Compounds and Methods Targeting Epiregulin

Abstract

The present invention relates to epiregulin antibodies, compositions comprising them, and methods of making and / or using the antibodies and / or compositions for chronic pain disorders such as chronic osteoarthritis pain or chronic diabetic peripheral neuropathy pain or chronic low back pain.

Description

Technical Field

[0001] The present invention relates to compounds, pharmaceutical compositions and methods comprising antibodies directed against human epiregulin, and their use in the treatment of pain, including nociceptive, neuropathic and mixed pain, and in particular in the treatment of osteoarthritis (OA) pain or diabetic peripheral neuropathy pain (DPNP) or chronic low back pain (CLBP).

Background Art

[0002] Chronic pain is classified into different categories of nociceptive, neuropathic and mixed based on its mechanism. Nociceptive pain is caused by stimuli that can damage or actually cause damage to tissues other than nerve cells. This activates nociceptive receptors in the peripheral sensory system. Pain due to osteoarthritis is a typical example of somatic nociceptive pain. Neuropathic pain is caused by damage or disease of the central or peripheral nervous system, leading to sensory system maladaptive hypersensitivity. Pain due to diabetic peripheral neuropathy is a typical example of peripheral neuropathic pain. Conditions that exhibit characteristics of both nociceptive and neuropathic pain, such as chronic low back pain, are classified as mixed pain.

[0003] Chronic pain is a very prevalent condition that has a major impact on society. In 2016, an estimated 20.4% of the adult population in the United States experienced chronic pain, defined as pain on most or every day in the past six months, based on data from the National Health Interview Survey. An estimated 8% of the population had chronic pain that substantially limited their life or work activities almost every day or every day in the past six months. As a result, chronic pain is a major cause of healthcare costs, with the annual cost of chronic pain management in the United States in 2010 estimated at approximately $635 billion. Despite its high disease burden and social impact, the management of chronic pain is currently unsatisfactory. Non-pharmacological therapies alone are not sufficient to relieve pain or improve function, and the available pharmacological therapies are diverse, with marginal benefits and significant safety risks. Currently, the drugs most frequently used to reduce the most common types of chronic pain are acetaminophen, non-steroidal anti-inflammatory drugs, and opioids. Gabapentinoids, other anti-seizure drugs (such as sodium valproate, carbamazepine, or lamotrigine), and some antidepressants (such as tricyclic drugs or duloxetine) can be used for specific pain disorders. Current pharmacological medical facilities typically provide a low level of effectiveness and have tolerance problems and / or harmful side effects. Opioids are effective for acute pain but are associated with a high risk of abuse and the potential for severe adverse reactions, so treatment options for chronic pain are limited. The physical, emotional, and economic impact of chronic pain on patients and society, combined with the lack of effective and tolerable treatment options, represents a significant unmet medical need.

[0004] Epiregulin is a member of the epidermal growth factor receptor (EGFR) family of ligands that includes seven ligands: TGF-α (TGFA), epiregulin (EREG), EGF, heparin-binding EGF (HB-EGF), epigen (EPGN), amphiregulin (AREG), and betacellulin (BTC) (Schneider MR, Wolf E. The epidermal growth factor receptor ligands at a glance. J Cell Physiol. 2009;218(3):460-466). In addition to EGFR (ErbB1), there are three additional receptors (ErbB2, ErbB3, and ErbB4) within this family of receptors, of which ErbB3 and ErbB4 can bind to ligands of the neuregulin family as well as select members of the EGFR family of ligands (epiregulin, betacellulin, and HB-EGF). These ligands are synthesized as transmembrane proteins that are proteolytically cleaved to produce soluble ligands that can act in a paracrine or autocrine manner to regulate various biological processes. Epiregulin can bind to both EGFR and ErbB4 and induce signaling via homodimerization or ligand-induced heterodimerization with ErbB2 or ErbB3.

[0005] Epiregulin signaling contributes to a wide range of physiological states such as inflammation, wound healing, and angiogenesis (Riese et al. Epiregulin: Roles in Normal Physiology and Cancer, Semin Cell Dev Biol. 2014, 0:49-56). Epiregulin signaling via EGFR receptor homo- or hetero-dimer pairs enables the activation of numerous downstream signaling pathways such as ERK, MAPK, AP1, PI3K, JAK / STAT, and NFKB. Activation of pathways such as JAK / STAT and NFKB has been well demonstrated in driving inflammation, while activation of AP1 signaling drives the activation of c-FOS and c-JUN, markers of neuronal activation. Recently published data in preclinical models have associated the role of epiregulin with the regulation of chronic pain characterized by neuronal inflammation and neuronal activation, suggesting the role of the epiregulin signaling pathway in regulating inflammation and neuronal activation as a potential mechanism in chronic pain disorders. Reports have shown that the EGFR pathway is involved in the etiology of neuropathic pain (Kersten et al., Epidermal growth factor receptor-inhibition (EGFR-I) in the treatment of neuropathic pain. Br J Anaesth. 2015;115(5):761-767). However, targeting the receptor with EGFR antibodies or EGFR tyrosine kinase inhibitors has been found to be associated with a high incidence of gastrointestinal (GI) and skin adverse reactions, thereby limiting their potential use in chronic pain disorders.

[0006] Antibodies that bind to both TGFα and epiregulin are disclosed in WO 2012 / 138510, along with methods of treating diabetic nephropathy. LY3016859 is a monoclonal antibody that binds to epiregulin and transforming growth factor α (TGF-α), which has been tested in a Phase 1 clinical trial and evaluated for safety, pharmacokinetics, pharmacodynamics, and efficacy in healthy subjects and patients with diabetic nephropathy (see Sloan-Lancaster, et al., Evaluation of the Safety, Pharmacokinetics, Pharmacodynamics, and Efficacy After Single and Multiple Dosings of LY3016859 in Healthy Subjects and Patients With Diabetic Nephropathy, Clinical Pharmacology in Drug Development 2018, 7(7)759-772). Sloan-Lancaster et al. described that LY3016859 has a higher affinity for TGFα than for epiregulin, and that administration of LY3016859 did not result in any apparent effect on nephropathy disease-related biomarkers (see also Beidler, et al., J. Pharmacology and Experimental Therapeutics, 2014, 349(2):330-343). Non-linear kinetics suggestive of target-mediated drug disposition were seen, with high doses required for high levels of soluble target engagement. Notably, high frequencies of anti-LY3016859 antibodies were observed in both studies reported, but there was no clear effect on pharmacokinetics or target engagement as shown by dose- and time-dependent increases in circulating epiregulin measured in drug tolerance assays. Antibodies that are more selective for epiregulin, have higher affinity, and are less likely to induce anti-drug antibody responses are an important unmet therapeutic need for the treatment of chronic pain indications.There remains an unmet need for alternative and / or improved treatments for chronic pain disorders, including nociceptive, neuropathic and mixed pain, and in particular for the treatment of osteoarthritis or diabetic peripheral neuropathy or chronic low back pain, and / or for the treatment of therapy-resistant pain. SUMMARY OF THE INVENTION

[0007] Embodiments of the present disclosure provide 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. According to some embodiments, the invention provides an antibody comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), the LCVR comprising complementarity determining regions (CDRs), LCDR1, LCDR2 and LCDR3, and the HCVR comprising CDRs, HCDR1, HCDR2 and HCDR3, which are selected from the group of CDR combinations provided in Table 1. The sequence identifiers used herein are listed in Table 1 and the sequences are provided in the amino acid and nucleotide sequence listings provided herein. Antibody 1 is a high-affinity fully human immunoglobulin G4 (IgG4) monoclonal antibody that binds to residues in the C-terminal region of human epiregulin and prevents the binding of human epiregulin to EGFR and the activation of EGFR. Antibody 1 is an improved anti-epiregulin antibody for human therapy, which has a combination of advantageous properties including enhanced affinity, selectivity, reduced risk of off-target and unwanted activities, reduced risk of immunogenicity, high efficacy and long duration of action, and other desirable properties, blocks epiregulin, and provides an improved means for treating pain and chronic pain disorders.

[0008] TABLE 1

[0009] Accordingly, embodiments of the present disclosure also provide an antibody comprising an LCVR having the amino acid sequence of SEQ ID NO: 4 and an HCVR having the amino acid sequence of SEQ ID NO: 3.

[0010] According to other embodiments, the present disclosure also provides an antibody comprising an LCVR having the amino acid sequence of SEQ ID NO: 4, an HCVR having the amino acid sequence of SEQ ID NO: 3, and a hinge region and an Fc region selected from SEQ ID NO: 51 and SEQ ID NO: 52.

[0011] In another embodiment, the present disclosure also provides an antibody comprising an LC having the amino acid sequence of SEQ ID NO: 2 and an HC having the amino acid sequence of SEQ ID NO: 1. According to other embodiments, the present disclosure also provides an antibody comprising an LC and an HC having an amino acid sequence having at least 95% homology with the amino acid sequences of an LC having the amino acid sequence of SEQ ID NO: 2 and an HC having the amino acid sequence of SEQ ID NO: 1.

[0012] As used herein, "Antibody 1" refers to an antibody having the HCDR1 amino acid sequence of SEQ ID NO: 5, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 7, the LCDR1 amino acid sequence of SEQ ID NO: 8, the LCDR2 amino acid sequence of SEQ ID NO: 9, the LCDR3 amino acid sequence of SEQ ID NO: 10, the HCVR amino acid sequence of SEQ ID NO: 3, the 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 being encoded by the HC DNA sequence of SEQ ID NO: 11, or being encoded by the LC DNA sequence of SEQ ID NO: 12. The framework and CDR sequences in each of the antibodies whose sequences are shown herein are annotated using annotation rules consistent with the method of North, et al.. J. Mol. Biol. 2011:406:228-256 unless otherwise specified.

[0013] The carboxy-terminal portion of each HC defines a constant region that mainly bears the effector function. In some embodiments of the present invention, the antibody has one or more modifications in the constant region of each HC that reduces the effector function. Preferably, the embodiment of the present invention is an IgG4 antibody, and thus contains an IgG4 Fc region, or an Fc region derived from human IgG4, for example, a modified IgG4 Fc region.

[0014] According to some embodiments, modifications and amino acid substitutions in the constant regions of both HCs that reduce the effector function are introduced into the IgG4 hinge and Fc regions. Thus, some embodiments have modifications in the constant regions of both HCs that include the amino acid alanine at both residues 229 and 230 (EU index positions 234 - 235) (exemplified in the HC of antibody 1 and shown in SEQ ID NO: 52), and further modifications in the constant regions of both HCs that promote stability and include the amino acid proline at residue 223 (EU index position 228) (exemplified in the HC of antibody 1 and shown in SEQ ID NO: 51), and a deletion of the amino acid lysine at residue 442 (EU index position 447) (exemplified in the HC of SEQ ID NO: 1).

[0015] The antibodies of the present invention have the following characteristics: 1) high binding affinity and desirable association and dissociation rates, 2) potency in neutralizing human epiregulin to achieve a pain relief response and in vivo efficacy, 3) being potent enough as a monotherapy for the treatment and / or prevention of pain disorders, 4) a sustained duration of action, 5) a sufficiently limited injection site reaction, 6) acceptably low immunogenicity (i.e., sufficiently non-immunogenic in humans), 7) reduction of adverse skin rash reactions, and / or 8) heat stability, solubility, low self-association, and pharmacokinetic characteristics including but not limited to desirable in vivo stability, physical and chemical stability, which are acceptable for the development and / or use in the treatment of pain disorders, such as chronic pain including nociceptive, neuropathic, and mixed pain, and in particular the treatment of chronic osteoarthritis pain or chronic diabetic peripheral neuropathy pain or chronic low back pain, and having a combination of characteristics that are particularly advantageous over prior art anti-epiregulin antibodies, including but not limited to one or more of the above.

[0016] Embodiments of the present invention provide a significant advance over the prior art by providing methods useful in the treatment, downregulation or remission of pain disorders via epiregulin neutralization, using antibodies against human epiregulin, their compositions, and the pharmacologically advantageous anti-human epiregulin antibodies provided in the embodiments described herein. The anti-human epiregulin antibodies of the present invention can preferably alleviate pain symptoms and improve pain pathophysiology, particularly through inhibition of the pain response in chronic pain disorders and pain states. Clinical use of such antibodies may lead to long-term remission of the pain-related disorders being treated.

[0017] Furthermore, there is a need for diagnostic anti-human epiregulin antibodies that are specific for human epiregulin, have improved binding affinity, and demonstrate enhanced sensitivity in human epiregulin determination, as well as improved enzyme-linked immunosorbent assay (ELISA) assay conditions that result in minimal interference and broad dilution linearity. According to some aspects of the present disclosure, provided are anti-human epiregulin antibodies that include human epiregulin neutralizing antibodies that bind to the human epiregulin provided by SEQ ID NO: 21. "Epiregulin" or "human epiregulin" refers to the human epiregulin protein. As used herein, epiregulin refers to the mature epiregulin peptide. Epiregulin (also known as EREG, EPR) is a 46-amino acid protein belonging to the epidermal growth factor (EGF) family of peptide hormones. Epiregulin is produced as a 162-amino acid transmembrane epiregulin precursor, which is cleaved to release a 46-amino acid mature peptide of the following sequence: "VSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVRCEHFFL" (SEQ ID NO: 21) (see, for example, Toyoda, et al., Molecular cloning of mouse epiregulin, a novel epidermal growth factor-related protein, expressed in the early stage of development. FEBS Lett. 1995;377:403-7). Human epiregulin (SEQ ID NO: 22), as described and prepared in Example 2, can be used, for example, in the in vitro experiments described herein. References to the ability of the antibodies described herein to bind to and / or neutralize the human epiregulin protein also refer to their ability to bind to and neutralize human epiregulin in in vitro experiments.

[0018] As used herein, the term "human anti-epiregulin antibody" or "anti-human epiregulin antibody" refers to an antibody that binds to human epiregulin and, when administered in vitro or in vivo, results in an epiregulin activity neutralization and / or blocking response such as at least one significantly decreased activity. For example, a desired reduction of epiregulin signaling as demonstrated by a change in an epiregulin-responsive molecule or cellular endpoint. As used herein, the terms "signaling" and "signal transduction" and "epiregulin-mediated" when in reference to epiregulin refer to cellular and / or intercellular reactions resulting from the activity of epiregulin.

[0019] As used herein, the term "antibody" refers to an immunoglobulin molecule that binds to an antigen. Embodiments of antibodies include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, or conjugated antibodies. Antibodies can be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and of any subclass (e.g., IgG1, IgG2, IgG3, IgG4). An exemplary antibody is an immunoglobulin G (IgG)-type antibody composed of four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked via interchain disulfide bonds. The LC is classified as kappa or lambda, each of which is characterized by a specific constant region. Embodiments of the invention can include IgG1 or IgG4 antibodies and can further include a kappa light chain or a lambda light chain. Preferably, the antibodies of the invention include a light chain constant region that is a kappa constant region.

[0020] HC is classified as gamma, mu, alpha, delta, or epsilon, and defines the antibody isotype as IgG, IgM, IgA, IgD, or IgE, respectively. The amino-terminal portion of each of the four polypeptide chains contains a variable region of about 100 to 125 or more amino acids that is mainly involved in antigen recognition. The carboxy-terminal portion of each of the four polypeptide chains contains a constant region that is mainly involved in effector functions. Each heavy chain is composed of a heavy-chain variable region (VH) and a heavy-chain constant region. The constant region of the heavy chain contains CH1, CH2, and CH3 domains. CH1 follows HCVR, and CH1 and HCVR form the heavy-chain portion of the antigen-binding (Fab) fragment, which is part of the antibody that binds to the antigen. CH2 follows the hinge region and precedes CH3. CH3 follows CH2 and is at the carboxy terminus of the heavy chain. The constant region of the light chain contains one domain, CL. CL follows LCVR, and CL and LCVR form the light-chain portion of Fab.

[0021] The antibodies of the present invention include IgG heavy chains (HCs) that can be further classified into subclasses, such as IgG1, IgG2, IgG3, IgG4, and embodiments of the present disclosure can include one or more modifications in the constant region of each HC that, for example, enhance or reduce effector function. As used herein, the term "Fc region" refers to the region of an antibody that includes the CH2 and CH3 domains of the antibody heavy chain. Optionally, the Fc region can include a portion or the entire hinge region of the antibody heavy chain. IgG1 is known to induce antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and the Fc mutations described herein can reduce aggregation, reduce or enhance ADCC or CDC activity (or other functions), and / or modify the pharmacokinetics of the antibody. Embodiments of the anti-human EREG antibodies described herein have reduced binding to FcγR and C1q receptors, thereby reducing or eliminating cytotoxicity that can be induced by antibodies having a wild-type IgG Fc region. Thus, according to some embodiments, the mutations are introduced into the Fc region at the positions described herein. By sufficiently reducing or eliminating the effector function of such anti-human EREG antibodies containing a modified Fc region, patient safety can be improved, and in combination with other properties described herein, a therapeutic agent can be provided that has an improved profile of useful activity while avoiding undesirable activity.

[0022] When expressed in a particular biological system, the antibody is glycosylated in the Fc region. Typically, glycosylation occurs in the Fc region of the antibody at highly conserved N-glycosylation sites. The N-glycan typically binds to asparagine. The antibody can also be glycosylated at other positions. The antibodies of the present disclosure are monoclonal antibodies. Monoclonal antibodies are antibodies derived from a single copy or clone (e.g., including any eukaryotic, prokaryotic, or phage clone), and are not defined by the method by which they are produced. Monoclonal antibodies can be produced, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic technology, such as CDR grafting, or a combination of such technologies or other technologies known in the art. The present disclosure contemplates that the antibodies of the present invention are human or humanized antibodies. In the context of monoclonal antibodies, the terms "human" and "humanized" are well known to those of skill in the art (Weiner LJ, J. Immunother. 2006;29:1-9, Mallbris L, et al., J. Clin. Aesthet. Dermatol. 2016;9:13-15). Exemplary embodiments of the antibodies of the present disclosure also include antibody fragments or antigen-binding fragments that include at least a portion of an antibody that retains the ability to specifically interact with an antigen, such as Fab, Fab’, F(ab’)2, Fv fragments, scFv antibody fragments, disulfide-bonded Fv (sdFv), Fd fragments, and linear antibodies.

[0023] The amino-terminal portions of each LC and HC contain a variable region of approximately 100 to 120 amino acids that is mainly responsible for antigen recognition via the CDRs contained therein. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), in which more conserved regions called framework regions (FRs) are interspersed. The CDRs are exposed on the surface of the protein and are important regions of the antibody for antigen-binding specificity. Each VH and VL is composed of three CDRs and four FRs, and is arranged from the amino terminus to the carboxy terminus in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In this specification, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3", 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 of an antibody to bind to a specific antigen is greatly influenced by the six CDRs.Assignment of amino acid residues to CDRs can be performed by well-known schemes, including those described in Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins", Journal of Molecular Biology, 196, 901-917 (1987), Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (the international ImMunoGeneTics database, available at www.imgt.org, see Lefranc et al., Nucleic Acids Res. 1999;27:209-212).

[0024] For the purposes of the present disclosure, unless otherwise specified, the North CDR definition is used for the anti-epiregulin antibodies described herein, as well as for the amino acid assignments to the CDR domains within the LCVR region and the HCVR region. Table 2 below provides the CDR sequences of antibody 1 and / or the antibodies of the present disclosure, generated using the SAbPred / ANARCI library (Dunbar and Deane, "ANARCI: antigen receptor numbering and receptor classification", Bioinformatics, 32, 298 - 300 (2016)), based on the conventions of North, Kabat, Chothia and / or IMGT, respectively.

[0025]

Table 2

[0026] Embodiments of the antibodies of the present disclosure have a combination of several pharmacologically useful and important activities and, in some respects, can bind to human epiregulin with high affinity and high specificity for human epiregulin and have other useful properties. As used herein, the term "binds" is intended to mean the ability of a protein or molecule to form an attractive interaction with another protein or molecule, unless otherwise indicated, which results in the proximity of two proteins or molecules as determined by common methods known in the art. As used herein, the phrase "specifically binds" refers to the affinity of an anti-epiregulin antibody for human epiregulin and is determined at about pH 7.4 by common methods known in the art, including the use of MSD-SET (solution equilibrium titration) described herein, unless otherwise indicated, of about 2×10 -9 M or less, preferably about 2×10 -11 M or less, even more preferably about 2×10 -11 M to about 2×10 -12 M of K Dis intended to mean. The phrase "specifically binds" also indicates the relative affinity of an anti-epiregulin antibody for human epiregulin as compared to other antigens and in particular the EGFR ligand TGFα, and the affinity for human epiregulin results in specific recognition of human epiregulin and lack of binding to other EGFR ligands tested.

[0027] Embodiments of the antibodies of the present disclosure can be expressed and produced from constructs containing the sequences of the present embodiments by various techniques known in the art. The terms "nucleic acid" or "polynucleotide" as used interchangeably herein refer to polymers of nucleotides, including single-stranded and / or double-stranded nucleotide-containing molecules such as DNA, cDNA, and RNA molecules incorporating natural nucleotides, modified nucleotides, and / or nucleotide analogs. The polynucleotides of the present disclosure can also include, for example, substrates incorporated therein by DNA or RNA polymerase or synthetic reactions. The DNA molecules of the present disclosure are DNA molecules comprising non-naturally occurring polynucleotide sequences encoding polypeptides having the amino acid sequence of at least one of the polypeptides (e.g., heavy chain, light chain, variable heavy chain, and variable light chain) in the antibodies of the present invention.

[0028] Isolated DNA encoding the HCVR or LCVR region can be converted into a full-length heavy chain gene by operably linking DNA encoding the HCVR or LCVR, respectively, to another DNA molecule encoding a heavy or light chain constant region to form a heavy or light chain, respectively. The sequences of the heavy chain constant region genes of humans and other mammals are known in the art. DNA fragments encompassing these regions can be obtained, for example, by standard PCR amplification.

[0029] The polynucleotides of the present disclosure can be expressed in a host cell after the sequence is operably linked to an expression control sequence. Expression vectors are typically replicable in a host organism, either as an episome or as an integrated part of the host chromosomal DNA. Generally, expression vectors contain selectable markers, such as tetracycline, neomycin, and dihydrofolate reductase, to enable the detection of those cells transformed with the desired DNA sequence. Vectors containing the polynucleotide sequence of interest (e.g., a polynucleotide encoding a polypeptide of an antibody and an expression control sequence) can be introduced into host cells by well-known methods that vary depending on the type of cell host.

[0030] The antibodies of the present disclosure can be readily produced in mammalian cells, non-limiting examples of which include CHO, NS0, HEK293, or COS cells. Host cells are cultured using techniques well known in the art. Mammalian antibody expression typically results in glycosylation. Antibody glycosylation is typically either N-linked or O-linked. N-linked glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of a sugar, such as N-acetylgalactosamine, galactose, or xylose, to a hydroxy amino acid. Typically, glycosylation occurs in the Fc region of the antibody at highly conserved N-glycosylation sites (e.g., position 297 in IgG1 according to IMGT or EU index numbering). Glycosylation sites can be modified to alter glycosylation (e.g., to block or reduce glycosylation, or to change the amino acid sequence to generate additional or diverse glycosylation).

[0031] Expression of antibodies in mammals from IgG subclasses can result in the clipping of C-terminal amino acids from one or both heavy chains. For example, in the case of IgG1 antibodies, one or two C-terminal amino acids can be removed. In the case of IgG1 antibodies, if a C-terminal lysine is present, it can be trimmed or excised from the heavy chain during expression. Additionally, the penultimate glycine can also be trimmed or excised from the heavy chain.

[0032] Expression of antibodies in mammals can also result in the modification of N-terminal amino acids. For example, if the most N-terminal amino acid of the heavy or light chain is glutamine, it can be modified to pyroglutamic acid.

[0033] The antibodies of the present disclosure, or pharmaceutical compositions containing the same, can be administered by parenteral routes, non-limiting examples of which are subcutaneous administration and intravenous administration. The antibodies of the present disclosure can be administered to a patient in a single dose or multiple doses together with a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical compositions of the present disclosure can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), A. Loyd et al., Pharmaceutical Press), and contain the antibodies disclosed herein, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0034] Use of the antibody embodiments of the present disclosure: According to some embodiments, the anti-epiregulin antibodies of the present disclosure are useful for the treatment of pain disorders. As used herein, the term "pain disorder" refers to an undesirable condition resulting from excessive and / or chronic pain states, where epiregulin inhibition results in a more tonic and less pathological pain state. Exemplary pain disorders contemplated for treatment with the antibodies of the present disclosure described herein include chronic pain, including nociceptive, neuropathic, and mixed pain, and in particular, osteoarthritis pain or diabetic peripheral neuropathy pain or low back pain, and in particular the treatment of chemotherapy-induced peripheral neuropathy in chronic pain states.

[0035] According to other embodiments of the invention, the anti-epiregulin antibodies are useful for diagnostic use in epiregulin-mediated pain disorders. In some embodiments, the pain disorder is at least one of osteoarthritis (OA) pain, diabetic peripheral neuropathy pain (DPNP), or chronic low back pain (CLBP). In some more specific embodiments, the pain disorder is osteoarthritis (OA).

[0036] The present disclosure further provides a pharmaceutical composition comprising the anti-epiregulin antibody of the present disclosure and one or more pharmaceutically acceptable carriers, diluents, or excipients. Further, the present disclosure provides a method of treating a pain disorder such as osteoarthritis pain, diabetic peripheral neuropathy pain, or chronic low back pain, the method comprising administering the pharmaceutical composition of the present disclosure to a patient in need thereof.

[0037] In addition, the present disclosure provides a method of treating an epiregulin-mediated disease. More specifically, the present invention provides a method of treating a chronic pain disorder such as osteoarthritis pain, diabetic peripheral neuropathy pain, or chronic low back pain, the method comprising administering an effective amount of the anti-epiregulin antibody of the present disclosure to a patient in need thereof.

[0038] The present disclosure also provides the anti-epiregulin antibodies of the present disclosure for use in therapy. More specifically, the present disclosure provides the anti-epiregulin antibodies of the present disclosure for use in the treatment of chronic pain disorders such as osteoarthritis pain or painful diabetic neuropathy or chronic low back pain.

[0039] In certain embodiments, the present disclosure provides the use of the anti-epiregulin antibodies of the present disclosure or compositions thereof in the manufacture of a medicament for the treatment of one or more chronic pain disorders such as osteoarthritis pain or painful diabetic neuropathy or chronic low back pain.

[0040] The antibodies of the present disclosure are useful for the identification of chronic pain disorders in which epiregulin may contribute to the etiology of the disorder. In a further embodiment, the present disclosure provides a method of treating a chronic pain disorder in a patient. Such a method comprises contacting a patient sample with an anti-epiregulin antibody and detecting binding between human epiregulin and the antibody in the patient sample, and diagnosing that the patient has, is at risk of having, is in need of treatment for, and / or is at risk of symptoms associated with an epiregulin-mediated disorder if the presence of epiregulin in the patient sample is detected above a reference value observed in non-affected individuals. According to some more specific embodiments of the treatment methods provided herein, such a method further comprises determining a reference value comprising contacting a control standard with a first antibody that binds to a first epitope region of epiregulin that is the same as that used when contacting the patient sample, contacting the control standard with a second antibody that has a detectable label and binds to a second epitope region of epiregulin that is the same as that used when contacting the patient sample, and detecting a signal provided by the detectable signal. In some specific embodiments, the anti-epiregulin antibody comprises a combination of LC and HC CDRs provided in Table 1. In a further embodiment, the second antibody comprises a combination of LCVR and HCVR provided in Table 1. In certain embodiments, the chronic pain disorder is one of osteoarthritis pain or painful diabetic peripheral neuropathy or chronic low back pain. In some embodiments, the patient sample is one of CSF, blood, serum, tissue lysate, or plasma. According to some embodiments, the method further comprises contacting the patient sample with a second anti-epiregulin antibody that binds to a second epitope region of epiregulin and has a detectable label, and detecting a signal provided by the detectable signal. In a further embodiment, the second antibody comprises a combination of LC and HC CDRs provided in Table 1. In a further embodiment, the second antibody comprises a combination of LCVR and HCVR provided in Table 1. According to certain embodiments, the first and second anti-epiregulin antibodies cannot be put together in a vial.

[0041] According to some embodiments, the present disclosure provides a method for detecting epiregulin in a patient sample, the method comprising contacting the patient sample with a first antibody that binds to a first epitope region of epiregulin, contacting the patient sample with a second antibody that binds to a second epitope region of epiregulin and has a detectable label, and detecting a signal provided by the detectable label. In some embodiments, the patient sample is one of blood, serum, tissue lysate, or plasma. According to some more specific embodiments, the first epitope region of epiregulin partially overlaps with the second epitope region of epiregulin. Further, in some embodiments, the steps of contacting with the first and second antibodies occur simultaneously. In some specific embodiments, 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 LCVR and HCVR provided in Table 1.

[0042] According to some embodiments of the present disclosure, a method for quantifying epiregulin in a patient sample is provided. Such a method includes contacting the patient sample with a first antibody that binds to a first epitope region of epiregulin, contacting the patient sample with a second antibody that binds to a second epitope region of epiregulin and has a detectable label, detecting a signal provided by the detectable label, contacting a control standard with the first antibody that binds to the same first epitope region of epiregulin (as that used when contacting the patient sample), contacting the control standard with the second antibody that binds to the same second epitope region of epiregulin (as that used when contacting the patient sample) and has a detectable label, and detecting a signal provided by the detectable signal. In some embodiments, the patient sample is one of blood, serum or plasma, or a tissue lysate. According to some more specific embodiments, the first epitope region of epiregulin partially overlaps with the second epitope region of epiregulin. Further, in some embodiments, the steps of contacting with the first and second antibodies occur simultaneously. In some specific embodiments, the first antibody includes a combination of LC and HC CDRs provided in Table 1. In a further embodiment, the first antibody includes a combination of LCVR and HCVR provided in Table 1. In some specific embodiments, the second antibody includes a combination of LC and HC CDRs provided in Table 1. In a further embodiment, the second antibody includes a combination of LCVR and HCVR provided in Table 1.

[0043] According to some embodiments, a method for diagnosing an epiregulin-mediated disease or disorder is provided. Such a method includes contacting a patient sample with an anti-epiregulin antibody and detecting a binding between epiregulin and the antibody in the patient sample. According to some specific embodiments, the method for diagnosing includes diagnosing that the patient has, is at risk of having, requires treatment for, and / or is at risk of symptoms associated with an epiregulin-mediated disorder if the presence of epiregulin in the patient sample is detected as exceeding a reference value. According to some more specific embodiments, such a method includes a step of determining a reference value, which includes contacting a control standard with a first antibody that binds to a first epitope region of epiregulin that is the same as that used when contacting the patient sample, a step of contacting the control standard with a second antibody that has a detectable label and binds to a second epitope region of epiregulin that is the same as that used when contacting the patient sample, and a step of detecting a signal provided by the detectable signal. In some embodiments, the first antibody includes a combination of LC and HC CDRs provided in Table 1. In a further embodiment, the antibody includes a combination of LCVR and HCVR provided in Table 1. Some embodiments of the method for diagnosing an epiregulin-mediated disease provided herein further include contacting the patient sample with a second anti-epiregulin antibody that binds to a second epitope region of epiregulin and has a detectable label, and a step of detecting a signal provided by the detectable label. In some specific embodiments, the anti-epiregulin antibody includes a combination of LC and HC CDRs provided in Table 1. In a further embodiment, the antibody includes a combination of LCVR and HCVR provided in Table 1. According to a specific embodiment, the first epitope region of epiregulin partially overlaps with the second epitope region of epiregulin. According to a particular embodiment, the first and second antibodies cannot be put together in a vial. According to a further embodiment, the reference value is established from plasma, body fluid or tissue lysate of healthy volunteers and / or is as determined by one of ordinary skill in the art for an appropriate reference group and sample source.In a further embodiment, the pain disorder is one of osteoarthritis pain, diabetic peripheral neuropathy pain, or chronic low back pain.

[0044] In one embodiment, the present disclosure is a method for determining the level of human epiregulin in a body fluid sample, comprising: (a) contacting the body fluid sample with an anti-human epiregulin diagnostic monoclonal antibody or an antigen-binding fragment thereof that specifically binds to human epiregulin comprising an amino acid sequence as set forth in SEQ ID NO: 21, wherein the antibody or antigen-binding fragment thereof comprises a light chain complementarity-determining region LCDR1, LCDR2, and LCDR3 each comprising an amino acid sequence as set forth in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively, and a heavy chain complementarity-determining region HCDR1, HCDR2, and HCDR3 each comprising an amino acid sequence as set forth in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively; (b) optionally, removing any non-specifically bound monoclonal antibody or antigen-binding fragment thereof; and (c) detecting and / or quantifying the amount of the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human epiregulin. Preferably, the body fluid sample is a blood, serum or plasma sample, or a cerebrospinal fluid sample, and the contacting occurs ex vivo.

[0045] In embodiments of the present disclosure, the patient is a human diagnosed with a medical risk, condition, or disorder, such as one of the diseases or disorders described herein, who is in need of treatment with an antibody described herein. If the disorders that can be treated by the methods of the present invention are known by established, accepted classifications, such as osteoarthritis pain or painful diabetic neuropathy or chronic low back pain, those classifications can be found in various well-known medical books. For example, the 5th Edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) or similar books provide diagnostic tools for identifying specific disorders described herein (see, e.g., Scholz, et al., The IASP classification of chronic pain for ICD-11: chronic neuropathic pain. Pain. 2019 January; 160(1): 53-59, and Treede et al., Chronic pain as a symptom or a disease: the IASP Classification of Chronic Pain for the International Classification of Diseases (ICD-11), PAIN: 2019 January 160: 19-27). Also, the 10th Edition of the International Classification of Diseases (ICD-10) provides classifications for specific disorders described herein. Those skilled in the art will recognize that there are alternative nomenclatures, taxonomies, and classification systems for the diseases and disorders described herein, including those described in DSM-5 and ICD-10 or ICD-11, and that terms and classification systems evolve with the advancement of medical science.

[0046] Accordingly, the term "treating" (or "treat" or "treatment") is intended to refer to all processes by which the progression or severity of an existing symptom, disorder, condition or disease described herein can be delayed, interrupted, inhibited, controlled, alleviated, stopped, reduced or reversed, but is not necessarily intended to indicate complete elimination of all disorders or disease symptoms. Treatment includes administration of a protein or nucleic acid or vector or composition for the treatment of a disease, disorder or condition in a patient, particularly a human. Treatment includes administration of an antibody of the present disclosure for the treatment of a disease or disorder in a human who would benefit from a reduction in epiregulin activity, and the treatment provides for (a) inhibiting further progression of the disease, i.e., arresting its onset, (b) alleviating the disease, i.e., causing regression of the disease or disorder, or reducing its symptoms or complications, and / or (c) preventing onset of symptoms of the disease. Treatment as defined and used herein clearly includes reduction in the incidence of pain, remission of pain or one or more symptoms of pain, alleviation of pain or one or more symptoms of pain, delay in the onset of pain. In some situations, treatment also includes treatment of pain, but does not necessarily modify the underlying disease or condition that causes the pain. As used herein, "therapy-resistant pain" is defined as pain that is refractory to two or more conventional monotherapy and / or dual therapy treatment regimens.

[0047] As used herein, "reducing the incidence of" pain means either reducing the duration and / or frequency of pain (e.g., including delaying or increasing the time to pain symptoms in an individual). As will be understood by those skilled in the art, individuals can vary with respect to their responses to treatment. Treatment of pain also includes reducing the severity of pain, as well as reducing the need for and / or amount (e.g., exposure thereto) of other drugs and / or therapies commonly used in this condition (e.g., including opioids). "Remitting" pain or one or more symptoms of pain (such as osteoarthritis pain) means a decrease or improvement in one or more symptoms of pain as compared to the case where the antibody or composition of the present disclosure is not administered. "Remitting" also includes shortening or reducing the duration of symptoms. "Alleviating" pain or one or more symptoms of pain (such as osteoarthritis pain) means reducing the degree of one or more undesirable clinical signs of pain in an individual or population of individuals treated with the antibody or composition according to the present disclosure. As used herein, "delaying the onset of" pain means advancing, interfering with, delaying, decelerating, stabilizing and / or postponing the progression of pain such as osteoarthritis pain. This delay can be of various lengths depending on the medical history and / or the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay can in effect encompass prevention in that the individual does not develop pain. A method of "delaying" the onset of symptoms can be a method that reduces the likelihood of symptoms developing within a given time frame and / or reduces the degree of symptoms within a given time frame as compared to not using the method. Such comparisons are typically based on clinical studies using a statistically significant number of subjects.

[0048] "Effective amount" means the amount of the anti-human epiregulin antibody of the present disclosure, or a pharmaceutical composition comprising such an antibody, that will induce a biological or medical response of a tissue, system or human, or a desired therapeutic effect thereof, as required by the treating medical professional. As used herein, the term "effective response" of a patient, or responsiveness of a patient to treatment, refers to the clinical or therapeutic benefit conferred on the patient upon administration of an antibody of the present disclosure. The effective amount of an antibody can vary depending on factors such as the individual's medical condition, age, gender and weight, as well as the ability of the antibody to induce the desired response in the individual. Such desired responses include any one or more of a reduction in the level of chronic pain or an improvement in the signs or symptoms of a pain disorder. The effective amount can be readily determined by one of ordinary skill in the art by use of known techniques and by observing the results obtained under similar circumstances. The effective amount of the anti-human epiregulin antibody of the present disclosure can be administered as a single dose or as multiple doses. Further, the effective amount of the antibody of the present invention can be administered as multiple doses of an amount less than the effective amount if administered only once. In determining the effective amount for a patient, several factors are considered by the treating physician, including but not limited to the patient's size (e.g., weight or mass), body surface area, age and general health, the particular disease or disorder involved, the degree or involvement or severity of the disease or disorder, the response of the individual patient, the particular compound administered, the mode of administration, the bioavailability characteristics of the formulation administered, the dosing regimen selected, the use of concomitant medications, and other relevant circumstances known to the attending physician. The dose (including but not limited to subcutaneous, intramuscular and / or intravenous) can be from about 0.5 mg / kg to about 50 mg / kg. However, doses less than or greater than the doses mentioned herein are also contemplated, particularly in view of considerations of dosage known to those of ordinary skill in the art and / or described herein. The progress of a patient during treatment can be monitored by periodic evaluation and the dose adjusted accordingly as needed.

[0049] Possible advantages of the methods disclosed herein are that in patients suffering from chronic pain disorders, or with an acceptable safety profile including adverse events such as tolerance, toxicity, and / or anti-drug antibody responses, significant and / or long-term relief is provided, and thus the patient has the potential to benefit from the overall treatment method. More specifically, the antibodies of the present disclosure provide effective treatment while avoiding clinically undesirable adverse events such as skin rash (Li T, Perez-Soler R. Skin toxicities associated with epidermal growth factor receptor inhibitors. Target Oncol. 2009 Apr;4(2):107-19), and / or anti-drug antibody responses. The effectiveness of the treatment of the present disclosure can be measured by various endpoints commonly used when evaluating the treatment of various pain disorders. For example, other approaches for determining the effectiveness of any particular therapy of the present disclosure, including evaluations known to those skilled in the art, may be optionally employed.

Brief Description of the Drawings

[0050]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Example

[0051] The following examples are provided to illustrate the claimed invention and are not intended to limit it. The results of the following assays demonstrate that the exemplified monoclonal antibodies and / or their antigen-binding fragments of the present disclosure bind to and / or neutralize human epiregulin and can thus be used for the treatment of epiregulin-mediated disorders described herein.

[0052] Example 1: Generation, Expression, and Purification of Antibodies A panel of human anti-epiregulin antibodies is obtained by immunizing humanized mice with recombinant EREG (human and cynomolgus monkey) 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, and the resulting library is subjected to multiple rounds of selection while reducing antigen concentration, reducing the time to antigen association, and / or increasing the dissociation period to isolate clones with improved affinity. The sequences of the individual variants are determined and used to construct a combinatorial library, which is subjected to additional rounds of selection while increasing stringency to identify additive or synergistic pairings of mutations between individual CDR regions. The individual combinatorial clones are sequenced and their binding characteristics are determined. This screening is performed against human or mouse epiregulin to increase the affinity for one or more selected species (e.g., antibody 1 against human epiregulin). Counter-screening may be performed against other EGFR ligands to maintain selectivity after manipulation. The selected antibody can be mutagenized to repair post-translational modifications such as methionine oxidation while maintaining binding affinity for epiregulin. Additionally, framework (FW) and CDR substitutions can be made to the antibody to reduce the potential for immunogenic risk and return these sequences to their germline state.

[0053] An engineered and / or optimized anti-epiregulin antibody, referred to herein as antibody 1, is obtained, which has an amino acid sequence of the variable regions of the heavy and light chains, the complete heavy and light chain amino acid sequences, and a nucleotide sequence encoding the same as those listed in the section entitled "Sequence Listing of Amino Acids and Nucleotide Sequences" below. The sequence numbers corresponding to these sequences, as well as the CDR amino acid sequences of the light and heavy chains, are shown in Table 1.

[0054] Expression and purification: The exemplary anti-epiligrin antibodies of the present disclosure can be expressed and purified essentially as follows. Antibody 1 is expressed in a suitable host cell such as HEK293 or CHO, which is transfected either transiently or stably using an expression system for secreting Antibody 1 using an optimal predetermined HC:LC vector ratio, or a single vector system encoding both HC and LC. The expression plasmid contains cDNA versions of the LC and HC genes of Antibody 1 (e.g., the DNA sequence of SEQ ID NO: 11 encoding the HC of the exemplary Antibody 1 presented in Table 1, and the DNA sequence encoding the LC amino acid sequence according to Table 1, e.g., the DNA sequence of SEQ ID NO: 12 encoding the LC of the exemplary Antibody 1 presented in Table 1), and is expressed from commonly used and suitable constructs such as those based on the major immediate-early promoter of human cytomegalovirus.

[0055] The medium in which the antibody of the present invention is secreted can be purified by conventional techniques such as a mixed-mode method of ion exchange and hydrophobic interaction chromatography. For example, the medium can be applied to a protein A or protein G column and eluted therefrom using conventional methods, and a mixed-mode method of ion exchange and hydrophobic interaction chromatography can also be used. Soluble aggregates and multimers can be effectively removed by common techniques including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The product can be immediately frozen at, for example, -70°C, refrigerated, or lyophilized. Various methods of protein purification can be employed, such methods being known in the art and described, for example, in Deutscher, Methods in Enzymology 182:83-89 (1990), and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994). Antibody 1 can be immediately frozen at -70°C, stored at 2-8°C for several months, lyophilized, or stored at 4°C for immediate use. The amino acid sequence numbers of the exemplified human antibodies of the present invention are shown in Table 1.

[0056] Example 2: Characterization of Anti-Epiregulin Antibody Epiregulin reagent is prepared as a fusion protein, cleaved, and purified. The cleaved epiregulin is a mature soluble form containing extra amino acids compared to the native form, and this is used in in vitro binding and neutralization assays (SEQ ID NOs: 22-25). The human epiregulin (EREG) fusion (SEQ ID NO: 27) is also used in the binding assay (capture reagent), and the full-length epiregulin sequence with a single mutation that reduces shedding (SEQ ID NO: 26) is used in cell-based binding and effector function assays.

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

[0058] Sequence of antigen: Cleaved human epiregulin (SEQ ID NO: 22) GPGVSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVRCEHFFLG

[0059] Cleaved cynomolgus epiregulin (SEQ ID NO: 23) GPGVSITKCNSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVRCEHFYLG

[0060] Cleaved rat epiregulin (SEQ ID NO: 24) GPGVLITKCSSDMDGYCLHGHCIYLVDMSEKYCRCEVGYTGLRCEHFFLG

[0061] Cleaved rabbit epiregulin (SEQ ID NO: 25) GPGVSITKCGSDMNGYCLHGQCIYLVDMSENYCRCEVGYTGVRCEHFFLG

[0062] Full-length human epiregulin (membrane-bound with T111P mutation) (SEQ ID NO: 26) MTAGRRMEMLCAGRVPALLLCLGFHLLQAVLSTTVIPSCIPGESSDNCTALVQTEDNPRVAQVSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVRCEHFFLTVPQPLSKEYVALTVILIILFLITVVGSTYYFCRWYRNRKSKEPKKEYERVTSGDPELPQV

[0063] Monomeric Fc human Epiregulin (SEQ ID NO: 27) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVRCEHFFLG

[0064] The sequences of mature Epiregulin from various species are generally known in the art, and reference sequences are, for example, as follows: mature soluble human Epiregulin reference sequence (NP_001423.1 63-108), mature soluble cynomolgus monkey (also 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 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), EPGN (NP_001257918.1), AREG (NP_001648.1), BTC (NP_001720.1), EGF (NP_001954.2), HB-EGF (NP_001936.1).

[0065] Binding affinity for human epiregulin The solution-phase equilibrium binding affinity of antibody 1 for human (SEQ ID NO: 22), cynomolgus monkey (SEQ ID NO: 23), rat (SEQ ID NO: 24), and rabbit (SEQ ID NO: 25) epiregulin is measured at 37 °C by MSD solution equilibrium titration (MSD-SET) assay. (See, for example, Darling RJ, Brault P-A (2005) Kinetic Exclusion Assay Technology: Characterization of Molecular Interactions. Assay and Drug Development Technologies 2:647-657.)

[0066] The MSD plates are read using an MSD SI6000 instrument (Meso Scale Discovery, Rockville, MD). The MSD assay plates are prepared as follows. A multiarray 96-well plate (Meso Scale Discovery, P / N L15XA-3) is coated overnight at 2-8 °C with 30 μl of a 1 μg / ml solution of monomeric Fc-human epiregulin fusion in PBS. After coating, the plate is washed three times with PBST and then blocked for 1 hour at room temperature with shaking in 3% blocker A (diluted from 5% blocker A (Meso Scale Discovery, P / N R93AA-1) in PBS).

[0067] Prepare the SET sample in 1% blocker A at pH 6.0 and pH 7.4. Dilute the antibody to either 20 and 200 pM (pH 7.4) or 200 pM and 2 nM (pH 6.0). Dilute epiregulin serially in a total of 12 dilutions centered around approximately twice the antibody concentration. This is achieved by using starting concentrations of 10 and 100 nM (pH 7.4) or 100 nM and 1000 nM (pH 6.0), then diluting this 10-fold once, diluting serially 2-fold nine times, and then diluting 10-fold for the final dilution. Combine the epiregulin titration and fixed-concentration antibody solutions 1:1 to prepare the SET solution. Incubate the SET solution at 37 °C for approximately 96 hours to allow the binding to reach equilibrium.

[0068] Transfer 100 μl of the SET solution to duplicate columns of the prepared MSD plate and incubate at room temperature for 10 minutes to capture free antibody. After incubation, wash the plate three times with PBST, then add 100 μl of 1 μg / ml biotinylated goat anti-human kappa primary antibody (Southern Biotech, catalog 2060-08) in 1% blocker A to all wells. Incubate this on a plate shaker at room temperature for 60 minutes. Then, wash the plate three times with PBST, and then add 100 μl of 1 μg / ml SULFO streptavidin (Meso Scale Discovery, P / N R32AD-1) in 1% blocker A to all wells. Incubate this on a plate shaker at room temperature for 30 minutes. Then, wash the plate three times with PBST, and then add 100 μl of 1X Read Buffer T (diluted from 4X Read Buffer T (Meso Scale Discovery, P / N R93AA-1) with water) and then read the plate. The dissociation constant (K D ) and the least common multiplier (LCM) ligand correction factor are globally fitted to the equilibrium binding equation from the MSD-SET data using non-linear regression. This is done for each pair of fixed antibody concentrations at a given pH.

[0069] At pH 7.4, antibody 1 binds to human epiregulin with an affinity (K D ) of 14.9 pM, to cynomolgus monkey epiregulin with a K D of 26.8 pM, to rat epiregulin with a K D of 30.2 pM, and to rabbit epiregulin with a K D of 24.0 pM. At pH 6.0, antibody 1 binds to human epiregulin with a K D of 195 pM, to cynomolgus monkey epiregulin with a K D of 328 pM, to rat epiregulin with a K D of 286 pM, and to rabbit epiregulin with a K D of 330 pM. The pH selectivity of antibody 1 (K D at pH 6.0 / K D at pH 7.4) is 13.0 for human epiregulin, 12.2 for cynomolgus monkey epiregulin, 9.5 for rat epiregulin, and 13.7 for rabbit epiregulin. pH-dependent antigen binding can provide improvements in both pharmacokinetics and the duration of target neutralization (see Vincent, K.J. and M. Zurini (2012). “Current strategies in antibody engineering: Fc engineering and pH-dependent antigen binding, bispecific antibodies and antibody drug conjugates.” Biotechnol J 7(12):1444-1450), and antibody 1 and / or the antibodies of the present disclosure exhibit advantageous pH selectivity for epiregulin binding, suggesting that they provide useful pharmacokinetics and epiregulin neutralization properties.

[0070]

Table 3

[0071] Results are K DReport as the average. The error estimate is calculated as the standard deviation of independent replicates. The affinity of antibody 1 for human EREG at pH 7.4 is approximately 70-fold improved in affinity compared to LY3016859, which could result in a significantly lower dose of antibody 1 required to achieve equivalent EREG neutralization in vivo. The ability to use a lower dose of antibody 1 means that the required inhibition can be achieved with fewer antibodies, and as a result, this pharmaceutical product can be accepted for subcutaneous administration, which is clinically important for accessibility, tolerability, and compliance, compared to antibodies that require intravenous dosing. This is particularly advantageous in the context of chronic dosing as required for the chronic pain indication described herein.

[0072] Ligand specificity of antibody 1: The specificity of antibody binding to EGF family ligands can be tested by enzyme-linked immunosorbent assay (ELISA) and / or other common methods well known to those skilled in the art.

[0073] The ELISA reagent can be prepared as follows. The ligands tested are human epigen (R&D 6629-EP-025 / CF), epiregulin (prepared and purified as described in Example 2 herein and / or by methods known in the art (SEQ ID NO: 22)), amphiregulin (R&D 262-AR-100 / CF), betacellulin (R&D 261-CE-010 / CF), EGF (R&D 236-EG-200), HB-EGF (R&D 259-HE-050 / CF), and TGFα (R&D 239-A-100). The ligands are biotinylated with 10-fold excess biotin on ice for 2 hours using Thermo Pierce Sulfo-NHS-LC-biotin (A39257), and then frozen at -80°C in small aliquots for individual use.

[0074] The ELISA assay can be performed as follows. A Greiner 96-well high-binding plate (650061) is coated overnight at 4°C with 50 μl / well of NeutrAvidin (Thermo-Pierce 31000) at 1 μg / ml in PBS. The coating solution is removed the next day, and 100 μl / well of Pierce Blocker Casein (37528) is added and incubated at room temperature for at least 30 minutes to block the plate. After blocking, the plate is washed three times with PBS + 0.05% Tween20 to remove residual blocking buffer. The biotinylated ligand is diluted in a separate PCR plate (Eppendorf 951020443) at a constant concentration of 20 nM in Pierce Casein Blocker over 12 wells per ligand, and no antigen control is placed in the last column. The ligand is transferred to the blocked assay plate and incubated at room temperature for 1 hour. The assay plate is washed three times with PBS + 0.05% Tween20. The antibody to be tested is serially diluted at a 1:3 dilution across the plate (12 wells) starting at 5 μg / mL (LY3016859) or 10 μg / ml (antibody 1) in Pierce Casein Blocker, with a final volume of 50 μl / well, and incubated at room temperature for 1 hour. The assay plate is washed three times with PBS + 0.05% Tween20. The anti-human kappa-AP secondary antibody (Southern Biotech 2060-04) is added at 50 μl / well at a 1:2000 dilution in PBS + 0.05% Tween20 and incubated at room temperature for 1 hour. The assay plate is washed three times with PBS + 0.05% Tween20. Finally, 50 μl of pNPP substrate (Sigma-Aldrich N2765) is added and allowed to develop color until sufficient color is present, and read at 405 nm with a Spectramax Plus 384 plate reader by Molecular Devices.

[0075] Figure 1 shows the ELISA results of the ligand selectivity of antibody 1. Antibody 1 demonstrates selectivity for binding to human epiregulin, as determined in the above ligand specificity ELISA, but does not demonstrate appreciable binding to other EGF family ligands epigen, amphiregulin, betacellulin, EGF, HB-EGF, and TGFα. The selective and specific binding to epiregulin, rather than other EGF family ligands, indicates that antibody 1 can achieve the desired effect of blocking epiregulin signaling while avoiding the clinically undesirable effects of binding to other EGF family ligands such as TGFα or others. The lack of binding to TGFα may reduce the dose required to effectively inhibit epiregulin. This selectivity may avoid undesirable clinical adverse events such as skin rash that have been observed with pan-EGFR inhibitors.

[0076] Physicochemical properties: With respect to the therapeutic antibody product attributes, including chemical stability, solubility, and viscosity, antibody 1 exhibits a desirable combination of physicochemical properties for use as a human therapeutic.

[0077] Stability: The stability of Antibody 1 is evaluated at a high concentration (about 100 mg / ml) formulated in 5 mM histidine (pH 6.0) + 280 mM mannitol + 0.05% (w / v) polysorbate-80. Concentrated samples are incubated at 5 °C and 35 °C for a period of 4 weeks. After incubation, the samples are analyzed by size exclusion chromatography (SEC) for the percent high molecular weight (HMW%), by capillary electrophoresis (CE-SDS) for fragmentation, and by LC-MS peptide mapping for chemical modifications (e.g., deamidation, isomerization, or oxidation). After 4 weeks at 35 °C, Antibody 1 shows a 0.4% ΔHMW% (change in percentage of high molecular weight), a 0.6% Δfragment% (change in percentage of fragment), and no CDR chemical modification exceeding 0.5%.

[0078] The photo-stability under the same formulation conditions is evaluated by a total exposure of 40 watt-hours / m at 25 °C 2 to combined UV light and visible light at 240 klux. After incubation, the samples are analyzed by SEC for HMW% and by LC-MS peptide mapping for chemical modifications. Antibody 1 shows a 6.1% ΔHMW% measured by SEC compared to the unexposed control and no CDR chemical modification exceeding 0.7%.

[0079] The freeze / thaw stability under the same formulation conditions is evaluated using three repeated slow, controlled temperature cycles that mimic the freeze / thaw conditions of a large bulk of the drug substance placed at -70 °C. Antibody 1 shows a 1.0% ΔHMW% measured by SEC after three freeze-thaw cycles.

[0080] These results indicate that Antibody 1 has favorable physical and chemical stability sufficient to facilitate the development of solution formulations and use as a therapeutic agent.

[0081] Solubility: Solubility is evaluated by concentrating 100 mg of Antibody 1 to a volume of approximately 0.5 ml using a 30 kDa molecular weight cut-off centrifugal filter (e.g., Amicon U.C. filter, Millipore, catalog number UFC903024). The final concentration of the sample was measured by UV absorbance at 280 nm using a Solo VPE spectrophotometer (C Technologies, Inc).

[0082] Antibody 1 exhibits solubility of 175 mg / ml or more in 5 mM histidine pH 6 buffer and 168 mg / ml or more in PBS pH 7.4. No phase separation or cold precipitation is observed even after storage at 5 °C or -5 °C for one week. These results indicate that Antibody 1 exhibits sufficient solubility to enable high-dose administration.

[0083] Viscosity The viscosities of Antibody 1 and LY3016859 are analyzed at 15 °C at a concentration of approximately 125 mg / ml using VROC Initium (RheoSense). Antibody 1 exhibits a viscosity of 6.4 cP at 133 mg / ml in 5 mM histidine + 280 mM mannitol at pH 6, and LY3016859 exhibits a viscosity of 12.2 cP at 132 mg / mL in 5 mM histidine + 280 mM mannitol at pH 6. These results indicate that Antibody 1 exhibits a reduced viscosity compared to LY3016859 and a viscosity low enough to enable high-dose administration. The ability to use higher concentrations of Antibody 1, in combination with the need for lower doses, means that the required effective dose can be achieved in a smaller volume for delivery, which means that the drug product can be acceptable for subcutaneous administration, which is clinically important for accessibility, tolerability, and compliance compared to drugs that must be antibodies requiring intravenous dosing.

[0084] Example 3: In Vitro Functional Characterization of Anti-Epiregulin Antibody Neutralization of Epiregulin In Vitro: Neutralization of epiregulin activity by an antibody of the present disclosure, such as Antibody 1, can be evaluated by one or more of the epiregulin-induced cell-based response assays as described below.

[0085] The antibodies of the present disclosure are tested for their ability to neutralize epiregulin activity in two independent functional assays. Neutralization of epiregulin activity by Antibody 1 of the present disclosure can be evaluated by one or more cell-based activity assays that utilize the downstream signaling pathways of the EGFR family of receptors as described below. The mean half-maximal inhibitory concentration (IC 50 ) of the neutralization assays (C166-AP1-Luc and TGW-pERK) is calculated with SigmaPlot or GraphPad Prism. In GraphPad Prism, the IC 50 is calculated by performing a non-linear regression analysis of the transformed antibody concentration. For assays that are only performed in duplicates or triplicates across one plate, the IC 10 and the standard deviation (SD) are determined by calculating the SD from the IC 50 of each replicate and the mean IC 50 of the replicates. For the analysis of compounds across various plates in duplicates or triplicates, the IC 50 is calculated for each plate and the SD is determined across the plates. 50

[0086] Inhibition of epiregulin-induced response in the C166-AP1-luciferase functional assay: The ability of the antibodies of the present disclosure to neutralize human epiregulin-induced luciferase reporter activity can be evaluated in C166 cells (C166-AP1-Luc) that overexpress a luciferase reporter driven by AP1 activation. C166 cells are an endothelial-derived cell line that expresses all four EGFR families of receptors with the highest levels of EGFR expression. To test antibody 1 activity, C166-AP1-Luc cells are grown in growth medium [DMEM (Gibco 12430-047), 10% FBS (Gibco 10082147), Anti / Anti (Gibco 15240062) and 2 μg / ml Puro (1000X 2 mg / ml puromycin hydrochloride (Calbiochem, catalog number 540411)], dissociated in 0.05% trypsin-PBS, and seeded at 50,000 cells per ml in a tissue culture-treated 96-well plate at 50 μl per well in serum-free medium (growth medium without FBS). The cells are treated for 6 hours with 50 μl of epiregulin (human (SEQ ID NO: 22), cynomolgus monkey (SEQ ID NO: 23), rat (SEQ ID NO: 24) and rabbit (SEQ ID NO: 25) as described hereinabove) at a final concentration of 100 ng / ml (16 - 18.5 nM) and serial dilutions of the concentration of antibody 1. After incubation, the cells are lysed with 100 μl of Promega(™) One-Glo(™) Luciferase solution (Promega(™), catalog number E6120) for 3 minutes. Luminescence is read on a Perkin Elmer Wallace 1420 Victor2(™) microplate reader. The reduction in relative fluorescence units (RFU) shown in Table 4 below reflects the ability of antibody 1 to neutralize epiregulin activity in various related species tested, including human (SEQ ID NO: 22), cynomolgus monkey (SEQ ID NO: 23), rat (SEQ ID NO: 24) and rabbit (SEQ ID NO: 25). The IC 50 value for antibody 1 in the neutralization of human epiregulin is 8.7 nM, SD 2.3, n = 5 plates, 2 - 3 replicates / plate (Table 4). Neutralization across related species is for cynomolgus monkey (IC 50 2.1 nM, SD 0.08, n = 3 replicates), rat (IC50 2.5 nM, SD 0.27, n = 3 replicates), rabbit (IC 50 3.6 nM, SD 0.14, n = 3 replicates) as demonstrated by testing against. Assay validation of Antibody 1 and reduced IC when compared to LY3016859 (Antibody 1 of WO 2012 / 138510) 50 was compared and confirmed with the neutralization of human epiregulin in this assay using LY3016859 (Table 4). Human, rabbit, cynomolgus monkey and rabbit epiregulin used in these studies were generated as described hereinabove. LY3016895 neutralizes human epiregulin with an IC 50 , SD 11.7, n = 8 plates, 2 - 3 replicates / plate, cynomolgus monkey (IC 50 8.6 nM, SD 0.27, n = 2 plates, 2 - 3 replicates / plate), rat (IC 50 720 nM, SD 10, n = 2 plates, 2 - 3 replicates / plate) and rabbit (IC 50 11.0 nM, SD 1.15, n = 2 plates, 2 - 3 replicates / plate). Statistical validation of the epiregulin-induced C166-AP1-Luc assay determined that the MSR was 1.24, the 95% CI of MSR (MSR = minimum significant ratio) was 1.15, 1.43, and the potency analysis of Antibody 1 was 8.4 + / - 1.3. This assay demonstrates the in vitro neutralization of epiregulin-induced functional cellular responses by Antibody 1, the ability to inhibit the activity of epiregulin from various species, and the potential of the neutralizing effect of Antibody 1 against epiregulin in vivo.

[0087]

Table 4

[0088] For neutralization against human epiregulin, IC from replicates over 5 plates for Antibody 1 and 8 plates for LY3016859 50The mean and SD were determined by averaging. For neutralization against rat, cynomolgus monkey, and rabbit epiregulin, triplicate ICs from one plate for each species were 50 averaged and the SD for antibody 1 was determined. For neutralization against rat, cynomolgus monkey, and rabbit epiregulin, duplicate ICs across two different plates of LY3016859 were 50 averaged to generate the IC 50 .

[0089] The data in Table 4 demonstrate that antibody 1 can effectively neutralize human epiregulin-induced luciferase reporter activity (IC 50 = 8.7 nM), with an IC 50 approximately 3-fold lower than that of LY3016895 (IC 50 = 25.9 nM). Cross-reactivity against related species of epiregulin was confirmed by testing neutralization against rat (IC 50 = 2.5 nM), cynomolgus monkey (IC 50 = 2.1 nM), and rabbit (IC 50 = 3.6 nM) epiregulin. These data support the ability of antibody 1 to neutralize human epiregulin-mediated signaling and treat human diseases in which epiregulin-mediated signaling contributes to the etiology of pain disorders, and particularly OA, DPNP, and CLBP.

[0090] Selectivity of antibody 1 for epiregulin neutralization in the C166-AP1-Luc assay: The selectivity of antibody 1 against the neutralization of epiregulin can be further demonstrated by testing the activities of all seven EGFR ligands (epigen, TGFα, BTC, EGF, HB-EGF, epiregulin, and amphiregulin) in the C166-AP1-Luc assay (the ligands can be purchased, for example, from R&D systems, epiregulin number 1195-EP-CF, betacellulin number 261-CE-CF, EGF number 236-EG-CF, TGFα number 239-A-CF, HB-EGF number 259-HE-CF, epigen number 6629-EP-CF, and amphiregulin number 262-AR-CF). These seven EGFR ligands demonstrate AP1 activation with a 2- to 5-fold increase in luciferase activity depending on the ligand. The EC 50 of the EGFR ligand is determined, as described above herein, by performing a dose response of each ligand in the C166-AP1-Luc cell line. The ligand concentration for stimulation is determined by using the EC 50 -EC 80 (betacellulin 0.8 nM, EGF 1.3 nM, TGFα 2.5 nM, HB-EGF 0.52 nM, epiregulin 16 nM, epigen 333 nM, and amphiregulin 181 nM, epigen and amphiregulin were estimated EC50s due to the lack of a plateau on the curve). Antibody 1 has an IC 50Demonstrated the ability to neutralize epiregulin-induced luciferase activity in C166-AP1-Luc cells. In contrast, inhibition of betacellulin-, EGF-, TGFα-, HB-EGF-, epigen- or amphiregulin-induced luciferase activity was not demonstrated in the C166-AP1-Luc assay (see Table 5 below). The data in Table 5 also support the pharmacological selectivity of antibody 1, which specifically neutralizes the activity of epiregulin but not that of other family members, betacellulin, EGF, TGFα, HB-EGF, epigen, and amphiregulin. This selectivity is important in that antibody 1 selectively intervenes in the epiregulin response and thus avoids the potential for adverse effects that could be caused by less specific agents such as LY3016859, which can block other EGFR ligands.

[0091]

Table 5

[0092] Ability of antibody 1 to neutralize epiregulin-induced responses in the TGW neuroblastoma cell line: Epiregulin neutralization is further evaluated by measuring epiregulin-induced ERK phosphorylation in the TGW neuroblastoma cell line. TGW cells express all four members of the EGFR family of receptors, and EGFR expression is at the highest level. In this assay, TGW cells are grown on collagen 1-coated plates in growth medium [DMEM high glucose with NAPYR (Gibco #11995-065), 1X NEAA (Gibco #11140-050), Glutamax (Gibco #35050-061), and 1X Anti-Anti (Gibco #15240-062)]. Cells are dissociated in 0.05% trypsin-PBS and seeded at 100,000 cells / 100 μl in serum-free medium (growth medium with 0.1% BSA replacing FBS) in a 96-well collagen 1-coated 96-well plate (Corning #354407). In TGW cells, epiregulin treatment (80 ng / ml for 5 minutes) induces an increase in phosphorylation of ERK (Meso Scale Diagnostics, LLC, Phospho-ERK1 / 2 Whole Cell Lysate Kit #K151DWD) more than 5-fold compared to unstimulated cells. Phospho-ERK activation is determined by chemiluminescence induction and shown as relative fluorescence units (RFU). Neutralization of antibody 1 is determined by incubating human epiregulin with serially diluted antibody 1 for 20 minutes prior to cell stimulation. Data are obtained by reading the plate on a Meso Scale Diagnostics SECTOR Imager. Reduction of the epiregulin-induced phosphor-ERK1 / 2 signal by antibody 1 treatment is shown in Table 6, which reflects the ability of antibody 1 to neutralize human epiregulin. The IC 50 value for antibody 1 in neutralizing human epiregulin-induced pERK induction in TGW cells was observed to be 3.4 nM, SD 0.7 (n = 2 replicates), which is the IC 50is lower than LY3016859, which is 14.6 nM with an SD of 0.7 (n = 2 replicates). The data in Table 6 provide evidence of the ability of Antibody 1 to block human epiregulin-induced phospho-ERK1 / 2 activation in TGW cells. This data demonstrates the ability of the antibodies of the present disclosure, and particularly Antibody 1, to neutralize human epiregulin using two different cell lines with independent readouts, and supports the therapeutic use of these antibodies in the treatment of epiregulin-mediated disorders such as chronic pain disorders.

[0093]

Table 6

[0094] Example 4: In Vivo Functional Characterization of Epiregulin in Contact Allodynia Induced in CD-1 Mice The ability of epiregulin and TGFα, biologically active molecules, to induce contact allodynia can be evaluated in male CD-1 mice after plantar injection. The mice are acclimated in an animal facility with controlled temperature and light for at least 7 days before the experiment, with free access to water and food. On the test day, the animals are transported to the laboratory and allowed to acclimate in their home cages for 20 minutes, then placed in a transparent plastic chamber on an elevated metal mesh floor and allowed to acclimate for 1 hour. The hind paw withdrawal response to mechanical stimulation is determined by using a calibrated von Frey filament applied to the plantar surface of the hind paw from beneath the cage through the openings in the metal mesh floor. The measurements are achieved using the von Frey filament in an up-and-down test paradigm. The force range of the eight graded filaments is from 0.04 g to 6 g. The hind paw withdrawal threshold is measured and calculated. Based on the baseline values of the hind paw withdrawal threshold, the animals are randomized into dosing groups, which are PBS, 30 ng of epiregulin, 300 ng of epiregulin, 30 ng of TGFα, or 300 ng of TGFα. (Mouse epiregulin (SEQ ID NO: 28) and mouse TGFα (SEQ ID NO: 29) reagents were prepared as described herein, or for TGFα, prepared as purified by His-tagging and standard chromatographic techniques (immobilized metal ion affinity chromatography or IMAC, followed by size exclusion chromatography or SEC). The animals then receive an intraplantar injection into the hind paw of a 20 μl solution of PBS, or fresh diluted epiregulin or TGFα. The hind paw withdrawal threshold is evaluated at 2 hours and 4 hours after injection. The data are shown in Table 7 below.

[0095]

Table 7

[0096] The effects of epiregulin compared to TGFα are shown in Table 7 above, indicating that in this pain model, epiregulin induces a pain response as shown by a reduction in the withdrawal threshold, while TGFα does not. Antibody 1 is considered to embody a more selective and specific pain therapeutic compared to LY3016859, which recognizes both epiregulin and TGFα. The lack of binding to TGFα by antibody 1 of the present disclosure is considered advantageous with respect to avoiding off-target effects, reducing immunogenicity, and improving or reducing the dosage required for therapeutic use.

[0097] Example 5: Characterization of the potential immunogenicity of Antibody 1 Antibody 1 is an improved therapeutic antibody in several combined aspects, including enhanced affinity for human epiregulin, improved specificity for epiregulin compared to other EGFR ligands, improved biophysical properties compared to LY3016859, and a fully human sequence (the latter of which is thought to provide a potential for reduced immunogenicity).

[0098] Dendritic Cell (DC) internalization assay Monocyte-derived DC Culturing (MDDC) According to the standard protocol, CD14+ monocytes are isolated from peripheral blood mononuclear cells (PBMCs), cultured, and differentiated into DCs. Briefly, PBMCs are isolated from LRS-WBCs using density gradient centrifugation with Ficoll (#17-1440-02, GE Healthcare) and Sepmate 50 (#15450, STEMCELL Technologies). CD14+ monocytes are isolated using positive selection with a CD14+ microbead kit (#130-050-201, Miltenyi Biotec) according to the manufacturer's manual. Then, the cells are cultured at 1 million / ml for 6 days with 1000 units / ml of GM-CSF and 600 units / ml of IL-4, adding L-glutamine and 25 mM of HEPES, and supplemented with 10% FBS, 1 mM of sodium pyruvate, 1× penicillin-streptomycin, 1× non-essential amino acids, and 55 μM of 2-mercaptoethanol in RPMI medium (hereinafter referred to as complete RPMI medium or medium purchased from Life Technologies) to induce immature dendritic cells (MDDCs). The medium is changed twice on days 2 and 5. On day 6, the cells are gently collected with a cell scraper and used for the experiment. MDDCs are visually characterized by dendritic morphology under a microscope and the expression of CD14, CD11c, and HLA-DR by flow cytometry. The ability to respond to LPS treatment is confirmed by measuring the increase in CD80, CD83, and CD86 using flow cytometry.

[0099] Binding of Fab-TAMRA-QSY7 The F(ab’)2 fragment goat anti-human IgG (Jackson ImmunoResearch) is dual-labeled with QSY7-NHS and TAMRA-SE (Molecular Probes) to obtain Fab-TAMRA-QSY7, which is used as a universal probe for tracking the internalization of test substances. Each vial of F(ab’)2 (about 1 ml at 1.3 mg / ml) is concentrated to about 2 mg / ml by centrifuging at 14,000 rcf for 2 minutes using an Amico Ultra-0.5 centrifugal filter device (#UFC501096, Millipore). The pH is adjusted to basic (>pH 8) with 10% (v / v) 1 M sodium bicarbonate, 6.8 μl of a 10 mM QSY-NHS stock solution in DMSO is added, and mixed. The reaction vial is kept in the dark at room temperature for 30 minutes. The intermediate product, Fab-QSY7, is purified by centrifuging at a relative centrifugal force (RCF) of 1000 for 2 minutes using a Zeba Spin desalting column (#89890, Thermo Scientific). The concentration and degree of labeling (DOL) are calculated by measuring the absorbance at 280 nm and 560 nm with a NanoDrop (ThermoFisher). Then, Fab-QSY7 is concentrated to about 2 mg / ml by centrifuging at 14,000 rcf for 2 minutes again using the Amico Ultra-0.5 centrifugal filter device. After adjusting the pH with 10% (v / v) 1 M sodium bicarbonate, 4.3 μl of a 15 mM TAMRA-SE stock solution in DMSO is added and mixed. After 30 minutes in the dark at room temperature, the final product, Fab-TAMRA-QSY7, is purified and collected using a Zeba Spin desalting column by centrifuging at 1000 rcf for 2 minutes. The concentration and DOL are quantified again by reading the absorbance at 280 nm, 555 nm, and 560 nm with a NanoDrop spectrophotometer. Using this protocol, about 300 μl of Fab-TAMRA-QSY7 at about 1.5 mg / ml containing about 2 QSY7s and 2 TAMRAs per F(ab’)2 is obtained.

[0100] Standardized internalization assay by FACS Normalize each test molecule to 1 mg / ml in PBS and then further dilute it to 8 μg / ml in complete RPMI medium. Dilute Fab-TAMRA-QSY7 to 5.33 μg / ml in complete RPMI medium. Mix the antibody and Fab-TAMRA-QSY7 in equal amounts and incubate for 30 minutes at 4 °C in the dark for complex formation. Resuspend MDDC in complete RPMI medium at 4 million / ml and seed 50 μl per well into a 96-well round-bottom plate supplemented with 50 μl of the antibody / probe complex. Incubate the cells in a CO2 incubator at 37 °C for 24 hours. Wash the cells with 2% FBS PBS and resuspend them in 100 μl of 2% FBS PBS containing the Cytox Green live / dead dye. Collect data on a BD LSR Fortessa X-20 and analyze it with FlowJo. Gate on live single cells and record the percentage of TAMRA-fluorescent positive cells as the readout.

[0101] Data Presentation and Statistical Analysis Molecules are tested in duplicates or triplicates against three or more donors. For each donor, consider the percentage of the TAMRA-positive population. To enable comparison of molecules with data from different donors, the normalized internalization index (NII) is used. The internalization signal is normalized against the IgG1 isotype (NII = 0) and the internal positive control PC (NII = 100) using the following formula.

[0102]

Equation

[0103]

Table 8

[0104] (See, for example, Wen, Y., Cahya, S., Zeng, W. et al. Development of a FRET-Based Assay for Analysis of mAbs Internalization and Processing by Dendritic Cells in Preclinical Immunogenicity Risk Assessment. AAPS J 22, 68 (2020)).

[0105] MAPP assay (MHC-related peptide proteomics) method: Primary human dendritic cells from 10 normal human donors were prepared from buffy coats by isolation of CD-14 positive cells as described, and differentiated into immature dendritic cells by incubation at 37 °C, 5% CO2 for 3 days with 20 ng / ml IL-4 and 40 ng / ml GM-CSF in complete RPMI medium containing 5% serum replacement (Thermo Fisher Scientific, catalog number A2596101) (Knierman et al., "The Human Leukocyte Antigen Class II Immunopeptidome of the SARS-CoV-2 Spike Glycoprotein", Cell Reports, 33, 108454 (2020)). On day 4, 3 micromolar of the test antibody was added to approximately 5×10 6 cells, and after 5 hours of incubation, the fresh medium containing 5 mg / ml of LPS was replaced to transform the cells into mature dendritic cells. The next day, the mature cells were lysed in 1 ml of RIPA buffer containing protease inhibitor and DNAse. The lysate was stored at -80 °C until sample analysis.

[0106] Using an automated liquid handling system, isolate HLA-II molecules from thawed lysates using biotinylated anti-pan HLA class II antibody (clone Tu39). Elute the bound receptor-peptide complex with 5% acetic acid, 0.1% TFA. Pass the eluted MHC-II peptides through a pre-washed 10k MWCO filter to remove high molecular weight proteins. The isolated MHC-II peptides are analyzed by nano LC / MS using a Thermo easy 1200 nLC-HPLC system equipped with a Thermo LUMOS mass spectrometer. For this separation, a 75 μm × 7 cm YMC-ODS C18 column was used, with a flow rate of 250 nL / min, and a gradient of 65 minutes with 0.1% aqueous formic acid as solvent A and 80% acetonitrile with 0.1% formic acid as solvent B. Mass spectrometry was performed in full scan mode with a resolution of 240,000, followed by a 3-second data-dependent MS / MS cycle consisting of ion trap type rapid scan with HCD and EThcD fragmentation.

[0107] Peptide identification is generated by an in-house proteomics pipeline (Higgs et al., "Label-free LC-MS method for the identification of biomarkers", Methods in Molecular Biology, 428, 209-230 (2008)) using multiple search algorithms without enzymatic search parameters against a bovine / human database containing the test antibody sequence. Process the sample identification files using a KNIME workflow. Align the peptides identified from the test substance to the parental sequence. Create a summary of all donors annotating the percentage of donors presenting non-germline residues, the number of different regions presenting peptides with non-germline residues, and the depth of peptide presentation in each region with non-germline residues. An increase in the degree of presentation of non-germline peptides is associated with an increased risk of immunogenicity. The results in Table 8 show a decrease in the presentation of non-germline peptides for Antibody 1, and thus a decrease in the risk of immunogenicity for Antibody 1, when compared to LY3016859.

[0108]

Table 9

[0109] Example 6. Demonstration of in vivo binding to membrane epiregulin after dosing in rats. Epiregulin exists both in a membrane-bound form and in a soluble form generated by membrane cleavage by ADAM (A disintegrin and metalloprotease) proteases. The ability of the antibodies of the present disclosure to bind to membrane-bound epiregulin in vivo, for example, after peripheral dosing by subcutaneous injection, can be evaluated by isolating tissues ex vivo and subsequently applying standard immunofluorescence staining methods described herein and / or known to those of skill in the art. Positive and specific immunofluorescent labeling of the epithelial layer of the rat tongue can be demonstrated using Antibody 1.

[0110] Rat tongues were obtained from euthanized male Sprague Dawley rats (body weight 140 - 215 grams) after various doses (0.1 - 100 mg / kg, subcutaneous) of the antibody being tested containing antibody 1, or control or comparative antibodies, at various times after dosing (3 - 28 days after dosing), and can be rapidly frozen on dry ice. Frozen coronal 20 - micron sections of tongue tissue were thawed, washed in PBS for 3 minutes, and fixed in 4% paraformaldehyde for 10 minutes. The slides were washed in Super Sensitive Wash Buffer (HK583 - 5K, Biogenex), and then blocked with Power block (HK065 - 5K, Biogenex) + 0.1% Triton x - 100 for 10 minutes. The slides were then washed and incubated at room temperature for 15 minutes with Alexa Fluor 488 goat anti - human IgG (A11013, Life Technologies, 13.3 μg / ml), and a labeled human / mouse IgG1 chimeric antibody herein referred to as "antibody 1 VR chimera" (SEQ ID NOs: 64 and 65) having the variable regions of antibody 1 (SEQ ID NOs: 3 and 4) and mouse constant regions (DyLight 650 labeled). The slides were washed three times and Prolong Gold anti - fade reagent was applied in front of the coverslip. Fluorescent images at 488 and 650 nm wavelengths from each animal were taken using a Keyence BZ800, using the same exposure settings for all images. The green or red signals within the positively stained epithelial layer were quantified for average intensity by taking four random target regions using ImageJ - Win64 software. The green signal results from bound antibody 1, or control antibody, or reference comparative antibody (dosed in vivo), and the red signal is unbound epiregulin, and the detection antibody competes with the bound drug and stains only unbound free epiregulin. The data obtained were normalized by using the ratio of the Alexa Fluor 488 goat anti - human IgG signal (green) to the remaining antibody 1 VR chimera signal (red).

[0111] Figure 2 shows that a significant dose-dependent increase in the binding of membrane epiregulin is observed in vivo 3 days after dosing with antibody 1 at a dose of 1 mg / kg or higher. Figure 3 shows that there is persistent binding of membrane epiregulin in vivo for at least 14 days after a single administration of 10 mg / kg of antibody 1.

[0112] Figure 4 demonstrates that for antibody 1, the extent of membrane epiregulin bound in vivo is higher compared to a comparative antibody with an epiregulin binding potency of less than 1 / 50. Figure 4 shows that the improved affinity of antibody 1 also leads to an improvement in the binding of membrane-bound epiregulin ligand in vivo.

[0113] Example 7: Epitope Mapping of Antibody 1 Antibody 1 is a human IgG4 antibody that selectively binds to and neutralizes the EGFR ligand family member epiregulin (EREG) with high affinity and selectivity for other EGFR ligand family members, as well as favorable epiregulin neutralizing activity and pharmacokinetic properties. These properties, together with the additional stability and viscosity and solubility properties described herein, result in an antibody with improved attributes and a combination of a favorable therapeutic agent that neutralizes EREG activity. Epitope mapping studies were performed to determine the specific amino acids in human EREG required for the binding and specificity of antibody 1. Key amino acids were identified from the crystal structure of the antibody 1 Fab / EREG complex, and the contributions of these amino acids were further characterized through mutagenesis and binding studies. The contributions of these amino acids provide the structural basis for the specificity of antibody 1 for EREG compared to other EGFR ligand family members. These results provide information about 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 cross-referenced as follows: 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: 56), mono Fc human EREG H78N (SEQ ID NO: 55), mono Fc human EREG H78A (SEQ ID NO: 54), mono Fc human EREG F106K (SEQ ID NO: 61), mono Fc human EREG F106A (SEQ ID NO: 60), mono Fc human EREG Y98A (SEQ ID NO: 58), mono Fc human EREG L77F (SEQ ID NO: 53), mono Fc human EREG R102A (SEQ ID NO: 59).

[0115] Both the antibody 1 Fab fragment and human EREG were produced in transient CHO expression and purified by standard techniques. Complexes were prepared by adding a 30% molar excess of EREG to antibody 1 Fab and then purified by size exclusion chromatography to remove excess free EREG. Antibody 1 Fab binding to EREG was first probed by Western blot analysis, showing that antibody 1 Fab can recognize denatured, non-reduced EREG but not denatured, reduced EREG in this assay. This strongly suggests a conformational epitope that depends on the EREG disulfide bond.

[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 complex was crystallized and the 1.8 Å structure was solved by molecular replacement using Phaser 2.8.3 and then refined with Refmac 5.8.0258. In the crystal, antibody 1 Fab’ binds to the opposite face of the epiregulin dimer. The side chains of epiregulin Leu77 and His78 are at the center of the epitope, Leu77 is buried in a hydrophobic pocket, and the His78 side chain is at the center of a hydrogen bond network. The side chain of antibody 1 heavy chain Arg50 is a hydrogen bond donor to the hydroxyl of heavy chain Tyr52, and the hydroxyl of heavy chain Tyr52 is a hydrogen bond donor to the deprotonated His78 side chain N δ1 is a hydrogen bond donor to the hydrogen bond acceptor of the protonated His78 side chain N ε2 is a hydrogen bond donor to a water molecule, which is a hydrogen donor to the backbone carbonyls of heavy chain Leu109 and light chain Tyr107. Antibody 1 residues are numbered according to the IMGT convention. Additional identified epitope contacts include the side chains of Glu95, Tyr98, Arg102 and Phe106, and the backbones of Leu77, Val96 and Glu104. The structure clearly demonstrates that the antibody 1 epitope is conformational and not linear, confirming the above Western results.

[0117] Mutant epiregulin ELISA using antibody 1 Fab To understand their relative contributions to both binding and selectivity, key epitope residues were further characterized by site-directed mutagenesis. The mono-Fc EREG fusion (see above) was diluted to 1 μg / mL in PBS and 100 μL was added to each well to coat ELISA plates (Greiner catalog number 655061) overnight at 5 °C. Variants were grouped by columns on the plate, with the wild type coated on three replicate columns and the point mutants coated on single replicate columns. After coating, the plates were washed three times per well with 200 μL of PBST and then blocked for 1 hour at room temperature on a plate shaker using casein blocking buffer (Thermo catalog number 37528). After blocking, the plates were washed as before. Antibody 1 Fab fragment was diluted to 1 μg / mL in casein blocking buffer and then serially diluted three-fold in blocking buffer for a total of seven concentrations, with blocking buffer as the blank. The Fab dilution series was added to the prepared ELISA plates at 100 μL per well and incubated for 1 hour at room temperature on a plate shaker. The plates were washed as before and then 100 μL of goat anti-human kappa HRP secondary antibody (Southern Biotech catalog number 2060-05) diluted 1:8,000 in blocking buffer was added to each well. The plates were incubated with the secondary antibody for 1 hour at room temperature on a plate shaker and then washed as before. TMB substrate was prepared (catalog number 34021) and 100 μL was added to all wells. The plates were incubated statically at room temperature for 3 - 5 minutes and then 100 μL of 1N HCl was added to all wells to stop the reaction. The plates were read at 450 nm using a plate reader. ELISA data subtracted for the blank was plotted in GraphPad Prism 9 with antibody concentration on the X-axis and OD450 on the Y-axis. A curve was generated by fitting the data to a "sigmoidal, 4PL, X is concentration" model using non-linear regression.

[0118] Point mutants were evaluated by ELISA using epiregulin mutants coated on plates. An antibody 1 Fab dilution series was added, and the signal was measured with an anti-kappa secondary (see Figure 5). All mutants, except Y98A, showed decreased binding. H78A nearly knocked out binding, and Y98A showed no detectable binding at the concentrations tested.

[0119] SPR analysis of antibody 1 Fab fragment binding to monomeric Fc human EREG mutants Using a Biacore 8K instrument and reagents (Cytiva), SPR analysis of antibody 1 Fab fragments binding to both monomeric Fc human EREG mutants was performed. Low levels of monomeric Fc human EREG mutants were immobilized in the sample flow cell (Fc2) of a CM5 S series sensor chip (Cytiva P / N BR100530) using an amine coupling kit (Cytiva P / N BR100050) and the “low level CM5 coupling” method in Biacore 8K control software. The running buffer was either 1X HBS-EP+ pH 7.4 (prepared from 20X HBS-EP+, Teknova P / N H8022) or 1X MBS-EP+ pH 6.0 (10 mM MES + 150 mM NaCl + 3 mM EDTA + 0.05% Tween20), and the running temperature was 37 °C. Biacore experiments were performed in three independent experiments using a single replicate of each dilution within each independent experiment. SPR data, subtracted for reference and blank, were analyzed using Biacore Insight Evaluation Version 3.0 (Cytiva) using either a “1:1 binding” kinetic model (EREG variant) or a “steady state affinity” model to determine the KD value.

[0120] Antibody 1 Fab was diluted to 300 nM (pH 7.4) or 900 nM (pH 6.0) in running buffer and then serially diluted three-fold to a total of seven dilutions. The Fab was injected into all flow cells for 240 seconds, followed by dissociation for 1800 seconds at a flow rate of 50 μL / min. The chip surface was regenerated by injecting 7 M guanidine at 100 μL / min for 2 x 30 seconds. Data subtracted for reference was collected as sample flow cell minus reference flow cell (Fc2 - Fc1) in each of the eight channels, and then the data subtracted for reference was further subtracted for the buffer blank.

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

[0122]

Table 10

[0123] The results in Table 9 show various weaker affinities for mutants at pH 7.4, ranging from 3-fold for R102A to over 1000-fold for F106K.

[0124] Human EGFR ligand epitope alignment In the context of the EGFR ligand sequence alignment (Figure 6), this data indicates that E95 and F106 are important epitope positions for EREG selectivity. The positions of Y98 and R102, which are 100% conserved, are important for binding but do not contribute to selectivity. L77 and H78 are also important for binding and affect selectivity, but they are highly conserved across EGFR ligands and are thus not EREG-specific. These results not only demonstrate the structural basis for the binding and excellent selectivity of antibody 1 for EREG, but also provide information on ligands and species selectivity from epitope mapping studies.

[0125] Sequence Listing of Amino Acid and Nucleotide Sequences Heavy Chain of Antibody 1 (SEQ ID NO: 1) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPAGKGLEWIGRIYPSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGLVMDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0126] Light Chain of Antibody 1 (SEQ ID NO: 2) EIVLTQSPGTLSLSPGERATLSCRASQSVEFSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQYGTNPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0127] HCVR of Antibody 1 (SEQ ID NO: 3) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPAGKGLEWIGRIYPSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGLVMDVWGQGTLVTVSS

[0128] LCVR of Antibody 1 (SEQ ID NO: 4) EIVLTQSPGTLSLSPGERATLSCRASQSVEFSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQYGTNPFTFGQGTKVEIK

[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)

[0136] DNA encoding the light chain of antibody 1 (SEQ ID NO: 12) GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGTCTGTGGAATTCAGCTACTTAGCCTATGGTGCATCCAGCAGGGCCACTTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCACCAGTACGGAACAAACCCGTTCACATTCGGGCAGGGAACCAAGGTTGAAATAAAGCGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC

[0137] Human Epiregulin (SEQ ID NO: 21) VSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVRCEHFFL

[0138] Human Epiregulin construct (SEQ ID NO: 22) GPGVSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVRCEHFFLG

[0139] Cynomolgus Epiregulin (SEQ ID NO: 23) VSITKCNSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVRCEHFYL

[0140] Rat Epiregulin (SEQ ID NO: 24) VLITKCSSDMDGYCLHGHCIYLVDMSEKYCRCEVGYTGLRCEHFFL

[0141] Rabbit Epiregulin (SEQ ID NO: 25) VSITKCGSDMNGYCLHGQCIYLVDMSENYCRCEVGYTGVRCEHFFL

[0142] Full-length Human Epiregulin (membrane-bound, with T111P mutation) (SEQ ID NO: 26) MTAGRRMEMLCAGRVPALLLCLGFHLLQAVLSTTVIPSCIPGESSDNCTALVQTEDNPRVAQVSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVRCEHFFLTVPQPLSKEYVALTVILIILFLITVVGSTYYFCRWYRNRKSKEPKKEYERVTSGDPELPQV

[0143] Monomeric Fc Human Epiregulin (SEQ ID NO: 27) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVRCEHFFLG

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

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

[0146] HCDR1 of Antibody 1 (Kabat) (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 of Antibody 1 (Kabat) (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 of Antibody 1 (IMGT) (SEQ ID NO: 43) GGSISSYY

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

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

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

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

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

[0164] IgG4PAA hinge region (SEQ ID NO: 51) ESKYGPPCPPCP

[0165] IgG4PAA Fc region (SEQ ID NO: 52) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0166] Mono Fc huEREG L77F (SEQ ID NO: 53) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSITKCSSDMNGYCFHGQCIYLVDMSQNYCRCEVGYTGVRCEHFFLG

[0167] Mono Fc huEREG H78A (SEQ ID NO: 54) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSITKCSSDMNGYCLAGQCIYLVDMSQNYCRCEVGYTGVRCEHFFLG

[0168] Mono Fc huEREG H78N (SEQ ID NO: 55) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSITKCSSDMNGYCLNGQCIYLVDMSQNYCRCEVGYTGVRCEHFFLG

[0169] Mono Fc huEREG E95A (SEQ ID NO: 56) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCAVGYTGVRCEHFFLG

[0170] Mono Fc huEREG E95H (SEQ ID NO: 57) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCHVGYTGVRCEHFFLG

[0171] Mono Fc huEREG Y98A (SEQ ID NO: 58) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGATGVRCEHFFLG

[0172] Mono Fc huEREG R102A (SEQ ID NO: 59) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVACEHFFLG

[0173] Mono Fc huEREG F106A (SEQ ID NO: 60) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVRCEHAFLG

[0174] Mono Fc huEREG F106K (SEQ ID NO: 61) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVRCEHAKLG

[0175] Antibody 1 Fab HC (SEQ ID NO: 62) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPAGKGLEWIGRIYPSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGLVMDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK

[0176] Antibody 1 Fab LC (SEQ ID NO: 63) EIVLTQSPGTLSLSPGERATLSCRASQSVEFSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQYGTNPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTQGTTSVTKSFNRGEC

[0177] Antibody 1 VR chimeric h / mIgG1 HC (SEQ ID NO: 64) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPAGKGLEWIGRIYPSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGLVMDVWGQGTLVTVSSASTKGPSVFPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0178] Antibody 1 VR chimeric h / m kappa LC (SEQ ID NO: 65) EIVLTQSPGTLSLSPGERATLSCRASQSVEFSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQYGTNPFTFGQGTKVEIKRTVAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0179] Human epigen / EPGN (SEQ ID NO: 66) KFSHLCLEDHNSYCINGACAFHHELEKAICRCFTGYTGERCEHLTLT

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

[0181] Human Heparin-Binding EGF / HBEGF (SEQ ID NO: 68) KKRDPCLRKYKDFCIHGECKYVKELRAPSCICHPGYHGERCHGLSL

[0182] Human Betacellulin / BTC (SEQ ID NO: 69) GHFSRCPKQYKHYCIKGRCRFVVAEQTPSCVCDEGYIGARCERVDLFY

[0183] Human Transforming Growth Factor Alpha / TGFα (SEQ ID NO: 70) VVSHFNDCPDSHTQFCFHGTCRFLVQEDKPACVCHSGYVGARCEHADLLA

[0184] Human Epidermal Growth Factor / EGF (SEQ ID NO: 71) NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKW

Claims

1. An antibody that binds to human epiregulin, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, and the VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises SEQ ID NO: 5, HCDR2 comprises SEQ ID NO: 6, HCDR3 comprises SEQ ID NO: 7, LCDR1 comprises SEQ ID NO: 8, LCDR2 comprises SEQ ID NO: 9, LCDR3 comprises SEQ ID NO: 10, an antibody.

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

4.

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

2.

4. A nucleic acid comprising the sequences of SEQ ID NOs: 11 and 12.

5. A vector comprising the nucleic acid according to claim 4.

6. The vector according to claim 5, wherein the vector comprises a first nucleic acid sequence of SEQ ID NO: 11 and a second nucleic acid sequence of SEQ ID NO:

12.

7. A composition comprising 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.

8. A cell comprising the vector according to claim 5 or 6.

9. A cell comprising 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.

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

11. A process for producing an antibody, comprising culturing the cell according to any one of claims 8 to 10 under conditions such that the antibody is expressed, and recovering the expressed antibody from the culture medium.

12. An antibody produced by the process according to claim 11.

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

14. A pharmaceutical composition for the treatment of a pain disorder, comprising a therapeutically effective amount of an antibody according to any one of claims 1 to 3 or 12 or a pharmaceutical composition according to claim 13.

15. The pharmaceutical composition according to claim 14, wherein the pain disorder is selected from the group consisting of osteoarthritis pain, diabetic peripheral neuropathy pain and chronic low back pain.

16. The pharmaceutical composition according to claim 15, wherein the pain disorder is osteoarthritis pain.

17. The pharmaceutical composition according to claim 15, wherein the pain disorder is diabetic peripheral neuropathy pain.

18. The pharmaceutical composition according to claim 15, wherein the pain disorder is chronic low back pain.

19. The pharmaceutical composition according to claim 15, wherein the pain disorder is therapy-resistant.

20. A medicament for use in therapy, comprising an antibody according to any one of claims 1 to 3 or 12.

21. A medicament for use in the treatment of a pain disorder, comprising an antibody according to any one of claims 1 to 3 or 12.

22. The medicament according to claim 21, wherein the pain disorder is selected from the group consisting of osteoarthritis pain, diabetic peripheral neuropathy pain, and chronic low back pain.

23. The medicament according to claim 21, wherein the pain disorder is osteoarthritis pain.

24. The medicament according to claim 21, wherein the pain disorder is diabetic peripheral neuropathy pain.

25. The medicament according to claim 21, wherein the pain disorder is chronic low back pain.

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

27. The use according to claim 26, wherein the pain disorder is selected from the group consisting of osteoarthritis pain, diabetic peripheral neuropathy pain, and chronic low back pain.

28. A method for determining the human epiregulin level in a body fluid sample, comprising: (a) contacting the body fluid sample with the antibody according to any one of claims 1 to 3 or an antigen-binding fragment thereof; (b) optionally removing any monoclonal antibody or antigen-binding fragment thereof that binds non-specifically; (c) detecting and / or quantifying the amount of a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human epiregulin.

29. The method according to claim 28, wherein the body fluid sample is a blood, serum or plasma sample, or a cerebrospinal fluid sample, and the contacting occurs ex vivo.

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