Compounds and methods targeting epiregulin

Abstract

To provide epiregulin antibodies, compositions comprising the same, and methods for making and / or using the antibodies and / or compositions thereof for chronic pain disorders such as chronic osteoarthritis pain, chronic diabetic peripheral neuropathy pain, or chronic low back pain.SOLUTION: An antibody that binds human epiregulin is provided, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), in which the VH comprises heavy chain complementarity determining regions (HCDR) HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDR) LCDR1, LCDR2, and LCDR3, and the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise specific sequences.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the treatment of pain, including nociceptive, neuropathic and mixed pain, and in particular to the treatment of deformity. Osteoarthritis (OA) pain or diabetic peripheral neuropathy pain heral neuropathy pain (DPNP) or chronic low back pain (CLBP) Compounds and pharmaceutical compositions comprising antibodies directed against human epiregulin in the treatment of Related to products and methods and their uses. [Background technology]

[0002] Chronic pain can be divided into different categories based on its mechanism: nociceptive, neuropathic, and mixed. Nociceptive pain can damage tissue other than nerve cells. or actually damaging stimuli, which can lead to the invasion of the peripheral sensory system. Nociceptive receptors are activated. Osteoarthritis pain is a typical example of somatic nociceptive pain. Neuropathic pain is a common example of pain caused by damage or disease of the central or peripheral nervous system. This leads to maladaptive hypersensitivity of the sensory nervous system. Pain due to diabetic peripheral neuropathy is This is a typical example of peripheral neuropathic pain. It is a type of nociceptive pain and neuropathic pain, such as chronic low back pain. Conditions that exhibit characteristics of both forms of disabling pain are classified as mixed pain.

[0003] Chronic pain is a highly prevalent condition that has a significant impact on society. 2016 By 2020, an estimated 20.4% of the U.S. adult population had been diagnosed with HIV in the past six years, based on data from the National Health Survey. They experienced chronic pain, defined as pain most days of the month or every day. A significant 8% reported chronic pain that limited their life or work activities almost every day or every day in the past six months. As a result, chronic pain has become a leading cause of medical costs, with The annual cost of chronic pain management in the United States was estimated at approximately $635 billion. Despite its societal impact, chronic pain management is currently unsatisfactory. alone is not sufficient to relieve pain or improve function, and there are a variety of drug therapies available. They offer little benefit and pose significant safety risks. Currently, the most common types The drugs most frequently used to relieve chronic pain are acetaminophen, nonsteroidal anti-inflammatory drugs, and steroids. Gabapentinoids, other anticonvulsants ( sodium divalproate, carbamazepine, or lamotrigine), and some antidepressants Medications (such as tricyclics or duloxetine) are available for certain pain disorders. Current Pharmacology Conventional medical devices typically offer a low level of efficacy and have tolerability issues and / or harmful effects. Opioids are effective for acute pain, but they have a high risk of abuse and can cause serious side effects. Treatment options for chronic pain are limited due to the potential for serious adverse reactions. The physical, emotional and economic impact of chronic pain on This, combined with the lack of adequate medical care, represents a significant unmet medical need.

[0004] Epiregulin has seven ligands: TGF-α (TGFA), epiregulin (ER EG), EGF, heparin-binding EGF (HB-EGF), epidermal growth factor (EGF) A linker containing EPGN, amphiregulin (AREG) and betacellulin (BTC) Epidermal growth factor receptor (EGFR) family of genes Members of the Epidemiology Society (Schneider MR, Wolf E. mal growth factor receptor ligands at a glance.J Cell Physiol.2009;218(3):460-46 6) In addition to EGFR (ErbB1), there are three additional receptors within this family of receptors. There are three types of ERbB receptors (ErbB2, ErbB3, and ErbB4), of which ErbB3 and ErbB 4. Selection of the neuregulin family of ligands and the EGFR family of ligands Can bind to members (epiregulin, betacellulin, and HB-EGF) These ligands act in a paracrine or autocrine manner to mediate various biological processes. proteolytically cleaved to produce soluble ligands that can regulate membrane processes. Epiregulin is synthesized as a transmembrane protein. Binds and homodimerizes or forms ligand-induced heterodimers with ErbB2 or ErbB3 Signaling can be induced via activation.

[0005] Epiregulin signaling is involved in a wide range of physiological processes, including inflammation, wound healing, and angiogenesis. contributes to the condition (Riese et al. Epiregulin: Roles in Normal Physiology and Cancer,Semin Cell Dev Biol. 2014, 0:49-56). EGFR receptor homo- or heterodimers Epiregulin signaling via pairs of ERK, MAPK, AP1, and PI3K , potentially activating multiple downstream signaling pathways, including JAK / STAT and NFKB. Activation of pathways such as JAK / STAT and NFKB plays a key role in driving inflammation. As previously demonstrated, activation of AP1 signaling is a marker of neuronal activation. -FOS and c-JUN activation. Recently published data in preclinical models The role of epiregulin in chronic pain, characterized by neuronal inflammation and activation, has been investigated. It has been implicated in pain modulation and is associated with inflammation and neuropathic pain as possible mechanisms in chronic pain disorders. These findings suggest a role for the epiregulin signaling pathway in regulating neuronal activation. According to a study, the EGFR pathway has been shown to be involved in the pathogenesis of neuropathic pain (K ersten et al., Epidermal growth factor re ceptor-inhibition(EGFR-I)in the treatmen t of neuropathic pain.Br J Anaesth.2015; 115(5):761-767). However, the receptor is not regulated by EGFR antibodies or EGFR chimeric antibodies. Targeting phosphoinositide kinase inhibitors may improve gastrointestinal (GI) and skin function. have been found to be associated with a high incidence of adverse reactions, which may contribute to the Their possible uses are limited.

[0006] Antibodies that bind to both TGFα and epiregulin are useful in treating diabetic nephropathy. LY3016859 is disclosed in WO 2012 / 138510. Pyregulin and transforming growth factor α (TGF-α) It is a monoclonal antibody that binds to , which is being tested in a phase 1 clinical trial and The safety, pharmacokinetics, pharmacodynamics, and efficacy in healthy subjects and patients with diabetic nephropathy were evaluated. (Sloan-Lancaster, et al., Evaluation n of the Safety,Pharmacokinetics,Pharmac odynamics,and Efficacy After Single and Multiple Dosings of LY3016859 in Healthy Subjects and Patients With Diabetic Nep hropathy,Clinical Pharmacology in Drug D (See Development 2018,7(7)759-772). n-Lancaster et al. reported that LY3016859 acts more effectively on TGFα than on epiregulin. Furthermore, LY3016859 administration increased the number of nephropathy-related biomarkers. (Beidle r,et al.,J.Pharmacology and Experimental See also Therapeutics, 2014, 349(2):330-343 Nonlinear kinetics suggestive of target-mediated drug disposition were observed, with high levels of soluble target association. Of note, both reported studies showed a high incidence of Circulating anti-LY3016859 antibodies were observed, as measured in drug tolerance assays. Pharmacokinetics or target association as indicated by dose- and time-dependent increases in epiregulin There was no clear effect on epiregulin for the treatment of chronic pain indications. These compounds are more selective, have higher affinity, and are more likely to induce anti-drug antibody responses. Low antibody levels represent a significant unmet therapeutic need. Chronic pain disorders, including idiopathic and mixed pain, and especially osteoarthritis or diabetic peripheral nerve pain Alternative and / or alternative for the treatment of cardiac disorders or chronic low back pain and / or for the treatment of therapy-resistant pain There remains an unmet need for improved and / or curative treatments. Summary of the Invention

[0007] Embodiments of the present disclosure relate to novel anti-human epiregulin antibodies, pharmaceutical compositions thereof, and Methods of using these antibodies and compositions in the treatment of pain and chronic pain disorders are provided. According to some embodiments, the present invention provides a light chain variable region an antibody comprising a heavy chain variable region (LCVR) and a heavy chain variable region (HCVR) and the LCVR provides a complementarity determining region (CDR ), LCDR1, LCDR2 and LCDR3, and HCVR comprises CDR, HCDR1, HCDR2 and HCDR3, which are CDR combinations of the group provided in Table 1 The sequence identifiers used herein are listed in Table 1 and the sequences are selected from the The amino acid and nucleotide sequences are provided in the accompanying drawings. Antibody 1 is a high affinity, complete It is a human immunoglobulin G4 (IgG4) monoclonal antibody, It binds to residues in the C-terminal region of human epiregulin, and inhibits the binding of human epiregulin to EGFR. Antibody 1 is an improved anti-epileptic drug for human therapy. The resulting antibody has enhanced affinity, selectivity, and protection against off-target and unwanted targets. reduced risk of unexpected activity, reduced risk of immunogenicity, high potency and long duration of action, and have a combination of advantageous properties, including blocking epiregulin, and other desirable properties; Improved means for treating pain and chronic pain disorders are provided.

[0008] [Table 1]

[0009] Thus, embodiments of the present disclosure also include a method for producing a LCVR having the amino acid sequence of SEQ ID NO:4. and an antibody comprising an LCVR and an HCVR, wherein the HCVR has the amino acid sequence of SEQ ID NO: 3. provide.

[0010] According to other embodiments, the present disclosure also provides a method for producing a LCVR comprising the steps of: HCVR has the amino acid sequence of SEQ ID NO: 3, and the hinge region and Fc region have the amino acid sequence of SEQ ID NO: Also provided are antibodies comprising an LCVR and an HCVR selected from SEQ ID NO: 51 and SEQ ID NO: 52.

[0011] In another embodiment, the present disclosure also provides a LC having the amino acid sequence of SEQ ID NO: 2 and SEQ ID NO: Also provided is an antibody comprising an HC having the amino acid sequence of SEQ ID NO: 1. According to another embodiment, the present disclosure also has the LC having the amino acid sequence of SEQ ID NO:2 and the LC having the amino acid sequence of SEQ ID NO:1 LC and HC have an amino acid sequence that is at least 95% identical to the amino acid sequence of HC. Antibodies comprising the HC and HC are also provided.

[0012] As used herein, "Antibody 1" refers to the HCDR1 amino acid sequence of SEQ ID NO: 5, HCDR2 amino acid sequence of SEQ ID NO: 6, HCDR3 amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8 the LCDR1 amino acid sequence of SEQ ID NO: 9, the LCDR2 amino acid sequence of SEQ ID NO: 10 DR3 amino acid sequence, HCVR amino acid sequence of SEQ ID NO: 3, LCVR amino acid sequence of SEQ ID NO: 4 the HC amino acid sequence of SEQ ID NO: 1, the LC amino acid sequence of SEQ ID NO: 2, or is encoded by the HC DNA sequence of SEQ ID NO: 11 or the LC DNA sequence of SEQ ID NO: 12 The term "antibody" refers to an antibody encoded by a DNA sequence. The framework and CDR sequences in this application are those of North, et al., unless otherwise specified. Annotation rules consistent with the method of .J.Mol.Biol.2011:406:228-256 It is annotated using

[0013] The carboxy-terminal portion of each HC defines a constant region primarily responsible for effector function. In some embodiments, the antibody comprises a constant region of each HC that reduces effector function. Preferably, embodiments of the present invention are IgG4 antibodies, and Thus, an IgG4 Fc region, or an Fc region derived from human IgG4, such as a modified IgG4 Contains the Fc region.

[0014] According to some embodiments, the constant regions of both HCs have reduced effector function. Modifications in and amino acid substitutions are introduced into the IgG4 hinge and Fc regions. Some embodiments comprise a nucleotide sequence at residues 229 and 230 (positions 234-235 of the EU index). ) and modifications in the constant regions of both HCs containing the amino acid alanine (in the HC of Antibody 1) exemplified and shown in SEQ ID NO: 52), as well as residue 223 (position 228 in the EU index) Further modifications in the constant regions of both HCs that promote stability include the amino acid proline ( HC of Antibody 1 and set forth in SEQ ID NO: 51), as well as residue 442 (EU index It has a deletion of the amino acid lysine at position 447 (exemplified by HC in SEQ ID NO: 1).

[0015] The antibodies of the invention have the following properties: 1) high binding affinity and desirable association and dissociation rates; 2) in neutralizing human epiregulin to achieve pain relief responses and in vivo efficacy 3) sufficiently potent as monotherapy for the treatment and / or prevention of pain disorders. 4) sustained duration of action; 5) well-limited injection site reactions; 6) tolerable 6) low immunogenicity (i.e., sufficiently non-immunogenic in humans), 7) no adverse skin rash reactions 8) reduction of pain disorders, e.g., nociceptive, neuropathic and mixed pain and in particular chronic osteoarthritis pain or chronic diabetic peripheral nerve pain. A thermotherapy approved for development and / or use in the treatment of disabling pain or chronic low back pain. Desirable properties include, but are not limited to, quality, solubility, low self-association, and pharmacokinetic characteristics. In vivo stability, physical and chemical stability, and are believed to have a particularly advantageous combination of properties over prior art anti-epiregulin antibodies. can be done.

[0016] Embodiments of the present invention include antibodies to human epiregulin, compositions thereof, and the antibodies described herein. and the use of pharmacologically advantageous anti-human epiregulin antibodies provided in the embodiments described herein. , and methods useful in treating, downregulating, or ameliorating pain disorders via epiregulin neutralization - Patents.com The present invention provides a significant advance over the prior art by providing an anti-human epitope. The regulin antibodies preferably inhibit pain responses, particularly in chronic pain disorders and pain states. Through this, pain symptoms can be alleviated and pain pathophysiology can be improved. Clinical use may lead to long-term relief of the pain-related disorders being treated.

[0017] Furthermore, it is specific for human epiregulin, has improved binding affinity, and is Diagnostic anti-human epiregulin antibodies demonstrating enhanced sensitivity in pyregulin determination and an improved enzyme-linked immunosorbent assay providing minimal interference and wide dilution linearity - Patents.com (enzyme-linked immunosorbent assay, ELISA) assay conditions According to some embodiments of the present disclosure, there is a human epithelial cell line given by SEQ ID NO: 21. Anti-human epiregulin antibodies, including human epiregulin neutralizing antibodies, that bind to epiregulin have been proposed. "Epiregulin" or "human epiregulin" refers to human epiregulin protein. As used herein, epiregulin refers to the mature epiregulin peptide. Epiregulin (also known as EREG or EPR) is a peptide hormone It is a 46 amino acid protein that belongs to the epidermal growth factor (EGF) family of proteins. Regulin is produced as a 162 amino acid transmembrane epiregulin precursor, which is cleaved It is cut into the following array: “VSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTG VRCEHFFL" (SEQ ID NO: 21) (e.g., ,Toyoda,et al.,Molecular cloning of mice e epiregulin, a novel epidermal growth fa ctor-related proteins,expressed in the ea rly stage of development.FEBS Lett.1995; 377:403-7). Human epidermal growth factor receptor 1 (E1) as described and prepared in Example 2 Regulin (SEQ ID NO: 22) was used, for example, in the in vitro experiments described herein. The antibodies described herein can bind to human epiregulin protein. Reference to the ability to inhibit and / or neutralize the human epidermal growth factor receptor (EGFR) in in vitro experiments is It also refers to their ability to bind and neutralize phosphorus.

[0018] As used herein, "human anti-epiregulin antibody" or "anti-human epiregulin" refers to "Epiregulin antibodies" are antibodies that bind to human epiregulin and, when administered in vitro or in vivo, Epiregulin activity neutralizing and / or blocking response, such as at least one significantly reduced activity For example, antibodies that induce changes in epiregulin-responsive molecules or cellular endpoints. The desired reduction in epiregulin signaling is evidenced by the formation of In this case, the terms "signaling" and "transduction pathway" and "epiregulin-mediated" are used. The term, when referring to epiregulin, refers to cellular and / or molecular mechanisms that result from the activity of epiregulin. Refers to intercellular reactions.

[0019] As used herein, the term "antibody" refers to an immunoglobulin molecule that binds to an antigen. Antibody embodiments include monoclonal antibodies, polyclonal antibodies, human antibodies, Antibodies include humanized, chimeric, or conjugated antibodies. Antibodies can be of any class ( For example, IgG, IgE, IgM, IgD, IgA), and any subclass (e.g., Exemplary antibodies include IgG1, IgG2, IgG3, and IgG4. one polypeptide chain: two heavy chains cross-linked via interchain disulfide bonds Immunoglobulin G (IgG) is composed of a hydroxylase (HC) and two light chains (LC). LCs are classified as kappa or lambda, which Each of them is characterized by a particular constant region. Preferably, the antibody of the present invention comprises: The light chain constant region is a kappa constant region.

[0020] HCs are classified as gamma, mu, alpha, delta, or epsilon, respectively. The antibody isotype is defined as IgG, IgM, IgA, IgD, or IgE. The amino-terminal portion of each polypeptide chain is approximately 100-125 amino acids, which are primarily involved in antigen recognition. The carboxy-terminal portion of each of the four polypeptide chains contains a variable region of at least 100 amino acids. Each heavy chain contains a constant region that is primarily responsible for effector function. The heavy chain constant region consists of CH1, CH2, and CH3 domains. CH1 is located after HCVR, and CH1 and HCVR bind to the antigen. CH2 forms the heavy chain portion of the antigen-binding (Fab) fragment, which is the part of an antibody that binds to the antibody. , after the hinge region and before CH3. CH3 is after CH2 and The light chain constant region contains one domain, CL. CL is located at the carboxy-terminus of the LCV. Following the R, the CL and LCVR form the light chain portion of the Fab.

[0021] The antibodies of the present invention may be further subdivided into subclasses, for example, IgG1, IgG2, IgG3, and IgG4. and embodiments of the present disclosure include IgG HCs that can be classified as, for example, effectors. Each HC may contain one or more modifications in its constant region that enhance or reduce its function. The term "Fc region" as used refers to an antibody comprising the CH2 and CH3 domains of the antibody heavy chain. Optionally, the Fc region refers to a portion of the hinge region of an antibody heavy chain or the hinge region of an antibody heavy chain. IgG1 is a cytotoxic molecule that inhibits antibody-dependent cell cytotoxicity. xicity, ADCC) and complement-dependent cytotoxicity, The Fc mutations described herein are known to induce CDC, reduce aggregation, and Reduces or enhances DCC or CDC activity (or other function), and / or antibodies The anti-human EREG antibody embodiments described herein may alter the pharmacokinetics of FcγR. and C1q receptor binding, thereby reducing the binding of wild-type IgG Fc regions This reduces or eliminates the cytotoxicity that can be induced by antibodies that According to embodiments, mutations are introduced into the Fc region at the positions described herein. and (c) sufficiently reducing or eliminating the effector function of such anti-human EREG antibodies. This can improve patient safety and, in combination with other properties described herein, , therapeutic agents with an improved profile of useful activity while avoiding undesirable activity can be provided.

[0022] When expressed in a particular biological system, antibodies are glycosylated in the Fc region. Typically, glycosylation occurs on the Fc region of antibodies at highly conserved N-glycosylation sites. N-glycans typically occur at asparagine-linked sites. Antibodies may also bind to other sites. The antibodies of the present disclosure are monoclonal antibodies. A cloned antibody is a single copy or clone (e.g., any eukaryotic, prokaryotic, or plant-derived antibody). Antibodies derived from recombinant human antibodies (including recombinant human antibodies) and defined by the method by which they are produced Monoclonal antibodies are produced by, for example, hybridoma technology, recombinant technology, or pharmaceuticals. display technology, synthetic technology, e.g., CDR grafting, or any such technology or technology in the art. The present disclosure provides a method for producing antibodies of the present invention by combining the antibodies of the present invention with other techniques known in the art. In the context of monoclonal antibodies, "human" is intended to be a human or humanized antibody. The terms "humanization" and "humanized" are well known to those skilled in the art (Weiner LJ, J. Imm unother.2006;29:1-9, Mallbris L,et al.,J. Clin.Aesthet.Dermatol.2016;9:13-15). Exemplary embodiments of antibodies also include Fab, Fab', F(ab')2, Fv fragments. , scFv antibody fragments, disulfide-linked Fv (sdFv), Fd fragments and At least a portion of an antibody that retains the ability to specifically interact with an antigen, such as a linear antibody. and the like.

[0023] The amino-terminal portion of each LC and HC mainly recognizes antigens via the CDRs contained therein. The VH and VL regions contain a variable region of approximately 100 to 120 amino acids that is responsible for the Interspersed with more conserved regions called framework regions (FR), It can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs). These VH domains are exposed on the surface of the protein and are important regions of the antibody for antigen-binding specificity. and VL consists of three CDRs and four FRs, FR1, CDR1, FR 2, CDR2, FR3, CDR3, FR4 are arranged in this order from the amino terminus to the carboxy terminus. As used herein, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2 and HCDR3." The CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3," and the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3." The CDRs contain most of the residues that form specific interactions with the antigen. The functional ability to bind to an antigen is largely determined by the six CDRs. The CDR assignments are based on the Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest”, National Institutes of Health,Bethesda,M (1991)), Chothia (Chothia et al., “Canoni cal structures for the hypervariable reg ions of immunoglobulins”, Journal of Mole cular Biology,196,901-917(1987), Al-Lazik ani et al., “Standard conformations for t he canonical structures of immunoglobuli ns”, Journal of Molecular Biology, 273, 927 -948(1997)), North(North et al., “A New Cl ustering of Antibody CDR Loop Conformati ons”, Journal of Molecular Biology, 406, 22 8-256(2011)), or IMGT (the international Im MunoGeneTics database, available at www.imgt.org; Lefranc et al., Nucleic Acids Res.1999;27 :209-212). It can be applied.

[0024] For purposes of this disclosure, unless otherwise specified, the anti-epiregulin antibodies described herein and the assignment of amino acids to CDR domains within the LCVR and HCVR regions. The North CDR definitions are used. Table 2 below shows the SAbPred / ANARC I library (Dunbar and Deane, "ANARCI:antigen receptor numbering and receptor classif ication”,Bioinformatics,32,298-300(2016) ), respectively, North, Kabat, Chothia and / or The CDR sequences of Antibody 1 and / or the antibodies of the disclosure are provided according to the IMGT convention.

[0025] [Table 2]

[0026] Antibody embodiments of the present disclosure possess a combination of several pharmacologically useful and important activities. In some respects, it has high affinity for human epiregulin and As used herein, "binding" refers to a molecule that is capable of binding with specificity and has other useful properties. The term "bind" refers to an attractive interaction with another protein or molecule, unless otherwise indicated. The term "protein" is intended to mean the ability of a protein or molecule to form a complex, which is understood by those skilled in the art. The proximity of two proteins or molecules is also determined by common methods known in the art. As used herein, the phrase "specifically binds" refers to a human epithelial cell. The term "anti-epiregulin" refers to the affinity of an anti-epiregulin antibody for epiregulin, and unless otherwise indicated, is used herein Common methods known in the art include the use of MSD-SET (solution equilibrium titration) as described Approximately 2 × 10 determined at approximately pH 7.4 by the method -9 Less than M, preferably about 2 × 10 -11 M, and even more preferably less than about 2×10 -11 M ~ approx. 2×10 -12 K of M D I mean The phrase "specifically binds" also refers to binding to other antigens, and in particular to E. Anti-epilegulin activity against human epiregulin compared to the GFR ligand TGFα The relative affinities of the antibodies to human epiregulin were also shown. This results in specific recognition of the EGFR ligand and lack of binding to other EGFR ligands tested.

[0027] Antibody embodiments of the present disclosure can be prepared by isolating antibodies known in the art from constructs comprising the sequences of the present embodiments. The term "nucleic acid" or "polynucleotide" refers to a molecule that can be expressed and produced by a variety of techniques. The terms "natural nucleotides," "modified nucleotides," and "nucleotides" are used interchangeably herein. and / or nucleotide analogues, such as DNA, cDNA and RNA molecules refers to polymers of nucleotides, including single-stranded and / or double-stranded nucleotide-containing molecules. The disclosed polynucleotides also can be used in a variety of applications, including, for example, DNA or RNA polymerases or synthetic ribozymes. The DNA molecules of the present disclosure may also contain a substrate incorporated therein by reaction. at least one of the polypeptides (e.g., heavy chain, light chain, variable heavy chain, and variable light chain) in A non-naturally occurring polynucleotide encoding a polypeptide having an amino acid sequence It is a DNA molecule containing the sequence

[0028] Isolated DNA encoding an HCVR or LCVR region is referred to as an HCVR or LCVR region, respectively. The DNA encoding the heavy or light chain constant region can be operably linked to another DNA molecule encoding the heavy or light chain constant region. and converting them into full-length heavy chain genes by linking them to form heavy or light chains, respectively. The sequences of heavy chain constant region genes of humans and other mammals are known in the art. DNA fragments encompassing these regions can be isolated, for example, by standard PCR amplification. Therefore, it can be obtained.

[0029] The polynucleotides of the disclosure can be expressed in host cells after the sequences are operably linked to an expression control sequence. Expression vectors are typically expressed either episomally or intracellularly in the host. capable of replicating in the host organism either as an integral part of the host's DNA. Expression vectors are used to permit detection of those cells transformed with the desired DNA sequences. For this purpose, selectable markers such as tetracycline, neomycin, and dihydrofolate reductase are used. A polynucleotide sequence of interest (e.g., encoding an antibody polypeptide) is The vector containing the polynucleotide and expression control sequence is selected depending on the type of cell host. The vector can be introduced into the host cell by well-known methods that vary depending on the circumstances.

[0030] The antibodies of the present disclosure can be readily produced in mammalian cells, a non-limiting example of which is Host cells include CHO, NS0, HEK293 or COS cells. Mammalian antibody expression is typically achieved by glycosylation. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked glycosylation refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of sugars, such as N-acetylgalactosylation, to hydroxyamino acids. Glycosylation refers to the attachment of amino acids, galactose, or xylose to a protein. Typically, glycosylation is highly Conserved N-glycosylation sites (e.g., IMGT or EU index numbering) Glycosylation occurs in the Fc region of antibodies at position 297 in IgG1 (according to the IgG1A / IgG2A / IgG3A / IgG4A / IgG5A / IgG6A / IgG7A / IgG8A / IgG9A / IgG1 ... The glycosylation site can be modified to alter glycosylation (e.g., to block glycosylation). or altering the amino acid sequence to provide additional or varied glycosylation. Generate).

[0031] Expression of antibodies in mammals from the IgG subclass involves the C-terminal fragments from one or both heavy chains. This can result in clipping of amino acids, e.g., in the case of IgG1 antibodies, one or The two C-terminal amino acids can be removed. In the case of IgG1 antibodies, the C-terminal lysine is present. In this case, the heavy chain may be truncated or cut off during expression. The third glycine may also be truncated or deleted from the heavy chain.

[0032] Expression of an antibody in a mammal can also result in modifications of the N-terminal amino acid. For example, Or if the most N-terminal amino acid of the light chain is glutamine, it is modified to pyroglutamic acid. obtain.

[0033] The antibody of the present disclosure or a pharmaceutical composition comprising the same can be administered parenterally. Non-limiting examples of this are subcutaneous administration and intravenous administration. It may be administered to a patient in single or multiple doses together with an acceptable carrier, diluent, or excipient. The pharmaceutical compositions of the present disclosure can be prepared by methods well known in the art (e.g., Reming et al., on:The Science and Practice of Pharmacy, 22nd ed.(2012), A. Loyd et al., Pharmaceuti and the antibodies disclosed herein, and The composition may comprise one or more pharmaceutically acceptable carriers, diluents or excipients.

[0034] Uses of antibody embodiments of the present disclosure: According to some embodiments, the anti-epiregulin antibodies of the present disclosure are useful for treating pain disorders. As used herein, the term "pain disorder" refers to excessive and / or chronic pain. refers to the unwanted condition resulting from sexual pain conditions, where epiregulin inhibition is more effective. The antibodies of the present disclosure described herein result in a more homeostatic and less pathological pain state. Exemplary pain disorders contemplated for treatment with steroids include nociceptive, neuropathic, and mixed pain. chronic pain, including osteoarthritis pain or diabetic peripheral neuropathy pain or low back pain as well as the treatment of chemotherapy-induced peripheral neuropathy, especially in chronic pain states.

[0035] According to another embodiment of the present invention, the anti-epiregulin antibody is In some embodiments, the pain disorder is osteoarthritis (OA). A) Pain or diabetic peripheral neuropathy pain (DPNP) or chronic low back pain (CLBP) In some more specific embodiments, the pain disorder is at least one of: It is osteoarthritis (OA).

[0036] The present disclosure provides a method for producing a composition comprising administering to a subject an anti-epiregulin antibody of the present disclosure and one or more pharmaceutically acceptable carriers; Further provided is a pharmaceutical composition comprising a diluent or excipient. 1. A method for treating pain disorders such as chronic low back pain, diabetic peripheral neuropathy pain, or chronic low back pain. and administering the pharmaceutical composition of the present disclosure to a patient in need thereof.

[0037] Additionally, the present disclosure provides methods for treating epiregulin-mediated diseases. In particular, the present invention provides a method for treating osteoarthritis pain, diabetic peripheral neuropathy pain, chronic lower back pain, and the like. A method of treating a chronic pain disorder, comprising administering an effective amount of an anti-epiregulin antibody of the present disclosure to a subject to a patient in need thereof.

[0038] The present disclosure also provides an anti-epiregulin antibody of the present disclosure for use in therapy. Specifically, the present disclosure relates to the treatment of osteoarthritis pain, diabetic peripheral neuropathy pain, or chronic lower back pain. The present disclosure provides an anti-epiregulin antibody for use in treating chronic pain disorders such as rheumatoid arthritis, ... and rheumatoid arthritis.

[0039] In certain embodiments, the present disclosure provides a method for treating osteoarthritis pain or diabetic peripheral neuropathy pain or The present disclosure in the manufacture of a medicament for the treatment of one or more chronic pain disorders, such as chronic low back pain. The present invention provides the use of an anti-epiregulin antibody or a composition thereof.

[0040] The antibodies of the present disclosure may be used to treat chronic pain disorders in which epiregulin may contribute to the pathogenesis of the disorder. In a further embodiment, the present disclosure provides a method for treating a chronic pain disorder in a patient. Such methods include contacting a patient sample with an anti-epiregulin antibody and detecting the presence of an antibody against the patient. detecting binding between human epiregulin and the antibody in the patient sample; The presence of epiregulin is detected above baseline levels observed in unaffected individuals. If the patient has an epiregulin-mediated disorder, is at risk for it, or needs treatment, and diagnosing the patient as being at risk for and / or associated with the condition. According to some more specific embodiments of the methods of treatment provided herein, such The method involves contacting the control with the same epiregulin as that used in contacting the patient sample. a reference value comprising the further step of contacting the antibody with a first antibody that binds to the first epitope region. and determining a control standard having a detectable label and contacting the control standard with the patient sample. The cells were then conjugated with a second antibody that binds to a second epitope region of the same epiregulin as the one used. and detecting a signal provided by the detectable signal. In some specific embodiments, the anti-epiregulin antibody further comprises a In a further embodiment, the second antibody comprises a combination of LC and HC CDRs as provided. In certain embodiments, chronic LCVR and HCVR combinations are provided in Table 1. Pain disorders include osteoarthritis pain, diabetic peripheral neuropathy pain, and chronic low back pain. In some embodiments, the patient sample is CSF, blood, serum, tissue lysate, or the like. In some embodiments, the method further comprises: a second anti-epiregulin antibody that binds to a second epitope region of epiregulin and has a detectable label; contacting the pyregulin antibody with a signal provided by a detectable signal; In a further embodiment, the second antibody is one of the antibodies provided in Table 1. In a further embodiment, the second antibody comprises a combination of LC and HC CDRs comprising: The combinations of LCVR and HCVR provided in Table 1 are included. According to certain embodiments, The first and second anti-epiregulin antibodies are not bottled together.

[0041] According to some embodiments, the present disclosure provides a method for detecting epiregulin in a patient sample. a patient sample, a first antibody that binds to a first epitope region of epiregulin, and contacting the patient sample with a second epitope region of epiregulin; contacting the antibody with a second antibody having a detectable label; and detecting a signal provided by the assay. In some embodiments, the patient sample is one of blood, serum, tissue lysate, or plasma. According to a more specific embodiment, the first epitope region of epiregulin is In some embodiments, the second epitope region of the urin overlaps with the second epitope region of the urin. The steps of contacting with the first and second antibodies occur simultaneously. In one embodiment, the first antibody comprises a combination of LC and HC CDRs provided in Table 1. In a further embodiment, the first antibody comprises a combination of LCVRs and HCVRs provided in Table 1. This includes.

[0042] According to some embodiments of the present disclosure, a method for quantifying epiregulin in a patient sample Such a method comprises: sequestering a patient sample with a first epitope region of epiregulin; contacting the patient sample with a first antibody that binds to a second antibody of epiregulin; contacting the antibody with a second antibody that binds to the avian epitope region and has a detectable label; Detecting the signal provided by the detectable label and a control (e.g., a patient the first epitope region of the same epiregulin as that used to contact the subject sample and a control (when contacting the patient sample) with a first antibody that binds to binds to a second epitope region of the same epiregulin (as used in contacting the antibody with a second antibody having a label and detecting the detected signal provided by the second antibody; In some embodiments, the patient sample comprises detecting a signal generated by the blood sample. The sample may be one of: a liquid, serum or plasma, or a tissue lysate. According to one embodiment, the first epitope region of epiregulin is Furthermore, in some embodiments, the first and second epitope regions overlap. The steps of contacting with the antibody occur simultaneously. The antibody of claim 1 comprises a combination of LC and HC CDRs provided in Table 1. Further embodiments In one embodiment, the first antibody comprises a combination of LCVRs and HCVRs provided in Table 1. In some specific embodiments, the second antibody comprises the LC and HC CDRs provided in Table 1. In a further embodiment, the second antibody comprises a combination of the LCVRs and Includes combinations of HCVR and HCVR.

[0043] According to some embodiments, a method for diagnosing an epiregulin-mediated disease or disorder is provided. Such methods include contacting a patient sample with an anti-epiregulin antibody; and detecting binding between epiregulin and the antibody in the patient sample. According to certain embodiments, the method of diagnosis is based on the presence of epiregulin in a patient sample. If detected above the threshold, the patient is at risk for an epiregulin-mediated disorder. Diagnose a condition that requires treatment and / or is at risk for associated conditions According to some more specific embodiments, such methods include: The first epitope region of epiregulin is the same as that used in contacting the patient sample. determining a reference value, comprising contacting the antibody with a first antibody that binds to the corresponding region; The reference standard has a detectable label and is the same epitaxial layer as that used in contacting the patient sample. contacting with a second antibody that binds to a second epitope region of regulin; and detecting and detecting a signal provided by the possible signal. In this embodiment, the first antibody comprises a combination of LC and HC CDRs provided in Table 1. In further embodiments, the antibody comprises a combination of LCVRs and HCVRs provided in Table 1. Some of the methods provided herein for diagnosing epiregulin-mediated diseases include In one embodiment, the patient sample is subjected to a detection assay that binds to a second epitope region of epiregulin. contacting the antibody with a second anti-epiregulin antibody having a detectable label; and detecting a signal provided by the signal. In some embodiments, the anti-epiregulin antibody comprises a combination of LC and HC CDRs as provided in Table 1. In further embodiments, the antibody comprises a combination of LCVRs and HCVRs provided in Table 1. According to a specific embodiment, the first epitope region of epiregulin is According to a particular embodiment, the second epitope region of pyregulin partially overlaps with the second epitope region of pyregulin. The first and second antibodies are not bottled together. According to a further embodiment, the reference value is: Established from plasma, body fluids or tissue lysates of healthy volunteers and / or suitable As determined by one of skill in the art for the appropriate reference group and sample source. In this article, the pain disorder is defined as osteoarthritis pain, diabetic peripheral neuropathy pain, or chronic low back pain. It is one of them.

[0044] In one embodiment, the present disclosure provides a method for determining human epiregulin levels in a bodily fluid sample. (a) subjecting a body fluid sample to a human epithelial cell line comprising an amino acid sequence as set forth in SEQ ID NO: 21; A diagnostic monoclonal antibody for anti-human epiregulin that specifically binds to epiregulin or its antibody contacting the antibody or antigen-binding fragment thereof with an antigen-binding fragment thereof; Light chain complementarity determinations comprising the amino acid sequences SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively Regions LCDR1, LCDR2 and LCDR3, and amino acid sequence, SEQ ID NO: 5, SEQ ID NO: Heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 containing SEQ ID NO:6 and SEQ ID NO:7, respectively (b) contacting the DR3-containing antibody with the DR3-containing antibody; and (b) optionally, removing any non-specifically bound antibody. (c) removing clonal antibodies or antigen-binding fragments thereof; and The amount of a monoclonal antibody or antigen-binding fragment thereof that specifically binds to phospholipid is detected and Preferably, the body fluid sample is blood, a serum or plasma sample, or a cerebrospinal fluid sample, and said contacting is performed ex vivo. This occurs.

[0045] In an embodiment of the present disclosure, the patient is in need of treatment with the antibodies described herein. Having a medical risk, condition, or disorder, such as one of the diseases or disorders listed in the subdivision The disorders that can be treated by the methods of the present invention include osteoarthritis pain. or into established and accepted classifications such as diabetic peripheral neuropathy pain or chronic low back pain. Thus, where known, these classifications can be found in various well-known medical textbooks. Diagnostic and Statistical Manual of Men The 5th edition of the Department of Mental Disorders (DSM-5) or similar text is incorporated herein by reference. provide diagnostic tools for identifying specific disorders of al.,The IASP classification of chronic p ain for ICD-11:chronic neuropathic pain. Pain.2019 January;160(1):53-59, and Treede et al., Chronic pain as a symptom or a di sease:the IASP Classification of Chronic Pain for the International Classificati on of Diseases(ICD-11),PAIN:2019 January 160:19-27). Also see International C The International Classification of Diseases, 10th Edition (ICD-10) Classifications are provided for the specific disorders described in the specification. Those skilled in the art will understand the DSM-5 and IC For the diseases and disorders described herein, including those listed in ICD-10 or ICD-11 There are alternative nomenclatures, nosologies and classification systems, and terminology and classification systems are used in medical science. You will realize that it evolves with progress.

[0046] Thus, the term "treating" (or "treating" or "treatment") is used herein. The progression or severity of any pre-existing symptom, disorder, condition or disease described in the subdivision is delayed, interrupted, arrested or controlled. is intended to refer to any process that can control, mitigate, stop, reduce or reverse, but is not necessarily It is not intended to indicate complete elimination of all disorder or disease symptoms. The present invention relates to a method for the treatment of a disease, disorder or condition in a patient, particularly a human, by administering to the patient a protein or nucleic acid or The treatment includes administering a vector or composition to a subject who would benefit from reduced epiregulin activity. The present invention also provides a method for treating a disease or disorder in a subject, the method comprising administering an antibody of the present disclosure to the subject for the treatment of a disease or disorder in the subject, the treatment comprising: (a) administering to the subject an antibody of the present disclosure; (b) inhibiting further progression of the disease, i.e., arresting its onset; To ameliorate, i.e., to cause regression of a disease or disorder or to improve or reverse its symptoms. (c) provide for reducing complications and / or preventing the onset of symptomatic disease. Treating, as defined herein, includes reducing the incidence of pain, reducing pain or Amelioration of one or more symptoms of pain, relief of pain or one or more symptoms of pain, delay in onset of pain In some circumstances, treatment also includes, but is not necessarily limited to, the treatment of pain. It does not modify the underlying disease or condition causing the pain. "Anti-inflammatory pain" is pain that is refractory to two or more conventional monotherapy and / or dual therapy treatment regimens. It is defined as pain that is felt during or after a session.

[0047] As used herein, "reducing the incidence" of pain refers to reducing the duration and / or severity of pain. This means either reducing the frequency of pain (e.g., reducing the time to pain onset in an individual). As will be appreciated by those skilled in the art, an individual may Pain treatment also involves assessing the severity of pain and varying the severity of symptoms. reducing the risk of heart failure, as well as other drugs and / or therapies commonly used for this condition (e.g., This includes reducing the need for and / or amount of (e.g., exposure to) opiates, including opiates. "Ameliorating" pain (such as osteoarthritis pain) or one or more symptoms of pain refers to a reduction in one or more symptoms of pain compared to when an antibody or composition of the disclosure is not administered. "Ameliorating" means reducing or ameliorating the symptoms. "Relieving" pain (such as osteoarthritis pain) or one or more symptoms of pain is also included. and" refers to a reduction in pain in an individual or population of individuals treated with an antibody or composition according to the present disclosure. As used herein, "reducing the severity of one or more undesirable clinical signs" means "reducing the severity of one or more undesirable clinical signs." When used in a clinical setting, "delaying" the onset of pain means delaying the progression of pain, such as osteoarthritis pain. This means to postpone, impede, delay, slow down, stabilize and / or postpone. This may be of varying lengths of time depending on the history and / or individual being treated. As such, a sufficient or significant delay is, in effect, prophylaxis, in that the individual does not develop pain. A method of "delaying" the onset of a symptom may include, compared to not using the method: Reduce the likelihood of symptoms developing in a given time frame and / or Such a comparison may be a method of reducing the severity of symptoms. Such a comparison is typically performed using a statistically significant Based on clinical studies using a number of subjects.

[0048] "Effective amount" means the amount of a substance that has the desired effect on a tissue, system, or human life as desired by the treating medical professional. The present disclosure will induce a biological or medical response or desired therapeutic effect thereon. This means an amount of anti-human epiregulin antibody or a pharmaceutical composition containing such an antibody. As used herein, the term "effective response" of a patient or the patient's responsiveness to treatment is used. The term refers to the clinical or therapeutic benefit conferred to a patient upon administration of an antibody of the present disclosure. The effective amount will depend on the condition, age, sex and weight of the individual, as well as the amount of the antibody that will elicit the desired response in the individual. Such desired responses may vary depending on factors such as the level of chronic pain, or improvement of the signs or symptoms of a pain disorder. Efficacy can be determined by the use of known techniques and by observing results obtained under similar circumstances. The anti-human epiregulin antibody of the present disclosure can be easily determined by a person skilled in the art. can be administered in a single dose or multiple doses. Furthermore, an effective amount of an antibody of the present invention is If administered only once, multiple doses of an amount that is less than the effective amount may be administered. In determining an effective amount, the patient's size (e.g., weight or mass), body surface area, age, and General health condition, specific disease or disorder involved, extent or involvement or severity of the disease or disorder the degree of efficacy, individual patient response, the specific compound administered, the mode of administration, the bioanalysis of the administered formulation, availability characteristics, the chosen dosing regimen, the use of concomitant medications, and other factors known to the treating physician. Several factors, including but not limited to certain other relevant circumstances, may influence the decision of your doctor. Dosage (including but not limited to subcutaneous, intramuscular and / or intravenous) However, the dose may be from about 0.5 mg / kg to about 50 mg / kg. known and / or in light of the dosage considerations described herein, Doses lower or higher than those prescribed are also envisioned. The dose can be monitored by periodic evaluation and the dose adjusted accordingly, if necessary.

[0049] A potential benefit of the methods disclosed herein is that they may be beneficial for patients suffering from chronic pain disorders. or tolerated adverse events, including tolerability, toxicity, and / or anti-drug antibody responses. provide significant and / or long-lasting relief with a reasonable safety profile, thus enabling patients More specifically, the antibodies of the present disclosure may be used to treat skin Rash(Li T,Perez-Soler R.Skin toxicities as associated with epidermal growth factor re ceptor inhibitors.Target Oncol.2009 Apr; 4(2):107-19), and / or clinically undesirable adverse effects such as anti-drug antibody responses. The effectiveness of the treatments disclosed herein is in the treatment of various pain disorders while avoiding pain events. It can be measured by a variety of endpoints commonly used in evaluating treatments. For example, to determine the effectiveness of any particular therapy of the present disclosure, including assessments known to those of skill in the art, Other approaches for determining the quality of the data may optionally be adopted. [Brief explanation of the drawings]

[0050] [Figure 1] Figure 1 shows the ligand selectivity ELISA results for Antibody 1 (Panel A) and LY3016859 (Panel B), demonstrating selectivity of Antibody 1 for human epiregulin, but no appreciable binding to other EGF family ligands (epigen, amphiregulin, betacellulin, EGF, HB-EGF, and TGFα (TGFA)), as determined in the ligand specificity ELISA described in Example 2. In comparison, LY3016859 binds to both epiregulin and TGFα. [Figure 2] In vivo membrane epiregulin binding 3 days after administration of Antibody 1 (filled circles) compared to a control hIgG antibody (filled squares). [Figure 3] Pretreatment time dependent binding of membrane epiregulin in vivo after a subcutaneous dose of 10 mg / kg of Antibody 1 (closed squares) compared to a control hIgG antibody (open squares). [Figure 4] In vivo membrane epiregulin binding 3 days after administration of Antibody 1 (closed squares) compared to a comparison antibody (open circles) that is 50-fold less potent at binding epiregulin. [Figure 5] ELISA assessment of Fab binding of antibody 1 to the indicated mutations of epiregulin. [Figure 6]Alignment of the human EGFR ligand epitope (EGF-like domain) for Antibody 1. The ligand sequence is provided in the sequence listing for amino acid and nucleotide sequences. [Example]

[0051] The following examples are offered to illustrate, but not to limit, the claimed invention. The results of the following assays are not intended to be limiting unless otherwise specified. and their antigen-binding fragments bind to and / or inhibit human epiregulin. and thus can be used to treat epiregulin-mediated disorders as described herein. This demonstrates that:

[0052] Example 1: Antibody generation, expression and purification A panel of human anti-epiregulin antibodies was tested against recombinant EREG (human and cynomolgus monkey). immunized mice to identify antibodies that may be effective in neutralizing epiregulin signaling. Mutations are systematically introduced into the individual complementarity-determining regions (CDRs) of each antibody. Injecting a soluble antibody into the target cell reduces the antigen concentration, decreases the time to antigen association, and / or increases the dissociation period. The resulting library was subjected to multiple rounds of selection while allowing the target to undergo fusion. Clones are isolated. Individual variants are sequenced and used for combinatorial analysis. A library is constructed and subjected to additional rounds of selection with increasing stringency. To identify additive or synergistic mutation pairings between individual CDR regions. The combinatorial clones are sequenced and the binding characteristics are determined. Performed against human or mouse epiregulin and affinity for one or more selected species It is possible to increase the number of antibodies against human epiregulin (e.g., antibody 1 against human epiregulin). To maintain selectivity, counterscreening was performed against other EGFR ligands. The selected antibody may be mutagenized to maintain its binding affinity to epiregulin. However, post-translational modifications such as methionine oxidation can also be repaired. To reduce the risk of smearing, framework (FW) and CDR substitutions were performed. Antibodies can be modified to revert these sequences to their germline state.

[0053] An engineered and / or optimized anti-epiregulin antibody, referred to herein as Antibody 1, was obtained. This includes the amino acid sequences of the heavy and light chain variable regions, the complete heavy and light chain amino acid sequences, column, and in the section below entitled "Amino Acid and Nucleotide Sequence Listing." The nucleotide sequences corresponding to these sequences are identical to those described in The corresponding SEQ ID NOs and the CDR amino acid sequences of the light and heavy chains are shown in Table 1.

[0054] Expression and purification: The exemplified anti-epiregulin antibodies of the present disclosure are expressed and purified essentially as follows. Antibody 1 can be expressed in a suitable host cell, such as HEK293 or CHO. The cells are then cultured to secrete antibody 1 using the optimal predetermined HC:LC vector ratio. or a single vector system encoding both the HC and LC, either transiently or stably. The expression plasmids encode the LC and HC genes of Antibody 1. cDNA versions of the constructs (e.g., encoding the HC of exemplary Antibody 1 presented in Table 1) The DNA sequence of SEQ ID NO: 11, which is a sequence encoding the LC amino acid sequence according to Table 1. For example, the DNA of SEQ ID NO: 12 encoding the LC of exemplified antibody 1 presented in Table 1 sequence) and based on the major immediate early promoter of human cytomegalovirus It is commonly used and expressed from constructs suitable for this purpose.

[0055] The medium into which the antibody of the present invention is secreted is subjected to ion exchange and hydrophobic interaction chromatography. For example, the medium can be purified by conventional techniques such as the mixed mode method. applied to and eluted from a Protein A or Protein G column using the method of and mixed-mode methods of ion exchange and hydrophobic interaction chromatography can also be used. Soluble aggregates and multimers can be isolated by size exclusion, hydrophobic interactions, ionic Efficacy was achieved by common techniques including ion exchange, or hydroxyapatite chromatography. The product can be immediately frozen, e.g., at -70°C, refrigerated, or It may be lyophilized. Various methods of protein purification may be employed, such as: Methods in Enzymology 182:83-89(1990), and Scopes, Pro tein Purification:Principles and Practical e, 3rd Edition, Springer, NY (1994). 1 can be frozen immediately at -70°C, stored at 2-8°C for several months, or lyophilized. It may be dried or stored at 4°C for immediate use. The amino acid sequence numbers of the antibodies are shown in Table 1.

[0056] Example 2: Characterization of anti-epiregulin antibodies The epiregulin reagent is produced as a fusion protein, cleaved, and purified. Epiregulin is a mature soluble form that contains extra amino acids compared to the native form. Used in in vitro binding and neutralization assays (SEQ ID NOs: 22-25). Human epiregulin (EREG) fusion (SEQ ID NO: 27) was also used in binding assays (capture reagents) and A cell-based assay was performed to identify a full-length epiregulin sequence (SEQ ID NO: 26) with a single mutation that reduces the Used in binding and effector function assays of IgG.

[0057] Mature soluble epiregulin was expressed in transiently transfected CHO cell cultures. and HRV on monomeric human IgG4 Fc (F405Q / Y407E) (SEQ ID NO: 27). The fusion protein is expressed as a 3C protease-cleavable C-terminal fusion. The fusion is captured on an A affinity column and further purified by size exclusion chromatography. was cleaved with HRV3C protease and applied to a protein A affinity column to obtain monomeric Fc. and further purified by size exclusion chromatography to obtain mature soluble epiregulatory Get the .

[0058] Antigen sequence: Truncated human epiregulin (SEQ ID NO: 22) GPGVSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGY TGVRCEHFFLG

[0059] Truncated cynomolgus epiregulin (SEQ ID NO: 23) GPGVSITKCNSDMNGYCLHGQCIYLVDMSQNYCRCEVGY TGVRCEHFYLG

[0060] Truncated rat epiregulin (SEQ ID NO: 24) GPGVLITKCSSDMDGYCLHGHCIYLVDMSEKYCRCEVGY TGLRCEHFFLG

[0061] Truncated rabbit epiregulin (SEQ ID NO: 25) GPGVSITKCGSDMNGYCLHGQCIYLVDMSENYCRCEVGY TGVRCEHFFLG

[0062] Full-length human epiregulin (membrane bound with T111P mutation) (SEQ ID NO: 26) MTAGRRMEMLCAGRVPALLLCLGFHLLQAVLSTTVIPSC IPGESSDNCTALVQTEDNPRVAQVSITKCSSDMNGYCLHG QCIYLVDMSQNYCRCEVGYTGVRCEHFFLTVPQPLSKEYV ALTVILIILFLITVVGSTYYFCRWYRNNRKSKEPKKEYERV TSGDPELPQV

[0063] Monomeric Fc human epiregulin (SEQ ID NO: 27) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSIT KCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVRCEHF FLG

[0064] The sequences of mature epiregulin from various species are generally known in the art. The reference sequence is, for example, the following: mature soluble human epiregulin reference sequence ( NP_001423.1 63-108), mature soluble cynomolgus monkey (referred to herein as cyno) epiregulin reference sequence (XP_005555120.1 63 -108), mature soluble rat epiregulin reference sequence (NP_067721.1 56 -101) and the mature soluble rabbit epiregulin reference sequence (XP_008265968. 1 57-102). Other human EGFR ligand reference sequence numbers are as follows: Human TGFα (NP_003227.1), EREG (NP_001423.1), EP GN(NP_001257918.1), AREG(NP_001648.1), BTC (NP_001720.1), EGF(NP_001954.2), HB-EGF(NP _001936.1).

[0065] Binding affinity to human epiregulin Human (SEQ ID NO: 22), cynomolgus monkey (SEQ ID NO: 23), rat (SEQ ID NO: 24) and The solution phase equilibrium binding affinity of Antibody 1 to rabbit (SEQ ID NO: 25) epiregulin was determined using MS D solution equilibrium titration (MSD-SET) assay The measurement is performed at 37°C. (See, for example, Darling RJ, Brault PA (20 05)Kinetic Exclusion Assay Technology:Ch aracterization of Molecular Interactions .Assay and Drug Development Technologies 2:647-657.)

[0066] MSD SI6000 instrument (Meso Scale Discovery, Rockv Read the MSD plate using the MD (ille,MD). Prepare the following: Multi-array 96-well plate (Meso Scale Dis covery, P / N L15XA-3) was added to a 1000 ml of monomeric Fc-human epitaxial growth factor (E1) in PBS. Coat with 30 μl of a 1 μg / ml solution of the fusion protein overnight at 2-8°C. After incubation, the plate was washed three times with PBST, followed by 3% Blocker A (5% Blocker A ( in PBS from Meso Scale Discovery, P / N R93AA-1 Block in 1000µL of PBS (diluted) for 1 hour at room temperature with shaking.

[0067] SET samples are prepared in 1% Blocker A at pH 6.0 and pH 7.4. Either 20 and 200 pM (pH 7.4) or 200 pM and 2 nM (pH 6.0) Epiregulin was diluted 12 times in total, with the concentration of the antibody being approximately twice that of the antibody. This is 10 and 100 nM (pH 7.4) or 100 nM and 1000 nM. This was achieved by using a starting concentration of 100 mM (pH 6.0), which was then diluted 10-fold. Dilute, serially dilute 2-fold 9 times, then dilute 10-fold for the final dilution. The titrated and fixed concentration antibody solutions are combined 1:1 to prepare the SET solution. The mixture is incubated at 37°C for approximately 96 hours to allow binding to reach equilibrium.

[0068] Transfer 100 μl of SET solution to duplicate rows of the prepared MSD plate and incubate at room temperature for 10 minutes. After incubation, the plate is washed with PBS. Wash three times with T, then add 100 μl of 1 μg / ml biotinylated goat lecithin in 1% Blocker A. Anti-human kappa primary antibody (Southern Biotech, catalog 2060-08) Add 100 ml of PBS to all wells. Incubate this on a plate shaker at room temperature for 60 minutes. The plate is then washed three times with PBST, followed by 1 μL of PBS in 1% Blocker A. g / ml SULFO Streptavidin (Meso Scale Discovery Add 100 μl of the antibody (P / N R32AD-1) to all wells. Let this stand at room temperature for 30 minutes. Incubate on a plate shaker for 1 minute. Then wash the plate three times with PBST. Wash the tube with 100 μl of 1X Read Buffer T (4X Read Buffer T) and then add fer T (Meso Scale Discovery, P / N R93AA-1) The plate is read after adding the antibody (diluted in water). Dissociation constant (K D ) and least common multiplier (LCM) ligand correction coefficients The numbers were globally fitted to the equilibrium binding equation from the MSD-SET data using nonlinear regression. This is done for each pair of fixed antibody concentrations at a given pH.

[0069] At pH 7.4, Antibody 1 binds human epiregulin with an affinity (K D ) to join and cynomolgus monkey epiregulin with a K of 26.8 pM. D Binds to rat epiregulin K of 30.2 pM D binds to rabbit epiregulin with a K of 24.0 pM D Join with At pH 6.0, Antibody 1 binds human epiregulin with a K of 195 pM. D Combined with crabeater K of 328 pM for monkey epiregulin D and bound to rat epiregulin at 286 pM. K D binds to rabbit epiregulin with a K of 330 pM D Binds at pH 7.0. Antibody 1 pH selection (K at pH 6.0 D / K at pH 7.4 D ) is human epiregulin 13.0, crab The monkey epiregulin was 12.2, the rat epiregulin was 9.5, and the rabbit epiregulin The pH-dependent antigen binding is responsible for both the pharmacokinetics and the duration of target neutralization. Improvements can be provided (Vincent, KJ and M. Zurini (20 12).Current strategies in antibody engineering neering:Fc engineering and pH-dependent antigen binding, bispecific antibodies an d antibody drug conjugates.”Biotechnol J 7(12):1444-1450), Antibody 1 and / or the antibodies of the disclosure , which are believed to provide beneficial pharmacokinetic and epiregulin-neutralizing properties. It exhibits favorable pH selectivity for binding.

[0070] [Table 3]

[0071] Results are presented as a function of K from three independent replicates. D The error estimates are reported as the average of the independent The affinity of antibody 1 to human EREG at pH 7.4 is calculated as the standard deviation of replicates. showed approximately 70-fold improvement in affinity compared to LY3016859, showed that significantly lower doses of antibody 1 were required to achieve equivalent EREG neutralization in vivo. The ability to use lower doses of Antibody 1 may result in less antibody inhibition being required. This means that the drug product can be achieved without the need for intravenous administration. compared with subcutaneous administration, which is clinically important for accessibility, tolerability, and compliance. This is acceptable for chronic pain, such as that required for the chronic pain indications described herein. This is particularly advantageous in the context of medication.

[0072] Ligand specificity of Antibody 1: The specificity of antibody binding to EGF family ligands was determined using enzyme-linked immunosorbent assay (EL ISA) and / or other common methods known to those skilled in the art.

[0073] The preparation of the ELISA reagents can be carried out as follows. The tested ligands were human Epigen (R&D 6629-EP-025 / CF), Epiregulin (as used herein) Prepared and purified as described in Example 2 and / or by methods known in the art (SEQ ID NO: No. 22), amphiregulin (R&D 262-AR-100 / CF), beta-cellulosic acid (R&D 261-CE-010 / CF), EGF (R&D 236-EG-200) , HB-EGF (R&D 259-HE-050 / CF) and TGFα (R&D 23 The ligand was Thermo Pierce Sulfo-NH S-LC-Biotin (A39257) was used, with a 10-fold excess of biotin for 2 hours on ice. Biotinylated and then frozen at -80°C in small aliquots for individual use.

[0074] The ELISA assay can be performed as follows: High-binding plate (650061) was filled with 50 μl / well of 1 μg / ml nuclease in PBS. The membrane was coated overnight at 4°C with tetrahydroavidin (Thermo-Pierce 31000). The next day, remove the coating solution and apply 100 μl / well of Pierce Bloc Add ker Casein (37528) and incubate at room temperature for at least 30 minutes. After blocking, the plate is resuspended in PBS + 0.05% Tween 1000. Wash three times with EN20 to remove residual blocking buffer. Pierce Casein at a constant concentration of 20 nM across 12 wells per ligand In Blocker, separate PCR plates (Eppendorf 951020443 ) and the last row contains no antigen control. Transfer the plate to an assay plate and incubate at room temperature for 1 hour. +0.05% Tween 20. In VEGF Blocker, 5 μg / mL (LY3016859) or 10 μg / mL ( Starting with antibody 1), serially dilute 1:3 across the plate (12 wells) and The volume is 50 μl / well and incubated at room temperature for 1 hour. Wash three times with PBS + 0.05% Tween 20. (Uthern Biotech 2060-04) in PBS + 0.05% Tween 2 Add 50 μl / well of 1:2000 dilution in 0 and incubate for 1 hour at room temperature. The assay plate is washed three times with PBS + 0.05% Tween 20. Finally, Add 0 μl of pNPP substrate (Sigma-Aldrich N2765) and allow sufficient color Color was developed until α was present, and the resulting chromatogram was analyzed by SpectraM Read at 405 nm on an ax Plus 384 plate reader.

[0075] Figure 1 shows the ELISA results for the ligand selectivity of Antibody 1. Antibody 1 was able to bind to the above ligands. Selection for binding to human epiregulin as determined in a specificity ELISA Although other EGF family ligands, epigenetic factors, amphiregulin, and beta-secreta, have been demonstrated to be No significant binding was demonstrated to EGF, HB-EGF, or TGFα. The selective and specific binding to epiregulin, but not to other family ligands, indicates that antibody 1 , TGFα or other EGF family ligands, Achieving the desired effect of blocking epiregulin signaling while avoiding undesired effects The lack of binding to TGFα effectively inhibits epiregulin. This selectivity is comparable to that observed with pan-EGFR inhibitors. This may avoid undesirable clinical adverse events such as skin rashes that have been associated with this condition.

[0076] Physicochemical attributes: With respect to therapeutic antibody product attributes, including chemical stability, solubility, and viscosity, Antibody 1 is a human They exhibit a desirable combination of physicochemical attributes for use as therapeutic agents.

[0077] Stability: The stability of antibody 1 was confirmed in 5 mM histidine (pH 6.0) + 280 mM mannitol + 0. At high concentrations (approximately 100 mg / ml) formulated in 0.5% (w / v) polysorbate-80 The concentrated samples are incubated at 5°C and 35°C for a period of 4 weeks. After incubation, the samples were subjected to size exclusion chromatography. High molecular weight (HMW%) was measured by SEC (Sectional Atography). ) and fragmentation were investigated by capillary electrophoresis (CE-SDS). and chemical modifications (e.g., deamidation, isomerization, or After 4 weeks at 35°C, Antibody 1 had a ΔHMW% (high molecular weight) of 0.4%. 0.6% Δfragment% (change in percentage of fragment mass) The results show a CDR chemical modification of over 0.5% (change in percentage).

[0078] Photostability under the same formulation conditions was measured at 40 watt-hours / m at 25°C. 2 UV light and 240kl The sample is evaluated by a combined total exposure to visible light for 10 minutes. HMW% by EC and chemical modification by LC-MS peptide mapping Antibody 1 was analyzed for 100% IL-1 activity as measured by SEC compared to unexposed controls. The ΔHMW% of the nucleotides was 6.1% and did not show more than 0.7% CDR chemical modification.

[0079] Freeze / thaw stability under the same formulation conditions was assessed by freezing a large quantity of bulk drug substance at -70°C. Using slow, controlled temperature cycles repeated three times, mimicking freezing / thawing conditions Antibody 1 is measured by 1.0% SEC after three freeze-thaw cycles. ΔHMW% is shown.

[0080] These results suggest that Antibody 1 may facilitate the development of solution formulations and their use as therapeutic agents. It is shown to have sufficient and advantageous physical and chemical stability.

[0081] Solubility: Solubility was determined by filtering 100 mg of antibody 1 through a 30 kDa molecular weight cutoff centrifugal filter (e.g. For example, Amicon UC filters, Millipore, Cat. No. UFC90 The solution is concentrated to a volume of approximately 0.5 ml using a 3024 (Solo VPE) UV absorbance at 280 nm was measured using a spectrophotometer (C Technologies, Inc.). The final concentration of the samples was determined photometrically.

[0082] Antibody 1 was measured at ≥175 mg / ml in 5 mM histidine pH 6 buffer and PBS pH 7. It has a solubility of 168 mg / ml or more in .4. After storage at 5°C or -5°C for 1 week, No phase separation or cryoprecipitation was observed. These results demonstrate that Antibody 1 allows for high-concentration dosing. This indicates that the compound has sufficient solubility to

[0083] viscosity The viscosity of antibody 1 and LY3016859 was measured using VROC Initium (RheoSen Antibody 1 is analyzed at a concentration of approximately 125 mg / ml at 15°C using a pH Viscosity of 6.4 cP at 133 mg / ml in 5 mM histidine + 280 mM mannitol LY3016859 was prepared in 5 mM histidine + 280 mM mannitol at pH 6. The viscosity of antibody 1 was 12.2 cP at 32 mg / mL. These results indicate that antibody 1 It exhibits reduced viscosity compared to 6859 and viscosity low enough to allow for high concentration dosing. The ability to use higher concentrations of Antibody 1 combined with the need for lower doses Together, this means that the required effective dose can be achieved with a smaller volume for delivery, This means that the drug product is amenable to subcutaneous administration, which does not require intravenous administration. Compared to drugs that require antibodies, This is clinically important.

[0084] Example 3: In vitro functional characterization of anti-epiregulin antibodies Neutralization of epiregulin in vitro: Neutralization of epiregulin activity by antibodies of the present disclosure, such as Antibody 1, is achieved as described below. These responses can be assessed by one or more of the following epiregulin-induced cell-based response assays: This can be done.

[0085] The antibodies of the present disclosure neutralize epiregulin activity in two independent functional assays. The ability of antibody 1 of the present disclosure to neutralize epiregulin activity is tested as described below. one or more receptors that utilize downstream signaling pathways of the EGFR family of receptors, such as It can be assessed by cell-based activity assays. Neutralization assay (C166-A Mean half-maximal inhibitory concentrations (IC 50 ) is determined by Si Calculate with gmaPlot or GraphPad Prism. In ism, IC 50Log 10 Perform nonlinear regression analysis of transformed antibody concentrations Assays performed only in duplicate or triplicate across one plate are calculated by Regarding I, IC 50 and standard deviation (SD) are the IC values ​​of each replicate. 50 and the mean IC of the replicates 50 Determine by calculating the SD from the For analysis of compounds across various plates, IC for each plate 50 Calculate Determine the SD across the plate.

[0086] Epiregulin-induced responses in the C166-AP1-luciferase functional assay Inhibition: Ability of the antibodies of the present disclosure to neutralize human epiregulin-induced luciferase reporter activity Force C16 overexpressing a luciferase reporter driven by AP1 activation The expression of C166 cells can be evaluated in C166-AP1-Luc cells. Among those expressing all four EGFR family of receptors, with the highest levels of EGFR expression To test the activity of Antibody 1, C166-AP1-Luc cells were grown. Long medium [DMEM (Gibco 12430-047), 10% FBS (Gibco 1 0082147), Anti / Anti (Gibco 15240062) and 2 μg / ml Puro (1000X 2 mg / ml puromycin hydrochloride (Calbioch 540411)] and in 0.05% trypsin-PBS Dissociate and culture the tissue in serum-free medium (growth medium without FBS) at 50 μl per well. Seed cells at 50,000 cells per ml in a culture-treated 96-well plate. Epiregulin (as described herein) at a final concentration of 100 ng / ml (16-18.5 nM) As noted, human (SEQ ID NO: 22), cynomolgus monkey (SEQ ID NO: 23), rat (SEQ ID NO: No. 24) and rabbits (SEQ ID NO: 25)) and treated with serial dilutions of antibody 1 for 6 hours After incubation, the cells were resuspended in 100 μl Promega™ One-G Io™ Luciferase Solution (Promega™, Cat. No. E61 20) for 3 minutes. Luminescence was measured using a Perkin Elmer Wallace 142 Read on a Victor2™ microplate reader. The reduction in relative fluorescence units (RFU) was observed for human (SEQ ID NO: 22), Cynomolgus monkeys (SEQ ID NO: 23), rats (SEQ ID NO: 24) and rabbits (SEQ ID NO: 25) This reflects the ability of Antibody 1 to neutralize epiregulin activity in various relevant species tested, including IC of antibody 1 in neutralizing human epiregulin 50 The value is 8.7 nM, SD2 3, n = 5 plates, 2–3 replicates / plate (Table 4). Neutralization across related species was performed in cynomolgus monkeys (IC 50 2.1 nM, SD 0.08, n = 3 replicates), rat (IC 50 2.5 nM, SD 0.27, n = 3 replicates), rabbit (IC 50 3.6 nM, SD 0.14, n=3 replicates). Assay validation and low performance of antibody 1 compared to antibody 1 of Patent Publication No. 2012 / 138510 Reduced IC 50To confirm the efficacy of LY3016859 in human epithelial cells in this assay, The neutralization of the virus was confirmed by comparing it with that of the human, rabbit, and cynomolgus monkeys used in these studies (Table 4). Monkey and rabbit epiregulin were produced as described herein above. 895 inhibits human epiregulin with an IC of 25.9 nM 50 , SD11.7, n=8 plays In 2-3 replicates / plate, cynomolgus monkeys (IC 50 8.6nM, SD0.27, n = 2 plates, 2–3 replicates / plate), rats (IC 50 720nM, SD10, n = 2 plates, 2-3 replicates / plate) and rabbits (IC 50 11.0nM, SD1. 15, n = 2 plates, 2-3 replicates / plate). Statistical validation of the urin-induced C166-AP1-Luc assay revealed an MSR of 1.24 , 95%CI MSR (MSR = least significant ratio) was 1.15, 1.43, potency analysis of antibody 1 The β-amyloid ... In vitro neutralization of functional cellular responses and inhibiting activity of epiregulins from various species This demonstrates the ability and potential neutralizing effect of Antibody 1 against epiregulin in vivo.

[0087] [Table 4]

[0088] For neutralization against human epiregulin, 5 plates for antibody 1, and ICs from replicates across eight plates for LY3016859 50 By averaging The mean and SD were determined by the following: Rat, cynomolgus monkey, and rabbit epiregulin For neutralization against, triplicate ICs from one plate for each species were collected. 50 Average the anti The SD of antibody 1 was determined. For LY3016859, duplicate ICs across two different plates 50 Average Therefore, IC 50 was generated.

[0089] The data in Table 4 demonstrate that Antibody 1 inhibits the C166-AP1-Luc cell-based assay described above. In this study, we found that the α-glucanase-induced luciferase reporter activity was effectively neutralized. (IC 50 =8.7nM), LY3016895(IC 50 =25.9nM) Rimo IC 50 The cross-reactivity of epiregulin to related species is approximately three-fold lower. Response was measured in rats (IC 50 =2.5 nM), cynomolgus monkeys (IC 50 =2.1nM) and Rabbit (IC 50 This was confirmed by testing the neutralization of epiregulin (=3.6 nM). These data suggest that α-glucanase inhibits human epiregulin-mediated signaling and inhibits epiregulation. It has been suggested that insulin-mediated signaling is involved in pain disorders, and in particular in diseases such as OA, DPNP, and CLBP. This supports the ability of Antibody 1 to treat human diseases contributed by factors.

[0090] Selection of Antibody 1 for Epiregulin Neutralization in the C166-AP1-Luc Assay sex: The selectivity of Antibody 1 for neutralizing epiregulin was determined using the C166-AP1-Luc assay Seven EGFR ligands (epigenetic, TGFα, BTC, EGF, HB-EGF) This was further demonstrated by testing the activity of all three phospholipases (epiregulin, epiregulin, and amphiregulin). (The ligand can be, for example, epiregulin from R&D systems. No. 1195-EP-CF, Betacellulin No. 261-CE-CF, EGF No. 236 -EG-CF, TGFα number 239-A-CF, HB-EGF number 259-HE-CF, Purchased under Epigen number 6629-EP-CF and Amphiregulin number 262-AR-CF These seven EGFR ligands produce luciferases depending on the ligand. demonstrate AP1 activation accompanied by a 2- to 5-fold increase in enzyme activity. 50 were expressed in the C166-AP1-Luc cell line as described hereinabove. The ligand concentration for stimulation is determined by performing a dose response of each ligand. EC 50 -EC 80 (betacellulin 0.8 nM, E GF 1.3nM, TGFα 2.5nM, HB-EGF 0.52nM, epiregulation Epigen 16nM, epigen 333nM and amphiregulin 181nM, epigen and amphiregulin Antibody 1 was the estimated EC50 due to the lack of a plateau on the curve. , IC of 8.7 nM 50 Epiregulin-induced cytotoxicity in C166-AP1-Luc cells In contrast, C166-AP1-Luc upregulation demonstrated the ability to neutralize luciferase activity. In the case of betacelluarin, EGF, TGFα, HB-EGF, epigen or amphi No inhibition of regulin-induced luciferase activity is demonstrated (see Table 5 below). The data in Table 5 also demonstrate that the activity of epiregulin is specifically neutralized, but not that of other family members. Betacellulin, EGF, TGFα, HB-EGF, epigenetic and amphiphilic This also supports the pharmacological selectivity of Antibody 1, which does not neutralize the activity of gliomas. Antibody 1 selectively interferes with epiregulin responses and therefore inhibits other EGFR ligands. of the adverse effects that may be caused by less specific agents such as LY3016859, which may block This is important in that it is expected to avoid the possibility of

[0091] [Table 5]

[0092] Ability of Antibody 1 to Neutralize Epiregulin-Induced Responses in the TGW Neuroblastoma Cell Line. : Epiregulin neutralization inhibits epiregulin-induced ER proliferation in TGW neuroblastoma cell lines. TGW cells express the receptor EGFR phospholipase K, which is further assessed by measuring the phosphorylation of the receptor EGFR. All four of the markers were expressed, with EGFR expression at the highest level. TGW cells were cultured in growth medium [NAPYR (Gibco no. 11995-065), 1X NEAA (Gibco No. 11140-050), Glutamax (Gibco No. 3 5050-061) and 1X Anti-Anti (Gibco number 15240-062 ) in DMEM high glucose [with 0.1% COOH] on collagen 1-coated plates. Cells were dissociated in 0.05% trypsin-PBS and plated onto a 96-well plate containing one collagen plate. 100,000 in a 96-well plate (Corning #354407) Cells were seeded at 100 μl / 100 μl in serum-free medium (growth medium in which FBS was replaced with 0.1% BSA). In TGW cells, epiregulin treatment (80 ng / ml for 5 minutes) significantly increased the activity of unstimulated cells. Compared with the cells, ERK (Meso Scale Diagnostics, LLC, Ph ospho-ERK1 / 2 Whole Cell Lysate Kit#K151D Phospho-ERK activation was measured using chemiluminescence-induced phospho-ERK activation. The neutralization of antibody 1 was determined by induction and is expressed as relative fluorescence units (RFU). By incubating human epiregulin with serially diluted antibody 1 for 20 minutes, Meso Scale Diagnostics SECTOR Im Data are obtained by reading the plate on a scanner. The reduction of glucuron-induced phosphor-ERK1 / 2 signaling is shown in Table 6, which indicates that antibody 1 This reflects the ability of TGW cells to neutralize human epiregulin. IC of Antibody 1 in neutralizing urin-induced pERK induction 50 The value was 3.4nM, SD0. 7 (n=2 replicates), which is 50 was 14.6nM, SD0.7 (n=2 replicates) is lower than LY3016859. The data in Table 6 show that Antibody 1 Ability to block human epiregulin-induced phosphor-ERK1 / 2 activation in W cells This data utilizes two different cell lines with independent readouts. Thus, the antibodies of the present disclosure, and in particular antibody 1, have demonstrated the ability to neutralize human epiregulin and The present invention supports the therapeutic use of these antibodies in the treatment of epiregulin-mediated disorders, such as chronic pain disorders. Hold.

[0093] [Table 6]

[0094] Example 4: Epiregulation of tactile allodynia induced in CD-1 mice In vivo functional characterization of phosphorus Biologically active molecules epiregulin and TGFα induce tactile allodynia The ability of the mice to express the IL-1 receptor can be assessed in male CD-1 mice after intraplantar injection. The animals were housed in a temperature- and light-controlled animal facility with access to water and food for at least 7 days prior to the experiment. On the day of testing, animals were brought to the testing room and allowed to acclimate in their home cages for 20 minutes. After that, they were placed in a transparent plastic chamber on an elevated metal mesh floor and allowed to acclimate for 1 hour. Paw withdrawal responses to mechanical stimuli were measured by measuring the pressure of the rats under the cage through an opening in the metal mesh floor. using calibrated von Frey filaments applied to the plantar surface of the hind paw. Measurements were performed using a von Frey filament in a raising and lowering test paradigm. The force range of the eight graduated filaments is 0.04g to 6g. The paw withdrawal threshold is measured and calculated based on the baseline value of the paw withdrawal threshold. The animals were randomized into treatment groups, which consisted of PBS, epiregulin 30 ng, epiregulin 10 ng, and 300 ng of urin, 30 ng of TGFα, or 300 ng of TGFα (mouse epidermal growth factor receptor 1 (EGF)). Regulin (SEQ ID NO: 28) and mouse TGFα (SEQ ID NO: 29) reagents were prepared as described herein. Prepared as described, or His-tagged and standard chromatographic assays for TGFα. chromatographic techniques (immobilized metal ion affinity chromatography or IMAC), followed by It was prepared as purified by size exclusion chromatography (SEC). The animals were then injected with 20 μl of PBS or freshly diluted epiregulin or The rats received an intraplantar injection of TGFα into the hind paw. Paw withdrawal thresholds were measured at 2 and 4 hours after injection. The data are shown in Table 7 below.

[0095] [Table 7]

[0096] The effect of epiregulin compared to TGFα is shown in Table 7 above, and this pain model Epiregulin induces pain responses as indicated by a reduction in withdrawal threshold in rats Antibody 1 induces both epiregulin and TGFα. Compared with LY3016859, which recognizes the The lack of binding to TGFα by Antibody 1 of the present disclosure is believed to be due to off-target effects. avoidance of side effects, reduction of immunogenicity, and improvement or reduction of the dose that may be required for therapeutic use It is considered to be advantageous.

[0097] Example 5: Characterization of the immunogenic potential of Antibody 1 Antibody 1 has enhanced activity against human epiregulin, as supported by the following findings: Improved affinity for epiregulin compared to other EGFR ligands Specificity and improved biophysical properties compared to LY3016859, and fully human sequences (the latter of which is thought to offer the potential for reduced immunogenicity). This combination of features makes it an improved therapeutic antibody.

[0098] Dendritic cell (DC) internalization assay Monocyte-derived DC Culturing (MDDC) CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMCs) according to standard protocols. DCs are isolated from primary PBMCs (PBMCs), cultured, and differentiated into DCs. S-WBC to Ficoll (#17-1440-02, GE Healthcare) and Sepmate 50 (#15450, STEMCELL Technology Isolate PBMCs using density gradient centrifugation according to the manufacturer's instructions. CD14+ Microbead Kit (#130-050-201, Miltenyi CD14+ monocytes are isolated using positive selection with a PBS (Biotec). The cells were cultured at 1 million / ml with 1000 units / ml GM-CSF and 600 units / ml IL-4. The cells were cultured in 10% FB medium for 6 days, supplemented with L-glutamine and 25 mM HEPES. S, 1 mM sodium pyruvate, 1x penicillin-streptomycin, 1x nonessential RPMI medium (hereafter referred to as L) supplemented with amino acids and 55 μM 2-mercaptoethanol in complete RPMI medium (referred to as medium) purchased from Fieve Technologies Immature dendritic cells (MDDCs) are induced. The medium is changed twice on days 2 and 5. Day 6 Then, gently collect the cells with a cell scraper and use them in experiments. dendritic morphology and CD14, CD11c, and HLA-DR by flow cytometry Visually characterize cells for expression of .alpha.. The ability to respond to LPS treatment is assessed by flow cytometry. This was confirmed by measuring increases in CD80, CD83, and CD86 using do.

[0099] Binding of Fab-TAMRA-QSY7 F(ab')2 fragment goat anti-human IgG (Jackson ImmunoRes search) were used with QSY7-NHS and TAMRA-SE (Molecular Probe es) as a universal probe to follow the internalization of test substances The Fab-TAMRA-QSY7 used was obtained as F(ab')2 (approximately 1.3 mg / ml). Each vial of 1 ml was filtered through an Amico Ultra-0.5 centrifugal filter device (#UF Centrifuge at 14,000 rcf for 2 minutes using a 1000 rpm syringe (C501096, Millipore) The solution was concentrated to approximately 2 mg / ml by adding 10% (v / v) 1 M sodium bicarbonate. Adjust the pH to basic (>pH 8) with DMSO and add 6.8 ml of a 10 mM stock solution of QSY-NHS in DMSO. Add 1 μl of HCl and mix. Keep the reaction vial in the dark at room temperature for 30 minutes. Fab-QSY7 was desalted on a Zeba Spin Desalting Column (#89890, Thermo Scientific) RCF (relative centrifugal force) is calculated using the Purify by centrifugation at 400°C for 2 minutes. The concentration and degree of labeling were determined by measuring the absorbance at 280 nm and 560 nm using a shear. The degree of labeling (DOL) is then calculated. .5 by centrifugation at 14,000 rcf for 2 minutes using a centrifugal filter device. Concentrate Fab-QSY7 to approximately 2 mg / ml. 10% (v / v) 1 M sodium bicarbonate After adjusting the pH with 100, add 4.3 μl of a 15 mM TAMRA-SE stock solution in DMSO. After 30 minutes in the dark at room temperature, the final product Fab-TAMRA-QSY7 was purified. and desalting the lysate on a Zeba Spin column by centrifugation at 1000 rcf for 2 minutes. Collect using a NanoDrop spectrophotometer at 280 nm, 555 nm, and 560 nm. Quantify the concentration and DOL again by reading the absorbance at 100 nm. Using the formula, approximately two QSY7 and two TAMRA fragments per F(ab')2 fragment are obtained. Approximately 300 μl of 1.5 mg / ml Fab-TAMRA-QSY7 is obtained.

[0100] Standardized internalization assay by FACS Individual test molecules were normalized to 1 mg / ml in PBS and then diluted for a further 8 h in complete RPMI medium. Dilute Fab-TAMRA-QSY7 to 5.33 μg / ml in complete RPMI medium. The antibody and Fab-TAMRA-QSY7 were mixed in equal amounts and diluted to 100 μg / ml. Incubate at 4°C for 30 minutes in the dark for MDDC to form complete RPMI medium. Add 50 μl of antibody / probe complex resuspended at 4 million / ml to a 96-well round-bottom plate. Seed 50 μl per well in a CO2 incubator at 37°C. Incubate for 24 hours. Wash the cells with 2% FBS PBS and incubate with Cytox Grease. Resuspend in 100 μl of 2% FBS PBS containing Live / Dead dye. BD LSR Data was collected using a Fortessa X-20 and analyzed using FlowJo. Gating is performed and the percentage of TAMRA fluorescent positive cells is recorded as the readout.

[0101] Data presentation and statistical analysis The molecules are tested in duplicate or triplicate on three or more donors. For each donor, T Consider the proportion of AMRA-positive populations. To allow comparison, a normalized internalization index (NII) is used. Null was determined for the IgG1 isotype (NII = 0) and the internal positive control PC ( Normalize to NII=100).

[0102]

number

[0103] [Table 8]

[0104] (For example, Wen, Y., Cahya, S., Zeng, W. et al.Devel opment of a FRET-Based Assay for Analysis s of mAbs Internalization and Processing by Dendritic Cells in Preclinical Immun ogenicity Risk Assessment.AAPS J 22,68(2 020)

[0105] MAPP Assay (MHC-Associated Peptide Proteomics) Method: Primary human dendritic cells from 10 normal human donors were cultured as described, using CD-14 positive Cells were prepared from buffy coats by isolation and incubated in 5% serum replacement (ThermoFisher) Complete RPM containing Her Scientific catalog number A2596101 37 with 20 ng / ml IL-4 and 40 ng / ml GM-CSF in I medium. The cells were then incubated at 37°C, 5% CO2 for 3 days to differentiate into immature dendritic cells. (Knierman et al., “The Human Leukocyte An tigen Class II Immunopeptidome of the SA RS-CoV-2 Spike Glycoprotein”,Cell Report s,33,108454(2020)). On day 4, 3 micromolar test antibody was administered at approximately 5 × 1 0 6 5 mg / ml LPS was added to the cells to transform them into mature dendritic cells. Fresh medium containing 1000kJ / ml is replaced after 5 hours of incubation. The next day, mature cells are cultured in a 2000kJ / ml PBS containing 1000kJ / ml PBS. Lyse the lysate in 1 ml of RIPA buffer containing ribosomal enzyme inhibitors and DNAse. The samples are stored at -80°C until analysis.

[0106] Using an automated liquid handling system, biotinylated anti-pan-HLA class II antibody (clone T Isolate HLA-II molecules from the thawed lysate using a ELISA kit (u39). The MHC-II peptide complexes were eluted with 5% acetic acid, 0.1% TFA. The peptide was passed through a pre-washed 10k MWCO filter to remove high molecular weight proteins. The isolated MHC-II peptides were analyzed using a Thermo LUMOS mass spectrometer. A Thermo easy 1200 nLC-HPLC system equipped with The separation was performed using a 75 μm×7 cm YMC-ODS C18 column. The column was used at a flow rate of 250 nL / min, and 0.1% formic acid aqueous solution was used as solvent A, and B A 65-minute gradient was run with 80% acetonitrile with 0.1% formic acid as the solvent. Quantitative analysis was performed in full scan mode at a resolution of 240,000, followed by HCD and ETh. A 3-second rapid scan consisting of an ion trap with CD fragmentation A data-dependent MS / MS cycle is performed.

[0107] Peptide identification was performed by enzymatic searching against bovine / human databases containing test antibody sequences. Internal proteomics pipeline using parameter-free multiple search algorithms (Higgs et al., “Label-free LC-MS method f or the identification of biomarkers”,Met hods in Molecular Biology,428,209-230(20 08)) and generate the sample identification file using the KNIME workflow. The peptides identified from the test material are aligned to the parent sequence. Percentage of donors presenting residues, different regions presenting peptides with non-germline residues All donors annotated with the number and depth of peptide presentation in each region with non-germline residues. Summary: Increasing the extent of non-germline peptide presentation increases the risk of immunogenicity. The results in Table 8 show the relative efficacy of Antibody 1 compared to LY3016859. Reduced presentation of non-germline peptides and therefore reduced risk of immunogenicity to Antibody 1 show.

[0108] [Table 9]

[0109] Example 6. Demonstration of binding to membrane epiregulin in vivo after dosing in rats. Epiregulin is a membrane-bound and ADAM (A disintegrin and metalloprotease) A disintegrin and metalloprotease) occurs by cleavage of the membrane form by proteases The antibodies of the present disclosure exist in both in vivo and in soluble forms. The ability of the cells to bind to membrane-type epiregulin in vivo was assessed by isolating the tissue ex vivo and subsequently analyzing the cells as described herein. by applying standard immunofluorescence staining techniques described in the literature and / or known to those skilled in the art. Positive and specific immunofluorescent labeling of the epithelial layer of the rat tongue can be assessed by antibody 1. This can be demonstrated using

[0110] Rat tongues were prepared from euthanized male Sprague Dawley rats (weight 140–210 kg). 5 grams) to various doses of the tested antibodies, including Antibody 1, or a control or comparative antibody. (0.1–100 mg / kg, subcutaneously) at various times after dosing (3–28 days after dosing). Frozen coronal 20 micron sections of tongue tissue can be obtained and flash frozen on dry ice. Thaw, wash in PBS for 3 minutes, and fix in 4% paraformaldehyde for 10 minutes. Wash the ride with Super Sensitive Wash Buffer (HK583-5K , Biogenex), followed by washing in Power block (HK065-5K, Block with Biogenex) + 0.1% Triton x-100 for 10 minutes. The slides were then washed and stained with Alexa Fluor 488 goat anti-human IgG (A11 013, Life Technologies, 13.3 μg / ml), and antibody 1 The antibody contained variable regions (SEQ ID NOs: 3 and 4) and mouse constant regions (labeled with DyLight 650). 1 VR chimera (SEQ ID NOs: 64 and 65) Incubate the slides three times with the mouse IgG1 chimera for 15 minutes at room temperature. Wash and apply Prolong gold antifade reagent before coverslipping. Fluorescence images at 88 and 650 nm wavelengths were taken using a Keyence BZ800. The same exposure settings are used for the images. The red signals were analyzed using ImageJ-Win64 software in four random pairs. Quantify the average intensity by taking the area of ​​interest. The green signal represents the bound antibody. 1, or control antibody, or reference comparison antibody (administered in vivo), and the red signal is The detection antibody competes with the bound drug to remove the free, unbound epiregulin. The data obtained were analyzed using Alexa Fluor 488 goat anti-human leukocyte antigen (HA) staining. The ratio of the antibody IgG signal (green) to the remaining antibody 1 VR chimera signal (red) was calculated. Normalize by using

[0111] Figure 2 shows the binding of membrane epiregulin 3 days after administration of Antibody 1 at doses of 1 mg / kg or higher. Figure 3 shows that a significant dose-dependent increase in the number of steroid hormones is observed in vivo at 10 mg / kg. Sustained binding of membrane epiregulin in vivo for at least 14 days after a single dose of antibody 1 Indicates that there is a match.

[0112] Figure 4 shows a comparative antibody that is 50-fold less potent at binding epiregulin than Antibody 1. demonstrate a higher degree of bound membrane epiregulin in vivo compared to that in vivo. Figure 4 shows that the improved affinity of Antibody 1 also supports membrane-bound epiregulin ligand binding in vivo. This also shows that it can lead to improvements in

[0113] Example 7: Epitope mapping of Antibody 1 Antibody 1 has high affinity and selectivity with respect to other EGFR ligand family members, and EGFR ligand family with advantageous epiregulin-neutralizing activity and pharmacokinetic properties. A human IgG4 antibody that selectively binds to and neutralizes member epiregulin (EREG). These properties, along with the additional stability and viscosity and solubility properties described herein, Both have improved attributes and advantageous therapeutic agents that neutralize EREG activity. Epitope mapping studies were performed to identify the binding and specificity of Antibody 1. The specific amino acids in human EREG that are important were identified. The contribution of these amino acids was identified from the crystal structure of the REG complex and was confirmed by mutagenesis and binding studies. The contribution of these amino acids was further characterized through the analysis of other EGFR ligand families. Compared with the members, the structural basis of the specificity of Antibody 1 for EREG is provided. Results provide information on cross-reactivity and selectivity in relevant preclinical species .

[0114] The sequences of the test substances are provided in the sequence listing for amino acid and nucleotide sequences and are compared as follows: Cross-reference: human EREG (SEQ ID NO: 21), Antibody 1 Fab HC (SEQ ID NO: 62), Antibody 1 Fab LC (SEQ ID NO: 63), mono Fc human EREG (SEQ ID NO: 27), mono Fc human EREG E95H (SEQ ID NO: 57), mono Fc human EREG E95A (SEQ ID NO: No. 56), mono-Fc human EREG H78N (SEQ ID NO: 55), mono-Fc human EREG H78A (SEQ ID NO: 54), monoFc human EREG F106K (SEQ ID NO: 61), mono No-Fc human EREG F106A (SEQ ID NO: 60), mono-Fc human EREG Y98A ( SEQ ID NO: 58), monoFc human EREG L77F (SEQ ID NO: 53), monoFc human ER EG R102A (SEQ ID NO: 59).

[0115] Both the antibody 1 Fab fragment and human EREG were produced in transient CHO expression. The antibody was purified by standard techniques. A 30% molar excess of EREG was added to antibody 1 Fab. The complex was prepared by adding 100% PEG-400 to PEG-400, and then purified by size exclusion chromatography. The binding of antibody 1 Fab to EREG was first detected by ELISA. Western blot analysis showed that Antibody 1 Fab was We demonstrated that denatured and non-reduced EREG can be recognized, but denatured and reduced EREG cannot. This strongly suggests a conformational epitope dependent on EREG disulfide bonds.

[0116] Crystal structure of the Fab fragment of antibody 1 bound to human epiregulin The crystal structure of the Fab fragment of antibody 1 bound to human epiregulin was analyzed. The mixture was crystallized and a 1.8 Å structure was obtained by molecular replacement using Phaser 2.8.3. The crystal was analyzed using Refmac 5.8.0258 and then refined. Fab' binds to the opposite face of the epiregulin dimer. The side chains of His7 and His78 are in the center of the epitope, and Leu77 is buried in a hydrophobic pocket. The His78 side chain is at the center of the hydrogen bond network. The g50 side chain is a hydrogen bond donor to the heavy chain Tyr52 side chain hydroxyl, The yr52 side chain hydroxyl is linked to the deprotonated His78 side chain N δ1 Hydrogen bond acceptors The protonated His78 side chain N ε2 is a hydrogen bond to a water molecule This water molecule is transferred to the backbone carbonyls of heavy chain Leu109 and light chain Tyr107. The residues of antibody 1 are numbered according to the IMGT convention. The identified epitope contacts include Glu95, Tyr98, Arg102, and Phe106 The structure includes the side chains, and the backbone of Leu77, Val96, and Glu104. The epitope was clearly demonstrated to be conformational and not linear, and the Western Check the results.

[0117] Mutant epiregulin ELISA using antibody 1 Fab Important epitopes are identified to understand their relative contribution to both binding and selectivity. The amino acid residues were further characterized by point mutagenesis. Dilute the antibody (to be used) to 1 μg / mL in PBS and add 100 μL to each well to perform ELI. SA plates (Greiner Cat. No. 655061) were coated overnight at 5°C. The variants were grouped by columns on the plate, and the wild type was placed in three replicate columns. The point mutants were coated onto a single replicate column. The plate was washed three times per well with 200 μL of PBST, followed by casein blot. on a plate shaker using King's buffer (Thermo Cat. No. 37528) After blocking, the plate was washed in the same manner as before. Antibody 1 Fab fragment was diluted to 1 μg / mL in casein blocking buffer. , then serially diluted 3-fold in blocking buffer to give a total of seven concentrations. Buffer was used as a blank. Fab dilution series was applied to the prepared ELISA plate per well. 100 μL of each plate was added and incubated on a plate shaker at room temperature for 1 hour. The plate was washed as before and then lysed with 1:8,000 diluted 1:1 in blocking buffer. 100 μL of goat anti-human kappa HRP secondary antibody (Southern Biotech catalog) The plate was incubated with the secondary antibody at room temperature for 1 hour. The plate was incubated on a plate shaker for 1 hour and then washed as before. A plate was prepared (Cat. No. 34021) and 100 μL was added to all wells. Incubate the samples statically at room temperature for 3-5 minutes, then add 100 μL of 1N HCl to each well. The reaction was stopped by adding 1000 mg of ... Blank-subtracted ELISA data was plotted on the x-axis with antibody concentration and OD4 The data were plotted in GraphPad Prism 9 with 50 as the Y-axis. by fitting the data to a "Sigmoidal, 4PL, X is concentration" model using , the curve was generated.

[0118] Point mutants were analyzed by ELISA using epiregulin mutants coated on plates. A dilution series of antibody 1 Fab was added and the signal was compared with that of the anti-kappa secondary antibody. (See Figure 5). All mutants except Y98A showed reduced results. H78A nearly knocked out binding, and Y98A showed no detectable binding at the concentrations tested. No possible bonds were shown.

[0119] SPR analysis of Antibody 1 Fab fragment binding to mono-Fc human EREG variants Both mono- and human Fc-antigens were analyzed using a Biacore 8K instrument and reagents (Cytiva). SPR analysis of Antibody 1 Fab fragments binding to EREG mutants was performed. Mono-Fc human EREG mutants were synthesized using an amine coupling kit (Cytiva P / N BR100050) and "Low Level CM" in Biacore 8K control software Using the "5 coupling" method, CM5 S-series sensor chips (Cytiva P The electrophoresis buffer was immobilized in the sample flow cell (Fc2) of the electrophoresis buffer (Bio-Rad). 1X HBS-EP+pH7.4(20X HBS-EP+, Teknova P / N H8022) or 1X MBS-EP + pH 6.0 (prepared from 10 mM MES + 150 The electrophoresis temperature was 3 The Biacore experiments were performed in three independent experiments, with the temperature at 7°C. Biacore Insight Eval uation Version 3.0 (Cytiva) to measure "1:1 binding" kinetics. Using either the "EREG variant" model or the "steady-state affinity" model, Control-subtracted and blank-subtracted SPR data were analyzed to determine KD values. Ta.

[0120] Antibody 1 Fab was diluted to 300 nM (pH 7.4) or 900 nM (pH 6. 0) and then serially diluted 3-fold for a total of 7 dilutions. The cell was injected for 240 seconds, followed by dissociation for 1800 seconds at a flow rate of 50 μL / min. The cap surface was regenerated with 2 x 30 second injections of 7 M guanidine at 100 μL / min. The subtracted data was collected from the sample flow cell minus the reference flow cell in each of the eight channels. Data was collected as low cells (Fc2-Fc1) and then subtracted for the reference buffer. Blanks were subtracted.

[0121] The binding affinities of a subset of mutants were measured by Biacore at 37°C. are reported in Table 9 as the mean ± standard deviation of three independent replicate determinations. The values ​​are relative to the wild type (wt).

[0122] [Table 10]

[0123] The results in Table 9 show that at pH 7.4, the solubility ranged from 3 times for R102A to 10 times for F106K. The results show various weaker affinities for the mutants, up to over 100-fold.

[0124] Human EGFR ligand epitope alignment In the context of the EGFR ligand sequence alignment (Figure 6), this data supports the E95 and F106 are important epitope positions for EREG selectivity. The 0% conserved positions Y98 and R102 are important for binding but do not contribute to selectivity. L77 and H78 are also important for binding and affect selectivity, but These are highly conserved across EGFR ligands and are therefore EREG-specific These results demonstrate the binding and excellent selectivity of Antibody 1 to EREG. Not only will the structural basis for all of these be demonstrated, but also the ligand and species profiles from epitope mapping studies will be Information about selectivity is also obtained.

[0125] Sequence listing of amino acid and nucleotide sequences Antibody 1 heavy chain (SEQ ID NO: 1) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQ PAGKGLEWIGRIYPSGNTNYNPSLKSRVTISVDTSKNQFS LKLSSVTAADTAVYYCARGGLVMDVWGQGTLVTVSSASTK GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN VDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKGLPSSIEKTISKAKGQPREPQVYTLPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LG

[0126] Antibody 1 light chain (SEQ ID NO: 2) EIVLTQSPGTLSLSPGERATLSCRASQSVEFSYLAWYQQ KPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRL EPEDFAVYYCHQYGTNPFTFGQGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC

[0127] HCVR of Antibody 1 (SEQ ID NO: 3) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQ PAGKGLEWIGRIYPSGNTNYNPSLKSRVTISVDTSKNQFS LKLSSVTAADTAVYYCARGGLVMDVWGQGTLVTVSS

[0128] LCVR of Antibody 1 (SEQ ID NO: 4) EIVLTQSPGTLSLSPGERATLSCRASQSVEFSYLAWYQQ KPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRL EPEDFAVYYCHQYGTNPFTFGQGTKVEIK

[0129] HCDR1 of Antibody 1 (SEQ ID NO: 5) TVSGGSISSYYWS

[0130] HCDR2 of Antibody 1 (SEQ ID NO: 6) RIYPSGNTN

[0131] HCDR3 of Antibody 1 (SEQ ID NO: 7) ARGGLVMDV

[0132] LCDR1 of Antibody 1 (SEQ ID NO: 8) RASQSVEFSYLA

[0133] LCDR2 of Antibody 1 (SEQ ID NO: 9) YGASSRAT

[0134] LCDR3 of Antibody 1 (SEQ ID NO: 10) HQYGTNPFT

[0135] DNA encoding the heavy chain of Antibody 1 (SEQ ID NO: 11) CAGGTGCAGCTGCAGGAGTCGGGTCCAGGACTGGTGAAG CCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTG GCTCCATCAGTTCGTACTACTGGAGCTGGATTCGGCAGCC CGCAGGGAAGGGACTGGAGTGGATTGGGAGGATCTATCCG AGTGGGAACACCAACTACAACCCCTCCCTCAAGAGTCGAG TCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCT GAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTAT TACTGTGCGAGAGGAGGACTGGTGATGGACGTGTGGGGAC AGGGAACACTAGTGACCGTGAGTAGCGCCTCCACCAAGGG CCCATCGGTCTTCCCGCTAGCGCCCTGCTCCAGGAGCACC TCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACT ACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGC CCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAG TCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGC CCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGT AGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTT GAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCAC CTGAGGCCGCCGGGGGACCATCAGTCTTCCTGTTCCCCCC AAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAG GTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCG AGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCA TAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGC ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGG ACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAA CAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAA GCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGC CCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCT GACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCC GTGGAGTGGGAAAGCAATGGGCAGCCGGAGAACAACTACA AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT CCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAG GAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTC TGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCT GGGT

[0136] DNA encoding the light chain of antibody 1 (SEQ ID NO: 12) GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTG TCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTC AGTCTGTGGAATTCAGCTACTTAGCCTATGGTGCATCCAG CAGGGCCACTTGGTACCAGCAGAAACCTGGCCAGGCTCCC AGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCA TCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCA GTGTATTACTGTCACCAGTACGGAACAAACCCGTTCACAT TCGGGCAGGGAACCAAGGTTGAAATAAAGCGAACTGTGGC TGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAG TTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATA ACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGA TAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACA GAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCA CCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGT CTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCC GTCACAAAGAGCTTCAACAGGGGAGAGTGC

[0137] Human epiregulin (SEQ ID NO: 21) VSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGV RCEHFFL

[0138] Human epiregulin construct (SEQ ID NO: 22) GPGVSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGY TGVRCEHFFLG

[0139] Cynomolgus monkey epiregulin (SEQ ID NO: 23) VSITKCNSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGV RCEHFYL

[0140] Rat epiregulin (SEQ ID NO: 24) VLITKSSDMDGYCLHGHCIYLVDMSEKYCRCEVGYTGL RCEHFFL

[0141] Rabbit epiregulin (SEQ ID NO: 25) VSITKCGSDMNGYCLHGQCIYLVDMSENYCRCEVGYTGV RCEHFFL

[0142] Full-length human epiregulin (membrane-bound, with T111P mutation) (SEQ ID NO: 26) MTAGRRMEMLCAGRVPALLLCLGFHLLQAVLSTTVIPSC IPGESSDNCTALVQTEDNPRVAQVSITKCSSDMNGYCLHG QCIYLVDMSQNYCRCEVGYTGVRCEHFFLTVPQPLSKEYV ALTVILIILFLITVVGSTYYFCRWYRNNRKSKEPKKEYERV TSGDPELPQV

[0143] Monomeric Fc human epiregulin (SEQ ID NO: 27) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSIT KCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVRCEHF FLG

[0144] Truncated mouse EREG (SEQ ID NO: 28) GPGVQITKSSDMDGYCLHGQCIYLVDMREKFCRCEVGY TGLRCEHFFLG

[0145] Mouse TGFα His (SEQ ID NO: 29) VVSHFNKCPDSHTQYCFHGTCRFLVQEEKPACVCHSGYV GVRCEHADLLAGHHHHHH

[0146] HCDR1 (Kabat) of Antibody 1 (SEQ ID NO: 31) SYYWS

[0147] HCDR2 of Antibody 1 (Kabat) (SEQ ID NO: 32) RIYPSGNTNYNPSLKS

[0148] HCDR3 of Antibody 1 (Kabat) (SEQ ID NO: 33) GGLVMDV

[0149] LCDR1 (Kabat) of Antibody 1 (SEQ ID NO: 34) RASQSVEFSYLA

[0150] LCDR2 of Antibody 1 (Kabat) (SEQ ID NO: 35) GASSRAT

[0151] LCDR3 of Antibody 1 (Kabat) (SEQ ID NO: 36) HQYGTNPFT

[0152] HCDR1 of Antibody 1 (Chothia) (SEQ ID NO: 37) GGSISSY

[0153] HCDR2 of Antibody 1 (Chothia) (SEQ ID NO: 38) YPSGN

[0154] HCDR3 of Antibody 1 (Chothia) (SEQ ID NO: 39) GGLVMDV

[0155] LCDR1 of Antibody 1 (Chothia) (SEQ ID NO: 40) RASQSVEFSYLA

[0156] LCDR2 of Antibody 1 (Chothia) (SEQ ID NO: 41) GASSRAT

[0157] LCDR3 of Antibody 1 (Chothia) (SEQ ID NO: 42) HQYGTNPFT

[0158] HCDR1 (IMGT) of Antibody 1 (SEQ ID NO: 43) GGSISSYY

[0159] HCDR2 (IMGT) of Antibody 1 (SEQ ID NO: 44) IYPSGNT

[0160] HCDR3 (IMGT) of Antibody 1 (SEQ ID NO: 45) ARGGLVMDV

[0161] LCDR1 (IMGT) of Antibody 1 (SEQ ID NO: 46) QSVEFSY

[0162] LCDR2 (IMGT) of Antibody 1 (SEQ ID NO: 47) GAS

[0163] LCDR3 (IMGT) of Antibody 1 (SEQ ID NO: 48) HQYGTNPFT

[0164] IgG4 PAA hinge region (SEQ ID NO: 51) ESKYGPPCPPCP

[0165] IgG4PAA Fc region (SEQ ID NO: 52) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLG

[0166] MonoFc huEREG L77F (SEQ ID NO: 53) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSIT KCSSDMNGYCFHGQCIYLVDMSQNYCRCEVGYTGVRCEHF FLG

[0167] MonoFc huEREG H78A (SEQ ID NO: 54) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSIT KCSSDMNGYCLAGQCIYLVDMSQNYCRCEVGYTGVRCEHF FLG

[0168] MonoFc huEREG H78N (SEQ ID NO: 55) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSIT KCSSDMNGYCLNGQCIYLVDMSQNYCRCEVGYTGVRCEHF FLG

[0169] MonoFc huEREG E95A (SEQ ID NO: 56) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSIT KCSSDMNGYCLHGQCIYLVDMSQNYCRCAVGYTGVRCEHF FLG

[0170] MonoFc huEREG E95H (SEQ ID NO: 57) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSIT KCSSDMNGYCLHGQCIYLVDMSQNYCRCHVGYTGVRCEHF FLG

[0171] MonoFc huEREG Y98A (SEQ ID NO: 58) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSIT KCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGATGVRCEHF FLG

[0172] MonoFc huEREG R102A (SEQ ID NO: 59) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSIT KCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVACEHF FLG

[0173] MonoFc huEREG F106A (SEQ ID NO: 60) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSIT KCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVRCEHA FLG

[0174] MonoFc huEREG F106K (SEQ ID NO: 61) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSIT KCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVRCEHA KLG

[0175] Antibody 1 Fab HC (SEQ ID NO: 62) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQ PAGKGLEWIGRIYPSGNTNYNPSLKSRVTISVDTSKNQFS LKLSSVTAADTAVYYCARGGLVMDVWGQGTLVTVSSASTK GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN VDHKPSNTKVDKRVESK

[0176] Antibody 1 Fab LC (SEQ ID NO: 63) EIVLTQSPGTLSLSPGERATLSCRASQSVEFSYLAWYQQ KPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRL EPEDFAVYYCHQYGTNPFTFGQGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTQ GTTSVTKSFNRGEC

[0177] Antibody 1 VR chimeric h / mIgG1 HC (SEQ ID NO: 64) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQ PAGKGLEWIGRIYPSGNTNYNPSLKSRVTISVDTSKNQFS LKLSSVTAADTAVYYCARGGLVMDVWGQGTLVTVSSASTK GPSVFPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSG SLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNV AHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKP KDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTA QTQPREEQFNSTFRSVSELIMHQDWLNGKEFKCRVNSAA FPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTC MITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVY SKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0178] Antibody 1 VR chimeric h / m kappa LC (SEQ ID NO: 65) EIVLTQSPGTLSLSPGERATLSCRASQSVEFSYLAWYQQ KPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRL EPEDFAVYYCHQYGTNPFTFGQGTKVEIKRTVAAPTVSIF PPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNG VLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATH KTSTSPIVKSFNRNEC

[0179] Human Epigen / EPGN (SEQ ID NO: 66) KFSHLCLEDHNSYCINGACAFHHELEKAICRCFTGYTGE RCEHLTLT

[0180] Human amphiregulin / AREG (SEQ ID NO: 67) KKKNPCNAEFQNFCIHGECKYIEHLEAVTCKCQQEYFGE RCGEKSMK

[0181] Human heparin-binding epidermal growth factor / HBEGF (SEQ ID NO: 68) KKRDPCLRKYKDFCIHGECKYVKELRAPSCICHPGYHGE RCHGLSL

[0182] Human betacellulin / BTC (SEQ ID NO: 69) GHFSRCPKQYKHYCIKGRCRFVVAEQTPSCVCDEGYIGA RCERVDLFY

[0183] Human transforming growth factor alpha / TGFα (SEQ ID NO: 70) VVSHFNDCPDSHTQFCFHGTCRFLVQEDKPACVCHSGYV GARCEHADLLA

[0184] Human epidermal growth factor / EGF (SEQ ID NO: 71) NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIG ERCQYRDLKW

Claims

1. An antibody that binds to human epiregulin, the antibody comprising a heavy chain variable region (VH) and A light chain variable region (VL) and a heavy chain complementarity determining region (HCDR) HCDR1 , HCDR2 and HCDR3, and the VL comprises a light chain complementarity determining region (LCDR) LC comprising DR1, LCDR2 and LCDR3; the HCDR1 comprises SEQ ID NO: 5; said HCDR2 comprising SEQ ID NO: 6; the HCDR3 comprises SEQ ID NO: 7; the LCDR1 comprises SEQ ID NO: 8; the LCDR2 comprises SEQ ID NO: 9; An antibody wherein the LCDR3 comprises SEQ ID NO:

10.

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

4. body.

3. The antibody comprises a heavy chain (HC) comprising SEQ ID NO: 1 and a light chain (LC) comprising SEQ ID NO:

2. The antibody according to claim 1 or 2.

4. A nucleic acid comprising the sequence of SEQ ID NO: 11 or 12.

5. A vector comprising the nucleic acid of claim 4.

6. The vector comprises a first nucleic acid sequence of SEQ ID NO: 11 and a second nucleic acid sequence of SEQ ID NO:

12. The vector of claim 5 .

7. A first vector comprising the nucleic acid sequence of SEQ ID NO: 11 and a second vector comprising the nucleic acid sequence of SEQ ID NO:

12. A composition comprising a vector of the above.

8. A cell comprising the vector of claim 5 or 6.

9. A first vector comprising the nucleic acid sequence of SEQ ID NO: 11 and a second vector comprising the nucleic acid sequence of SEQ ID NO:

12. A cell containing the vector.

10. The cell of claim 8 or 9, wherein the cell is a mammalian cell.

11. A process for producing an antibody, comprising the steps of:

10. Culturing the cells of any one of claims 1 to 9, and recovering the expressed antibody from the culture medium. A process that includes:

12. 12. An antibody produced by the process of claim 11.

13. An antibody according to any one of claims 1 to 3 or 12; and a pharmaceutically acceptable excipient, diluent or carrier.

14. 1. A method of treating a pain disorder in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of the antibody according to any one of claims 1 to 3 or 12 or the antibody according to claim 1 4. A method comprising administering the pharmaceutical composition of claim 3.

15. The pain disorders include osteoarthritis pain, diabetic peripheral neuropathy pain, and chronic low back pain.

15. The method of claim 14, wherein the compound is selected from the group consisting of:

16. 16. The method of claim 15, wherein the pain disorder is osteoarthritis pain.

17. 16. The method of claim 15, wherein the pain disorder is diabetic peripheral neuropathic pain.

18. 16. The method of claim 15, wherein the pain disorder is chronic low back pain.

19. 16. The method of claim 15, wherein the pain disorder is therapy-resistant.

20. An antibody according to any one of claims 1 to 3 or 12 for use in therapy.

21. A compound according to any one of claims 1 to 3 or 12 for use in the treatment of pain disorders. The antibody or pharmaceutical composition of claim 13.

22. The pain disorders include osteoarthritis pain, diabetic peripheral neuropathy pain, and chronic low back pain.

22. The antibody or pharmaceutical composition of claim 21 , selected from the group consisting of:

23. 22. The antibody or pharmaceutical composition of claim 21, wherein the pain disorder is osteoarthritis pain.

24. The antibody or medicament of claim 21, wherein the pain disorder is diabetic peripheral neuropathic pain. composition.

25. The antibody or pharmaceutical composition of claim 21 , wherein the pain disorder is chronic low back pain.

26. A compound according to any one of claims 1 to 3 or 12 in the manufacture of a medicament for the treatment of pain disorders. Use of the antibody described in .

27. The pain disorders include osteoarthritis pain, diabetic peripheral neuropathy pain, and chronic low back pain.

27. The use according to claim 26, wherein the compound is selected from the group consisting of:

28. 1. A method for determining human epiregulin levels in a body fluid sample, comprising: (a) subjecting the body fluid sample to the anti-human epiregulin diagnostic test according to any one of claims 1 to 3; contacting the subject with a purified monoclonal antibody or antigen-binding fragment thereof; (b) optionally, any non-specifically bound monoclonal antibodies or their antigen binding. Removing the fragment; (c) A monoclonal antibody that specifically binds to human epiregulin or its antigen-binding fragment and detecting and / or quantifying the amount of fragment.

29. The body fluid sample is a blood, serum or plasma sample, or a cerebrospinal fluid sample, 29. The method of claim 28, wherein the causing occurs ex vivo.

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