COMPOUNDS AND METHODS TARGETING EPIREGULIN

Abstract

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

Claims

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

2. The antibody of claim 1, wherein the VH comprises SEQ ID NO: 3 and the VL comprises SEQ ID NO: 4.

3. The antibody of claim 1, 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 sequence of SEQ ID NO: 11 or 12.

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

6. The vector of 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 a nucleic acid sequence of SEQ ID NO: 11, and a second vector comprising a nucleic acid sequence of SEQ ID NO: 12.

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

9. A cell comprising a first vector comprising a nucleic acid sequence of SEQ ID NO: 11, and a second vector comprising a nucleic acid sequence of SEQ ID NO: 12.

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

11. A process of producing an antibody comprising culturing the cell of claim 8 under conditions such that the antibody is expressed and recovering the expressed antibody from the culture medium.

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

13. A pharmaceutical composition comprising the antibody of any one of claim 1-3, or 12, and a pharmaceutically acceptable excipient, diluent or carrier.

14. A method of treating a pain disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody of any one of claim 1-3, or 12.

15. A method of treating a pain disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 13.

16. The method of claim 14, wherein the pain disorder is selected from the group consisting of osteoarthritis pain, diabetic peripheral neuropathy pain, and chronic low back pain.

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

18. The method of claim 16, wherein the pain disorder is diabetic peripheral neuropathy pain.

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

20. The method of claim 16, wherein the pain disorder is therapy resistant.

21. A method of determining the human epiregulin level in a bodily fluid sample comprising: (a) contacting the bodily fluid sample with an anti-human epiregulin diagnostic monoclonal antibody, or antigen-binding fragment thereof, of any one of claims 1-3; (b) optionally, removing any non-specifically bound monoclonal antibody or, antigen-binding fragment thereof; and (c) detecting and/or quantifying the amount of monoclonal antibody, or antigen-binding fragment thereof, which is specifically bound to human epiregulin.

22. The method of claim 21, wherein said bodily fluid sample is a blood, serum or plasma sample, or cerebrospinal fluid sample, and said contacting occurs ex vivo.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] FIG. 1 shows ligand selectivity ELISA results for Antibody 1 (Panel A), and LY 3016859 (Panel B), and demonstrates Antibody 1 selectivity for human epiregulin, but no apparent binding to other EGF family ligands (Epigen, Amphiregulin, Betacellulin, EGF, HB-EGF, and TGFα (TGFA)) as determined in the ligand specificity ELISA described in Example 2. In comparison LY 3016859 binds to both epiregulin and TGFα.

[0050] FIG. 2. Binding of membrane epiregulin in vivo 3 days after administration of Antibody 1 (filled circles) compared to a control hIgG antibody (filled square).

[0051] FIG. 3. Pretreatment time dependent binding of membrane epiregulin in vivo following a 10 mg/kg subcutaneous dose of Antibody 1 (filled squares) compared to a control hIgG antibody (open squares).

[0052] FIG. 4. Binding of membrane epiregulin in vivo 3 days after administration of Antibody 1 (filled squares) compared to a >50×less potent epiregulin binding comparator antibody (open circles).

[0053] FIG. 5. ELISA assessment of Antibody 1 Fab binding to indicated mutations of epiregulin.

[0054] FIG. 6. Human EGFR ligand epitope (EGF-like domains) alignment for Antibody 1. Ligand sequences provided in the Listing of Amino Acid and Nucleotide Sequences.

EXAMPLES

[0055] The following examples are offered to illustrate, but not to limit, the claimed invention. The results of the following assays demonstrate that the exemplified monoclonal antibodies and/or antigen-binding fragments thereof of the present disclosure bind and/or neutralize human epiregulin and therefore may be used for treating epiregulin-mediated disorders described herein.

Example 1: Antibody Generation, Expression and Purification

[0056] A panel of human anti-epiregulin antibodies is obtained by immunization of a humanized mouse with recombinant EREG (human and cyno) to identify antibodies that could be effective at neutralizing epiregulin signaling. Mutations are systematically introduced into individual complementarity determining regions (CDRs) of each antibody and the resulting libraries are subjected to multiple rounds of selection with decreasing concentrations of antigen, decreased time to associate in antigen, and/or increasing periods of dissociation to isolate clones with improved affinities. The sequences of individual variants are determined and used to construct a combinatorial library which is subjected to an additional round of selection with increased stringency to identify additive or synergistic mutational pairings between the individual CDR regions. Individual combinatorial clones are sequenced, and binding characteristics are determined. This screening can be conducted against human, or mouse epiregulin to increase affinity against one or more selected species (for example Antibody 1 for human epiregulin). Counter-screening can be conducted against other EGFR ligands to maintain selectivity after engineering. Selected antibodies can also be mutagenized to fix post-translational modifications such as methionine oxidation, while retaining binding affinity to epiregulin. Additionally, framework (FW) and CDR substitutions can be made to the antibody to revert these sequences to their germline state in order to reduce potential immunogenicity risk.

[0057] Engineered and/or optimized anti-epiregulin antibodies referred to herein as Antibody 1 are obtained, having the amino acid sequences of the variable regions of the heavy chain and light chain, and the complete heavy chain and light chain amino acid sequences, and the nucleotide sequences encoding the same, as listed below in the section entitled “Listing of Amino Acid and Nucleotide Sequences”. The SEQ ID NO's corresponding to these sequences are shown in Table 1, as well as the light chain and heavy chain CDR amino acid sequences.

Expression and Purification:

[0058] The exemplified anti-epiregulin antibodies of the present disclosure can be expressed and purified essentially as follows. Antibody 1 is expressed in an appropriate host cell, such as HEK293 or CHO, either transiently or stably transfected with 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 for Antibody 1 (for example, a DNA sequence of SEQ ID NO:11 encoding a HC of exemplified Antibody 1 presented in Table 1, and a DNA sequence encoding a LC amino acid sequence according to Table 1, for example, a DNA sequence of SEQ ID NO:12 encoding a LC of exemplified Antibody 1 presented in Table 1); and are expressed from a commonly-used and suitable construct for this purpose, such as one based on human cytomegalovirus major immediate early promoters.

[0059] Medium, into which an antibody of the present invention has been secreted, may be purified by conventional techniques, such as mixed-mode methods of ion-exchange and hydrophobic interaction chromatography. For example, the medium may be applied to and eluted from a Protein A or G column using conventional methods; mixed-mode methods of ion-exchange and hydrophobic interaction chromatography may also be used. Soluble aggregate and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The product may be immediately frozen, for example at −70° C., refrigerated, or may be lyophilized. Various methods of protein purification may be employed, and such methods are 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 may be immediately frozen at −70° C. or stored at 2-8° C. for several months, or may be lyophilized, or preserved in 4° C. for immediate use. Amino acid SEQ ID NOs for exemplified human antibodies of the present invention are shown in Table 1.

Example 2: Characterization of the Anti-Epiregulin Antibodies

[0060] Epiregulin regents are made as fusion proteins, cleaved off, and purified. Cleaved epiregulin is the mature soluble form which includes extra amino acids relative to the native form and is used for in vitro binding and neutralization assays (SEQ ID NO's: 22-25). The human epiregulin (EREG) fusion (SEQ ID NO: 27) is also used in the binding assay (capture reagent), and a full length epiregulin sequence (SEQ ID NO: 26) is used for cell-based binding and effector function assays which has a single mutation to reduce shedding.

[0061] Mature, soluble epiregulin is expressed as a HRV3C protease cleavable C-terminal fusion on a monomeric human IgG4 Fc (F405Q/Y407E) (SEQ ID NO: 27) in a transiently transfected CHO cell culture. Fusion protein is captured with a Protein A affinity column and is further purified by size exclusion chromatography. The fusion is cleaved with HRV3C protease, flowed through a Protein A affinity column to remove the monomeric Fc, and further purified by size exclusion chromatography to yield mature, soluble epiregulin.

TABLE-US-00003 Sequences of antigens: Cleaved human epiregulin (SEQ ID NO: 22) GPGVSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVRCEHFFLG Cleaved cynomolgus monkey epiregulin (SEQ ID NO: 23) GPGVSITKCNSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVRCEHFYLG Cleaved rat epiregulin (SEQ ID NO: 24) GPGVLITKCSSDMDGYCLHGHCIYLVDMSEKYCRCEVGYTGLRCEHFFLG Cleaved rabbit epiregulin (SEQ ID NO: 25) GPGVSITKCGSDMNGYCLHGQCIYLVDMSENYCRCEVGYTGVRCEHFFLG Full length human epiregulin (membrane bound with T111P mutation) (SEQ ID NO: 26) MTAGRRMEMLCAGRVPALLLCLGFHLLQAVLSTTVIPSCIPGESSDNCTAL VQTEDNPRVAQVSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVR CEHFFLTVPQPLSKEYVALTVILIILFLITVVGSTYYFCRWYRNRKSKEPK KEYERVTSGDPELPQV Monomeric Fc human epiregulin (SEQ ID NO: 27) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSITKCSSDMNGYCLHGQCI YLVDMSQNYCRCEVGYTGVRCEHFFLG

[0062] 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 ligands reference sequence numbers are: 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).

Binding Affinity to Human Epiregulin

[0063] The solution phase equilibrium binding affinities of Antibody 1 to human (SEQ ID NO: 22), cynomolgus monkey (SEQ ID NO: 23), rat (SEQ ID NO: 24), and rabbit (SEQ ID NO: 25) epiregulin are measured by an MSD solution equilibrium titration (MSD-SET) assay at 37° C. (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.)

[0064] An MSD SI6000 instrument (Meso Scale Discovery, Rockville, Md.) is used for reading MSD plates. MSD assay plates are prepared as follows. A multi-array 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 a monomeric Fc-human epiregulin fusion in PBS. Plates are washed 3×in PBST following coating, then blocked for 1 hour at room temperature with shaking in 3% Blocker A (diluted in PBS from 5% Blocker A, Meso Scale Discovery, P/N R93AA-1).

[0065] SET samples are prepared in 1% Blocker A at pH 6.0 and at pH 7.4. Antibody is diluted to either 20 and 200 pM (pH 7.4) or 200 pM and 2 nM (pH 6.0). Epiregulin is serially diluted for a total of 12 dilutions centered around 2×the antibody concentration. This is accomplished by using starting concentrations of 10 and 100 nM (pH 7.4) or 100 nM and 1000 nM (pH 6.0) which are then 10×diluted once, 2×serially diluted 9 times, and then 10×diluted for the final dilution. Epiregulin titrations and fixed concentration antibody solutions are combined 1:1 to prepare the SET solutions. SET solutions are incubated at 37° C. for approximately 96 hours to allow binding to reach equilibrium. 100 μl of the SET solution is transferred to the prepared MSD plate in duplicate rows and incubated at room temperature for 10 minutes to capture free antibody. Following incubation, the plate is washed 3× with PBST, then 100 μl of 1 μg/ml biotinylated goat anti-human kappa primary antibody (Southern Biotech, Catalog 2060-08) in 1% Blocker A is added to all wells. This is incubated on a plate shaker at room temperature for 60 minutes. The plate is then washed 3× with PBST, then 100 μl of 1 μg/ml SULFO streptavidin (Meso Scale Discovery, P/N R32AD-1) in 1% Blocker A is added to all wells. This is incubated on a plate shaker at room temperature for 30 minutes. The plate is then washed 3× with PBST, then 100 μl of 1X Read Buffer T (diluted in water from 4X Read Buffer T, Meso Scale Discovery, P/N R93AA-1) is added before reading the plate. Dissociation constant (K.sub.D) and a least common multiplier (LCM) ligand correction factor to account for unknown active antigen fraction are globally fit from the MSD-SET data to an equilibrium binding equation using non-linear regression. This is done for each pair of fixed antibody concentrations at a given pH.

[0066] At pH 7.4, Antibody 1 binds to human epiregulin with an affinity (K.sub.D) of 14.9 pM, to cynomolgus monkey epiregulin with a K.sub.D of 26.8 pM, to rat epiregulin with a K.sub.D of 30.2 pM, and to rabbit epiregulin with a K.sub.D of 24.0 pM. At pH 6.0, Antibody 1 binds to human epiregulin with a K.sub.D of 195 pM, to cynomolgus monkey epiregulin with a K.sub.D of 328 pM, to rat epiregulin with a K.sub.D of 286 pM, and to rabbit epiregulin with a K.sub.D of 330 pM. The pH selectivity of Antibody 1 (pH 6.0 K.sub.D/pH 7.4 K.sub.D) 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 antibodies of the present disclosure exhibit advantageous pH selectivity for epiregulin binding which are conceived to provide useful pharmacokinetic and epiregulin neutralization properties.

TABLE-US-00004 TABLE 3 In Vitro Binding Affinities of Antibody 1 to epiregulin measured by MSD-SET at 37° C., at pH 7.4 and 6.0. K.sub.D values are the average ± standard deviation of 3 independent replicates. pH 7.4 pH 6.0 Selectivity Affinity Affinity (pH 6.0 K.sub.D/ Species (pM) (pM) pH 7.4 K.sub.D) Human 14.9 ± 2.9  195 ± 43  13.0 Cynomolgus 26.8 ± 10.3 328 ± 110 12.2 monkey Rat 30.2 ± 15.9 286 ± 47  9.5 Rabbit 24.0 ± 7.3  330 ± 34  13.7

[0067] Results are reported as the mean of the K.sub.D from 3 independent replicates. Error estimate is calculated as the standard deviation of the independent replicates. The affinity of Antibody 1 to human EREG at pH 7.4 represents an approximate 70-fold improvement in affinity, relative to LY3016859, which may result in a significantly lower dose of Antibody 1 needed to achieve equivalent EREG neutralization in vivo. The ability to use a lower dose of Antibody 1 means that necessary inhibition can be accomplished with less antibody, and as a result the drug product may be amenable to subcutaneous administration, which is clinically important for accessibility, tolerability, compliance, compared to an antibody that requires intravenous dosing. This is particularly advantageous in the context of chronic dosing such as is needed for the chronic pain indications described herein.

Ligand Specificity of Antibody 1:

[0068] Specificity of antibody binding to EGF family ligands can be tested by enzyme linked immune sorbent assay (ELISA) and/or other common methods well known to the skilled artisan.

[0069] ELISA reagent preparation can be conducted as follows. Ligands tested are human Epigen (R&D 6629-EP-025/CF), epiregulin ((SEQ ID NO: 22) prepared and purified as described herein in Example 2, and/or by methods known in the art), 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). Ligands are biotinylated with 10-fold excess biotin using Thermo Pierce Sulfo-NHS-LC-Biotin (A39257) for 2 hours on ice, then frozen at −80° C. in small aliquots for individual use.

[0070] ELISA assays can be run as follows. Greiner 96 well high binding plates (650061) are coated overnight at 4° C. with 50 μl/well Neutravidin (Thermo-Pierce 31000) at 1 μg/ml in PBS. The coating solution is removed the next day and 100 μl/well Pierce Blocker Casein (37528) is added and incubated at room temperature for 30 minutes or more to block the plates. After blocking, plates are washed 3 times with PBS plus 0.05% Tween20 to remove residual blocking buffer. Biotinylated ligands are diluted in a separate PCR plate (Eppendorf 951020443) in Pierce Casein Blocker at a constant concentration of 20 nM across 12 wells per ligand with a no antigen control in the last row. Ligands are transferred to blocked assay plate and incubated at room temperature for 1 hour. The assay plate is washed 3 times with PBS plus 0.05% Tween20. The antibody to be tested is serial diluted starting at 5 μg/mL (LY3016859) or 10 μg/ml (Antibody 1) in Pierce Casein Blocker with 1:3 dilutions across the plate (12 wells) with a final volume of 50 μl/well and incubated at room temperature for 1 hour. The assay plate is washed 3 times with PBS plus 0.05% Tween20. Anti-Human Kappa-AP secondary antibody (Southern Biotech 2060-04) is added at 50 μl/well at 1:2000 dilution in PBS plus 0.05% Tween20 and incubated at room temperature for 1 hour. The assay plate is washed 3 times with PBS plus 0.05% Tween20. Finally, 50 μl of pNPP substrate (Sigma-Aldrich N2765) is added and allowed to develop until sufficient color is present and read at 405 nm on a Spectramax Plus 384 plate reader by Molecular Devices.

[0071] FIG. 1 illustrates ligand selectivity ELISA results for Antibody 1. Antibody 1 demonstrates selectivity for binding human epiregulin, but no apparent binding to other EGF family ligands Epigen, Amphiregulin, Betacellulin, EGF, HB-EGF, and TGFα, as determined in the above ligand specificity ELISA. The selective and specific binding to epiregulin, and not other EGF family ligands, indicates that Antibody 1 may achieve the desired effects of blocking epiregulin signaling while avoiding clinically undesirable effects of binding to other EGF family ligands, such as TGFα or others. Lack of binding to TGFα may reduce the dose required in order to effectively inhibit epiregulin. This selectivity may avoid undesirable clinical adverse events such as skin rashes which have been observed for pan-EGFR inhibitors.

Physicochemical Attributes:

[0072] With respect to therapeutic antibody product attributes, including Chemical Stability, Solubility, and Viscosity, Antibody 1 exhibits a desirable combination of physicochemical attributes for use as a human therapeutic agent.

Stability:

[0073] Stability of Antibody 1 is assessed at high concentration (approximately 100 mg/ml) formulated in 5 mM histidine, pH 6.0, plus 280 mM mannitol plus 0.05% (w/v) polysorbate-80. Concentrated samples are incubated for a period of 4 weeks at 5° C. and 35° C. Following incubation, samples are analyzed for percent high molecular weight (% HMW) with size exclusion chromatography (SEC), for fragmentation by capillary electrophoresis (CE-SDS), and for chemical modification (for example deamidation, isomerization, or oxidation) by LC-MS peptide mapping. After 4 weeks at 35° C., Antibody 1 exhibited A % HMW (change in percentage of high molecular weight) of 0.4%, Δ% fragments (change in percentage of fragments) of 0.6%, and no CDR chemical modifications greater than 0.5%.

[0074] Photostability under the same formulation conditions is evaluated by a combined total exposure of 40 watt hour/m.sup.2 UV light and 240 klux hour visible light at 25° C. Following incubation, samples are analyzed for % HMW by SEC and for chemical modification by LC-MS peptide mapping. Antibody 1 exhibited A % HMW measured by SEC of 6.1% and no CDR chemical modifications greater than 0.7% relative to an unexposed control.

[0075] Freeze/thaw stability under the same formulation conditions is evaluated using a 3 repeated slow, controlled temperature cycle which mimics the freeze/thaw conditions of large volumes of bulk drug substance placed at −70° C. Antibody 1 exhibited A % HMW measured by SEC of 1.0% after the 3 freeze-thaw cycles.

[0076] These results indicate Antibody 1 possesses advantageous physical and chemical stability sufficient to facilitate development of solution formulations and use as a therapeutic agent.

Solubility:

[0077] Solubility is assessed by concentrating 100 mg of Antibody 1 with a 30 kDa molecular weight cut-off centrifugal filter (for example, Amicon U.C. filters, Millipore, catalog # UFC903024) to a volume of approximately 0.5 ml. The final concentration of the sample was measured by UV absorbance at 280 nm using a Solo VPE spectrophotometer (C Technologies, Inc).

[0078] Antibody 1 displays a solubility of greater than or equal to 175 mg/ml in 5 mM histidine pH 6 buffer and greater than or equal to 168 mg/ml in PBS pH 7.4. No phase separation or cryo-precipitation is observed at storage at 5° C. or at −5° C. after 1 week. These results indicate that Antibody 1 exhibits sufficient solubility to enable high concentration dosing.

Viscosity:

[0079] Viscosity of Antibody 1 and of LY3016859 are analyzed at 15° C. at an approximate concentration of 125 mg/ml using a VROC Initium (RheoSense). Antibody 1 exhibited a viscosity of 6.4 cP at 133 mg/ml in 5 mM histidine at pH 6 plus 280 mM mannitol, and LY3016859 exhibited a viscosity of 12.2 cP at 132 mg/mL in 5 mM histidine at pH 6 plus 280 mM mannitol. These results indicate that Antibody 1 exhibits reduced viscosity relative to LY3016859, and sufficiently low viscosity to enable high concentration dosing. The ability to use a higher concentration of Antibody 1, in combination with the need for a lower dose, means that necessary effective doses can be accomplished in a smaller volume for delivery, and that means the drug product may be amenable to subcutaneous administration, which is clinically important for accessibility, tolerability, compliance, compared to an agent that must be an antibody that requires intravenous dosing.

Example 3: In Vitro Functional Characterization of the Anti-Human Epiregulin Antibodies

Neutralization of Epiregulin In Vitro:

[0080] Neutralization of epiregulin activity by antibodies of the present disclosure, such as Antibody 1, may be assessed by one or more of the epiregulin-induced cell-based response assays as described below.

[0081] Antibodies of the present disclosure are tested for the ability to neutralize epiregulin activity in two independent functional assays. Neutralization of epiregulin activity by Antibody 1 of the present disclosure may be assessed by one or more cell-based activity assays utilizing downstream signaling pathways of the EGFR family of receptors as described below. Determination of mean half-maximum inhibitory concentration (IC.sub.50) for neutralization assays (C166-AP1-Luc and TGW-pERK) is calculated in SigmaPlot or GraphPad Prism. In GraphPad Prism IC.sub.50 is calculated by running non-linear regression analysis of Log.sub.10 transformed antibody concentration. For assays run only in duplicate or triplicate over 1 plate, IC.sub.50 and standard deviation (SD) is determined by calculating IC.sub.50 for each replicate and SD from averaged IC.sub.50 of replicates. For analysis of compounds across various plates in duplicate or triplicate, IC.sub.50 is calculated for each plate and SD determined across plates.

Inhibition of Epiregulin Induced Responses in C166-AP1-Luciferase Functional Assay:

[0082] The ability of antibodies of the present disclosure to neutralize human epiregulin-induced luciferase reporter activity can be assessed in C166 cells overexpressing a luciferase reporter driven by AP1 activation (C166-AP1-Luc). C166 cells are an endothelial derived cell line which express all 4 EGFR family of receptors with EGFR expression at highest level. For testing Antibody 1 activity, C166-AP1-Luc cells are grown in growth media [DMEM (Gibco 12430-047), 10% FBS (Gibco 10082147), Anti/Anti (Gibco 15240062) and 2 μg/ml Puro (1000×2 mg/ml Puromycin Dihydrochloride (Calbiochem, Cat #540411)] and dissociated in 0.05% trypsin-PBS and plated at 50,000 cells per 1 ml in tissue culture-treated 96 well plates at 50 μl per well in serum free media (growth media without FBS). Cells are treated with 50 μl of 100 ng/ml (16-18.5 nM) final concentration of epiregulin (human (SEQ ID NO: 22), cyno (SEQ ID NO: 23), rat (SEQ ID NO: 24) and rabbit (SEQ ID NO: 25), as described herein above) and serial dilutions of concentrations of Antibody 1 for 6 hr. Following incubation, cells are lysed for 3 minutes with 100 μl Promega™ One-Glo™ Luciferase solution (Promega™, Cat. # E6120). Luminescence is read on Perkin Elmer Wallace 1420 Victor2™ Microplate Reader. The reduction in relative fluorescence units (RFUs) shown in Table 4 below reflects the ability of Antibody 1 to neutralize epiregulin activity of various relevant species tested including human (SEQ ID NO: 22), cyno (SEQ ID NO: 23), rat (SEQ ID NO: 24) and rabbit (SEQ ID NO: 25)). The IC.sub.50 value for Antibody 1 in neutralizing human epiregulin is 8.7 nM, SD 2.3, n=5 plates, 2-3 replicates/plate (Table 4). Neutralization across relevant species is demonstrated by testing against cynomolgus (IC.sub.502.1 nM, SD 0.08, n=3 replicates), rat (IC.sub.502.5 nM, SD 0.27, n=3 replicates), rabbit (IC.sub.50 3.6 nM, SD 0.14, n=3 replicates). Assay validation and reduced IC.sub.50 of Antibody 1 as compared to LY3016859 (Antibody 1 of WO 2012/138510) was compared confirmed with neutralization of human epiregulin in this assay with LY3016859 (Table 4). Human, rabbit, cyno and rabbit epiregulin used in these studies were generated as described herein above. LY3016895 was demonstrated to neutralize human epiregulin with IC.sub.50 of 25.9 nM, SD 11.7, n=8 plates, 2-3 replicates/plate, cynomolgus (IC.sub.508.6 nM, SD 0.27, n=2 plates, 2-3 replicates/plate), rat (IC.sub.50720 nM, SD 10, n=2 plates 2-3 replicates/plate) and rabbit (IC.sub.5011.0 nM, SD 1.15, n=2 plates, 2-3 replicates/plate). Statistical validation of the epiregulin-induced C166-AP1-Luc assay determined the MSR as 1.24, 95% CI of MSR (MSR=minimal significant ratio) 1.15, 1.43 and potency analysis for Antibody 1 of 8.4+/−1.3. This assay demonstrates in vitro neutralization of epiregulin induced functional cellular responses by Antibody 1, and the ability to inhibit the activity of epiregulin from various species, and the potential for Antibody 1 neutralizing effects against epiregulin in vivo.

TABLE-US-00005 TABLE 4 Antibody 1 neutralization of epiregulin induced luciferase reporter activity in C166-AP1-Luciferase cells. Antibody 1 LY3016859 IC.sub.50 ± SD (nM) IC.sub.50 ± SD (nM) Human epiregulin 8.7 ± 2.3  25.9 ± 11.4 Cyno epiregulin 2.1 ± 0.1  8.6 ± 0.3 Rat epiregulin 2.5 ± 0.3 720 ± 10 Rabbit epiregulin 3.6 ± 0.1 11.0 ± 1.2

[0083] For neutralization against human epiregulin mean average and SD was determined by averaging IC.sub.50 from replicates across 5 plates for Antibody 1 and 8 plates for LY3016859. For neutralization against rat, cyno and rabbit epiregulin IC.sub.50 from triplicates from 1 plate for each species was averaged and SD determine for Antibody 1. For neutralization against rat, cyno and rabbit epiregulin IC.sub.50 was generated from averaging IC.sub.50 from duplicates across 2 different plates for LY3016859.

[0084] The data in Table 4 demonstrate that Antibody 1 can effectively neutralize human epiregulin-induced luciferase reporter activity in the above C166-AP1-Luc cell-based assay (IC.sub.5032 8.7 nM), with an approximately 3-fold lower IC.sub.50 than LY3016895 (IC.sub.50=25.9 nM). Cross-reactivity to relevant species of epiregulin was confirmed by testing neutralization against rat (IC.sub.5032 2.5 nM), cyno (IC.sub.5032 2.1 nM) and rabbit (IC.sub.5032 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 etiopathogenesis, such as pain disorders, and in particular OA, DPNP, and CLBP.

Selectivity of Antibody 1 to Epiregulin Neutralization in C166-AP1-Luc Assay:

[0085] Selectivity of Antibody 1 for neutralizing epiregulin can be further demonstrated by testing the activity of all 7 EGFR ligands (Epigen, TGFα, BTC, EGF, HB-EGF, epiregulin, and amphiregulin) in the C166-AP1-Luc assay (ligands can be purchased for example from R&D systems: epiregulin #1195-EP-CF, betacellulin #261-CE-CF, EGF #236-EG-CF, TGFα #239-A-CF, HB-EGF #259-HE-CF, Epigen #6629-EP-CF and amphiregulin #262-AR-CF). These 7 EGFR ligands demonstrate AP1 activation with a 2-5 fold increase in luciferase activity, depending on the ligand. The EC50 for EGFR ligands is determined by running dose responses of each ligand in C166-AP1-Luc cell line as described herein above. Ligand concentration for stimulation is determined by using the EC50-EC80 for each ligand (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 EC50 due to lack of plateau on curve). Antibody 1 demonstrates the ability to neutralize epiregulin-induced luciferase activity in C166-AP1-Luc cells with IC.sub.50 of 8.7 nM, and in contrast, fails to demonstrate inhibition of betacelluin, EGF, TGFα, HB-EGF, Epigen or amphiregulin-induced luciferase activity in C166-AP1-Luc assay (see Table 5 below). The data in Table 5 also support the pharmacological selectivity of Antibody 1 as specifically neutralizing the activity of epiregulin, but not other family members betacellulin, EGF, TGFα, HB-EGF, Epigen and amphiregulin. This selectivity is important in that Antibody 1 is expected to intervene selectively in epiregulin responses, and thus avoid possible adverse effects that could arise by less specific agents such as LY3016859 that may block other EGFR ligands.

TABLE-US-00006 TABLE 5 Specificity of Antibody 1 in neutralizing epiregulin in a C166-AP1-Luc assay. All 7 EGFR ligands are tested for EC.sub.50 calculation in a C166-AP-Luc assay. NA (no activity) indicates lack of an inhibition curve to determine an IC.sub.50 and thus lack of neutralization at highest concentration of antibody tested, 200 nM. For epiregulin neutralization an IC.sub.50 was calculated using an epiregulin reagent (SEQ ID NO. 22). Ligand Antibody 1 EC.sub.50 nM IC.sub.50 nM betacellulin 0.8 NA EGF 1.3 NA TGFα 2.5 NA HB-EGF 0.13 NA epiregulin 7.8 8.7 Epigen 429 NA amphiregulin 42.9 NA

Ability of Antibody 1 to Neutralize the Epiregulin Induced Responses in a TGW Neuroblastoma Cell Line:

[0086] Epiregulin neutralization is further assessed by measuring epiregulin-induced ERK phosphorylation in the TGW neuroblastoma cell line. TGW cells express all 4 of the EGFR family of receptors with EGFR expression being the highest level. In this assay, TGW cells are grown on collagen 1 coated plates in growth media [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 plated in serum free media (growth media with FBS replaced with 0.1% BSA) at 100,000 cells/100 μl in 96-well collagen 1 coated 96-well plates (Corning #354407). In TGW cells, epiregulin treatment (80 ng/ml for 5 minutes) induces over 5-fold increased phosphorylation of ERK (Meso Scale Diagnostics, LLC phospho-ERK1/2 Whole Cell Lysate Kit #K151 DWD) as compared to unstimulated cells. Phospho-ERK activation is determined by chemiluminescent induction and displayed as relative fluorescent unites (RFUs). Neutralization of Antibody 1 is determined by incubation of human epiregulin with Antibody 1 in serial dilutions for 20 minutes prior to stimulation of cells. Data is obtained by reading plates on Meso Scale Diagnostics SECTOR Imager. The reduction in epiregulin induced phosphor-ERK1/2 signal with Antibody 1 treatment is shown in Table 6 and reflects the ability of Antibody 1 to neutralize human epiregulin. The IC.sub.50 value for Antibody 1 in neutralizing human epiregulin induced pERK induction in TGW cells is observed to be 3.4 nM, SD 0.7, (n=2 replicates), which is lower than LY3016859, with an IC.sub.50 of 14.6 nM, SD 0.7, (n=2 replicates). The data in Table 6 provide evidence of the ability of the Antibody 1 to block human epiregulin-induced phosphor-ERK1/2 activation in TGW cells. This data demonstrates the ability of the antibodies of the present disclosure, and Antibody 1 in particular, to neutralize human epiregulin utilizing 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.

TABLE-US-00007 TABLE 6 Antibody 1 neutralizes the effect of human epiregulin induced phopho-ERK induction in TGW cells. Antibody 1 LY3016859 IC.sub.50 ± SD (nM) IC.sub.50 ± SD (nM) Human epiregulin 3.4 ±0.7 14.6 ± 0.7

Example 4: In Vivo Functional Characterization of Epiregulin in Induced Tactile Allodynia in CD-1 Mice

[0087] The ability of biologically active molecules epiregulin and TGFα to induce tactile allodynia can be assessed in male CD-1 mice following intraplantar injection. Mice are allowed to acclimate in a temperature and light controlled animal facility with ad libitum water and food for a period of at least 7 days prior to experimentation. On the testing days, animals are brought to the testing room, acclimate for 20 minutes in home cage prior to placing in clear plastic chambers on an elevated metal mesh floor and allowed to acclimate for one hour. Paw withdrawal responses to mechanical stimulations are determined by using calibrated von Frey filaments applied to the plantar surface of hind paw from underneath the cage through openings in the metal mesh floor. The measurements are accomplished with the use of von Frey filaments in an up-down testing paradigm. The force range of eight graded filaments is 0.04 g to 6 g. The paw withdrawal threshold is measured and calculated. Animals are randomized based on baseline value of paw withdrawal threshold to dosing groups, PBS, epiregulin 30 ng, epiregulin 300 ng, TGFα 30 ng, or TGFα 300 ng (murine epiregulin (SEQ ID NO: 28), and murine TGFα (SEQ ID NO: 29) reagents prepared as described herein, or for TGFα as His tagged and purified by standard chromatography techniques (immobilized metal ion affinity chromatography or IMAC followed by size exclusion chromatography or SEC). Then, animals receive hind paw intraplantar injection with 20 μl solution of PBS, or fresh diluted epiregulin or TGFα. Paw withdrawal thresholds are assessed at post-injection 2-hour and 4-hour time points. Data are shown in Table 7 below.

TABLE-US-00008 TABLE 7 Paw Withdrawal Threshold (g) mean ± SEM (n) Baseline 2 hrs 4 hrs Vehicle 2.55 ± 0.50 (10) 2.34 ± 0.36 (10) 2.27 ± 0.49 (10) 30 ng epiregulin 2.58 ± 0.46 (10) 1.91 ± 0.32 (10) 1.56 ± 0.34 (10) 300 ng epiregulin 2.58 ± 0.47 (10) 1.05 ± 0.49 (10) 1.32 ± 0.47 (10) 30 ng TGFα 2.57 ± 0.49 (10) 2.50 ± 0.52 (10) 3.13 ± 0.50 (10) 300 ng TGFα 2.61 ±0 .50 (10) 2.67 ± 0.53 (10) 2.40 ± 0.63 (10)

[0088] The effect of epiregulin as compared to TGFα are shown in Table 7 above, and indicate that in this pain model, epiregulin induces a painful response as indicated by a reduction in withdrawal threshold, whereas TGFα does not. Antibody 1 is believed to embody a more selective and specific pain therapeutic as compared to LY3016859 which recognizes both epiregulin and TGFα, and the lack of binding to TGFα by Antibody 1 of the present disclosure is considered to be advantageous with respect to avoiding off target effects, reducing immunogenicity, and improving or lowering the dose which may be needed for therapeutic uses.

Example 5: Characterization of Antibody 1 Immunogenicity Potential

[0089] Antibody 1 represents an improved therapeutic antibody in several combined respects, including enhanced affinity for human epiregulin, improved specificity for epiregulin relative to other EGFR ligands, and as compared to LY3016859, improved biophysical properties, and a fully human sequence, the latter of which is believed to provide reduced immunogenicity potential, as supported by the following findings.

Dendritic Cell (DC) Internalization Assay

Monocyte-Derived DC Culturing (MDDC)

[0090] CD14+monocytes are isolated from periphery blood mononuclear cells (PBMCs) and are cultured and differentiated into DC following standard protocols. Briefly, PBMCs are isolated using density-gradient centrifugation with Ficoll (#17-1440-02, GE Healthcare) and Sepmate 50 (#15450, STEMCELL Technologies) from LRS-WBC. CD14+monocytes are isolated using positive selection with a CD14+microbead kit (#130-050-201, Miltenyi Biotec) following the manufacturer's manual. Cells are then cultured at 1 million/ml with 1000 unit/ml GM-CSF and 600 unit/ml IL-4 for 6 days to drive to immature dendritic cells (MDDC) in RPMI medium with L-glutamine and 25 mM HEPES supplemented with 10% FBS, 1 mM sodium pyruvate, 1x penicillin-streptomycin, 1 x non-essential amino acids, and 55 μm 2-mercaptoethanol (hereafter referred to complete RPMI medium or medium, purchased from Life Technologies). The medium is changed twice, on day 2 and day 5. On day 6, cells are gently collected with a cell scraper and used for experiment. MDDC are characterized visually for dendritic morphology by microscope and for expression of CD14, CD11 c, and HLA-DR by flow cytometry. Their ability to respond to LPS treatment is confirmed by measuring upregulation of CD80, CD83, and CD86 using flow cytometry.

Conjugation of Fab-TAMRA-QSY7

[0091] A F(ab′)2 fragment goat anti-human IgG (Jackson ImmunoResearch) is double-labeled with QSY7-NHS and TAMRA-SE (Molecular Probes) to obtain Fab-TAMRA-QSY7 used as a universal probe to track test article internalization. Each vial of F(ab′)2 (approximately 1 ml at 1.3 mg/ml) is concentrated to about 2 mg/ml by centrifugation at 14,000 rcf for 2 minutes with the 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, and 6.8 μl QSY-NHS stock solution at 10 mM in DMSO is added and mixed. The reaction vial is kept in dark at room temperature for 30 min. The intermediate product, Fab-QSY7, is purified with Zeba Spin desalting column (#89890, Thermo Scientific) by centrifugation at 1000 relative centrifugal force (RCF) for 2 min. The concentration and degree of labeling (DOL) are calculated by measuring the absorbance at 280 nm and 560 nm on a NanoDrop (ThermoFisher). Fab-QSY7 is then concentrated to about 2 mg/ml by centrifugation at 14,000 rcf for 2 min with Amico Ultra-0.5 centrifugal filter device again. After pH adjustment with 10% (v/v) 1 M sodium bicarbonate, 4.3 μl of 15 mM TAMRA-SE stock solution in DMSO are added and mixed. After 30 min. at room temperature in the dark, the final product Fab-TAMRA-QSY7 is purified and collected using a Zeba Spin desalting column by centrifugation at 1000 rcf for 2 min. The concentration and DOL are again quantitated by reading the absorbance at 280 nm, 555 nm, and 560 nm on a NanoDrop Spectrophotometer. Using this protocol, about 300 μl of Fab-TAMRA-QSY7 at around 1.5 mg/ml with approximately two QSY7 and two TAMRA per F(ab′)2 are obtained.

Standardized Internalization Study by FACS

[0092] Individual test molecules are normalized to 1 mg/ml with PBS and then further diluted to 8 μg/ml in complete RPMI medium. Fab-TAMRA-QSY7 is diluted to 5.33 μg/ml in complete RPMI medium. The antibody and Fab-TAMRA-QSY7 are mixed with equal volume and incubated for 30 min at 4° C. in dark for complex formation. MDDC are resuspended at 4 million/ml in complete RPMI medium and seeded at 50 μl per well in a 96-well round-bottom plate, to which 50 μl of the antibody/probe complex is added. Cells are incubated for 24 h at 37° C. in a CO.sub.2 incubator. Cells are washed with 2% FBS PBS and resuspended in 100 μl 2% FBS PBS with Cytox Green live/dead dye. Data are collected on a BD LSR Fortessa X-20 and analyzed in FlowJo. Live single cells are gated and percent of TAMRA fluorescence positive cells is recorded as the readout.

Data Presentation and Statistical Analysis

[0093] Molecules are tested on three or more donors in duplicate or triplicate. The percent of TAMRA-positive population is considered for each donor. To allow the comparison of molecules with data generated from different donors, a normalized internalization index (NII) is used. The internalization signal is normalized to IgG1 isotype (NII=0) and an internal positive control PC (NII=100) using the formula:

[00001]$100\times \frac{X_{\mathrm{TAMRA}}-\mathrm{IgG}1{\mathrm{isotype}}_{\mathrm{TAMRA}}}{{\mathrm{PC}}_{\mathrm{TAMRA}}-\mathrm{IgG}1{\mathrm{isotype}}_{\mathrm{TAMRA}}}$

where X.sub.TAMRA, IgG1 isotype.sub.TAMRA, and PC.sub.TAMRA are the percent of TAMRA-positive population for the test molecule X, IgG1 isotype, and PC respectively. Data are analyzed in JMP® 14.1.0 or Graphpad Prism 8.1.2. Mean of the percent of TAMRA-positive population and NII are calculated and reported. Increased internalization in antigen presenting cells such as DCs is associated with increased immunogenicity risk. Results in Table 9 demonstrate decreased DC internalization of Antibody 1 relative to LY3016859, and therefore a decreased immunogenicity risk.

TABLE-US-00009 TABLE 9 DC internalization Results Test Antibody % TAMRA+ NII LY3016859 10.3 16.9 Antibody 1 2.3 1.3
(See e.g. 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 J22, 68 (2020))

MAPPS Assay (MHC-Associated Peptide Proteomics) Methods:

[0094] Primary human dendritic cells from 10 normal human donors are prepared from buffy coats by isolation of CD-14 positive cells and differentiated into immature dendritic cells by incubation with 20 ng/ml IL-4 and 40 ng/ml GM-CSF in complete RPMI media containing 5% Serum Replacement (Thermo Fisher Scientific, cat#A2596101) for 3 days at 37° C. and 5% CO.sub.2 as described (Knierman et al., “The Human Leukocyte Antigen Class II Immunopeptidome of the SARS-CoV-2 Spike Glycoprotein”, Cell Reports, 33, 108454 (2020)). Three micromolar of test antibody is added to approximately 5×10.sup.6 cells on day 4 and fresh media containing 5 mg/ml of LPS to transform the cells into mature dendritic cells is exchanged after 5-hour incubation. The matured cells are lysed in 1 ml of RIPA buffer with protease inhibitors and DNAse the following day. The lysates are stored at −80° C. until sample analysis.

[0095] An automated liquid handling system is used to isolate the HLA-II molecules from thawed lysate using biotinylated anti-pan HLA class II antibody (clone Tu39). The bound receptor-peptide complex is eluted with 5% acetic acid, 0.1% TFA. The eluted MHC-II peptides are passed over a prewashed 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 with a Thermo LUMOS mass spectrometer. The separation used a 75 μm ×7 cm YMC-ODS C18 column for 65-minute gradient with a 250 nL/min flow rate and 0.1% formic acid in water as A solvent and 80% acetonitrile with 0.1% formic acid as B solvent. Mass spectrometry is run in full scan mode with 240,000 resolution followed by a 3 second data dependent MS/MS cycle comprised of ion trap rapid scans with HCD and EThcD fragmentation.

[0096] Peptide identifications are generated by an internal 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 with no enzyme search parameter against a bovine/human database containing the test antibody sequences. A KNIME workflow is used to process the identification files for the samples. Peptides identified from the test articles are aligned against the parent sequence. A summary is created for all donors that annotates the percent of donors that display non-germline residues, the number of different regions that display peptides with non-germline residues and the depth of peptide display at each region with non-germline residues. Increases in the extent of display of non-germline peptides is associated with increased risk for immunogenicity. Results in Table 8 show decreased display of non-germline peptides for Antibody 1 compared to LY3016859, and therefore a decreased risk of immunogenicity for Antibody 1.

TABLE-US-00010 TABLE 8 MAPPs Results Total # of non- Total # of non- Test % # of germline residues germline peptides Antibody Donors clusters from all clusters from all clusters LY3016859 60% 3 15 20 Antibody 1 20% 2 5 5

Example 6. Demonstration of Binding to Membrane Epiregulin In Vivo after Dosing in Rats

[0097] Epiregulin exists in both a membrane bound form, and a soluble form that occurs with cleavage of the membrane form by ADAM (A disintegrin and metalloprotease) proteases. The ability of antibodies of the present disclosure to bind to the membrane form of epiregulin in vivo after peripheral dosing, for example by subcutaneous injection, may be assessed by ex vivo isolation of tissue followed by adaptation of standard immunofluorescent staining methods as described herein and/or known to the skilled artisan. Positive and specific immunofluorescent labelling of the epithelial layer of the rat tongue can be demonstrated with Antibody 1.

[0098] Rat tongue can be obtained from euthanized Male Sprague Dawley rats (weighing 140-215 grams) after various doses (0.1 to 100 mg/kg, subcutaneous) of antibodies to be tested including Antibody 1, or control or comparator antibodies, and at various times after dosing (3-28 days after dosing), and flash frozen on dry ice. Frozen coronal 20 micron sections of tongue tissue are thawed, washed in PBS for 3 minutes, and fixed in 4% paraformaldehyde for 10 minutes. Slides are washed in Super Sensitive Wash Buffer (HK583-5K, Biogenex) and then blocked with Power block (HK065-5K, Biogenex) plus 0.1% Triton x-100 for 10 minutes. Slides are then washed and incubated for 15 minutes at room temperature with Alexa Fluor 488 goat anti-human IgG (A11013, Life Technologies, 13.3 μg/ml), and a labeled human/mouse IgG1 chimera, referred to herein as “Antibody 1 VR chimera” (SEQ ID's 64 and 65), having the variable regions (SEQ ID's 3 and 4) of Antibody 1 and murine constant regions (DyLight 650 labeled). Slides are washed 3 times and Prolong gold antifade reagent applied prior to coverslips. The fluorescent images at 488 and 650 nm wavelength from each animal are captured utilizing a Keyence BZ800 using the same exposure settings for all images. The green or red signal within the positively stained epithelial layer is quantified utilizing the ImageJ-Win64 software for average intensity by taking four random areas of interest. The green signal arises from bound Antibody 1, or control antibody, or reference comparator antibody (dosed in vivo), and the red signal is unbound epiregulin, where the detection antibody competes with bound drug, and only stains free epiregulin and not bound. Resulting data are normalized by utilizing the ratio of Alexa Fluor 488 goat anti-human IgG signal (green) over the remaining Antibody 1 VR chimera signal (red).

[0099] FIG. 2 shows that 3 days after dosing with Antibody 1, at doses >1 mg/kg, a significant dose dependent increase in binding of membrane epiregulin is observed in vivo. FIG. 3 shows that after a single dose of 10 mg/kg of Antibody 1, there is a sustained binding of membrane epiregulin in vivo for at least 14 days. FIG. 4 demonstrates a higher degree of bound membrane epiregulin in vivo, for Antibody 1, compared to a >50×less potent epiregulin binding comparator antibody. FIG. 4 shows the improvements in Antibody 1 affinity also translate to improved membrane-bound epiregulin ligand binding in vivo.

Example 7: Epitope Mapping of Antibody 1

[0100] Antibody 1 is a human IgG4 antibody that selectively binds and neutralizes the EGFR ligand family member epiregulin (EREG) with high affinity, and selectivity with respect to other EGFR ligand family members, and advantageous epiregulin neutralizing activity and pharmacokinetic properties. These properties, along with additional stability and viscosity and solubility properties described herein, result in an antibody with a combination of improved attributes and an advantageous therapeutic agent to neutralize EREG activity. Epitope mapping studies were performed to determine the specific amino acids in human EREG required for Antibody 1 binding and specificity. Critical amino acids were identified from the crystal structure of the Antibody 1 Fab/EREG complex, and contribution of these amino acids were further characterized through mutagenesis and binding studies. The contributions of these amino acids provide a structural basis for the specificity of Antibody 1 for EREG as compared to other EGFR ligand family members. These results also inform the cross-reactivity and selectivity in relevant pre-clinical species.

[0101] Sequences for test materials are provided in the Listing of 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).

[0102] Antibody 1 Fab fragment and human EREG were both produced in transient CHO expression and purified by standard techniques. Complex was prepared by adding 30% molar excess of EREG to Antibody 1 Fab, then purified by size exclusion chromatography to remove excess free EREG. Antibody 1 Fab binding to EREG was first probed by Western blot analysis and showed that Antibody 1 Fab can recognize the denatured, non-reduced, but not the denatured, reduced EREG in this assay. This strongly suggests a conformational epitope dependent on the EREG disulfide bonds.

Crystal Structure of the Fab Fragment of Antibody 1 Bound to Human Epiregulin

[0103] The crystal structure of the Fab fragment of Antibody 1 bound to human epiregulin was solved. The complex was crystallized and the 1.8A structure was solved by molecular replacement using Phaser 2.8.3 and subsequently refined with Refmac 5.8.0258. In the crystal, Antibody 1 Fab's bind on opposite faces of an epiregulin dimer. The epiregulin Leu77 and His78 sidechains are at the center of the epitope with Leu77 buried in a hydrophobic pocket and His78 sidechain centered in a network of hydrogen bonds. The Antibody 1 heavy chain Arg50 sidechain is a hydrogen bond donor to the heavy chain Tyr52 sidechain hydroxyl, the heavy chain Tyr52 sidechain hydroxyl is a hydrogen bond donor to the deprotonated His78 sidechain N.sup.δ1 hydrogen bond acceptor, the protonated His78 sidechain N.sup.ε2 is as a hydrogen bond donor to a water molecule, and this water molecule is as a hydrogen donor to the heavy chain Leu109 and light chain Tyr107 backbone carbonyls. Antibody 1 residues are numbered by IMGT convention. Additional identified epitope contacts include Glu95, Tyr98, Arg102, and Phe106 sidechains and Leu77, Val96, and Glu104 backbone. The structure clearly demonstrates that the Antibody 1 epitope is conformational and not linear, confirming the Western results above.

Mutant Epiregulin ELISA with Antibody 1 Fab

[0104] Key epitope residues were further characterized by point mutagenesis to understand their relative contribution both to binding and to selectivity. Mono Fc EREG fusions (see above) were diluted to 1 μg/mL in PBS, and 100 μL was added to each well to coat an ELISA plate (Greiner Cat#655061) overnight at 5° C. Variants were grouped by column on the plate with wild type coated in 3 replicate columns and the point mutants coated in single replicate columns. Following the coat, the plate was washed 3×200 μL per well with PBST, then blocked at RT for 1 hour on a plate shaker using casein blocking buffer (Thermo Cat#37528). After blocking, the plate was washed as before. Antibody 1 Fab fragment was diluted to 1 μg/mL in casein blocking buffer, then 3×serially diluted in blocking buffer for a total of 7 concentrations and a blocking buffer blank. The Fab dilution series was added to the prepared ELISA plate, 100 μL per well, and incubated at RT for 1 hour on a plate shaker. The plate was washed as before, then 100 μL of goat anti-human kappa HRP secondary (Southern Biotech Cat#2060-05) diluted 1:8,000 in blocking buffer was added to each well. The plate was incubated with secondary antibody for 1 hour at RT on a plate shaker, then washed as before. TMB substrate was prepared (Thermo Cat#34021), and 100 μL was added to all wells. Plate was incubated statically at RT for 3-5 minutes, then 100 μL of 1N HCl was added to all wells to stop the reaction. The plate was read at 450 nm on a plate reader. Blank subtracted ELISA data were plotted in GraphPad Prism 9 with antibody concentration on the X-axis and OD450 on the Y-axis. Curves were generated by fitting the data using non-linear regression to the ‘Sigmoidal, 4PL, X is concentration’ model.

[0105] Point mutants were evaluated by ELISA with epiregulin mutants coated on the plate, Antibody 1 Fab dilutions series added, and signal measured by anti-kappa secondary (see FIG. 5). With exception to Y98A, all mutants showed diminished binding. H78A nearly knocked out binding, and Y98A did not exhibit detectable binding at the concentrations tested.

SPR Analysis of Antibody 1 Fab Fragment Binding to Mono Fc Human EREG Mutants

[0106] A Biacore 8K instrument and reagents (Cytiva) were used for the SPR analysis of Antibody 1 Fab fragment binding to both mono Fc human EREG mutants. Low levels of mono Fc human EREG mutants were immobilized in the sample flow cells (Fc2) of a CMS S series sensor chip (Cytiva P/N BR100530) using an amine coupling kit (Cytiva P/N BR100050) and the “Low Level CMS Coupling” method in Biacore 8K control software. Running buffer was 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% Tween 20), and running temperature was 37° C. Biacore experiments were performed in three independent experiments with single replicates of each dilution within each independent experiment. Reference subtracted, blank subtracted SPR data was analyzed using either the ‘1:1 binding’ kinetic model (EREG variants) or the ‘Steady State Affinity’ models with Biacore Insight Evaluation Version 3.0 (Cytiva) to determine a KD value.

[0107] Antibody 1 Fab was diluted in running buffer to 300 nM (pH 7.4), or 900 nM (pH 6.0), then 3×serially diluted down for a total of 7 dilutions. Fab was injected over all flow cells for 240 seconds followed by an 1800 second dissociation at a flow rate of 50 μL/min. Chip surface was regenerated with 2×30 second injections of 7M guanidine at 100 μL/min. Reference subtracted data was collected as sample flow cell minus reference flow cell (Fc2-Fc1) in each of the 8 channels, and then the reference subtracted data was buffer blank subtracted.

[0108] Binding affinity of a subset of the mutants was measured by Biacore at 37° C. Results are reported in Table 9 as the mean±standard deviation of 3 independent replicate measurements. “rel” indicates relative to wild type (wt).

TABLE-US-00011 TABLE 9 Antibody 1 binding affinity to wild type and mutant EREG at pH 6.0 and pH 7.4 at 37° C. pH 7.4 K.sub.D pH 6.0 K.sub.D Fold Fold K.sub.D pH EREG pH 7.4, 37° C. Change pH 6.0, 37° C. Change 6.0/K.sub.D Variant SEQ ID NO K.sub.D (pM) (rel to wt) K.sub.D (pM) (rel to wt) pH 7.4 wt (wild 27 51.8 ± 5.6 1 508 ± 6 1 9.8 type) L77F 53 26,500 ± 1,600 511 197,000 ± 6,000  388 7.4 H78N 55 29,300 ± 1,500 565 25,600 ± 2,200 50 0.9 E95A 56 19,600 ± 1,700 379 81,400 ± 500   160 4.2 E95H 57 7,180 ± 420  139 47,300 ± 700   93 6.6 R102A 59 177 ± 43 3 1,930 ± 140  4 10.9 F106A 60 12,500 ± 500   241 77,100 ± 3,600 152 6.2 F106K 61 54,600 ± 3,100 1,050 293,000 ± 16,000 577 5.4

[0109] Results in Table 9 show a range of weaker affinities for mutants of 3-fold for R102A up to >1000 fold for F106K at pH 7.4.

Human EGFR Ligand Epitope Alignment

[0110] In context of EGFR ligand sequence alignment (FIG. 6), the data show that E95 and F106 are the key epitope positions for EREG selectivity. The 100% conserved Y98 and R102 positions, while important for binding, are not contributing to selectivity. L77 and H78 are also critical for binding and impact selectivity, but they are highly conserved across the EGFR ligands and therefore not as EREG specific. These results demonstrate not only the structural basis for binding and exquisite selectivity of Antibody 1 for EREG, but also inform ligand and species selectivity from the epitope mapping studies.

TABLE-US-00012 Listing of Amino Acid and Nucleotide Sequences Heavy Chain of Antibody 1 (SEQ ID NO: 1) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPAGKGLEWIGRI YPSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGLVM DVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKP SNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG Light Chain of Antibody 1 (SEQ ID NO: 2) EIVLTQSPGTLSLSPGERATLSCRASQSVEFSYLAWYQQKPGQAPRLLIYG ASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQYGTNPFTFGQG TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC HCVR of Antibody 1 (SEQ ID NO: 3) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPAGKGLEWIGRI YPSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGLVM DVWGQGTLVTVSS LCVR of Antibody 1 (SEQ ID NO: 4) EIVLTQSPGTLSLSPGERATLSCRASQSVEFSYLAWYQQKPGQAPRLLIYG ASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQYGTNPFTFGQG TKVEIK HCDRI of Antibody 1 (SEQ ID NO: 5) TVSGGSISSYYWS HCDR2 of Antibody 1 (SEQ ID NO: 6) RIYPSGNTN HCDR3 of Antibody 1 (SEQ ID NO: 7) ARGGLVMDV LCDR1 of Antibody 1 (SEQ ID NO: 8) RASQSVEFSYLA LCDR2 of Antibody 1 (SEQ ID NO: 9) YGASSRAT LCDR3 of Antibody 1 (SEQ ID NO: 10) HQYGTNPFT DNA Encoding the Heavy Chain of Antibody 1 (SEQ ID NO: 11) CAGGTGCAGCTGCAGGAGTCGGGTCCAGGACTGGTGAAGCCTTCGGAGACC CTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTTCGTACTACTGG AGCTGGATTCGGCAGCCCGCAGGGAAGGGACTGGAGTGGATTGGGAGGATC TATCCGAGTGGGAACACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACC ATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTG ACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGGAGGACTGGTGATG GACGTGTGGGGACAGGGAACACTAGTGACCGTGAGTAGCGCCTCCACCAAG GGCCCATCGGTCTTCCCGCTAGCGCCCTGCTCCAGGAGCACCTCCGAGAGC ACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG GTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCC TCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCC AGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGC CCACCCTGCCCAGCACCTGAGGCCGCCGGGGGACCATCAGTCTTCCTGTTC CCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACG TGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGG TACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG CAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAG GACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTC CCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAG CCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAG GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTG GAGTGGGAAAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCC GTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGAC AAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAG GCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGT DNA Encoding the Light Chain of Antibody 1 (SEQ ID NO: 12) GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAA AGAGCCACCCTCTCCTGCAGGGCCAGTCAGTCTGTGGAATTCAGCTACTTA GCCTATGGTGCATCCAGCAGGGCCACTTGGTACCAGCAGAAACCTGGCCAG GCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCA GACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGC AGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCACCAGTACGGAACA AACCCGTTCACATTCGGGCAGGGAACCAAGGTTGAAATAAAGCGAACTGTG GCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCC AAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACC CTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA GTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGA GAGTGC Human epiregulin (SEQ ID NO: 21) VSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVRCEHFFL Human epiregulin construct (SEQ ID NO: 22) GPGVSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVRCEHFFLG Cynomolgus monkey epiregulin (SEQ ID NO: 23) VSITKCNSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVRCEHFYL Rat epiregulin (SEQ ID NO: 24) VLITKCSSDMDGYCLHGHCIYLVDMSEKYCRCEVGYTGLRCEHFFL Rabbit epiregulin (SEQ ID NO: 25) VSITKCGSDMNGYCLHGQCIYLVDMSENYCRCEVGYTGVRCEHFFL Full length human epiregulin (membrane bound, with T111P mutation) (SEQ ID NO: 26) MTAGRRMEMLCAGRVPALLLCLGFHLLQAVLSTTVIPSCIPGESSDNCTAL VQTEDNPRVAQVSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVR CEHFFLTVPQPLSKEYVALTVILIILFLITVVGSTYYFCRWYRNRKSKEPK KEYERVTSGDPELPQV Monomeric Ec human epiregulin (SEQ ID NO: 27) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSITKCSSDMNGYCLHGQCI YLVDMSQNYCRCEVGYTGVRCEHFFLG Cleaved mouse EREG (SEQ ID NO: 28) GPGVQITKCSSDMDGYCLHGQCIYLVDMREKFCRCEVGYTGLRCEHFFLG Mouse TGFa His (SEQ ID NO: 29) VVSHFNKCPDSHTQYCFHGTCRFLVQEEKPACVCHSGYVGVRCEHADLLAG HHHHHH HCDR1 of Antibody 1 (Kabat) (SEQ ID NO: 31) SYYWS HCDR2 of Antibody 1 (Kabat) (SEQ ID NO: 32) RIYPSGNTNYNPSLKS HCDR3 of Antibody 1 (Kabat) (SEQ ID NO: 33) GGLVMDV LCDR1 of Antibody 1 (Kabat) (SEQ ID NO: 34) RASQSVEFSYLA LCDR2 of Antibody 1 (Kabat) (SEQ ID NO: 35) GASSRAT LCDR3 of Antibody 1 (Kabat) (SEQ ID NO: 36) HQYGTNPFT HCDR1 of Antibody 1 (Chothia) (SEQ ID NO: 37) GGSISSY HCDR2 of Antibody 1 (Chothia) (SEQ ID NO: 38) YPSGN HCDR3 of Antibody 1 (Chothia) (SEQ ID NO: 39) GGLVMDV LCDR1 of Antibody 1 (Chothia) (SEQ ID NO: 40) RASQSVEFSYLA LCDR2 of Antibody 1 (Chothia) (SEQ ID NO: 41) GASSRAT LCDR3 of Antibody 1 (Chothia) (SEQ ID NO: 42) HQYGTNPFT HCDRI of Antibody 1 (IMGT) (SEQ ID NO: 43) GGSISSYY HCDR2 of Antibody 1 (IMGT) (SEQ ID NO: 44) IYPSGNT HCDR3 of Antibody 1 (IMGT) (SEQ ID NO: 45) ARGGLVMDV LCDRI of Antibody 1 (IMGT) (SEQ ID NO: 46) QSVEFSY LCDR2 of Antibody 1 (IMGT) (SEQ ID NO: 47) GAS LCDR3 of Antibody 1 (IMGT) (SEQ ID NO: 48) HQYGTNPFT IgG4PAA hinge region (SEQ ID NO: 51) ESKYGPPCPPCP IgG4PAA Fc region (SEQ ID NO: 52) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLG Mono Fc huEREG L77F (SEQ ID NO: 53) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSITKCSSDMNGYCFHGQCI YLVDMSQNYCRCEVGYTGVRCEHFFLG Mono Fc huEREG H78A (SEQ ID NO: 54) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSITKCSSDMNGYCLAGQCI YLVDMSQNYCRCEVGYTGVRCEHFFLG Mono Fc huEREG H78N (SEQ ID NO: 55) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSITKCSSDMNGYCLNGQCI YLVDMSQNYCRCEVGYTGVRCEHFFLG Mono Fc huEREG E95A (SEQ ID NO: 56) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSITKCSSDMNGYCLHGQCI YLVDMSQNYCRCAVGYTGVRCEHFFLG Mono Fc huEREG E95H (SEQ ID NO: 57) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSITKCSSDMNGYCLHGQCI YLVDMSQNYCRCHVGYTGVRCEHFFLG Mono Fc huEREG Y98A (SEQ ID NO: 58) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSITKCSSDMNGYCLHGQCI YLVDMSQNYCRCEVGATGVRCEHFFLG Mono Fc huEREG R102A (SEQ ID NO: 59) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSITKCSSDMNGYCLHGQCI YLVDMSQNYCRCEVGYTGVACEHFFLG Mono Fc huEREG F106A (SEQ ID NO: 60) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSITKCSSDMNGYCLHGQCI YLVDMSQNYCRCEVGYTGVRCEHAFLG Mono Fc huEREG F106K (SEQ ID NO: 61) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFQLESRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGGGGGSGGGGSLEVLFQGPGVSITKCSSDMNGYCLHGQCI YLVDMSQNYCRCEVGYTGVRCEHAKLG Antibody 1 Fab HC (SEQ ID NO: 62) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPAGKGLEWIGRI YPSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGLVM DVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKP SNTKVDKRVESK Antibody 1 Fab LC (SEQ ID NO: 63) EIVLTQSPGTLSLSPGERATLSCRASQSVEFSYLAWYQQKPGQAPRLLIYG ASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQYGTNPFTFGQG TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTQGTTSV TKSFNRGEC Antibody 1 VR chimera h/mIgG1 HC (SEQ ID NO: 64) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPAGKGLEWIGRI YPSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGLVM DVWGQGTLVTVSSASTKGPSVFPLAPGSAAQTNSMVTLGCLVKGYFPEPVT VTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPAS STKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVV VDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWL NGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSL TCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSN WEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK Antibody 1 VR chimera h/mKappa LC (SEQ ID NO: 65) EIVLTQSPGTLSLSPGERATLSCRASQSVEFSYLAWYQQKPGQAPRLLIYG ASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQYGTNPFTFGQG TKVEIKRTVAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDG SERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTS PIVKSFNRNEC human epigen/EPGN (SEQ ID NO: 66) KFSHLCLEDHNSYCINGACAFHHELEKAICRCFTGYTGERCEHLTLT human amphiregulin/AREG (SEQ ID NO: 67) KKKNPCNAEFQNFCIHGECKYIEHLEAVTCKCQQEYFGERCGEKSMK human heparin binding epidermal growth factor/HBEGF (SEQ ID NO: 68) KKRDPCLRKYKDFCIHGECKYVKELRAPSCICHPGYHGERCHGLSL human betacellulin/BTC (SEQ ID NO: 69) GHFSRCPKQYKHYCIKGRCRFVVAEQTPSCVCDEGYIGARCERVDLFY human transforming growth factor alpha/TGFa (SEQ ID NO: 70) WSHFNDCPDSHTQFCFHGTCRFLVQEDKPACVCHSGYVGARCEHADLLA human epidermal growth factor/EGF (SEQ ID NO: 71) NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKW