a luciferase gene under the control of the NF-kB promoter. The assay uses a NF-κB-luciferase reporter signal as a readout of recombinant IL-1β-induced signaling. Neutralization of IL-1β is then quantified by measuring the level of reduction in luciferase activity after titration of anti-IL-1β antibodies. Alternatively, another in vitro neutralization assay, such as the HEK-Blue cell-based assay, is described in detail below.

[0090] Specifically, HEK-Blue TM IL-1β cells were grown in T-75 flasks in 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. TM , 100μg / mL Zeocin TMand 200 μg / mL hygromycin B gold) until 90% confluence. ++ and Mg ++ ) Wash the cells twice and incubate in 1 mL PBS for 2 minutes. Then, detach the cells by tapping the side of the flask and add 10 mL of test medium (DMEM, 4.5 g / l glucose, 2 mM L-glutamine, 10% (v / v) heat-activated FBS (56°C for 30 minutes), 50 U / mL penicillin, 50 μg / mL streptomycin, 100 μg / mL normocin). TM ) resuspended cells, counted cells and diluted to 0.33 × 10 6 cells / mL. Recombinant human or cynomolgus macaque IL-1β and test article were prepared to the desired concentration in assay medium. 40 μL of antibody (5× concentration) was mixed with 10 μL of IL-1β (20× concentration, final concentration of the assay was 4 pM) in a BioCoat poly-D-lysine plate (Corning 354461) and incubated at room temperature for 30 minutes. 150 μL of HEK-Blue TM IL-1β cell suspension was prepared at 0.33×10 6 Cells / mL were distributed into each well of the poly-D-lysine plate containing the antibody and IL-1β mixture. The plates were incubated overnight at 37°C, 5% CO2, and 90% relative humidity. The next day, 25 μL of culture medium from the poly-D-lysine plate was transferred to a Costar assay plate (Corning 3695). 75 μL of QUANTI-Blue detection solution (Invivogen product catalog # rep-qb1, rep-qb2) preheated to 37°C was added to the assay plate. The assay plate was covered and incubated at 37°C for 1 hour, then read at OD on a plate reader (SpectraMax Plus, Molecular Device). 650 The data were normalized and expressed as percent inhibition of 4 pM IL-1β: 0% inhibition = 4 pM IL-1β, 100% inhibition = 0 pM IL-1β. Recombinant human IL-1β Stimulation of HEK-Blue TM IL-1β cell activity. The relative potency of neutralizing antibodies was calculated using a 4-parameter logistic fit and expressed as IC50 values.

[0091] Table 2: In vitro neutralization of human or cynomolgus monkey IL-1β

[0092]

[0093] NA: Not applicable

[0094] Example 4: In vivo neutralization of human IL-1β

[0095] Human IL-1β can bind to and stimulate mouse IL-1 receptors, resulting in an increase in mouse cytokine IL-6 in serum. Time and dose range studies were conducted to determine the optimal dose of human IL-1β and the optimal time for mouse IL-6 induction. In order to test the in vivo neutralization activity of the antibodies disclosed herein, the optimized protocol is described below. Specifically, male C57BL / 6 mice from Envigo were used for research at approximately 9 weeks of age. Mice were fed a normal diet (Harlan Teklad diet, 2014) and randomly assigned to treatment groups (n=5-8 / group) by body weight. The antibodies disclosed herein and control antibodies were dissolved in saline and administered subcutaneously at the indicated dose levels. Twenty-four hours later, human IL-1β was dissolved in saline and administered intraperitoneally at a dose level of 1 μg / kg. Two hours later, blood samples were collected by retro-orbital bleeding and then centrifuged at 2000g for 3 minutes to separate serum samples.

[0096] Mouse IL-6 levels in serum were determined using the V-PLEX Mouse IL-6 Kit (Meso Scale Discovery, catalog # K152QXD-2) according to the manufacturer's instructions. Briefly, the MSD plate was washed 3 times with 150 μL of wash buffer. 50 μL of pre-prepared calibrators (serial dilutions), controls, and test samples (1:10 dilution) were transferred to the appropriate wells on the plate and then shaken at room temperature for 2 hours (500-1000 rpm). The plate was washed 3 times with 150 μL of wash buffer. Then 25 μL of detection antibody solution was added to each well and then shaken at room temperature (500-1000 rpm) for 2 hours. The plate was washed 3 times with 150 μL of wash buffer. 150 μL of 2× read buffer was added to each well. The plate was immediately read on the MSDSQ120 plate reader. The IL-6 concentration of the test sample was analyzed from the calibration curve using a 4-parameter logistic fit.

[0097] Isotype-matched control antibody (IgG4-PAA) was used as a negative control for the study. Data were calculated as percentage inhibition compared to the mean IL-6 level of the control group. One-way ANOVA was used to assess the statistical significance of the mean difference (Dunnett post hoc analysis was performed using JMP11 software). The antibodies disclosed herein dose-dependently blocked the effect of human IL-1β stimulating the increase in mouse IL-6 mediated by mouse IL-1 receptor (Tables 3-1, 3-2).

[0098] Table 3-1: Neutralization of human IL-1β in vivo using Antibody I

[0099]

[0100] Table 3-2: Neutralization of human IL-1β in vivo using Antibody II

[0101]

[0102] Example 5: Binding affinity measurement of Antibody I and Antibody II by MSD-SET

[0103] The affinity of antibody I, antibody II and canakinumab for human IL-1β was measured by MSD (Meso Scale Discovery) electrochemiluminescence assay. First, an equilibrium mixture of antibody and human IL-1β was established; in the mixture, the antibody concentration was kept constant at 1pM, 10pM and 100pM, while the titration concentration of the ligand ranged from 0.9nM to 0.00004nM (2.5-fold dilution between concentrations). The equilibrium mixture was placed in a sealed non-binding 96-well plate and placed at 37°C for 72 hours.

[0104] MSD gold streptavidin plates were used to detect free antibodies in the equilibrium mixture. The MSD plates were first blocked with blocking buffer (PBS + 1% BSA) for 1 hour on an oscillator set at 800 rpm, and then washed 3 times with washing buffer PBST (PBS + 0.05% Tween 20). The plates were coated with biotinylated human IL-1β and then washed 3 times with PBST. The equilibrium mixture was added to the coated plates, incubated at room temperature with shaking for 2.5 minutes, and immediately washed 3 times with PBST. Goat anti-human Sulfo-TAG antibody was added to the plates and incubated at room temperature with shaking for 1 hour. After washing three more times, MSD reading buffer at a concentration of 1:2 diluted in MilliQ water was added to the wells. The plates were then immediately read using an MSD Sector Imager SI6000 instrument.

[0105] For data evaluation, readouts from the MSD instrument are imported into a custom Excel- or GraphPad Prism 8-based evaluation program, which automatically plots the titration data and calculates K D values ​​and statistical parameters.

[0106] Table 4: Binding affinity of antibodies (ligand: human IL-1β, 37°C)

[0107]

[0108] Example 6: Immunogenicity Risk Assessment

[0109] The relative risk of clinical immunogenicity of Antibody II was characterized using in silico and ex vivo methods by comparison with antibodies representing US 7,714,120 (herein US'120) and canakinumab, as shown below.

[0110] Dendritic cell (DC) internalization assay

[0111] This assay evaluates the ability of human DC to internalize test antibodies. CD14+ cells are cultured with IL-4 and GM-CSF and differentiated into immature DCs. Test antibodies, isotype controls or positive controls are pre-incubated with detection agents (Fab-QSY7-TAMRA) at a ratio of 1:1 to form a complex and then added to the culture. Cell culture for one day. After internalization and cleavage, positive TAMRA signals are detected by flow cytometry, and normalized internalization indexes are calculated using IgG1-EN isotype controls and anti-CXCR antibodies.

[0112] MAPPS assay (MHC-associated peptide proteomics)

[0113] MAPPS analyzes MHC-II presented peptides on human dendritic cells previously treated with test antibodies. CD14+ cells isolated from PBMCs of normal human donors were cultured and differentiated into immature DCs by incubation with IL-4 and GM-CSF. On day 4, the culture medium was replaced with fresh culture medium containing the test antibodies. On day 5, LPS was added to convert the cells into mature DCs. On day 6, the cells were lysed in RIPA buffer containing protease inhibitors. MHC-II complexes were immunoprecipitated using biotinylated anti-MHC-II antibodies coupled to streptavidin beads. The bound complexes were eluted and filtered. The separated MHC-II peptides were analyzed by mass spectrometry. Peptide identification was generated by an internal proteomics pipeline using an enzyme-free search algorithm and a bovine / human database with additional test sequences to determine the percentage of donors showing MHC-II peptides from the complementarity determining regions of the test candidates. The KNIME workflow was used to process the identification files of the samples. The peptides identified from the test article were compared with the parental sequence.

[0114] In silico TCEM (T cell exposed motif) analysis

[0115] This analysis evaluates the likelihood that a particular peptide cluster identified by MAPPS will activate CD4+T cells. Peptide sequences containing non-germline residues identified by MAPPS were entered into the Immune Epitope Database (IEDB) Analysis Resource MHCII Binding Prediction page. The prediction method recommended by IEDB was selected. The prediction assumes that the 27 most common HLA-DR, -DP and -DQ alleles cover a large portion of the human population. Each input sequence equal to or greater than 15 residues in length is divided into overlapping 15-mers offset by 1 amino acid to span the entire sequence. For each peptide, a percentile ranking is generated by comparing the score of the peptide with the scores of five million random 15-mers selected from the SWISSPROT database. Amino acids located at the putative P-1, P2, P3, P5, P7 and P8 positions of the register generate TCEMs, and risks are defined based on the presence of non-germline residues at these positions. The likelihood of non-germline residues and core binding to multiple alleles is reported in a graphical rendering and considered for immunogenicity risk assessment.

[0116] MS serum binding

[0117] This assay evaluates off-target binding of test candidates to serum proteins. Test antibodies are coated on Immulon4HBX microplates. After blocking, human serum is added and incubated overnight. Bound proteins are eluted, reduced, alkylated, and digested. Peptides are analyzed by mass spectrometry. Peptide and protein identifications are generated by an in-house proteomics pipeline using a search algorithm with trypsin and a human database with attached test antibody sequences. Ions are quantified by in-house proteomics tools (Chrom-Alignment, Metaconsense, and Quant) and analyzed in JMP using the One-Way Analysis / Each Pair, Student's t-Test platform. The log2auc of the ions is analyzed using JMP: Fit Y with X / Compare Means / All Pairs, Tukey HSD for Each Ion.

[0118] T cell proliferation assay

[0119] This assay evaluates the ability of a test antibody or test MAPPS peptide to activate CD4+T cells by inducing cell proliferation. PBMCs depleted of CD8+T cells were prepared and labeled with CFSE. Each sample was tested with a medium control, keyhole limpet hemocyanin (KLH; a positive clinical benchmark control), a test antibody or a test MAPPS peptide. The cultures were incubated for 7 days. On day 7, samples were analyzed by flow cytometry.

[0120] Pre-existing reactivity (ACE bridge format)

[0121] This assay evaluates the presence of pre-existing antibodies against a test antibody in human serum. Diluted serum is captured overnight on a plate coated with a biotinylated test antibody. On the next day, the captured reactive proteins are eluted with acid and then neutralized in the presence of the biotinylated and ruthenated test antibody. If anti-drug antibodies are present, they will bridge the labeled test antibody and form a complex. The complex is captured by a streptavidin-coated mesoscale plate and the resulting signal is referred to as the Layer 1 signal (expressed as electrochemiluminescence). This signal is confirmed in Layer 2 by adding an excess of unlabeled test antibody in the detection step, which results in inhibition of the Layer 1 signal. The presence of pre-existing anti-drug antibodies is represented as the 90th percentile of Layer 2 inhibition. 个 The size of the percentile.

[0122] Table 5. Summary of immunogenicity risk assessment

[0123]

[0124]

[0125] Abbreviations: ACE = acid capture elution; CDR = complementarity determining region; DC = dendritic cell; H1 = VH CDR1; H2 = VH CDR2; H3 = VH CDR3; L1 = VL CDR1; L2 = VL CDR2; MAPP = MHC-associated peptide proteomics; MHC = major histocompatibility complex; MS = mass spectrometry; T2 = layer 2; TCEM = T cell exposed motif; VH = variable heavy chain; VL = variable light chain; VHFR3 = variable heavy chain framework 3

[0126] Example 7: Epitope mapping of anti-human IL-1β antibody II

[0127] X-ray crystallography was used to obtain a high resolution structure of the IL-1β antibody II Fab complex. IL-1β SEQ ID NO: 14 and Antibody II were expressed, purified and mixed to form the IL 1β antibody II Fab complex. The complex was crystallized using commonly known techniques. See Vonrhein, C. et al., Biological Crystallography 67, 293-302 (2011), Evans, P. R. & Murshudov, G. N., Biological Crystallography 69, 1204-1214 (2013), McCoy, A. J. et al., Journal of Applied Crystallography 40, 658-674 (2007), Emsley, P., Lohkamp, ​​B., Scott, W. G. & Cowtan, K., Biological Crystallography 66, 486-501 (2010), Murshudov, G. N. et al., Biological Crystallography 67, 355-367 (2011), Winn, M. D. et al., Biological Crystallography 67, 235-242 (2011) and Williams, C. J. et al., Protein Science 27, 293-315(2018).

[0128] Epitope displayed on antibody II The IL-1 β amino acid residues within the scope include R120, E153, K219, E221, N224, M264, Q265, F266 and S268 (according to SEQ ID NO: 1).

[0129] Amino acid and nucleotide sequences

[0130] SEQ ID NO: 1: (human IL-1β preprotein or pre-hIL-1β)

[0131]

[0132] SEQ ID NO: 2: (HC of Antibody I and Antibody II)

[0133]

[0134] Where X1 is E or pE

[0135] Where X2 is M or L

[0136] SEQ ID NO: 3: (HC DNA of Antibodies I and II)

[0137]

[0138]

[0139] SEQ ID NO: 4: (LC of Antibody I and Antibody II)

[0140]

[0141] SEQ ID NO: 5: (LC DNA of Antibody I and Antibody II)

[0142]

[0143] SEQ ID NO: 6: (VH of Antibody I and Antibody II)

[0144]

[0145] Where X1 is E or pE

[0146] Where X2 is M or L

[0147] SEQ ID NO: 7: (HCDR1 of Antibody I and Antibody II)

[0148] AASGFTFSDHYMS

[0149] SEQ ID NO: 8: (HCDR2 of Antibody I and Antibody II)

[0150] YISSSGSTIYYADSVKG

[0151] SEQ ID NO: 9: (HCDR3 of Antibody I and Antibody II)

[0152] AREADSSGYYYVGVDV

[0153] SEQ ID NO: 10: (VL of Antibody I and Antibody II)

[0154]

[0155] SEQ ID NO: 11: (LCDR1 of Antibody I and Antibody II)

[0156] RASQSISSYLN

[0157] SEQ ID NO: 12: (LCDR2 of Antibody I and Antibody II)

[0158] YGASSDQ

[0159] SEQ ID NO: 13: (LCDR3 of Antibody I and Antibody II)

[0160] QQGYYFPPT

[0161] SEQ ID NO: 14 (mature human IL-1β; residues 117-269 of SEQ ID NO: 1)

[0162]

[0163] SEQ ID NO: 15 (HC of Antibody I)

[0164]

[0165] Where X1 is E or pE

[0166] SEQ ID NO: 16: (HC of Antibody II)

[0167]

[0168] Where X1 is E or pE

[0169] SEQ ID NO: 17: (VH of Antibody I)

[0170]

[0171] Where X1 is E or pE

[0172] SEQ ID NO: 18: (VH of Antibody II)

[0173]

[0174] Where X1 is E or pE

[0175] SEQ ID NO: 19: (canakinumab HC)

[0176]

[0177] SEQ ID NO: 20: (canakinumab LC)

[0178] Sequence Listing <110> Eli Lilly and Company <120> Anti-IL-1β Antibody <130> X22586 <150> US 62 / 963,327 <151> January 20, 2020 <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 269 <212> PRT <213> Homo sapiens <400> 1 Met Ala Glu Val Pro Glu Leu Ala Ser Glu Met Met Ala Tyr Tyr Ser 1 5 10 15 Gly Asn Glu Asp Asp Leu Phe Phe Glu Ala Asp Gly Pro Lys Gln Met 20 25 30 Lys Cys Ser Phe Gln Asp Leu Asp Leu Cys Pro Leu Asp Gly Gly Ile 35 40 45 Gln Leu Arg Ile Ser Asp His His Tyr Ser Lys Gly Phe Arg Gln Ala 50 55 60 Ala Ser Val Val Val Ala Met Asp Lys Leu Arg Lys Met Leu Val Pro 65 70 75 80 Cys Pro Gln Thr Phe Gln Glu Asn Asp Leu Ser Thr Phe Phe Pro Phe 85 90 95 Ile Phe Glu Glu Glu Pro Ile Phe Phe Asp Thr Trp Asp Asn Glu Ala 100 105 110 Tyr Val His Asp Ala Pro Val Arg Ser Leu Asn Cys Thr Leu Arg Asp 115 120 125 Ser Gln Gln Lys Ser Leu Val Met Ser Gly Pro Tyr Glu Leu Lys Ala 130 135 140 Leu His Leu Gln Gly Gln Asp Met Glu Gln Gln Val Val Phe Ser Met 145 150 155 160 Ser Phe Val Gln Gly Glu Glu Ser Asn Asp Lys Ile Pro Val Ala Leu 165 170 175 Gly Leu Lys Glu Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp 180 185 190 Lys Pro Thr Leu Gln Leu Glu Ser Val Asp Pro Lys Asn Tyr Pro Lys 195 200 205 Lys Lys Met Glu Lys Arg Phe Val Phe Asn Lys Ile Glu Ile Asn Asn 210 215 220 Lys Leu Glu Phe Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr 225 230 235 240 Ser Gln Ala Glu Asn Met Pro Val Phe Leu Gly Gly Thr Lys Gly Gly 245 250 255 Gln Asp Ile Thr Asp Phe Thr Met Gln Phe Val Ser Ser 260 265 <210> 2 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs <220> <221> MISC_FEATURE <222> (1) <223> Xaa at position 1 is E or pyroglutamic acid <220> <221> MISC_FEATURE <222> (118)..(118) <223> Xaa at position 118 is M or L <400> 2 Xaa Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp His 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Ala Asp Ser Ser Gly Tyr Tyr Tyr Tyr Val Gly Val Asp Val 100 105 110 Trp Gly Gln Gly Thr Xaa Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val 195 200 205 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 210 215 220 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 260 265 270 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 445 Gly <210> 3 <211> 1347 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 3 gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgaggctc 60 tcctgtgcag cctctggatt caccttcagt gaccactaca tgagctggat ccgccaggct 120 ccagggaagg ggctggagtg ggtttcatac attagtagta gtggtagtac catatactac 180 gcagactctg tgaagggccg attcaccatc tccagggaca acgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac acggcggtgt actactgcgc cagagaggct 300 gacagcagcg gatactacta cgtgggcgta gacgtatggg gtcagggtac aatggtcacc 360 gtctcctcag ccagcaccaa gggcccatcg gtcttcccac tagcgccctg ctccaggagc 420 acctccgaga gcacagccgc cctgggctgc ctggtcaagg actacttccc cgagccggtg 480 acggtgtcgt ggaactcagg agccctgacc agcggcgtgc acaccttccc ggctgtccta 540 cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc 600 acgaagacct acacctgcaa cgtagatcac aagcccagca acaccaaggt ggacaagaga 660 gttgagtcca aatatggtcc cccatgccca ccctgcccag cacctgaggc cgccggggga 720 ccatcagtct tcctgttccc cccaaaaccc aaggacactc tcatgatctc ccggacccct 780 gaggtaacgt gcgtggtggt ggacgtgagc caggaagacc ccgaggtcca gttcaactgg 840 tacgtggatg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagttcaac 900 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaacggcaag 960 gagtacaagt gcaaggtctc caacaaaggc ctcccgtcct ccatcgagaa aaccatctcc 1020 aaagccaaag ggcagccccg agagccacag gtgtacaccc tgcccccatc ccaggaggag 1080 atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctaccc cagcgacatc 1140 gccgtggagt gggaaagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200 ctggactccg acggctcctt cttcctctac agcaggctaa ccgtggacaa gagcaggtgg 1260 caggagggga atgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca 1320 cagaagagcc tctccctgtc tctgggt 1347 <210> 4 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 4 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ser Asp Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Tyr Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 5 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 5 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctatggt gcatccagtg atcaaagtgg ggtcccatca 180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg caacttacta ctgtcagcaa ggatactact tccctcctac ttttggcgga 300 gggaccaagg ttgagatcaa acgaaccgtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gc 642 <210> 6 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <220> <221> MISC_FEATURE <222> (1) <223> Xaa at position 1 is E or pyroglutamic acid <220> <221> MISC_FEATURE <222> (118)..(118) <223> Xaa at position 118 is M or L <400> 6 Xaa Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp His 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Ala Asp Ser Ser Gly Tyr Tyr Tyr Tyr Val Gly Val Asp Val 100 105 110 Trp Gly Gln Gly Thr Xaa Val Thr Val Ser Ser 115 120 <210> 7 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs <400> 7 Ala Ala Ser Gly Phe Thr Phe Ser Asp His Tyr Met Ser 1 5 10 <210> 8 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs <400> 8 Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 9 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs <400> 9 Ala Arg Glu Ala Asp Ser Ser Gly Tyr Tyr Tyr Tyr Val Gly Val Asp Val 1 5 10 15 <210> 10 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs <400> 10 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ser Asp Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Tyr Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 11 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 11 Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn 1 5 10 <210> 12 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs <400> 12 Tyr Gly Ala Ser Ser Asp Gln Ser 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs <400> 13 Gln Gln Gly Tyr Tyr Phe Pro Pro Thr 1 5 <210> 14 <211> 153 <212> PRT <213> Homo sapiens <400> 14 Ala Pro Val Arg Ser Leu Asn Cys Thr Leu Arg Asp Ser Gln Gln Lys 1 5 10 15 Ser Leu Val Met Ser Gly Pro Tyr Glu Leu Lys Ala Leu His Leu Gln 20 25 30 Gly Gln Asp Met Glu Gln Gln Val Val Phe Ser Met Ser Phe Val Gln 35 40 45 Gly Glu Glu Ser Asn Asp Lys Ile Pro Val Ala Leu Gly Leu Lys Glu 50 55 60 Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp Lys Pro Thr Leu 65 70 75 80 Gln Leu Glu Ser Val Asp Pro Lys Asn Tyr Pro Lys Lys Lys Met Glu 85 90 95 Lys Arg Phe Val Phe Asn Lys Ile Glu Ile Asn Asn Lys Leu Glu Phe 100 105 110 Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ala Glu 115 120 125 Asn Met Pro Val Phe Leu Gly Gly Thr Lys Gly Gly Gln Asp Ile Thr 130 135 140 Asp Phe Thr Met Gln Phe Val Ser Ser 145 150 <210> 15 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs <220> <221> MISC_FEATURE <222> (1) <223> Xaa at position 1 is E or pyroglutamic acid <400> 15 Xaa Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp His 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Ala Asp Ser Ser Gly Tyr Tyr Tyr Val Gly Val Asp Val 100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val 195 200 205 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 210 215 220 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 260 265 270 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 445 Gly <210> 16 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs <220> <221> MISC_FEATURE <222> (1) <223> Xaa at position 1 is E or pyroglutamic acid <400> 16 Xaa Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp His 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Ala Asp Ser Ser Gly Tyr Tyr Tyr Val Gly Val Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val 195 200 205 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 210 215 220 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 260 265 270 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 445 Gly <210> 17 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs <220> <221> MISC_FEATURE <222> (1) <223> Xaa at position 1 is E or pyroglutamic acid <400> 17 Xaa Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp His 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Ala Asp Ser Ser Gly Tyr Tyr Tyr Val Gly Val Asp Val 100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 18 <211> 123 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> synthetic construction <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa at position 1 is E or pyroglutamic acid <400> 18 Xaa Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp His 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Ala Asp Ser Ser Gly Tyr Tyr Tyr Tyr Val Gly Val Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 19 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs <400> 19 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Val Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ile Ile Trp Tyr Asp Gly Asp Asn Gln Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Arg Thr Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 <210> 20 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 20 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Ser 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Ala Tyr Tyr Cys His Gln Ser Ser Ser Leu Pro Phe 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210

Claims

1. An antibody that binds to human IL-1β protein, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementary determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, and the VL comprises light chain complementary determining regions (LCDRs) LCDR1, LCDR2 and LCDR3, wherein The HCDR1 consists of AASGFTFSDHYMS (SEQ ID NO: 7), The HCDR2 consists of YISSSGSTIYYADSVKG (SEQ ID NO: 8), The HCDR3 consists of AREADSSGYYYVGVDV (SEQ ID NO: 9), The LCDR1 consists of RASQSISSYLN (SEQ ID NO: 11), The LCDR2 consists of YGASSDQS (SEQ ID NO: 12), and The LCDR3 consists of QQGYYFPPT (SEQ ID NO: 13).

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

10.

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

10.

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

10.

5. The antibody of claim 1 or 2, wherein the antibody comprises a heavy chain (HC) comprising SEQ ID NO: 2 and a light chain (LC) comprising SEQ ID NO:

4.

6. The antibody of claim 1 or 2, wherein the antibody comprises a heavy chain (HC) comprising SEQ ID NO: 15 and a light chain (LC) comprising SEQ ID NO:

4.

7. The antibody of claim 1 or 2, wherein the antibody comprises a heavy chain (HC) comprising SEQ ID NO: 16 and a light chain (LC) comprising SEQ ID NO:

4.

8. The antibody of claim 1, wherein the antibody comprises a heavy chain (HC) comprising amino acids 2-449 of SEQ ID NO:2 and a light chain (LC) comprising SEQ ID NO:

4.

9. The antibody of claim 1 or 2, wherein the antibody comprises a HC consisting of SEQ ID NO: 2 and a LC consisting of SEQ ID NO:

4.

10. The antibody of claim 1 or 2, wherein the antibody has a human IgG1 or IgG4 isotype.

11. The antibody of claim 10, wherein the antibody has a human IgG4 isotype.

12. A composition comprising (a) a vector comprising a first nucleic acid sequence encoding SEQ ID NO: 2 and a second nucleic acid sequence encoding SEQ ID NO: 4; or (b) a first vector comprising a nucleic acid sequence encoding SEQ ID NO:2 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:

4.

13. A host cell comprising (a) a vector comprising a first nucleic acid sequence encoding SEQ ID NO: 2 and a second nucleic acid sequence encoding SEQ ID NO: 4; or (b) a first vector comprising a nucleic acid sequence encoding SEQ ID NO:2 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:

4.

14. The host cell of claim 13, wherein the cell is a mammalian cell.

15. A method of producing an antibody, the method comprising culturing the cell of any one of claims 13-14 under conditions such that the antibody is expressed and recovering the expressed antibody from the culture medium.

16. An antibody produced by culturing the cell of any one of claims 13 to 14 under conditions allowing expression of the antibody and recovering the expressed antibody from the culture medium.

17. A pharmaceutical composition comprising the antibody of any one of claims 1-11 and 16 and a pharmaceutically acceptable excipient, diluent or carrier.

18. A pharmaceutical composition comprising the antibody of any one of claims 1-11 and 16 for use in treating inflammatory diseases.

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