ANTI-FERROPORTIN 1 MONOCLONAL ANTIBODIES AND USES THEREOF

Abstract

Provided are monoclonal antibodies and antigen-binding fragments thereof that bind to, and inhibit the activity of human FPN1, and which are effective in maintaining or increasing the transport of iron out of mammalian cells and/or maintaining or increasing the level of serum iron, reticulocyte count, red blood cell count, hemoglobin, and/or hematocrit in a subject in vivo.

Description

[0001] The present invention relates to antibodies that bind ferroportin 1 (FPN1) and their use in treating anemia.

[0002] Iron is an essential trace element that is required for numerous cellular functions. In mammals, the supply of iron to the body is regulated to match the body's iron requirements at the level of iron absorption by duodenal enterocytes. Iron transport across the basolateral membrane of the enterocyte is thought to be mediated by ferroportin 1 (also known as iron-regulated transporter 1 (IREG-1), metal transporter protein 1 (MTP1) and SLC40A1), hereafter referred to as FPN1.

[0003] FPN1 is now known to be a receptor for hepcidin, a polypeptide hormone made by the liver in response to iron stores and inflammation. Binding of mature hepcidin to FPN1 leads to the internalization and degradation of FPN1, preventing cellular iron export, and it is a major controlling factor of systemic iron homeostasis.

[0004] U.S. Pat. No. 7,166,448 discloses, inter alia, nucleotide sequences encoding human FPN1 proteins, human FPN1 proteins having iron transport function, and a rabbit polyclonal antiserum generated to a peptide consisting of the C-terminal 19 amino acids of the human FPN1. PCT International Patent Application Publication No. WO2009/094551 describes ferroportin Mabs and methods of using them for treating disorders of iron homeostasis. More specifically, WO2009/094551 describes rodent and fully human monoclonal antibodies to various epitopes of human FPN1 proteins.

[0005] In view of the involvement of FPN1 in iron transport and the association of FPN1 mutations with diseases of iron homeostasis, there exists a need for therapeutically useful FPN1 antagonists that bind with high affinity to an extracellular epitope of FPN1 and, upon binding, inhibit mature hepcidin-mediated FPN1 internalization, thereby maintaining or enhancing export of iron from intracellular stores. Additionally, targeting FPN1 therapeutically with a Mab, or antigen-binding fragment thereof, with the goal of inhibiting the binding of mature hepcidin to an extracellular epitope of FPN1 must be accomplished precisely enough so that the antibody doesn't significantly perturb the efflux of cellular iron and/or induce internalization upon binding to its target. Additionally, an anti-FPN antibody intended for use in human medical therapy must exhibit sufficient pharmacokinetic and pharmacodynamic characteristics, including in vivo stability and/or elimination half life to allow for their therapeutic use. One or more of the anti-human FPN1 antibodies disclosed herein specifically bind human FPN1, including at least one peptide fragment thereof selected from the group consisting of:

TABLE-US-00001 a) ₄₀₃SPFEDIRSRFIQGESITPTK₄22; (SEQ ID NO: 12) b) ₄₀₆EDIRSRFIQGESIT₄19; (SEQ ID NO: 13) c) ₄₀₉RSRFIQGESITPTK₄22; (SEQ ID NO: 14) d) ₄₀₃SPFEDIRSRFIQG₄15; (SEQ ID NO: 15) e) ₄₀₉RSRFIQGESIT₄19; (SEQ ID NO: 16) and f) ₄₀₉RSRFIQG₄15, (SEQ ID NO: 95)

block the binding of hepcidin to ferroportin, potently inhibit hepcidin activity in vitro, elevate serum iron levels in a dose-dependent manner in vivo, and have acceptable solubility, in vivo stability, and elimination half life characteristics, making them useful agents for treating and/or preventing anemia in a subject in need of such treatment by administration via intravenous infusion or, even perhaps, via subcutaneous injection.

[0006] Thus, among its various aspects, the present invention provides:

[0007] Monoclonal antibodies, or antigen-binding fragments thereof, which specifically bind to human ferroportin 1 consisting of the amino acid sequence shown in SEQ ID NO: 1 at an epitope comprising amino acids localized to one or more amino acid sequences selected from the group consisting of:

TABLE-US-00002 a. ₄₀₃SPFEDIRSRFIQGESITPTK₄22; (SEQ ID NO: 12) b. ₄₀₆EDIRSRFIQGESIT₄19; (SEQ ID NO: 13) c. ₄₀₉RSRFIQGESITPTK₄22; (SEQ ID NO: 14) d. ₄₀₃SPFEDIRSRFIQG₄15; (SEQ ID NO: 15) e. ₄₀₉RSRFIQGESIT₄19; (SEQ ID NO: 16) and f. ₄₀₉RSRFIQG₄15. (SEQ ID NO: 95)

[0008] In some embodiments, the present invention provides Mabs, or antigen-binding fragments thereof, comprising a LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NOs: 37, 129, 22, 107, 118, and 120, respectively, and the Mab, or antigen-binding fragment, binds human FPN 1 consisting of the amino acid sequence shown in SEQ ID NO: 1 at an epitope comprising an amino acid or amino acids localized to an amino acid sequence as shown in SEQ ID NO: 12 with a K_D of about 100 nM or less as determined by surface plasmon resonance (SPR), preferably, at 25° C. for Mabs and 37° C. for Fabs.

[0009] In some embodiments, the Mab, or antigen-binding fragment thereof, comprises six CDRs selected from the group consisting of:

[0010] (i) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NOs: 20, 32, 33, 30, 31, and 19, respectively;

[0011] (ii) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NOs: 42, 32, 33, 30, 43, and 19, respectively;

[0012] (iii) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NOs: 42, 27, 22, 23, 41, and 19, respectively;

[0013] (iv) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NOs: 42, 32, 33, 23, 41, and 19, respectively;

[0014] (v) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NOs: 20, 21, 22, 17, 18, and 19, respectively;

[0015] (vi) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NOs: 20, 27, 29, 23, 24, and 19, respectively;

[0016] (vii) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NOs: 37, 27, 22, 23, 41, and 19, respectively;

[0017] (viii) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NOs: 170, 171, 172, 182, 173, and 19, respectively;

[0018] (ix) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NOs: 37, 127, 22, 23, 116, and 19, respectively;

[0019] (x) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NOs: 37, 125, 22, 23, 110, and 19, respectively;

[0020] (xi) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NOs: 37, 122, 22, 23, 110, and 19, respectively;

[0021] (xii) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NOs: 37, 129, 22, 107, 118, and 120, respectively;

[0022] (xiii) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NOs: 37, 128, 22, 105, 41, and 119, respectively;

[0023] (xiv) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NOs: 37, 174, 22, 175, 176, and 120, respectively;

[0024] (xvi) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NOs: 37, 128, 22, 105, 117, and 19, respectively; and

[0025] (xvii) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NOs: 37, 177, 22, 23, 112, and 19, respectively, and binds human FPN1 consisting of the amino acid sequence shown in SEQ ID NO: 1 at an epitope consisting of or consisting essentially of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of:

TABLE-US-00003 a) ₄₀₃SPFEDIRSRFIQGESITPTK₄22 (SEQ ID NO: 12) b) ₄₀₆EDIRSRFIQGESIT₄19; (SEQ ID NO: 13) c) ₄₀₉RSRFIQGESITPTK₄22; (SEQ ID NO: 14) d) ₄₀₃SPFEDIRSRFIQG₄15; (SEQ ID NO: 15) e) ₄₀₉RSRFIQGESIT₄19; (SEQ ID NO: 16) and f) ₄₀₉RSRFIQG₄15 (SEQ ID NO: 95)

with a K_D of less than about 100 nM as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs.

[0026] In particular embodiments, the Mabs, or antigen-binding fragment thereof, of the present invention, inhibit hepcidin-induced internalization and/or degradation of human FPN1, thereby maintaining or increasing 1) the transport of iron out of cells, 2) the level of serum iron, 3) reticulocyte count, 4) red blood cell count, 5) hemoglobin, and/or 6) hematocrit in a subject, preferably, a human subject, by at least about 10% compared to that of said subject in the absence of said Mab, or antigen-binding fragment thereof.

[0027] In another aspect, polynucleotides comprising a nucleotide sequence encoding anti-human FPN1 Mabs, or antigen-binding fragments thereof, of the present invention are provided.

[0028] In another aspect, pharmaceutical compositions are provided comprising any of the Mabs, or antigen-binding fragments thereof, described herein and a pharmaceutically acceptable carrier, diluent, or excipient. Also provided is 1) the use of the Mabs, or antigen-binding fragments thereof, disclosed herein in therapy, preferably, a therapy for treating or preventing anemia, 2) the use of the Mabs, or antigen-binding fragments thereof, disclosed herein in combination therapy, preferably, a combination therapy for treating or preventing anemia, 3) the use of the Mabs, or antigen-binding fragments thereof, disclosed herein for treating or preventing anemia, maintaining or increasing serum iron levels, reticulocyte count, red blood cell count, hemoglobin, and/or hematocrit in a subject, preferably, a human, 4) the use of the Mabs, or antigen-binding fragments thereof, disclosed herein for the manufacture of a medicament for treating or preventing anemia, maintaining or increasing serum iron levels, reticulocyte count, red blood cell count, hemoglobin, and/or hematocrit in a subject, preferably, a human, and 5) the use of the Mabs, or antigen-binding fragments thereof, disclosed herein in the manufacture of a medicament for use in combination therapy for treating or preventing anemia, increasing serum iron levels, reticulocyte count, red blood cell count, hemoglobin, and/or hematocrit in a human, wherein said medicament is to be administered in combination with one or more ESA or other therapeutic agent or therapeutic treatment conventionally employed to treat anemia, maintain or increase serum iron levels, reticulocyte count, red blood cell count, hemoglobin, and/or hematocrit in a human.

[0029] In yet another aspect, methods are provided for 1) maintaining or increasing serum iron levels, reticulocyte count, red blood cell count, hemoglobin, and/or hematocrit in a subject, preferably, a human, comprising administering to a subject in need thereof an effective amount of a Mab, or antigen-binding fragment thereof, disclosed herein, 2) treating or preventing anemia, including, but not limited to anemia of chronic disease, anemia of cancer, and anemia of inflammation, comprising administering to a subject, preferably, a human, in need thereof an effective amount of a Mab, or antigen-binding fragment thereof, disclosed herein, 3) a method of treating or preventing anemia, including, but not limited to anemia of chronic disease, anemia of cancer, and anemia of inflammation, comprising administering to a subject, preferably, a human patient in need thereof an effective amount of a combination of Mabs, or antigen-binding fragments thereof, disclosed herein, or a mixture of at least one Mab and at least one antigen-binding fragment disclosed herein, and 4) any one of the foregoing methods 1-3, further comprising administering to said human patient an ESA, or other therapeutic agent or therapeutic treatment administered to maintain or increase serum iron levels, reticulocyte count, red blood cell count, hemoglobin, and/or hematocrit in a human.

[0030] In yet another aspect, methods are provided for inhibiting mature hepcidin-induced internalization and degradation of FPN1, comprising contacting said FPN1 and an effective amount of at least one Mab, or antigen-binding fragment thereof, disclosed herein.

[0031] Also provided is a method of decreasing the binding of mature human hepcidin to human FPN1 comprising contacting said human FPN1 and an effective amount of at least one Mab, and/or antigen-binding fragment thereof, disclosed herein.

[0032] Also provided is a method of decreasing the amount of FPN1 proteins that are internalized by a cell expressing FPN1 proteins, comprising administering to a human patient in need thereof an effective amount of at least one of the Mabs, and/or antigen-binding fragments thereof, disclosed herein.

[0033] FIG. 1 depicts sequences of various peptides comprising fragments of the immunogen used to generate anti-FPN1 antibodies and the results of peptide-antibody binding experiments using the peptides to define the epitope of anti-FPN1 Mab 34A9. Underlined amino acids denote actual ferroportin sequences. Mab 34A9 binds to peptides FpnE3a, 060719Z, 0708L4C, and 0708L4D, which all contain the common amino acid sequence of RSRFIQG (SEQ ID NO:95).

[0034] FIG. 2A shows the amino acid sequences of fully human light chain framework O2 with interspersed CDRs. The four framework regions are labeled as FRL1, 2, 3, and 4 (SEQ ID NOs: 74, 75, 76, and 77, respectively).

[0035] FIG. 2B shows the amino acid sequence of the human heavy chain framework VH1-69 with interspersed CDRs. The four framework regions are labeled FRH1-4 (SEQ ID NOs: 78-81, respectively).

[0036] FIG. 3A shows the amino acid sequences of the human light chain framework O18 with interspersed CDRs The four framework regions are labeled as FRL1, 2, 3, and 4 (SEQ ID NOs: 74, 75, 82, and 77, respectively). Residues different from O2 residues are in bold and underlined

[0037] FIG. 3B shows the amino acid sequence of the human heavy chain framework VH1-18 with interspersed CDRs. The four framework regions are labeled FRH1, 2, 3, and 4 (SEQ ID NOs: 83, 79, 84, and 81, respectively). Residues different from VH1-69 residues are in bold and underlined

[0038] FIG. 4A shows the amino acid sequences of the human light chain framework L12 with interspersed CDRs The four framework regions are labeled as FRL1, 2, 3, and 4 (SEQ ID NOs: 85, 75, 86, and 77, respectively). Residues different from O2 residues are in bold and underlined

[0039] FIG. 4B shows the amino acid sequence of the human heavy chain framework VH1-46 with interspersed CDRs. The four framework regions are labeled FRH1, 2, 3, and 4 (SEQ ID NOs: 83, 79, 87, and 81, respectively). Residues different from VH1-69 residues are in bold and underlined.

[0040] FIG. 5 shows the amino acid sequences of the human light chain framework L1 with interspersed CDRs The four framework regions are labeled as FRL1, 2, 3, and 4 (SEQ ID NOs: 74, 168, 76, and 169, respectively). Residues different from O2 residues are in bold and underlined. The germline sequence for FR1 region of human light chain framework L12 is as shown in SEQ ID NO:85; a variation wherein the amino acid sequence of the FR1 region of the human light chain framework L1 is as shown in SEQ ID NO:74 is contemplated in certain embodiments of the present invention.

[0041] FIG. 6 shows a graph of serum hepcidin levels in male Cynomolgus monkeys after administration of control murine IgG1 or murine Mab 1G9 as a single I.V. dose of 30 mg/kg. Data are from individual animals.

[0042] FIG. 7 shows a graph of serum iron levels in male Cynomolgus monkeys after administration of control murine IgG1 or murine Mab 1G9 administered as a single I.V. dose of 30 mg/kg. Data are from individual animals.

[0043] FIG. 8 shows the amino acid sequences and consensus amino acid sequence of preferred heavy chain variable regions for the antibodies, and antigen-binding fragments thereof, of the present invention. A period (.) indicates the amino acid in that position is identical to the corresponding amino acid of sequence number 1. CDRs are underlined and in bold for sequence number 1.

[0044] FIG. 9 shows the amino acid sequences and consensus amino acid sequence of preferred light chain variable regions for the antibodies, and antigen-binding fragments thereof, of the present invention. A period (.) indicates the amino acid in that position is identical to the corresponding amino acid of sequence number 1. CDRs are underlined and in bold for sequence number 1.

[0045] FIG. 10 depicts serum concentrations of Mab 4A10-3 and Mab Combi11 in Cynomolgus monkeys following a single i.v. bolus dose of 0.3, 1.0, or 3.0 mg/kg. Serum concentrations of Mab Combi11 at the 0.3 mg/kg dose were below detection limit of the assay (<20 ng/mL). Data are the mean±SD.

[0046] FIG. 11 depicts serum concentrations of Mab 4A10-3 and Mab Combi11 in Sprague Dawley rats following a single i.v. bolus dose of 3.0 mg/kg. Data are the mean±SD.

[0047] The following abbreviations are used herein: BCA: bicinchoninic acid, BSA: bovine serum albumin, CDR: complementarity determining region, DTT: dithiothreitol, DMEM: Dulbecco's Modified Eagle's medium, D-PBS: Dulbecco's phosphate-buffered saline, EDTA: ethylenediamine tetraacetic acid, ELISA: enzyme linked immunosorbent assay, ESA: erythropoiesis-stimulating agent, FAC: ferric ammonium citrate, FBS: fetal bovine serum, Fe:NTA: ferric nitrilotriacetate, FLU: fluorescence units, GFP: green fluorescent protein, I.V.: intravenous, IPTG: Isopropyl β-D-1-thiogalactopyranoside, IMAC: Immobilized Metal Ion Affinity Chromatography, Mab: monoclonal antibody, Mabs: monoclonal antibodies, OPD: o-phenylenediamine dihydrochloride, PBS: phosphate-buffered saline, PBST: phosphate-buffered saline Tween-20, SDS: sodium dodecyl sulfate, TBS: Tris-buffered saline, Tris: tris(hydroxymethyl) aminomethane, Triton-X: 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol t-octylphenoxypolyethoxyethanol polyethylene glycol tert-octylphenyl ether, Tween-20: polysorbate 20.

[0048] The Mabs, or antigen-binding fragments thereof, of the present invention bind an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to amino acids 403-422 (SEQ ID NO: 12) of the human FPN1 polypeptide having the amino acid sequence as shown in SEQ ID NO: 1. In preferred embodiments, these antibodies, or antigen-binding fragments thereof, inhibit the mature human hepcidin induced-internalization and/or degradation of human FPN1, thereby increasing transport of iron out of cells in vitro and in vivo and elevating serum iron levels in vivo. For example, in preferred embodiments, the antibodies of the present invention significantly decrease mature hepcidin-induced accumulation of ferritin within Caco-2 cells, a human enterocyte cell line that endogenously expresses FPN1, in vitro (see, Example 5).

[0049] A full-length antibody as it exists naturally is an immunoglobulin molecule comprising 2 heavy chains and 2 light chains interconnected by disulfide bonds. The amino terminal portion of each chain includes a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition via the CDRs contained therein. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.

[0050] The term "CDR" as used herein is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat, et al., J. Biol. Chem. 252, 6609-6616 (1977), Kabat, et al., Sequences of protein of immunological interest, (1991), and by Chothia, et al., J. Mol. Biol. 196:901-917 (1987) and by MacCallum, et al., J. Mol. Biol., 262:732-745 (1996) where the definitions include overlapping or subsets of amino acid residues when compared against each other.

[0051] The CDRs are interspersed with regions that are more conserved, termed framework regions ("FR"). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3" and the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3." The CDRs contain most of the residues which form specific interactions with the antigen. The numbering and positioning of CDR amino acid residues within the LCVR and HCVR regions is in accordance with well-known conventions (e.g., Kabat, (1991) and/or Chothia (1987)).

[0052] The phrase "Kabat numbering" as used herein is recognized in the art and refers to a system of numbering amino acid residues which are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain regions of an antibody (Kabat, et al., Ann. NY Acad. Sci., 190:382-93 (1971); Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991)).

[0053] As used herein, the term "monoclonal antibody" (Mab) refers to an antibody that is derived from a single copy or clone including, for example, any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Mabs of the present invention preferably exist in a homogeneous or substantially homogeneous population. Complete Mabs contain 2 heavy chains and 2 light chains. Monoclonal antibodies, or antigen-binding fragments thereof, of the present invention can be produced, for example, by recombinant technologies, phage display technologies, synthetic technologies, e.g., CDR-grafting, or combinations of such technologies, or other technologies known in the art.

[0054] As used herein, the phrase "antigen-binding fragments" of Mabs include, for example, Fab fragments, Fab' fragments, F(ab')₂ fragments, and single chain Fv fragments.

[0055] The term "antibody", or grammatical versions thereof, unless indicated otherwise, refers to Mabs, antigen-binding fragments thereof, as well as combinations thereof, including, for example, combinations of Fabs, and combinations of Mabs and Fabs. Additional antibodies exhibiting similar functional properties as the antibodies according to the present invention can be generated by conventional methods. For example, mice can be immunized with, for example, FPN1 expressing cells, FPN1 or fragments thereof, the resulting antibodies can be recovered and purified, and determination of whether they possess binding, pharmacokinetic, and functional properties similar to or the same as the antibodies disclosed herein can be assessed by the methods disclosed in Examples 3-14 herein below. Antigen-binding fragments can also be prepared by conventional methods well-known in the art. Methods for producing and purifying antibodies and antigen-binding fragments are also well known in the art.

[0056] As used herein, the phrase "specifically binds" refers to the ability of an antibody of the present invention to bind to a specified polypeptide or peptide, preferably, human ferroportin 1 consisting of the amino acid sequence shown in SEQ ID NO: 1, or a specified peptide fragment thereof, with a K_D less than about 1000 nM, less than about 100 nM, less than about 10 nM, less than about 1 nM, less than about 100 μM, or less than about 10 μM, as determined by affinity ELISA or SPR assays as described herein, for example, or similar assays known in the art.

[0057] Additionally, or alternatively, the phrase "specifically binds" in reference to an antibody of the present invention indicates that the ability of the antibody to bind to or detect human ferroportin 1 consisting of the amino acid sequence shown in SEQ ID NO: 1, or a peptide fragment thereof, is at least about 5 times greater, at least about 10 times greater, at least about 20 times greater, at least about 50 times greater, at least about 100 times greater, at least about 200 times greater, at least about 500 times greater, or at least about 1000 times greater than its ability to bind to or detect another polypeptide (e.g., heparin) or, optionally, another specified peptide fragment of human ferroportin 1.

[0058] The present invention also provides an isolated polynucleotide comprising a nucleotide sequence that encodes a Mab, or antigen-binding fragment thereof. In particular embodiments, the present invention also provides an isolated polynucleotide comprising a nucleotide sequence that encodes i) a heavy chain polypeptide having the amino acid sequence as shown in SEQ ID NOs: 50, 51, 52, 150, 152, 156, 160, and 164, and/or ii) a light chain polypeptide having the amino acid sequence as shown in SEQ ID NOs: 53, 54, 55, 151, 154, 158, 162, and 166.

[0059] In another embodiment, the present invention provides a recombinant expression vector comprising a polynucleotide that encodes a Mab, or antigen-binding fragment thereof. In particular embodiments, the present invention also provides a recombinant expression vector comprising a polynucleotide that encodes (i) a heavy chain polypeptide having the amino acid sequence as shown in SEQ ID NOs: 50, 51, 52, 150, 152, 156, 160, and 164, and/or ii) a light chain polypeptide having the amino acid sequence as shown in SEQ ID NOs: 53, 54, 55, 151, 154, 158, 162, and 166.

[0060] When used herein, the term "hepcidin" refers to any form of the hepcidin protein known to be present in mammals. When used herein, the term "mature hepcidin" refers to any mature, bioactive form of the hepcidin protein expressed in mammals. When used herein, the phrase "human hepcidin" refers to any form of the hepcidin protein present in humans. When used herein, the phrase "mature human hepcidin" any mature, bioactive form of the hepcidin protein known to be present in humans. Preferably, "mature human hepcidin" refers to "human hepcidin-25", a mature form of human hepcidin having the amino acid sequence as shown in SEQ ID NO: 91.

[0061] The term "bioactivity" in reference to mature hepcidin polypeptides such as hepcidin-25 includes, but is not limited to, specific binding of mature hepcidin to its receptor FPN1, one or more FPN1-mediated functions of mature hepcidin, such as a) mature hepcidin-induced internalization and/or degradation of FPN1 (see, e.g., Nemeth, E., et al., Science, 306:2090-2093, (2004)), b) mature hepcidin regulation of FPN1-mediated i) iron efflux, ii) serum iron levels, iii) reticulocyte count, iv) red blood cell count, v) hemoglobin levels, vi) hematocrit, vii) expression levels of hepcidin polypeptides, and/or viii) tissue distribution of hepcidin polypeptides.

[0062] The phrase "human engineered antibodies" refers to certain antibodies disclosed herein as well as antibodies that have binding and functional properties according to the invention similar to the antibodies disclosed herein, and that have framework regions that are substantially human or fully human surrounding CDRs derived from a non-human antibody, or antigen-binding fragment thereof, disclosed herein. "Framework region" or "framework sequence" refers to any one of framework regions 1 to 4. Human engineered antibodies and antigen-binding fragments thereof encompassed by the present invention include molecules wherein any one or more of framework regions 1 to 4 is substantially or fully human, i.e., wherein any of the possible combinations of individual substantially or fully human framework regions 1 to 4, is present. For example, this includes molecules in which framework region 1 and framework region 2, framework region 1 and framework region 3, framework region 1, 2, and 3, etc., are substantially or fully human. Substantially human frameworks are those that have at least about 80% sequence identity to a known human germline framework sequence. Preferably, the substantially human frameworks have at least about 85%, about 90%, or about 95% sequence identity to a known human germline framework sequence.

[0063] Fully human frameworks are those that are identical to a known human germline framework sequence. Human framework germline sequences can be obtained from ImMunoGeneTics (IMGT) via their website http://imgt.cines.fr, or from The Immunoglobulin Facts Book by Marie-Paule Lefranc and Gerard Lefranc, Academic Press, 2001, ISBN 012441351. For example, germline light chain frameworks can be selected from the group consisting of: All, A17, A18, A19, A20, A27, A30, L1, L11, L12, L2, L5, L15, L6, L8, O12, O2, and O8, and germline heavy chain framework regions can be selected from the group consisting of: VH2-5, VH2-26, VH2-70, VH3-20, VH3-72, VH1-46, VH3-9, VH3-66, VH3-74, VH4-31, VH1-18, VH1-69, VI-13-7, VH3-11, VH3-15, VH3-21, VH3-23, VH3-30, VH3-48, VH4-39, VH4-59, and VH5-51. Preferably, germline light chain frameworks are selected from the group consisting of O2, O18, and L12, L1 and germline heavy chain framework regions are selected from the group consisting of VH1-69, VH1-18, or VH1-46. More preferably, germline light chain frameworks are selected from the group consisting of O2 and L1, and germline heavy chain frameworks are selected from the group consisting of VH1-69 and VH1-18. Most preferably, the germline light chain framework is O2 and the germline heavy chain framework region is VH1-69.

[0064] In addition to the human engineered antibodies disclosed herein, human engineered antibodies exhibiting similar functional properties as the antibodies according to the present invention can be generated using several different methods. The antibodies specifically disclosed herein can be used as templates or parent antibodies to prepare additional antibodies. In one approach, the CDRs of a parent antibody are grafted into a human framework that has a high sequence identity with the parent antibody framework. The sequence identity of the new framework will generally be at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the sequence of the corresponding framework in the parent antibody. This grafting may result in a reduction in binding affinity compared to that of the parent antibody. If this is the case, the framework can be back-mutated to the parent framework at certain positions based on specific criteria disclosed by Queen, et al., Proc. Natl. Acad. Sci. USA., 88:2869 (1991). Additional references describing methods useful in humanizing mouse antibodies include U.S. Pat. Nos. 4,816,397; 5,225,539, and 5,693,761; computer programs ABMOD and ENCAD as described in Levitt, J., Mol. Biol. 168:595-620 (1983); and the method of Winter and co-workers (Jones, et al., Nature 321:522-525 (1986); Riechmann, et al., Nature, 332:323-327 (1988); and Verhoeyen, et al., Science, 239:1534-1536 (1988).

[0065] The identification of residues to consider for back-mutation can be carried out as follows:

[0066] When an amino acid falls under the following category, the framework amino acid of the human germ-line sequence that is being used (the "acceptor framework") is replaced by a framework amino acid from a framework of the parent antibody (the "donor framework"): [0067] (a) the amino acid in the human framework region of the acceptor framework is unusual for human frameworks at that position, whereas the corresponding amino acid in the donor immunoglobulin is typical for human frameworks at that position; [0068] (b) the position of the amino acid is immediately adjacent to one of the CDRs; or [0069] (c) any side chain atom of a framework amino acid is within about 5-6 angstroms (center-to-center) of any atom of a CDR amino acid in a three dimensional immunoglobulin model.

[0070] When each of the amino acids in the human framework region of the acceptor framework and a corresponding amino acid in the donor framework is generally unusual for human frameworks at that position, such amino acid can be replaced by an amino acid typical for human frameworks at that position. This back-mutation criterion enables one to recover the activity of the parent antibody.

[0071] Another approach to generating human engineered antibodies exhibiting similar functional properties to the antibodies disclosed herein involves randomly mutating amino acids within the grafted CDRs without changing the framework, and screening the resultant molecules for binding affinity and other functional properties that are as good as or better than those of the parent antibodies. Single mutations can also be introduced at each amino acid position within each CDR, followed by assessing the effects of such mutations on binding affinity and other functional properties. Single mutations producing improved properties can be combined to assess their effects in combination with one another.

[0072] Further, a combination of both of the foregoing approaches is possible. After CDR grafting, one can back-mutate specific framework regions in addition to introducing amino acid changes in the CDRs. This methodology is described in Wu, et al., J. Mol. Biol. 294:151-162 (1999). Preferably, amino acid substitution within the frameworks is restricted to one, two, or three positions within any one or more of the light chain and/or heavy chain framework regions disclosed herein (i.e., framework regions shown in FIGS. 2-5). Preferably, amino acid substitution within the CDRs is restricted to one, two, or three positions within any one or more of the three light chain and/or heavy chain CDRs. Combinations of the various changes described within the framework regions and the CDRs are also contemplated herein. In particular embodiments of this aspect of the invention, all light and heavy chain variable region framework regions of such human engineered Mabs, or antigen-binding fragments thereof, are fully human.

[0073] Tables 1A and 1B below depict the CDR amino acid sequences and consensus amino acid sequences of preferred antibodies, or antigen-binding fragments thereof, of the present invention. Tables 2A and 2B below depict the CDR amino acid sequences and consensus amino acid sequences of more preferred antibodies, or antigen-binding fragments thereof, of the present invention.

TABLE-US-00004 TABLE 1A Fab HCDR1 HCDR2 HCDR3 34A9 GYAFTNFLIE TINPETGGTKYNEKFRG EFFDY (SEQ ID NO: 17) (SEQ ID NO: 18) (SEQ ID NO: 19) 1B1 GYAFTSFLIE (SEQ ID NO: 18) (SEQ ID NO: 19) (SEQ ID NO: 23) 1D2 (SEQ ID NO: 17) TINPRTGGTKYNEKFRG (SEQ ID NO: 19) (SEQ ID NO: 24) 1E3 (SEQ ID NO: 17) TINPKTGGTKYNEKFRG (SEQ ID NO: 19) (SEQ ID NO: 25) 2A6 (SEQ ID NO: 17) TINPETGGTKYNAKFRG (SEQ ID NO: 19) (SEQ ID NO: 26) 2H10 (SEQ ID NO: 17) (SEQ ID NO: 18) (SEQ ID NO: 19) 3A8 (SEQ ID NO: 17) (SEQ ID NO: 18) (SEQ ID NO: 19) 2G9 (SEQ ID NO: 17) (SEQ ID NO: 18) (SEQ ID NO: 19) 1A3 (SEQ ID NO: 23) (SEQ ID NO: 25) (SEQ ID NO: 19) 2E2 (SEQ ID NO: 23) (SEQ ID NO: 24) (SEQ ID NO: 19) 2A5 (SEQ ID NO: 23) (SEQ ID NO: 25) (SEQ ID NO: 19) 2B2 (SEQ ID NO: 23) (SEQ ID NO: 25) (SEQ ID NO: 19) 1D1 (SEQ ID NO: 23) TINPKTGGTKYNAKFRG (SEQ ID NO: 19) (SEQ ID NO: 34) 1E2 (SEQ ID NO: 23) (SEQ ID NO: 24) (SEQ ID NO: 19) hu-1 (SEQ ID NO: 23) (SEQ ID NO: 24) (SEQ ID NO: 19) 1G9 (SEQ ID NO: 23) (SEQ ID NO: 24) (SEQ ID NO: 19) huG1 (SEQ ID NO: 23) TSNPRTGGTKYNEKFRG (SEQ ID NO: 19) (SEQ ID NO: 35) huA2 (SEQ ID NO: 23) TINPRTGGTKYKEKFRG (SEQ ID NO: 19) (SEQ ID NO: 36) huA3 (SEQ ID NO: 23) (SEQ ID NO: 24) (SEQ ID NO: 19) huB3 (SEQ ID NO: 23) (SEQ ID NO: 24) (SEQ ID NO: 19) huD3 (SEQ ID NO: 23) (SEQ ID NO: 24) (SEQ ID NO: 19) huH5 (SEQ ID NO: 23) (SEQ ID NO: 24) (SEQ ID NO: 19) huH6 (SEQ ID NO: 23) (SEQ ID NO: 24) (SEQ ID NO: 19) 1D5 (SEQ ID NO: 23) TSNPRTGGTKYKEKFRG (SEQ ID NO: 19) (SEQ ID NO: 41) 2G5 (SEQ ID NO: 23) (SEQ ID NO: 41) (SEQ ID NO: 19) 3D8 (SEQ ID NO: 23) (SEQ ID NO: 41) (SEQ ID NO: 19) *C1 GYAFTX¹FLIE TINPX²TGGTKYNX³KFRG (SEQ ID NO: 19) (SEQ ID NO: 30) (SEQ ID NO: 31) *C2 (SEQ ID NO: 30) TX⁶NPX²TGGTKY (SEQ ID NO: 19) X⁷X³KFRG (SEQ ID NO: 43) *C3 (SEQ ID NO: 23) (SEQ ID NO: 41) (SEQ ID NO: 19) *C4 (SEQ ID NO: 23) (SEQ ID NO: 41) (SEQ ID NO: 19) *C5 (SEQ ID NO: 30) (SEQ ID NO: 43) (SEQ ID NO: 19)

TABLE-US-00005 TABLE 1B Fab LCDR1 LCDR2 LCDR3 34A9 RASKSISKYLA AGSTLHS QQHNEYPYT (SEQ ID NO: 20) (SEQ ID NO: 21) (SEQ ID NO: 22) 1B1 (SEQ ID NO: 20) (SEQ ID NO: 21) (SEQ ID NO: 22) 1D2 (SEQ ID NO: 20) (SEQ ID NO: 21) (SEQ ID NO: 22) 1E3 (SEQ ID NO: 20) (SEQ ID NO: 21) (SEQ ID NO: 22) 2A6 (SEQ ID NO: 20) (SEQ ID NO: 21) (SEQ ID NO: 22) 2H10 (SEQ ID NO: 20) AGSKLHS (SEQ ID NO: 22) (SEQ ID NO: 27) 3A8 (SEQ ID NO: 20) AGSRLHS (SEQ ID NO: 22) (SEQ ID NO: 28) 2G9 (SEQ ID NO: 20) AGSTLHS FQHNEYPYT (SEQ ID NO: 21) (SEQ ID NO: 29) 1A3 (SEQ ID NO: 20) (SEQ ID NO: 21) (SEQ ID NO: 22) 2E2 (SEQ ID NO: 20) (SEQ ID NO: 21) (SEQ ID NO: 22) 2A5 (SEQ ID NO: 20) (SEQ ID NO: 27) (SEQ ID NO: 22) 2B2 (SEQ ID NO: 20) (SEQ ID NO: 27) (SEQ ID NO: 29) 1D1 (SEQ ID NO: 20) (SEQ ID NO: 21) (SEQ ID NO: 29) 1E2 (SEQ ID NO: 20) (SEQ ID NO: 21) (SEQ ID NO: 29) 1G9 (SEQ ID NO: 20) (SEQ ID NO: 27) (SEQ ID NO: 29) hu-1 (SEQ ID NO: 20) (SEQ ID NO: 27) (SEQ ID NO: 29) huG1 (SEQ ID NO: 20) (SEQ ID NO: 27) (SEQ ID NO: 29) huA2 (SEQ ID NO: 20) (SEQ ID NO: 27) (SEQ ID NO: 29) huA3 RASKSISKYTA (SEQ ID NO: 27) (SEQ ID NO: 29) (SEQ ID NO: 37) huB3 RASKSISKYSA (SEQ ID NO: 27) (SEQ ID NO: 29) (SEQ ID NO: 38) huD3 RASKSISKYAA (SEQ ID NO: 27) (SEQ ID NO: 29) (SEQ ID NO: 39) huH5 (SEQ ID NO: 20) (SEQ ID NO: 27) (SEQ ID NO: 22) huH6 (SEQ ID NO: 20) (SEQ ID NO: 27) HQHNEYPYT (SEQ ID NO: 40) 1D5 (SEQ ID NO: 39) (SEQ ID NO: 27) (SEQ ID NO: 22) 2G5 (SEQ ID NO: 38) (SEQ ID NO: 27) (SEQ ID NO: 22) 3D8 (SEQ ID NO: 37) (SEQ ID NO: 27) (SEQ ID NO: 22) *C1 (SEQ ID NO: 20) AGSX⁴LHS X⁵QHNEYPYT (SEQ ID NO: 32) (SEQ ID NO: 33) *C2 RASKSISKY X⁸A (SEQ ID NO: 32) (SEQ ID NO: 33) (SEQ ID NO: 42) *C3 (SEQ ID NO: 42) (SEQ ID NO: 27) (SEQ ID NO: 22) *C4 (SEQ ID NO: 42) (SEQ ID NO: 32) (SEQ ID NO: 33) *C5 (SEQ ID NO: 42) (SEQ ID NO: 32) (SEQ ID NO: 33) *Tables lA and 1B, consensus sequences 1-5 (C1-C5), wherein X₁ is N or S; X₂ is E, K, or R; X₃ is E or A; X₆ is S or I; X₇ is N or K, X₄ is T, K, or R; X₅ is F, H, Q; X₈ is L, T, S, or A.

TABLE-US-00006 TABLE 2A Fab HCDR1 HCDR2 HCDR3 3D8 GYAFTSFLIE TSNPRTGGTKYKEKFRG EFFDY (SEQ ID NO: 23) (SEQ ID NO: 41) (SEQ ID NO: 19) 1G9 (SEQ ID NO: 23) (SEQ ID NO: 24) (SEQ ID NO: 19) 2G2 GRAFTSFLIE (SEQ ID NO: 41) (SEQ ID NO: 19) (SEQ ID NO: 103) 2A1 GKAFTSFLIE (SEQ ID NO: 41) (SEQ ID NO: 19) (SEQ ID NO: 104) 4H2 GYRFTSFLIE (SEQ ID NO: 41) (SEQ ID NO: 19) (SEQ ID NO: 105) 4C2 GYAFRSFLIE (SEQ ID NO: 41) (SEQ ID NO: 19) (SEQ ID NO: 106) 4A11 (SEQ ID NO: 23) TSNPRTRGTKYKEKFRG (SEQ ID NO: 19) (SEQ ID NO: 108) 5A2 (SEQ ID NO: 23) TSNPRTGRTKYKEKFRG (SEQ ID NO: 19) (SEQ ID NO: 109) 4A10 (SEQ ID NO: 23) TSNPRTGGRKYKEKFRG (SEQ ID NO: 19) (SEQ ID NO: 110) 1E3 (SEQ ID NO: 23) TSNPRTGGTKYKTKFRG (SEQ ID NO: 19) (SEQ ID NO: 111) 1F10 (SEQ ID NO: 23) TSNPRTGGTKYKSKFRG (SEQ ID NO: 19) (SEQ ID NO: 112) 3D1 (SEQ ID NO: 23) TSNPRTGGTKYKWKFRG (SEQ ID NO: 19) (SEQ ID NO: 113) 1E4 (SEQ ID NO: 23) TSNPRTGGTKYKEVFRG (SEQ ID NO: 19) (SEQ ID NO: 114) 4H6 (SEQ ID NO: 23) TSNPRTGGTKYKEKFRR (SEQ ID NO: 19) (SEQ ID NO: 115) 1G3 (SEQ ID NO: 23) (SEQ ID NO: 41) EFFVY (SEQ ID NO: 119) 1B5 (SEQ ID NO: 23) (SEQ ID NO: 41) (SEQ ID NO: 19) L2.2 (SEQ ID NO: 23) (SEQ ID NO: 41) (SEQ ID NO: 19) L2.6 (SEQ ID NO: 23) (SEQ ID NO: 41) (SEQ ID NO: 19) 7C8 (SEQ ID NO: 23) (SEQ ID NO: 41) (SEQ ID NO: 19) 6H4 (SEQ ID NO: 23) (SEQ ID NO: 41) (SEQ ID NO: 19) 7E4 (SEQ ID NO: 23) (SEQ ID NO: 41) (SEQ ID NO: 19) Combi-11 (SEQ ID NO: 23) TSNPRTGRTKYKSKFRG (SEQ ID NO: 19) (SEQ ID NO: 116) 4A10-3 (SEQ ID NO: 23) (SEQ ID NO: 110) (SEQ ID NO: 19) L2.2-4 (SEQ ID NO: 23) (SEQ ID NO: 110) (SEQ ID NO: 19) 1F8 GYRFTSFLIE TSNPRTGRTKYKTKFRG (SEQ ID NO: 19) (SEQ ID NO: 105) (SEQ ID NO: 117) 1B7 GYRFTSFLIE (SEQ ID NO: 41) (SEQ ID NO: 119) (SEQ ID NO: 105) Com11GY (SEQ ID NO: 23) (SEQ ID NO: 112) (SEQ ID NO: 19) Consensus GX₁X₂FX₃SFLIE TSNPRTX₄X₅X₆KYKX₇ EFFX₁₀Y 6* (SEQ ID NO: 107) X₈FRX₉ (SEQ ID NO: 120) (SEQ ID NO: 118)

TABLE-US-00007 TABLE 2B Fab LCDR1 LCDR2 LCDR3 3D8 RASKSISKYTA AGSKLHS QQHNEYPYT (SEQ ID NO: 37) (SEQ ID NO: 27) (SEQ ID NO: 22) 1G9 (SEQ ID NO: 20) (SEQ ID NO: 27) (SEQ ID NO: 29) 2G2 (SEQ ID NO: 37) (SEQ ID NO: 27) (SEQ ID NO: 22) 2A1 (SEQ ID NO: 37) (SEQ ID NO: 27) (SEQ ID NO: 22) 4H2 (SEQ ID NO: 37) (SEQ ID NO: 27) (SEQ ID NO: 22) 4C2 (SEQ ID NO: 37) (SEQ ID NO: 27) (SEQ ID NO: 22) 4A11 (SEQ ID NO: 37) (SEQ ID NO: 27) (SEQ ID NO: 22) 5A2 (SEQ ID NO: 37) (SEQ ID NO: 27) (SEQ ID NO: 22) 4A10 (SEQ ID NO: 37) (SEQ ID NO: 27) (SEQ ID NO: 22) 1E3 (SEQ ID NO: 37) (SEQ ID NO: 27) (SEQ ID NO: 22) 1F10 (SEQ ID NO: 37) (SEQ ID NO: 27) (SEQ ID NO: 22) 3D1 (SEQ ID NO: 37) (SEQ ID NO: 27) (SEQ ID NO: 22) 1E4 (SEQ ID NO: 37) (SEQ ID NO: 27) (SEQ ID NO: 22) 4H6 (SEQ ID NO: 37) (SEQ ID NO: 27) (SEQ ID NO: 22) 1G3 (SEQ ID NO: 37) (SEQ ID NO: 27) (SEQ ID NO: 22) 1B5 (SEQ ID NO: 37) AGSKRHS (SEQ ID NO: 22) (SEQ ID NO: 121) L2.2 (SEQ ID NO: 37) AGSKLRS (SEQ ID NO: 22) (SEQ ID NO: 122) L2.6 (SEQ ID NO: 37) AGSKLVS (SEQ ID NO: 22) (SEQ ID NO: 123) 7C8 (SEQ ID NO: 37) AGSKLYS (SEQ ID NO: 22) (SEQ ID NO: 124) 6H4 (SEQ ID NO: 37) AGSKLHW (SEQ ID NO: 22) (SEQ ID NO: 125) 7E4 (SEQ ID NO: 37) AGSKLHY (SEQ ID NO: 22) (SEQ ID NO: 126) Combi-11 (SEQ ID NO: 37) AGSKRHW (SEQ ID NO: 22) (SEQ ID NO: 127) 4A10-3 (SEQ ID NO: 37) (SEQ ID NO: 125) (SEQ ID NO: 22) L2.2-4 (SEQ ID NO: 37) (SEQ ID NO: 122) (SEQ ID NO: 22) 1F8 (SEQ ID NO: 37) AGSKRYY (SEQ ID NO: 22) (SEQ ID NO: 128) 1B7 (SEQ ID NO: 37) (SEQ ID NO:128) (SEQ ID NO: 22) Com11GY (SEQ ID NO: 37) AGSKRHY (SEQ ID NO: 22) (SEQ ID NO: 177) Consensus RASKSISKYTA AGSKX₁₁X₁₂X₁₃ QQHNEYPYT 6* (SEQ ID NO: 37) (SEQ ID NO: 129) (SEQ ID NO: 22) *Tables 2A and 2B, consensus sequence 6, wherein X₁ is Y, R, or K; X₂ is A, or R; X₃ is T or R; X₄ is G or R; X₅ is G or R; X₆ is T, or R; X₇ is E, T, S, or W; X₈ is K or V; X₉ is G or R; X₁₀ is D or V; X₁₁ is L or R; X₁₂ is H, R, V, or Y; X₁₃ is S, W, or Y.

[0074] Even more preferred antibodies, or antigen-binding fragments, of the invention comprise a LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NOs: 170, 171, 172, 23, 173, and 19, respectively, or SEQ ID NOs: 170, 171, 172, 182, 173, and 19, respectively. Even more preferred antibodies, or antigen-binding fragments, of the invention comprise a LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NOs: 37, 174, 22, 175, 176, and 120, respectively. Based upon pharmacokinetic (e.g., see, Example 11) and pharmacodynamic (e.g., see Examples 10 and 12) characteristics as well as the functional properties of exemplary anti-FPN1 Mabs, or antigen-binding fragments thereof, (e.g., see, Examples 3 (epitope mapping), 4 (affinity), 5 (effect on ferritin concentration in cells in vitro), 6 (effect on binding of mature hepcidin by cells engineered to express a FPN1-GFP fusion in vitro), 7 and 9 (effect on hepcidin-induced internalization and degradation of FPN1 in vitro), and 8 (effect on serum iron levels in vivo)), the most preferred Mabs, or antigen-binding fragments thereof, of the present invention are Mabs 4A10-3, L2.2-4, and Com11GY, or antigen-binding fragments thereof. The amino acid sequences encoding the heavy chains, the light chains, the heavy and light chain variable regions, and the CDRs for Mabs 34A9, 1G9, 3D8, Combi11, 4A10-3, L2.2-4, 1B7, 1F8, and Com11GY are indicated below in Table 3(a) and Table 3(b) by reference to SEQ ID NOs.

TABLE-US-00008 TABLE 3(a) Heavy HC HC HC Mab Chain HCVR CDR1 CDR2 CDR3 34A9 50 44 17 18 19 1G9 51 45 23 24 19 3D8 52 46 23 41 19 Combi11 150 130 23 116 19 4A10-3 152 134 23 110 19 L2.2-4 156 138 23 110 19 1B7 160 146 105 41 119 1F8 164 142 105 117 19 Com11GY 179 178 23 112 19

TABLE-US-00009 TABLE 3(b) Light LC LC LC Mab Chain LCVR CDR1 CDR2 CDR3 34A9 53 47 20 21 22 1G9 54 48 20 27 29 3D8 55 49 37 27 22 Combi-11 151 132 37 127 22 4A10-3 154 136 37 125 22 L2.2-4 158 140 37 122 22 1B7 162 148 37 128 22 1F8 166 144 37 128 22 Com11GY 181 180 37 177 22

[0075] In some embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, disclosed herein may be used in combination with one or more ESAs in order to provide additional benefits with respect to increasing serum iron levels, increasing hematocrits, increasing hemoglobin levels, reducing the need for transfusion, and/or improving the functional status, productivity, and quality of life of anemic patients as compared to the administration of the ESA therapy alone. By the phrase "combination therapy" or "in combination with" it is meant an anti-FPN1 Mab, or antigen-binding fragment thereof, of the present invention is administered separately, simultaneously, or sequentially with another agent intended to increase serum iron levels, increase hematocrits, increase hemoglobin levels, reducing the need for transfusions, and/or improving the functional status, productivity, and quality of life of anemic patients as compared to the administration of the anti-FPN1 Mab, or antigen-binding fragment thereof, alone.

[0076] In some embodiments, an anti-FPN1 Mab, or antigen-binding fragment thereof, disclosed herein may be administered in lieu of ESA therapy in patients intolerant or unresponsive to ESA therapy.

[0077] The phrase "erythropoiesis stimulating agent" or "erythropoiesis stimulator" means a compound that directly or indirectly causes activation of the erythropoietin receptor, for example, by binding to and causing dimerization of the receptor or by stimulating endogenous erythropoietin expression. ESAs include erythropoietin and variants, analogs, or derivatives thereof that bind to and activate erythropoietin receptor; antibodies that bind to erythropoietin receptor and activate the receptor; or peptides that bind to and activate erythropoietin receptor; or small organic chemical compounds, optionally less than about 1000 Daltons in molecular weight, that bind to and activate erythropoietin receptor. ESAs include, but are not limited to, epoetin alfa, epoetin beta, epoetin delta, epoetin omega, epoetin iota, epoetin zeta, and analogs thereof, pegylated erythropoietin, carbamylated erythropoietin, mimetic peptides (including EMP1/hematide), mimetic antibodies and HIF inhibitors (see U.S. Patent Publication No. 2005/0020487). Exemplary ESAs include erythropoietin, darbepoetin, erythropoietin agonist variants, and peptides or antibodies that bind and activate erythropoietin receptor including compounds reported in U.S. Patent Application Publication Nos. 2003/0215444 and 2006/0040858 as well as erythropoietin molecules or variants or analogs thereof also known in the art. Erythropoietin includes, but is not limited to, a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 88. The term "epoetin", includes, but is not limited to, epoetin alfa, epoetin beta, epoetin delta, epoetin omega, epoetin iota, epoetin gamma, epoetin zeta, and the like. Additionally, an epoetin also includes any of the aforementioned epoetins which are chemically modified, e.g., with one or more water-soluble polymers such as, e.g., polyethylene glycol (including PEG-EPO-beta). Exemplary sequences, manufacture, purification and use of recombinant human erythropoietin are described in a number of patent publications, including, but not limited to, U.S. Pat. Nos. 4,703,008 and 4,667,016. Exemplary sequences, manufacture, purification and use of darbepoetin and other erythropoietin analogs are described in a number of patent publications, including Strickland et al., 91/05867, and PCT International Patent Application Publications Nos. WO 95/05465, WO 00/24893, and WO 01/81405. Derivatives of naturally occurring or analog polypeptides include those which have been chemically modified, for example, to attach water soluble polymers (e.g., pegylated), radionuclides, or other diagnostic or targeting or therapeutic moieties.

[0078] The term "erythropoietic activity" means activity to stimulate erythropoiesis as demonstrated in an in vivo assay, for example, the exhypoxic, polycythemic mouse assay (see, e.g., Cotes and Bangham, Nature, 191:1065 (1961)).

[0079] The term "epitope" refers to that portion of any molecule capable of being recognized by and bound by an antibody at one or more of the antibody's antigen-binding regions. Epitopes often consist of a chemically active surface grouping of molecules such as amino acids or sugar side chains, and have specific three dimensional structural characteristics as well as specific charge characteristics. The human FPN1 epitopes disclosed herein are presented in the context of the primary amino acid structure of FPN1 (SEQ ID NO: 1). However, some of the epitopes may be discontinuous rather than continuous as the amino acid residues, rather than being in continuous peptide linkage, may be in spatial proximity to each other as a consequence of the tertiary or quaternary structure of FPN1 and their resultant presentation on the surface of this molecule. Preferably, an anti-FPN1 Mab, or antigen-binding fragment thereof, of the present invention binds human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to the amino acid sequence as shown in SEQ ID NO: 9. More preferably, an anti-FPN1 Mab, or antigen-binding fragment thereof, of the present invention binds human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95. Even more preferably, an anti-FPN1 Mab, or antigen-binding fragment thereof, of the present invention binds human FPN1 consisting of the amino acid sequence shown in SEQ ID NO: 1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 but does not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0080] Antibodies of the present invention also bind Cynomolgus monkey FPN1 (SEQ ID NO: 3), facilitating obligatory pre-clinical safety and efficacy therapeutic drug development studies in one or more primate models.

[0081] The phrase "human ferroportin 1" or, alternatively, "human FPN1" refers to an iron transporting protein expressed in humans that has the amino acid sequence as shown in SEQ ID NO: 1, as well as to variants which retain the ability to export cellular iron in response to interaction with mature human hepcidin.

[0082] Preferably, an anti-FPN1 Mab, or antigen-binding fragment thereof, of the present invention comprises: [0083] 1) a light chain variable region and a heavy chain variable region as shown in SEQ ID NO: 136 and SEQ ID NO: 134, respectively; [0084] 2) a light chain variable region and a heavy chain variable region as shown in SEQ ID NO: 180 and SEQ ID NO: 178, respectively; or [0085] 3) a light chain variable region and a heavy chain variable region as shown in SEQ ID NO: 140 and SEQ ID NO: 138, respectively. Even more preferably, an anti-FPN1 Mab, or antigen-binding fragment thereof, of the present invention comprises: [0086] 1) a light chain and a heavy chain as shown in SEQ ID NO: 154 and SEQ ID NO: 152, respectively; [0087] 2) a light chain and a heavy chain as shown in SEQ ID NO: 181 and SEQ ID NO: 179, respectively; or [0088] 3) a light chain and a heavy chain as shown in SEQ ID NO: 158 and SEQ ID NO: 156, respectively. Even more preferably, the monoclonal antibody or antigen binding fragment of the invention comprises: [0089] 1) two light chain polypeptides and two heavy chain polypeptides, and wherein each of the light chain polypeptides have the amino acid sequence as shown in SEQ ID NO: 154 and each of the heavy chain polypeptides have the amino acid sequence as shown in SEQ ID NO: 152; [0090] 2) two light chain polypeptides and two heavy chain polypeptides, and wherein each of the light chain polypeptides have the amino acid sequence as shown in SEQ ID NO: 181 and each of the heavy chain polypeptides have the amino acid sequence as shown in SEQ ID NO: 179; or [0091] 3) two light chain polypeptides and two heavy chain polypeptides, and wherein each of the light chain polypeptides have the amino acid sequence as shown in SEQ ID NO: 158 and each of the heavy chain polypeptides have the amino acid sequence as shown in SEQ ID NO: 156. Even more preferably, the monoclonal antibody or antigen binding fragment of the invention binds human ferroportin 1 with a K_D less than about 10 nM as determined by surface plasmon resonance at 25° C. Even more preferably, the monoclonal antibody or antigen binding fragment of the invention comprises a Fab, wherein the Fab binds human ferroportin 1 with a K_D less than about 100 nM as determined by surface plasmon resonance at 37° C. Even more preferably, the Fab has a dissociation rate (k_off) between about 7.5×10^-3 s^-1 and about 9×10^-4 s^-1 as determined by SPR at 37° C. for human ferroportin 1. Even more preferably, the Fab binds human ferroportin 1 with a K_D of between about 100 nM to about 1 nM. Even more preferably, the Fab binds human ferroportin 1 with a K_D of between about 10 nM to about 0.5 nM. Even more preferably, the monoclonal antibody or antigen-binding fragment has an IC₅₀ between about 100 nM and about 1 nM in an in vitro assay of hepcidin-25 bioactivity. Even more preferably, the monoclonal antibody or antigen-binding fragment thereof has an IC₅₀ between about 100 nM and about 10 nM in an in vitro assay of hepcidin-25 bioactivity. Even more preferably, the hepcidin-25 bioactivity is ferroportin 1 internalization and/or degradation. Even more preferably, the monoclonal antibody or antigen-binding fragment thereof has an IC₅₀ between about 100 nM and about 1 nM in an in vivo assay of hepcidin-25 bioactivity. Even more preferably, the in vivo assay of hepcidin-25 bioactivity measures an IL-6-induced decrease in serum iron levels in a primate. Most preferably, the anti-FPN 1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0092] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention are characterized by binding human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 with a K_D less than about 100 nM, less than about 75 nM, less than about 50 nM, less than about 25 nM, or less than about 10 nM as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs. Preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0093] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention are characterized by binding human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 with a K_D between about 100 nM to about 0.5 nM, preferably, between about 100 nM to about 0.5 nM, more preferably, between about 75 nM to about 5 nM, even more preferably, between about 50 nM to about 10 nM, even more preferably, between about 15 nM to about 10 nM, even more preferably, between about 10 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.7 nM, even more preferably, between about 3 nM to about 0.7 nM, or most preferably, from about 3 nM to about 1 nM, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs. Preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0094] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention are characterized by binding human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 with a dissociation rate (k_off) for human ferroportin 1 between about 7.5×10^-3 s^-1 and about 1×10^-4 s^-1, preferably between about 2.5×10^-3 s^-1 and about 1×10^-4 s^-1, more preferably between about 1×10^-3 s^-1 and about 1×10^-4 s^-1, and even more preferably between about 5×10^-4 s^-1 and about 1×10^-4 s^-1, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs. Preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0095] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention are characterized by binding human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 with a K_D less than about 100 nM, less than about 75 nM, less than about 50 nM, less than about 25 nM, or less than about 10 nM and a dissociation rate (k_off) for human ferroportin 1 between about 7.5×10^-3 s^-1 and about 1×10^-4 s^-1, preferably between about 2.5×10^-3 s^-1 and about 1×10^-4 s^-1, more preferably between about 1×10^-3 s^-1 and about 1×10^-4 s^-1, and even more preferably between about 5×10^-4 s^-1 and about 1×10^-4 s^-1, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs. Preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0096] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention are characterized by binding human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 with a K_D between about 100 nM to about 0.5 nM, preferably, between about 100 nM to about 1 nM, more preferably, between about 75 nM to about 5 nM, even more preferably, between about 50 nM to about 10 nM, even more preferably, between about 15 nM to about 10 nM, even more preferably, between about 10 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.7 nM, even more preferably, between about 3 nM to about 0.7 nM, or most preferably, from about 3 nM to about 1 nM, and a dissociation rate (k_off) for human ferroportin 1 between about 7.5×10^-3 s^-1 and about 1×10^-4 s^-1, preferably between about 2.5×10^-3 s^-1 and about 1×10^-4 s^-1, more preferably between about 1×10^-3 s^-1 and about 1×10^-4 s^-1, and even more preferably between about 5×10^-4 s^-1 and about 1×10^-4 s^-1, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs. Preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0097] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention comprise a LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NOs: 37, 129, 22, 107, 118, and 120, respectively, and bind human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 with a K_D less than about 100 nM, less than about 75 nM, less than about 50 nM, less than about 25 nM, or less than about 10 nM as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs. Preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0098] In some embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention comprise a LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NOs: 37, 129, 22, 107, 118, and 120, respectively, and bind human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 with a K_D between about 100 nM to about 0.5 nM, preferably, between about 100 nM to about 1 nM, more preferably, between about 75 nM to about 5 nM, even more preferably, between about 50 nM to about 10 nM, even more preferably, between about 15 nM to about 10 nM, even more preferably, between about 10 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.7 nM, even more preferably, between about 3 nM to about 0.7 nM, or most preferably, from about 3 nM to about 1 nM as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs. Preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0099] In other embodiments, the present invention provides Mabs, or antigen-binding fragments thereof, comprising a LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NOs: 37, 129, 22, 107, 118, and 120, respectively, that bind human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 with a dissociation rate (k_off) for human ferroportin 1 between about 7.5×10^-3 s^-1 and about 1×10^-4 s^-1, preferably between about 2.5×10^-3 s^-1 and about 1×10^-4 s^-1, more preferably between about 1×10^-3 s^-1 and about 1×10^-4 s^-1, and even more preferably between about 5×10^-4 s^-1 and about 1×10^-4 s^-1, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs. Preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0100] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention comprise a LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NOs: 37, 129, 22, 107, 118, and 120, respectively, and bind human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 with a K_D less than about 100 nM, less than about 75 nM, less than about 50 nM, less than about 25 nM, or less than about 10 nM and a dissociation rate (k_off) for human ferroportin 1 between about 7.5×10^-3 s^-1 and about 1×10^-4 s^-1, preferably between about 2.5×10^-3 s^-1 and about 1×10^-4 s^-1, more preferably between about 1×10^-3 s^-1 and about 1×10^-4 s^-1, and even more preferably between about 5×10^-4 s^-1 and about 1×10^-4 s^-1, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs. Preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0101] In some embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention comprise a LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NOs: 37, 129, 22, 107, 118, and 120, respectively, and bind human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 with a K_D between about 100 nM to about 0.5 nM, preferably, between about 100 nM to about 1 nM, more preferably, between about 75 nM to about 5 nM, even more preferably, between about 50 nM to about 10 nM, even more preferably, between about 15 nM to about 10 nM, even more preferably, between about 10 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.7 nM, even more preferably, between about 3 nM to about 0.7 nM, or most preferably, from about 3 nM to about 1 nM and a dissociation rate (k_off) for human ferroportin 1 between about 7.5×10^-3 s^-1 and about 1×10^-4 s^-1, preferably between about 2.5×10^-3 s^-1 and about 1×10^-4 s^-1, more preferably between about 1×10^-3 s^-1 and about 1×10^-4 s^-1, and even more preferably between about 5×10^-4 s^-1 and about 1×10^-4 s^-1, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs. Preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0102] The term "inhibit" means the ability to antagonize, prohibit, prevent, restrain, slow, disrupt, eliminate, stop, reduce, or reverse the biological effects of FPN1 and/or bioactivity of mature hepcidin including, but not limited to, a mature human hepcidin bioactivity as measured herein in Examples 5-11, 13, or 14, for example.

[0103] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention are characterized by having an IC₅₀ between about 250 nM and about 25 nM, preferably between 100 nM and about 25 nM, or more preferably between 50 nM and about 25 nM in an in vivo assay of hepcidin-25 bioactivity. Preferably, the in vivo assay measures an IL-6-induced decrease in serum iron levels.

[0104] Additionally, or alternatively, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention are characterized by having an IC₅₀ between about 100 nM and about 1 nM, preferably, between about 75 nM and about 1 nM, more preferably, between about 50 nM and about 1 nM, even more preferably, between about 25 nM and about 1 nM in an in vitro assay of hepcidin-25 bioactivity. Preferably, the in vitro assay of hepcidin-25 bioactivity assay measures hepcidin-induced internalization and/or degradation of ferroportin 1. Even more preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0105] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention are characterized by binding human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 with a K_D less than about 100 nM, less than about 75 nM, less than about 50 nM, less than about 25 nM, or less than about 10 nM as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs and an IC₅₀ between about 250 nM and about 25 nM, preferably between 100 nM and about 25 nM, or more preferably between 50 nM and about 25 nM in an in vivo assay of hepcidin-25 bioactivity. Preferably, the in vivo assay measures an IL-6-induced decrease in serum iron levels. Even more preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0106] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention are characterized by binding human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 with a K_D between about 100 nM to about 0.5 nM, preferably, between about 100 nM to about 1 nM, more preferably, between about 75 nM to about 5 nM, even more preferably, between about 50 nM to about 10 nM, even more preferably, between about 15 nM to about 10 nM, even more preferably, between about 10 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.7 nM, even more preferably, between about 3 nM to about 0.7 nM, or most preferably, from about 3 nM to about 1 nM, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs and further characterized by having an IC₅₀ between about 250 nM and about 25 nM, preferably between 100 nM and about 25 nM, or more preferably between 50 nM and about 25 nM in an in vivo assay of hepcidin-25 bioactivity. Preferably, the in vivo assay measures an IL-6-induced decrease in serum iron levels. Even more preferably, the anti-FPN1 Mabs, or antigen-binding fragment, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0107] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention are characterized by binding human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 with a K_D between about 100 nM to about 0.5 nM, preferably, between about 100 nM to about 1 nM, more preferably, between about 75 nM to about 5 nM, even more preferably, between about 50 nM to about 10 nM, even more preferably, between about 15 nM to about 10 nM, even more preferably, between about 10 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.7 nM, even more preferably, between about 3 nM to about 0.7 nM, or most preferably, from about 3 nM to about 1 nM, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs, have a dissociation rate (k_off) for human ferroportin 1 between about 7.5×10^-3 s^-1 and about 1×10^-4 s^-1, preferably between about 2.5×10^-3 s^-1 and about 1×10^-4 s^-1, more preferably between about 1×10^-3 s^-1 and about 1×10^-4 s^-1, and even more preferably between about 5×10^-4 s^-1 and about 1×10^-4 s^-1, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs and are further characterized by having an IC₅₀ between about 250 nM and about 25 nM, preferably between 100 nM and about 25 nM, or more preferably between 50 nM and about 25 nM in an in vivo assay of hepcidin-25 bioactivity. Preferably, the in vivo assay measures an IL-6-induced decrease in serum iron levels. Even more preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0108] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention comprise a LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NOs: 37, 129, 22, 107, 118, and 120, respectively, and bind human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 with a K_D less than about 100 nM, less than about 75 nM, less than about 50 nM, less than about 25 nM, or less than about 10 nM as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs and a dissociation rate (k_off) for human ferroportin 1 between about 7.5×10^-3 s^-1 and about 1×10^-4 s^-1, preferably between about 2.5×10^-3 s^-1 and about 1×10^-4 s^-1, more preferably between about 1×10^-3 s^-1 and about 1×10^-4 s^-1, and even more preferably between about 5×10^-4 s^-1 and about 1×10^-4 s^-1, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs and are further characterized by having an IC₅₀ between about 250 nM and about 25 nM, preferably between 100 nM and about 25 nM, or more preferably between 50 nM and about 25 nM in an in vivo assay of hepcidin-25 bioactivity. Preferably, the in vivo assay measures an IL-6-induced decrease in serum iron levels. Even more preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0109] In some embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention comprise a LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NOs: 37, 129, 22, 107, 118, and 120, respectively, and bind human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 with a K_D between about 100 nM to about 0.5 nM, preferably, between about 100 nM to about 1 nM, more preferably, between about 75 nM to about 5 nM, even more preferably, between about 50 nM to about 10 nM, even more preferably, between about 15 nM to about 10 nM, even more preferably, between about 10 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.7 nM, even more preferably, between about 3 nM to about 0.7 nM, or most preferably, from about 3 nM to about 1 nM, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs and a dissociation rate (k_off) for human ferroportin 1 between about 7.5×10^-3 s^-1 and about 1×10^-4 s^-1, preferably between about 2.5×10^-3 s^-1 and about 1×10^-4 s^-1, more preferably between about 1×10^-3 s^-1 and about 1×10^-4 s^-1, and even more preferably between about 5×10^-4 s^-1 and about 1×10^-4 s^-1, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs and are further characterized by having an IC₅₀ between about 250 nM and about 25 nM, preferably between 100 nM and about 25 nM, or more preferably between 50 nM and about 25 nM in an in vivo assay of hepcidin-25 bioactivity. Preferably, the in vivo assay measures an IL-6-induced decrease in serum iron levels. Even more preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0110] In some embodiments of the present invention, the anti-FPN1 Mabs, or antigen-binding fragments thereof, are characterized by binding human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 with a K_D less than about 75 nM, less than about 50 nM, less than about 25 nM, or less than about 10 nM as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs and have an IC₅₀ between about 250 nM and about 25 nM, preferably between 100 nM and about 25 nM, or more preferably between 50 nM and about 25 nM in an in vivo assay of hepcidin-25 bioactivity and are further characterized by having an IC₅₀ between about 100 nM and about 1 nM, preferably, between about 75 nM and about 1 nM, more preferably, between about 50 nM and about 1 nM, even more preferably, between about 25 nM and about 1 nM in an in vitro assay of hepcidin-25 bioactivity. Preferably, the in vivo assay measures an IL-6-induced decrease in serum iron levels. Preferably, the in vitro assay of hepcidin-25 bioactivity assay measures hepcidin-induced internalization and/or degradation of ferroportin 1. Even more preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0111] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention are characterized by binding human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 with a K_D between about 100 nM to about 0.5 nM, preferably, between about 100 nM to about 1 nM, more preferably, between about 75 nM to about 5 nM, even more preferably, between about 50 nM to about 10 nM, even more preferably, between about 15 nM to about 10 nM, even more preferably, between about 10 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.7 nM, even more preferably, between about 3 nM to about 0.7 nM, or most preferably, from about 3 nM to about 1 nM, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs and have an IC₅₀ between about 250 nM and about 25 nM, preferably, between 100 nM and about 25 nM, or more preferably, between 50 nM and about 25 nM in an in vivo assay of hepcidin-25 bioactivity and are further characterized by having an IC₅₀ between about 100 nM and about 1 nM, preferably, between about 75 nM and about 1 nM, more preferably, between about 50 nM and about 1 nM, even more preferably, between about 25 nM and about 1 nM in an in vitro assay of hepcidin-25 bioactivity. Preferably, the in vivo assay measures an IL-6-induced decrease in serum iron levels. Preferably, the in vitro assay of hepcidin-25 bioactivity assay measures hepcidin-induced internalization and/or degradation of ferroportin 1. Even more preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0112] In some embodiments, the present invention provides Mabs, or antigen-binding fragments thereof, which bind human FPN1 consisting of the amino acid sequence shown in SEQ ID NO: 1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 and 1) a K_D between about 100 nM to about 0.5 nM, preferably, between about 100 nM to about 1 nM, more preferably, between about 75 nM to about 5 nM, even more preferably, between about 50 nM to about 10 nM, even more preferably, between about 15 nM to about 10 nM, even more preferably, between about 10 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.7 nM, even more preferably, between about 3 nM to about 0.7 nM, or most preferably, from about 3 nM to about 1 nM, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs, 2) an IC₅₀ between about 250 nM and about 25 nM, preferably between 100 nM and about 25 nM, or more preferably between 50 nM and about 25 nM in an in vivo assay of hepcidin-25 bioactivity, 3) an IC₅₀ between about 100 nM and about 1 nM, preferably, between about 75 nM and about 1 nM, more preferably, between about 50 nM and about 1 nM, even more preferably, between about 25 nM and about 1 nM in an in vitro assay of hepcidin-25 bioactivity, and 4) a dissociation rate (k_off) for human ferroportin 1 between about 7.5×10^-3 s^-1 and about 1×10^-4 s^-1, preferably between about 2.5×10^-3 s^-1 and about 1×10^-4 s^-1, more preferably between about 1×10^-3 s^-1 and about 1×10^-4 s^-1, and even more preferably between about 5×10^-4 s^-1 and about 1×10^-4 s^-1, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs. Preferably, the in vivo assay measures an IL-6-induced decrease in serum iron levels. Preferably, the in vitro assay of hepcidin-25 bioactivity assay measures hepcidin-induced internalization and/or degradation of ferroportin 1. Even more preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0113] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention comprise a LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NOs: 37, 129, 22, 107, 118, and 120, respectively, and are characterized by having an IC₅₀ between about 250 nM and about 25 nM, preferably between 100 nM and about 25 nM, or more preferably between 50 nM and about 25 nM in an in vivo assay of hepcidin-25 bioactivity. Preferably, the in vivo assay measures an IL-6-induced decrease in serum iron levels. Even more preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0114] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention comprise a LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NOs: 37, 129, 22, 107, 118, and 120, respectively, and are characterized by having an IC₅₀ between about 250 nM and about 25 nM, preferably between 100 nM and about 25 nM, or more preferably between 50 nM and about 25 nM in an in vivo assay of hepcidin-25 bioactivity and an IC₅₀ between about 100 nM and about 1 nM, preferably, between about 75 nM and about 1 nM, more preferably, between about 50 nM and about 1 nM, even more preferably, between about 25 nM and about 1 nM in an in vitro assay of hepcidin-25 bioactivity. Preferably, the in vivo assay measures an IL-6-induced decrease in serum iron levels. Preferably, the in vitro assay of hepcidin-25 bioactivity assay measures hepcidin-induced internalization and/or degradation of ferroportin 1. Even more preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0115] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention comprise a LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NOs: 37, 129, 22, 107, 118, and 120, respectively, and bind human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 with a K_D less than about 100 nM, less than about 75 nM, less than about 50 nM, less than about 25 nM, or less than about 10 nM as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs and an IC₅₀ between about 250 nM and about 25 nM, preferably between 100 nM and about 25 nM, or more preferably between 50 nM and about 25 nM in an in vivo assay of hepcidin-25 bioactivity. Preferably, the in vivo assay measures an IL-6-induced decrease in serum iron levels. Even more preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0116] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention comprise a LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NOs: 37, 129, 22, 107, 118, and 120, respectively, and bind human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 with a K_D between about 100 nM to about 0.5 nM, preferably, between about 100 nM to about 1 nM, more preferably, between about 75 nM to about 5 nM, even more preferably, between about 50 nM to about 10 nM, even more preferably, between about 15 nM to about 10 nM, even more preferably, between about 10 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.7 nM, even more preferably, between about 3 nM to about 0.7 nM, or most preferably, from about 3 nM to about 1 nM, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs and further characterized by having an IC₅₀ between about 250 nM and about 25 nM, preferably between 100 nM and about 25 nM, or more preferably between 50 nM and about 25 nM in an in vivo assay of hepcidin-25 bioactivity. Preferably, the in vivo assay measures an IL-6-induced decrease in serum iron levels. Even more preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0117] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention comprise a LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NOs: 37, 129, 22, 107, 118, and 120, respectively, and bind human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 with a K_D between about 100 nM to about 0.5 nM, preferably, between about 100 nM to about 1 nM, more preferably, between about 75 nM to about 5 nM, even more preferably, between about 50 nM to about 10 nM, even more preferably, between about 15 nM to about 10 nM, even more preferably, between about 10 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.7 nM, even more preferably, between about 3 nM to about 0.7 nM, or most preferably, from about 3 nM to about 1 nM, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs, have a dissociation rate (k_off) for human ferroportin 1 between about 7.5×10^-3 s^-1 and about 1×10^-4 s^-1, preferably between about 2.5×10^-3 s^-1 and about 1×10^-4 s^-1, more preferably between about 1×10^-3 s^-1 and about 1×10^-4 s^-1, and even more preferably between about 5×10^-4 s^-1 and about 1×10^-4 s^-1, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs and are further characterized by having an IC₅₀ between about 250 nM and about 25 nM, preferably between 100 nM and about 25 nM, or more preferably between 50 nM and about 25 nM in an in vivo assay of hepcidin-25 bioactivity. Preferably, the in vivo assay measures an IL-6-induced decrease in serum iron levels. Even more preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0118] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention comprise a LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NOs: 37, 129, 22, 107, 118, and 120, respectively, and bind human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 with a K_D less than about 100 nM, less than about 75 nM, less than about 50 nM, less than about 25 nM, or less than about 10 nM as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs and a dissociation rate (k_off) for human ferroportin 1 between about 7.5×10^-3 s^-1 and about 1×10^-4 s^-1, preferably between about 2.5×10^-3 s^-1 and about 1×10^-4 s^-1, more preferably between about 1×10^-3 s^-1 and about 1×10^-4 s^-1, and even more preferably between about 5×10^-4 s^-1 and about 1×10^-4 s^-1, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs and are further characterized by having an IC₅₀ between about 250 nM and about 25 nM, preferably between 100 nM and about 25 nM, or more preferably between 50 nM and about 25 nM in an in vivo assay of hepcidin-25 bioactivity. Preferably, the in vivo assay measures an IL-6-induced decrease in serum iron levels. Even more preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0119] In some embodiments of the present invention, the anti-FPN1 Mabs, or antigen-binding fragments thereof, comprise a LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NOs: 37, 129, 22, 107, 118, and 120, respectively, and bind human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 with a K_D less than about 75 nM, less than about 50 nM, less than about 25 nM, or less than about 10 nM as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs and have an IC₅₀ between about 250 nM and about 25 nM, preferably between 100 nM and about 25 nM, or more preferably between 50 nM and about 25 nM in an in vivo assay of hepcidin-25 bioactivity and are further characterized by having an IC₅₀ between about 100 nM and about 1 nM, preferably, between about 75 nM and about 1 nM, more preferably, between about 50 nM and about 1 nM, even more preferably, between about 25 nM and about 1 nM in an in vitro assay of hepcidin-25 bioactivity. Preferably, the in vivo assay measures an IL-6-induced decrease in serum iron levels. Preferably, the in vitro assay of hepcidin-25 bioactivity assay measures hepcidin-induced internalization and/or degradation of ferroportin 1. Even more preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0120] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention comprise a LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NOs: 37, 129, 22, 107, 118, and 120, respectively, and bind human FPN1 consisting of the amino acid sequence shown in SEQ ID NO:1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 with a K_D between about 100 nM to about 0.5 nM, preferably, between about 100 nM to about 1 nM, more preferably, between about 75 nM to about 5 nM, even more preferably, between about 50 nM to about 10 nM, even more preferably, between about 15 nM to about 10 nM, even more preferably, between about 10 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.7 nM, even more preferably, between about 3 nM to about 0.7 nM, or most preferably, from about 3 nM to about 1 nM, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs and have an IC₅₀ between about 250 nM and about 25 nM, preferably, between 100 nM and about 25 nM, or more preferably, between 50 nM and about 25 nM in an in vivo assay of hepcidin-25 bioactivity and are further characterized by having an IC₅₀ between about 100 nM and about 1 nM, preferably, between about 75 nM and about 1 nM, more preferably, between about 50 nM and about 1 nM, even more preferably, between about 25 nM and about 1 nM in an in vitro assay of hepcidin-25 bioactivity. Preferably, the in vivo assay measures an IL-6-induced decrease in serum iron levels. Preferably, the in vitro assay of hepcidin-25 bioactivity assay measures hepcidin-induced internalization and/or degradation of ferroportin 1. Even more preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0121] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention comprise a LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NOs: 37, 129, 22, 107, 118, and 120, respectively, and bind human FPN1 consisting of the amino acid sequence shown in SEQ ID NO: 1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 and 1) a K_D between about 100 nM to about 0.5 nM, preferably, between about 100 nM to about 1 nM, more preferably, between about 75 nM to about 5 nM, even more preferably, between about 50 nM to about 10 nM, even more preferably, between about 15 nM to about 10 nM, even more preferably, between about 10 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.7 nM, even more preferably, between about 3 nM to about 0.7 nM, or most preferably, from about 3 nM to about 1 nM, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs, 2) an IC₅₀ between about 250 nM and about 25 nM, preferably between 100 nM and about 25 nM, or more preferably between 50 nM and about 25 nM in an in vivo assay of hepcidin-25 bioactivity, 3) an IC₅₀ between about 100 nM and about 1 nM, preferably, between about 75 nM and about 1 nM, more preferably, between about 50 nM and about 1 nM, even more preferably, between about 25 nM and about 1 nM in an in vitro assay of hepcidin-25 bioactivity, and 4) a dissociation rate (k_off) for human ferroportin 1 between about 7.5×10^-3 s^-1 and about 1×10^-4 s^-1, preferably between about 2.5×10^-3 s^-1 and about 1×10^-4 s^-1, more preferably between about 1×10^-3 s^-1 and about 1×10^-4 s^-1, and even more preferably between about 5×10^-4 s^-1 and about 1×10^-4 s^-1, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs. Preferably, the in vivo assay measures an IL-6-induced decrease in serum iron levels. Preferably, the in vitro assay of hepcidin-25 bioactivity assay measures hepcidin-induced internalization and/or degradation of ferroportin 1. Even more preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0122] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention comprise a LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NOs: 37, 125, 22, 23, 110, and 19, respectively, and bind human FPN1 consisting of the amino acid sequence shown in SEQ ID NO: 1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 and are characterized by having a K_D between about 100 nM to about 0.5 nM, preferably, between about 100 nM to about 1 nM, more preferably, between about 75 nM to about 5 nM, even more preferably, between about 50 nM to about 10 nM, even more preferably, between about 15 nM to about 10 nM, even more preferably, between about 10 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.7 nM, even more preferably, between about 3 nM to about 0.7 nM, or most preferably, from about 3 nM to about 1 nM, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs, 2) an IC₅₀ between about 250 nM and about 25 nM, preferably between 100 nM and about 25 nM, or more preferably between 50 nM and about 25 nM in an in vivo assay of hepcidin-25 bioactivity, 3) an IC₅₀ between about 100 nM and about 1 nM, preferably, between about 75 nM and about 1 nM, more preferably, between about 50 nM and about 1 nM, even more preferably, between about 25 nM and about 1 nM in an in vitro assay of hepcidin-25 bioactivity, and 4) a dissociation rate (k_off) for human ferroportin 1 between about 7.5×10^-3 s^-1 and about 1×10^-4 s^-1, preferably between about 2.5×10^-3 s^-1 and about 1×10^-4 s^-1, more preferably between about 1×10^-3 s^-1 and about 1×10^-4 s^-1, and even more preferably between about 5×10^-4 s^-1 and about 1×10^-4 s^-1, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs. Preferably, the in vivo assay measures an IL-6-induced decrease in serum iron levels. Preferably, the in vitro assay of hepcidin-25 bioactivity assay measures hepcidin-induced internalization and/or degradation of ferroportin 1. Even more preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214. Even more preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0123] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention comprise a LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NOs: 37, 177, 22, 23, 112, and 19, respectively, and bind human FPN1 consisting of the amino acid sequence shown in SEQ ID NO: 1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 and are characterized by having 1) a K_D between about 100 nM to about 0.5 nM, preferably, between about 100 nM to about 1 nM, more preferably, between about 75 nM to about 5 nM, even more preferably, between about 50 nM to about 10 nM, even more preferably, between about 15 nM to about 10 nM, even more preferably, between about 10 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.7 nM, even more preferably, between about 3 nM to about 0.7 nM, or most preferably, from about 3 nM to about 1 nM, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs, 2) an IC₅₀ between about 250 nM and about 25 nM, preferably between 100 nM and about 25 nM, or more preferably between 50 nM and about 25 nM in an in vivo assay of hepcidin-25 bioactivity, 3) an IC₅₀ between about 100 nM and about 1 nM, preferably, between about 75 nM and about 1 nM, more preferably, between about 50 nM and about 1 nM, even more preferably, between about 25 nM and about 1 nM in an in vitro assay of hepcidin-25 bioactivity, and 4) a dissociation rate (k_off) for human ferroportin 1 between about 7.5×10^-3 s^-1 and about 1×10^-4 s^-1, preferably between about 2.5×10^-3 s^-1 and about 1×10^-4 s^-1, more preferably between about 1×10^-3 s^-1 and about 1×10^-4 s^-1, and even more preferably between about 5×10^-4 s^-1 and about 1×10^-4 s^-1, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs. Preferably, the in vivo assay measures an IL-6-induced decrease in serum iron levels. Preferably, the in vitro assay of hepcidin-25 bioactivity assay measures hepcidin-induced internalization and/or degradation of ferroportin 1. Even more preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0124] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention comprise a LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NOs: 37, 122, 22, 23, 110, and 19, respectively, and bind human FPN1 consisting of the amino acid sequence shown in SEQ ID NO: 1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 and are characterized by having 1) a K_D between about 100 nM to about 0.5 nM, preferably, between about 100 nM to about 1 nM, more preferably, between about 75 nM to about 5 nM, even more preferably, between about 50 nM to about 10 nM, even more preferably, between about 15 nM to about 10 nM, even more preferably, between about 10 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.7 nM, even more preferably, between about 3 nM to about 0.7 nM, or most preferably, from about 3 nM to about 1 nM, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs, 2) an IC₅₀ between about 250 nM and about 25 nM, preferably between 100 nM and about 25 nM, or more preferably between 50 nM and about 25 nM in an in vivo assay of hepcidin-25 bioactivity, 3) an IC₅₀ between about 100 nM and about 1 nM, preferably, between about 75 nM and about 1 nM, more preferably, between about 50 nM and about 1 nM, even more preferably, between about 25 nM and about 1 nM in an in vitro assay of hepcidin-25 bioactivity, and 4) a dissociation rate (k_off) for human ferroportin 1 between about 7.5×10^-3 s^-1 and about 1×10^-4 s^-1, preferably between about 2.5×10^-3 s^-1 and about 1×10^-4 s^-1, more preferably between about 1×10^-3 s^-1 and about 1×10^-4 s^-1, and even more preferably between about 1×10^-4 s^-1 and about 1×10^-4 s^-1, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs. Preferably, the in vivo assay measures an IL-6-induced decrease in serum iron levels. Preferably, the in vitro assay of hepcidin-25 bioactivity assay measures hepcidin-induced internalization and/or degradation of ferroportin 1. Even more preferably, the anti-FPN1 Mabs, or antigen-binding fragments thereof, do not bind one or more peptides selected from the group consisting of SEQ ID NOS: 98, 183-214.

[0125] In particular embodiments, the anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention comprise: [0126] a. a light chain and a heavy chain as shown in SEQ ID NO: 154 and SEQ ID NO: 152, respectively; [0127] b. a light chain and a heavy chain as shown in SEQ ID NO: 181 and SEQ ID NO: 179, respectively; or [0128] c. a light chain and a heavy chain as shown in SEQ ID NO: 158 and SEQ ID NO: 156, respectively, and bind human FPN1 consisting of the amino acid sequence shown in SEQ ID NO: 1 at an epitope comprising or consisting essentially of or consisting of an amino acid or amino acids localized to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, and 95 and are characterized by having 1) a K_D between about 100 nM to about 0.5 nM, preferably, between about 100 nM to about 1 nM, more preferably, between about 75 nM to about 5 nM, even more preferably, between about 50 nM to about 10 nM, even more preferably, between about 15 nM to about 10 nM, even more preferably, between about 10 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.5 nM, even more preferably, between about 5 nM to about 0.7 nM, even more preferably, between about 3 nM to about 0.7 nM, or most preferably, from about 3 nM to about 1 nM, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs, 2) an IC₅₀ between about 250 nM and about 25 nM, preferably between 100 nM and about 25 nM, or more preferably between 50 nM and about 25 nM in an in vivo assay of hepcidin-25 bioactivity, 3) an IC₅₀ between about 100 nM and about 1 nM, preferably, between about 75 nM and about 1 nM, more preferably, between about 50 nM and about 1 nM, even more preferably, between about 25 nM and about 1 nM in an in vitro assay of hepcidin-25 bioactivity, and 4) a dissociation rate (k_off) for human ferroportin 1 between about 7.5×10^-3 s^-1 and about 1×10^-4 s^-1, preferably between about 2.5×10^-3 s^-1 and about 1×10^-4 s^-1, more preferably between about 1×10^-3 s^-1 and about 1×10^-4 s^-1, and even more preferably between about 5×10^-4 s^-1 and about 1×10^-4 s^-1, as determined by SPR, preferably, at 25° C. for Mabs and 37° C. for Fabs. Preferably, the in vivo assay measures an IL-6-induced decrease in serum iron levels. Preferably, the in vitro assay of hepcidin-25 bioactivity assay measures hepcidin-induced internalization and/or degradation of ferroportin 1.

[0129] The term "treating" (or "treat" or "treatment") refers to slowing, interrupting, arresting, controlling, stopping, reducing, or reversing the progression or severity of a symptom, disorder, condition, or disease, but does not necessarily involve a total elimination of all disease-related symptoms, conditions, or disorders.

[0130] The term "preventing" (or "prevent" or "prevention") means prohibiting, restraining, or inhibiting the incidence or occurrence of a symptom, disorder, condition, or disease. Acute events and chronic conditions may be treated and prevented. In an acute event, an antibody or antigen-binding fragment thereof is administered at the onset of a symptom, disorder, condition, or disease, and is discontinued when the acute event ends. In contrast, a chronic symptom, disorder, condition, or disease is treated over a more protracted time frame.

[0131] A "disorder" is any condition that would benefit from treatment according to the present invention. The terms "disorder", "condition" and "disease" are used interchangeably herein and include chronic and acute mature hepcidin-promoted disorders, including, but not limited to, anemia including, but not limited to, anemia of chronic disease.

[0132] The term "anemia of chronic disease" refers to any anemia that develops as a result of, for example, extended infection, inflammation, and neoplastic disorders. The anemia which develops is often characterized by a shortened red blood cell life span and sequestration of iron in macrophages, which results in a decrease in the amount of iron available to make new red blood cells. Conditions associated with anemia of chronic disease include, but are not limited to, chronic bacterial endocarditis, osteomyelitis, rheumatic fever, ulcerative colitis, and neoplastic disorders. Further conditions include other diseases and disorders associated with infection, inflammation, and neoplasms, including, for example, inflammatory infections (e.g., pulmonary abscess, tuberculosis), inflammatory noninfectious disorders (e.g., rheumatoid arthritis, systemic lupus erythrematosus, Crohn's disease, hepatitis, inflammatory bowel disease), and various cancers, tumors, and malignancies (e.g., carcinoma, sarcoma, lymphoma). Anemia of chronic disease is associated with hypoferremia and reticuloendothelial cell iron sequestration.

[0133] Inflammatory cytokines are potent inducers of hepcidin expression, and hepcidin excess may play a key role in the pathogenesis of anemia in these patients (Weiss, et al., N. Engl. J. Med., 352:1011-1023 (2005); Pigeon. et al., J. Biol. Chem. 276:7811-7819 (2001); Nicolas, et al., J. Clin. Invest. 110:1037-1044 (2002); Nemeth, et al., J. Clin. Invest. 113:1271-1276 (2004); Nemeth, et al., Blood, 101:2461-2463 (2003); Lee, et al., Proc. Natl. Acad. Sci. USA., 102:1906-1910 (2005)). Inflammatory mediators such as IL-6 may regulate hepcidin expression through STAT3 (Wrighting, et al., Blood, 108:3204-3209 (2006); Verga Falzacappa, et al., Blood, 109:353-358 (2007); Pietrangelo et al., Gastroenterology, 132:294-300 (2007)). The data presented herein provide in vivo evidence that anti-FPN1 Mabs of the present invention increase serum iron levels.

[0134] Also provided by the present invention are methods of treating anemia comprising the administration of anti-FPN1 Mabs, or antigen-binding fragments thereof, of the present invention. In some embodiments, the method of treating anemia comprises the step of administering a pharmaceutical composition comprising an anti-FPN1 Mab, or antigen-binding fragment thereof, to a subject at risk for or exhibiting pathologies as described herein, e.g., anemia disorders, using standard administration techniques.

[0135] The phrase "effective amount" as used herein refers to an amount necessary (at dosages and for periods of time and for the means of administration) to achieve the desired therapeutic result. An effective amount of the antibody may vary according to factors such as the disease state, age, gender, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effect of the antibody, are outweighed by the therapeutically beneficial effects.

[0136] An effective amount is at least the minimal amount, but less than a toxic amount, of an active agent which is necessary to impart therapeutic benefit to a subject. Stated another way, an effective amount or therapeutically effective amount of an antibody of the invention is an amount which, in mammals, preferably humans, (i) increases serum iron levels, reticulocyte count, red blood cell count, hemoglobin, and/or hematocrit, or (ii) treats a disorder wherein the presence of mature hepcidin causes or contributes to an undesirable pathological effect, or (iii) decreases mature hepcidin bioactivity resulting in a beneficial therapeutic effect in a mammal, preferably a human, including, but not limited to, having anemia including, but not limited to, anemia of chronic disease, including, but not limited to, anemia resulting from infection, inflammation, and/or cancer. An effective amount of an antibody of the invention may be administered in a single dose or in multiple doses. Furthermore, an effective amount of an antibody of the invention may be administered in multiple doses of amounts that would be less than an effective amount if not administered more than once.

[0137] As is well known in the medical arts, dosages for any one subject depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, gender, time and route of administration, general health, and other drugs being administered concurrently. Dose may further vary depending on the type and severity of the disease. A typical dose can be, for example, in the range of about 1 mg to about 100 mg; preferably, about 2 mg to about 100 mg; more preferably, about 5 mg to about 100 mg; even more preferably, about 5 mg to about 50 mg, even more preferably, about 5 mg to about 25 mg; even more preferably, about 5 mg to about 20 mg; even more preferably, about 5 mg to about 15 mg; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. A daily parenteral dosage regimen can be from about 10 μg/kg to about 100 mg/kg, preferably, from about 100 μg/kg to about 100 mg/kg, more preferably, from about 1 mg/kg to about 100 mg/kg, even more preferably, from about 1 mg/kg to about 30 mg/kg, even more preferably, from about 3 mg/kg to about 30 mg/kg, or most preferably from about 3 mg/kg to about 30 mg/kg. Progress may be monitored by periodic assessment, and the dose adjusted accordingly.

[0138] These suggested amounts of anti-FPN1 antibody are subject to a great deal of therapeutic discretion. The key factor in selecting an appropriate dose and scheduling is the result obtained. Factors for consideration in this context include the particular disorder being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the antibody, the particular type of antibody, the method of administration, the scheduling of administration, and other factors known to medical practitioners.

[0139] The antibodies of the present invention can be used as medicaments in human medicine, administered by a variety of routes. Most preferably, such compositions are for parenteral administration. Such pharmaceutical compositions can be prepared by methods well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy, 19^th ed. (1995), Gennaro, A., et al., Mack Publishing Co. Accordingly, this invention also provides pharmaceutical compositions comprising one or more antibodies of the invention in combination with one or more pharmaceutically acceptable carriers, diluents, or excipients. In a particular embodiment, the pharmaceutical composition further comprises one or more other therapeutic agents.

[0140] The term parenteral as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, or intraperitoneal administration. Parenteral delivery by intravenous infusion or intravenous, intraperitoneal, or subcutaneous injection is preferred. Subcutaneous injection is most preferred. Suitable vehicles for such injections are well known in the art.

[0141] The pharmaceutical composition typically must be sterile and stable under the conditions of manufacture and storage in the container provided, including e.g., a sealed vial, syringe or other delivery device, e.g., a pen. Therefore, pharmaceutical compositions may be sterile filtered after making the formulation, or otherwise made microbiologically acceptable.

[0142] An antibody of the invention can be incorporated into a pharmaceutical composition suitable for administration to a human subject. An antibody of the invention may be administered to a human subject alone or in combination with a pharmaceutically acceptable carrier and/or diluent in single or multiple doses. Such pharmaceutical compositions are designed to be appropriate for the selected mode of administration, and pharmaceutically acceptable diluents, carrier, and/or excipients such as dispersing agents, buffers, surfactants, preservatives, solubilizing agents, isotonicity agents including but not limited to sodium chloride, stabilizing agents and the like are used as appropriate. Said compositions can be designed in accordance with conventional techniques disclosed in, for example, Remington, The Science and Practice of Pharmacy, 19^th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa. (1995) which provides a compendium of formulation techniques as are generally known to practitioners. Suitable carriers for pharmaceutical compositions include any material which, when combined with an antibody of the invention, retains the molecule's activity and is non-reactive with the subject's immune system.

[0143] The terms "subject" and "patient" used interchangeably herein, refer to a mammal, preferably, a human. In certain embodiments, the patient has a disorder that would benefit from a decreased level of mature hepcidin, a decrease in mature hepcidin bioactivity, and/or an increase in serum iron level, reticulocyte count, red blood cell count, hemoglobin, and/or hematocrit.

[0144] Administration of a FPN1 antibody compound alone may be useful in patients intolerant to one or more ESAs, either at any dose or only at high doses, due to, for example, undesirable side effects. A FPN1 Mab, or antigen-binding fragment thereof, of the present invention administered alone or in combination with an ESA, may also be useful in ESA-resistant patients who are incapable of reaching their hematocrit goals with ESAs alone, either at conventional or high doses.

[0145] A FPN1 Mab, or antigen-binding fragment thereof, of the present invention may also be administered when combination drug therapy, including the use of ESAs, is inadequate in allowing patients to reach their hematocrit goals.

[0146] In another embodiment, the present invention provides the use of a monoclonal antibody or an antigen-binding fragment thereof, of the present invention for the manufacture of a medicament for increasing serum iron levels, reticulocyte count, red blood cell count, hemoglobin, and/or hematocrit in a human.

[0147] In another embodiment, the present invention provides the use of the monoclonal antibody or an antigen-binding fragment thereof in the manufacture of a medicament for use in combination therapy for increasing serum iron levels, reticulocyte count, red blood cell count, hemoglobin, and/or hematocrit in a human, wherein said medicament is to be administered in combination with one or more ESAs selected from the group consisting of epoetin alfa, epoietin beta, darbepoetin alfa, hematide, methoxy polyethylene glycol-epoetin beta, or other therapeutic agent or therapeutic treatment conventionally employed to increase serum iron levels, reticulocyte count, red blood cell count, hemoglobin, and/or hematocrit in a human.

[0148] The following non-limiting examples illustrate various properties of the anti-FPN1 antibodies disclosed herein.

EXAMPLE 1

Production of Human Hepcidin-25

[0149] Human hepcidin-25 can be obtained from commercial sources (e.g., Peptide International (Louisville, Ky.)) or produced by a variety of synthetic or recombinant techniques known in the art. Alternatively, a fusion protein comprising the twenty-five amino acids of human hepcidin-25 sequence and having the amino acid sequence as shown in SEQ ID NO: 91 is expressed in E. coli. Inclusion bodies are isolated from 3 liters of E. coli expressing the human hepcidin fusion protein after a 3-6 hour induction with 1 mM IPTG at 37° C. The inclusion bodies are solubilized in buffer A (50 mM Tris and 8 M urea (pH 8.0)). The supernatant is passed over an IMAC column (20 mL resin). The column is washed with buffer A until the absorbance returned to baseline and the bound polypeptides are batch eluted from the column by 0.5 M imidazole in buffer A. The human hepcidin-25 fusion protein is pooled and reduced with 50 mM DTT. This fusion protein is then refolded by diluting pooled material into 2 M urea, 3 mM cysteine, 50 mM Tris (pH 8.0) to a final protein concentration less than 50 μg/mL. This material is stirred at room temperature and air oxidized for 48 hours. The oxidized polypeptides are passed over an IMAC column (20 mL) at a flow rate of 5 mL/min, and the human hepcidin-25 fusion protein is batch eluted from the column by 0.5 M imidazol in buffer A. The pooled fractions containing the human hepcidin-25 fusion protein are concentrated and passed over a Superdex 75 (GE Healthcare, XK26/60) sizing column equilibrated with 50 mM Tris, 4 M urea, pH 8.0, at a flow rate of 3 mL/min. The monomeric fusion protein is pooled and then diluted to 50 mM Tris, 2M urea, 5 mM CaCl₂, pH 8.0 and then is cleaved with enterokinase to produce human hepcidin-25 of SEQ ID NO: 1. Uncleaved human hepcidin-25 fusion protein is removed by passive IMAC chromatography (as outlined above). The flow through from the IMAC column is then passed over a C-18 Reversed Phase column at a flow rate of 4.0 mL/minute. The column is washed with 0.1% TFA in water until the absorbance returned to baseline and the bound polypeptides are eluted from the column with a linear gradient of acetonitrile from 20% to 40% with 0.1% TFA at a rate of 0.5%/min. Fractions which contain the human hepcidin-25 polypeptide are pooled and analyzed by N-terminal amino acid sequencing and matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). Polypeptides encoding rat, mouse, and Cynomolgus monkey hepcidin-25 and various N-terminally truncated forms of human hepcidin-25, including hepcidin-22 and hepcidin-20 were obtained commercially (e.g., Peptide International).

EXAMPLE 2

Generation of 34A9 Fab

[0150] Anti-FPN1 antibodies may be obtained by immunizing mice with an immunogenic peptide having the amino acid sequence as shown in SEQ ID NO: 11. More specifically, an immunogenic peptide comprising an OVA epitope linked by a peptide linker to the amino acid sequence shown in SEQ ID NO: 12, which is thought to be at least part of an extracellular loop of human FPN1, may be used to immunize mice. After immunization, mice spleens are harvested and spleen cells are sorted by Magnetic Activated Cell Sorting using a biotinylated peptide having the amino acid sequence shown in SEQ ID NO: 12 and streptavidin beads. RNA is isolated from antigen binding cells and converted into cDNA using oligo dT. Antibody heavy and light chain variable regions are obtained by PCR using antibody framework primers and cloned into a phage vector to make a Fab antibody library. The phage antibody library is screened with a biotinylated peptide, e.g., 100 nM, having the amino acid sequence shown in SEQ ID NO: 12. Positively binding clones are then characterized by DNA sequencing, Fabs expression and binding to the immunizing peptide and/or cells expressing human ferroportin. Fab 34A9 was identified following the procedure essentially as described above.

EXAMPLE 3

Epitope Mapping of Anti-FPN1 Mabs

[0151] Peptides containing partial sequences of the FPN1 related immunogen may be used in dot blot hybridization experiments to determine the epitopes of the mouse Mab 34A9. The following peptides may be synthesized and dissolved in water (underlined amino acids denote actual FPN1 amino acid sequence):

TABLE-US-00010 FpnE3a (SEQ ID NO: 96): GGSPFEDIRSRFIQGESITPTKGC 060719Z (SEQ ID NO: 97): GGSPFEDIRSRFIQGC 060719Y (SEQ ID NO: 98): GGIQGESITPTKIPEITTEGC 0708L4A (SEQ ID NO: 99): GGMPGSPLDLSVSPFEDGC 0708L4B (SEQ ID NO: 100): GGSPLDLSVSPFEDIRSGC 0708L4C (SEQ ID NO: 101): GGEDIRSRFIQGESITGC 0708L4D (SEQ ID NO: 102): GGRSRFIQGESITPTKGC

[0152] For each peptide, 3 μl of 1-5 μg/mL peptide is spotted onto a piece of nitrocellulose membrane and air dried. The membrane is blocked with blocking buffer (e.g., PBS containing 1% BSA), then incubated with 3-5 μg/mL FPN1 antibody in blocking buffer at room temperature for an hour. The membrane is washed three times, 5 min each, with 1×PBST (10 mM sodium phosphate, 150 mM NaCl, 0.1% Tween-20, pH 7.4) before it is incubated with IR700-labeled-Goat-anti-Mouse antibody according to manufacturer's protocol (LiCor, Inc; Lincoln, Nebr.). The membrane is washed three times, five minutes each, with 1×PBST, is imaged on Odyssey imaging system and Odyssey software (LiCor, Inc).

[0153] FIG. 1 indicates that Mab 34A9 binds to peptides FpnE3a, 060719Z, 0708L4C, and 0708L4D, all of which contain amino acids 409 to 415 of SEQ ID NO: 1. Mab 34A9 does not require the amino acids sequence of EDI as indicated by the binding to peptide 0708L4D. Mab 34A9 binds more weakly to peptide 060719Z than 0708L4C; the latter peptide contains amino acids 416-419 of FPN1 (SEQ ID NO: 1).

EXAMPLE 4

Affinities of Anti-FPN1 Fabs and Mabs as Determined by SPR

[0154] The affinities of FPN1 binding Fabs and Mabs may be determined on Biacore T100 and using 1:1 binding model in the Biacore T100 evaluation software (BIAcore® AB, Upsala, Sweden). Briefly, the T-REx system, a commercially available tetracycline-regulated expression system without viral transactivators (Invitrogen, Carlsbad, Calif.) is used for stable cell line generation in T-Rex HEK 293 cells. All growth conditions are described in the T-REx manual provided by Invitrogen. FPN1 is C-terminally fused with GFP. Expression of FPN1-GFP is induced by 1-10 ng/mL doxycycline for 1-24 hours. Induced cells are harvested by scraping off from flasks and then washed with 1×PBS. Cell pellets can be stored at -80° C. before use. About five million induced cells are resuspended in 10 mM phosphate buffer with 0.2% Tween-20 and protease inhibitors, e.g., Complete® Protease Inhibitor Cocktail Tablet (Roche Diagnostics Corp., Indianapolis, Ind.). Three cycles of freeze/thaw/sonication are used to lyse the cells. The lysate is diluted two-fold with Biacore running buffer and centrifuged to remove debris.

[0155] On Biacore T100, rabbit anti-GFP antibody or goat anti-GFP antibody is immobilized onto flow cell 1 to 4 of a CMS chip at 5000-15000 Rus. FPN1-GFP is captured onto flow cell 2, 3, or 4 from the lysate of induced cells. Flow cell 1 is used as reference. Then all flow cells are injected with different concentrations of antibodies to evaluate the binding and kinetics. Surface plasmon resonance based measurements using univalent antigen-binding fragments such as Fabs are generally preferred to those using multivalent antibodies in this assay format to minimize avidity effects. Tables 4a and 4b show the binding characteristics for anti-human FPN1 binding Fabs using rabbit anti-GFP antibody (Invitrogen, Carlsbad, Calif. (catalog #A11122)) and goat anti-GFP antibody (R&D Systems, Minneapolis, Minn. (catalog #AF4240)), respectively.

TABLE-US-00011 TABLE 4(a) Binding Kinetics of Fabs from FPN1 Antibodies to Human FPN1 (Determined by Biacore T100 at 37° C.) Fab Kon (M^-1s^-1) Koff (s^-1) Kinetic K_D (M) 34A9 6.321E+04 2.620E-03 4.145E-08 1G9 1.269E+05 8.974E-04 7.707E-09 3D8 1.920E+05 2.000E-03 1.042E-08

[0156] As shown in Table 4(a), the K_D for human FPN1 of the mouse Fab 34A9 is approximately 41 nM as determined by SPR at 37° C. in this assay format. The K_D for human FPN1 of the mouse Fab 1G9, an affinity matured form of the mouse Fab 34A9 is about 7.7 nM as determined by SPR at 37° C., an improvement in binding affinity of approximately 5-fold. Fab 3D8, a humanized form of the mouse Fab 1G9 having the human heavy chain framework VH1-69 and light chain framework O2, demonstrated a K_D for human FPN1 of about 10 nM as determined by SPR at 37° C. in this assay format.

TABLE-US-00012 TABLE 4(b) Binding Kinetics of Fabs from FPN1 Antibodies to Human FPN1 (Determined by Biacore T100 at 37° C.) Fab (n) Kon (M^-1s^-1) Koff (s^-1) Kinetic K_D (M) mouse 1G9 4 1.726E+05 4.968E-04 2.900E-09 human 3D8 3 3.284E+05 2.061E-03 6.293E-09 human 4A10-3 4 8.443E+05 1.483E-03 1.761E-09 human Combi11 3 2.309E+06 6.369E-03 2.395E-09 human L2.2-4 3 4.308E+05 7.905E-04 1.959E-09

[0157] As shown in Table 4(b), the K_D for human FPN1 of the mouse Fab 1G9, an affinity matured form of the mouse Fab 34A9 is about 2.9 nM as determined by SPR at 37° C. in this assay format. Fab 3D8, a humanized form of the mouse Fab 1G9 having the human heavy chain framework VH1-69 and light chain framework O2, demonstrated a K_D for human FPN1 of about 6.3 nM as determined by SPR at 37° C. in this assay format. Affinity matured Fabs 4A10-3, Combi11, and L2.2-4 demonstrated a K_D for human FPN1 between about 2.4 nM to about 1.8 nM as determined by SPR at 37° C. in this assay format.

[0158] Table 5 shows the binding characteristics for anti-human FPN1 binding Mabs using rabbit anti-GFP antibody (Invitrogen, Carlsbad, Calif. (catalog #A11122)).

TABLE-US-00013 TABLE 5 Binding Kinetics of MAbs to Human FPN1 (Determined by Biacore T100 at 25° C. or 37° C.) Mab Temp. Kon (M^-1s^-1) Koff (s^-1) Kinetic K_D (M) mouse 34A9 25° C. 6.901E+04 8.155E-05 1.182E-09 mouse 1G9 25° C. 1.348E+05 9.813E-05 7.281E-10 human 3D8 37° C. 3.252E+05 1.095E-03 3.366E-09

[0159] The K_D for human FPN1 of the mouse 34A9 Mab is approximately 1.1 nM as determined by SPR at 25° C. The K_D for human FPN1 of the mouse 1G9 Mab, an affinity matured form of the mouse 34A9 Mab is about 0.73 nM as determined by SPR at 25° C. A humanized form of the mouse 1G9 Mab, 3D8 Mab, having the human heavy and light chain frameworks, VH1-69 and O2, respectively, demonstrated a K_D for human FPN1 of about 3.4 nM as determined by SPR at 37° C.

[0160] The K_D for human FPN1, determined as described in this Example, illustrates the generation of antibodies to human FPN1 which bind human FPN1 with high affinity and, more specifically, bind to an epitope of FPN1 that is present even when human FPN1 is expressed by cells and localized to the cellular membrane.

EXAMPLE 5

In Vitro Assay of the Effects of FPN1 Mabs on Cellular Ferritin Levels

[0161] Caco-2 cells, a human enterocyte cell line, endogenously expressing FPN1 may be monitored for changes in ferritin. Ferritin concentration in the Caco-2 cells may be increased by adding an exogenous iron source and the concentration may be further augmented with the addition of hepcidin which prevents iron export. Accordingly, the effect of anti-human FPN1 antibodies on mature hepcidin modulated iron regulation in Caco-2 cells may be determined as follows.

[0162] Caco-2 cells are removed from the cell culture vessel using trypsin (Invitrogen, Carlsbad, Calif.). The cells are collected and washed in growth medium (e.g., DMEM+10% FBS+1% non-essential amino acids+1% antibiotics/antimycotic) and collected by gentle centrifugation. Cells are resuspended in culture medium and counted. Cell concentration is adjusted to 1×10⁶ cells/mL in growth medium and 2.5 μM Fe:NTA (prepared 1:4 molar ratio) is added to the cells. One hundred μl of cells are added to wells of a 96 flat well plate, followed by incubation for 24 hours at 37° C., 5% CO₂. Mouse IgG₁, a negative control, and two antibodies with different affinities to human FPN1 are prepared in growth medium at 6× the final concentration. Antibody dilutions (25 μL) or medium are added to wells in triplicate. The plates are incubated at room temperature for 15 minutes at which time 25 μL 5 μM Fe:NTA with or without 600 nM hepcidin (100 nM final concentration) are added to the appropriate wells. The cells are incubated for 24 hours 37° C., 5% CO₂ and then washed 3× with 200 μl Dulbecco's PBS and lysed in 50 μL radioimmunoprecipitation assay buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% SDS, 1% Triton-X100®, and 0.5% sodium deoxycholate) plus protease inhibitors, e.g., Complete® Protease Inhibitor Cocktail Tablet (Roche Diagnostics Corp., Indianapolis, Ind.), mixed and frozen at -70° C. until assayed for ferritin using an ELISA.

[0163] Briefly, microtiter plates are coated with 110 μL/well of 1 μg/mL anti-human ferritin (Leinco Technologies, St. Louis, Mo.) and incubated overnight at 4° C. The plates are washed 2 times with wash buffer (0.02 M Tris, 0.15M NaCl, 0.1% Tween 20, pH 7.4) and blocked with 150 μL of 1% casein in PBS (Thermo Fisher Scientific, Rockford, Ill.). The plates are incubated for 1 hour at room temperature. One hundred microliters (μL) of lysates and standards (human liver ferritin, Calbiochem/EMD Biosciences, La Jolla, Calif.) are added to the appropriate wells and incubated for 1 hour at room temperature. The plates are washed 3 times and bound ferritin is detected using an anti-ferritin-HRP conjugate (Leinco Technologies) at 1:2000 dilution at 100 μL per well and incubation for 1 hour at room temperature. The plates are washed 4 times and 100 μL OPD substrate (5 mg substrate tablet, dissolved in 12.5 mL of 0.1 M Na₂HPO₄, 0.05 M citric acid, pH 5.0 with 5 μL of 30% H₂O₂) is dispensed to all wells. The reaction is stopped with 100 μL 1 N HCl after 10 minutes. Absorbance at 490 nm (A₄₉₀) is read using an appropriate ELISA plate reader. The protein concentration in each sample is also measured using a BCA protein assay kit (Thermo Fisher Scientific). To account for possible well-to-well differences in cell number, ferritin concentration data are normalized to protein concentration and the effect of added antibodies is expressed as percent inhibition.

[0164] Experiments conducted as described in this Example indicate that the effects of human hepcidin-25 on ferritin concentration in the cells are inhibited by Mabs 34A9 and 1G9 (Table 6). Furthermore, the results show that the affinity of the anti-FPN1 Mab has direct implications on its functionality. More specifically, the lower affinity Mab 34A9, even at the highest concentration (200 μg/mL) only slightly inhibited mature hepcidin-induced effects (25% inhibition±0.5%), whereas the higher affinity Mab 1G9 exhibited marked inhibition in a dose-dependent matter.

TABLE-US-00014 TABLE 6 Ferritin (ng)/ % Sample protein (μg) STD Inhibition NTA:Fe only 5.11 0.25 NA NTA + Fe + hepcidin 9.95 0.86 NA 200 μg/mL mIgG₁ 9.40 0.25 11.6 100 μg/mL mIgG₁ 9.78 0.68 3.5 50 μg/mL mIgG₁ 10.06 0.96 0 25 μg/mL mIgG₁ 9.6 1.29 7.2 12.5 μg/mL mIgG₁ 10.01 1.11 0 6.25 μg/mL mIgG₁ 10.45 2.05 0 200 μg/mL Mab 1G9 6.07 0.34 80.2 100 μg/mL Mab 1G9 5.68 0.34 88.2 50 μg/mL Mab 1G9 7.11 0.54 58.7 12.5 μg/mL Mab 1G9 7.93 0.43 41.7 6.25 μg/mL Mab 1G9 8.02 0.45 39.9 200 μg/mL Mab 34A9 8.74 0.50 25 50 μg/mL Mab 34A9 9.65 0.80 6.2

[0165] This data illustrates that Mabs 1G9 and 34A9 block the ability of human hepcidin-25 to induce internalization and degradation of ferroportin and hence reduce iron exported from the cells.

EXAMPLE 6

Assay for the Inhibition of Human Hepcidin-25 Binding to FPN1

[0166] Stably transfected FPN-GFP/293 cells are plated in poly-D-lysine coated plates at 60,000 cells per well in 80 μL of assay medium (DMEM 11965, 10% dialyzed FBS, 20 μM FAC, penicillin-streptomycin) and incubated 4 hours at 37° C., 10% CO₂. Doxycycline is added to a final concentration of 11.2 nM to induce FPN1 expression. Doxycycline induced and un-induced control cells are incubated overnight at 37° C. Next, the growth media is discarded and replaced with test antibody or an isotype control antibody in 30 μL of assay medium or 30 μL of assay medium alone control and incubated at 37° C. for 1 hour. Next, 20 μL of biotinylated mature human hepcidin is added to the wells to a final concentration of 30 nM. The samples are set aside for 1 hour, at 37° C. before washing 4 times with 200 μL 2% FBS, D-PBS (Invitrogen, Carlsbad, Calif.). Next, 65 μL of lysing buffer (0.5% Triton X-100, 10 mM EDTA) are added and the plates are shaken for 10 minutes. Next, 50 μL of the solution in each well is transferred to individual wells of a streptavidin coated plate (60 μL of 2 μg/mL streptavidin in PBS, incubated at 4° C. overnight, washed 2 times (0.1% Tween 20, TBS), blocked with casein/PBS), and then incubated for one hour at room temperature. Next, the wells of the plate are washed 3 times (0.1% Tween 20, TBS) and 50 μL anti-human hepcidin-25 Mab 3.23 at 0.5 μg/mL is added and the samples are incubated one hour at room temperature. The anti-human hepcidin-25 Mab 3.23 is described in PCT International Patent Application Publication WO 2009/058797. Next, the plates are washed three times and 50 μL anti-human IgG-horseradish peroxidase (HRP) is added at 1:2000 dilution. After incubating one hour at room temperature, 50 μL of OPD substrate is added. The reaction is stopped with 100 μL 1 N HCl after 4 minutes. Absorbance at 490 nm (A₄₉₀) is read using an appropriate ELISA plate reader.

TABLE-US-00015 TABLE 7 % Inhibition Antibody concentration Antibody 1.2 uM 0.3 uM 0.75 uM 0.019 uM 1G9 Mean 58.1 55.5 33.7 17.5 SD 1.7 6.7 20.4 8.7 Antibody concentration 6.0 uM 1.2 uM 0.24 uM 0.048 uM 34A9 Mean 26.4 31.2 9.6 -7.2 SD 26.8 6.9 6.0 12.1 ms IgG1 Mean -3.4 0.5 -4.0 -11.0 SD 8.8 6.1 7.3 9.6

[0167] Data generated in experiments conducted essentially described in Example 6 demonstrate that Mabs 1G9 and 34A9 inhibit the ability of human hepcidin-25 to bind human FPN1.

EXAMPLE 7

Cell-based Assay for anti-FPN1 Antibody Inhibition of Hepcidin-25-induced Internalization and Degradation

[0168] An in vitro cell based assay may be used to measure the mature hepcidin neutralization activity of Mabs, or antigen-binding fragments thereof, directed against human FPN1. Such an assay may be based on mature hepcidin-induced internalization and degradation of its receptor, FPN1. For instance, a HEK 293 stable cell line is prepared that allows for the inducible expression of FPN1. FPN1 is C-terminally fused with GFP for tracking purposes. The inducible expression of the FPN1-GFP molecule is controlled using the T-REx system (Invitrogen, Carlsbad, Calif.). The FPN1-GFP coding sequence is cloned into pcDNA4/TO vector, which contains an inducible promoter and a Zeocin resistance marker. The resulting construct is transfected into T-REx-293 cells which expresses the regulatory protein required for doxycycline inducible expression. Zeocin resistant clones are tested for the inducible expression of FPN-GFP. Cell growth conditions are essentially as described in the manufacturer's user manual for the T-REx System (Invitrogen). Briefly, cells are grown in DMEM, 10% dialyzed FBS, 20 μM FAC, plus 5 μg/mL penicillin-streptomycin. Selection is maintained with 100 μg/mL zeocin and 5 μg/mL blasticidin. Cells are plated onto 96-well black/clear plates that are coated with poly-D-lysine. An Acumen Explorer HTS, high resolution fluorescent plate reader is used for reading the total fluorescence per well.

[0169] Following trypsinization, 96-well assay plate is seeded with 9,000 cells/well using the FPN1-GFP/TREx 293 stable cell line. Seeding volume per well is 80 μL. Cells are allowed to attach overnight. Early the next morning, 9 μL of 30 ng/mL doxycyline is added to each well to induce FPN1-GFP expression. After 8 hours of induction at 37° C., the medium is aspirated and the wells are washed carefully with 150 μL/well of PBS.

[0170] The desired treatments (e.g., human hepcidin-25 and/or test antibodies) are set up in a 96-well format for quick addition to an assay plate after washing. Final assay volume per well is 45 μL Immediately after adding the treatments, the assay plate is read using the Acumen Explorer (set at 550 volts in channel 1). This reading is generally the 0 hour reading and is used to normalize for cell number per well, which correlates with the total fluorescence units (FLU) per well. For mature human hepcidin-induced internalization and degradation of FPN1, the maximum effect is seen at 0.5 μM mature human hepcidin. The IC₅₀ of mature human hepcidin is approximately 10 nM. For anti-FPN1 antibody neutralization assays, the human hepcidin-25 concentration is kept at 120 nM and the anti-FPN1 Mabs were tested at 600 nM, 200 nM, 67 nM, 22 nM, and 7.4 nM. The plates are incubated for 24 hours, after which, they are read again, and the data is generated as the ratio of total FLU per well at 24 hours divided by the total FLU per well at 0 hours. All data points are done in quadruplicate. The percent (%) inhibition is determined by subtracting the values for 120 nM mature human hepcidin treatment, and then dividing the FPN1 antibody treated values by the no human hepcidin-25 treated value.

[0171] In an in vitro assay conducted essentially as described above, human hepcidin-25 bioactivity was neutralized with various anti-FPN1 Mabs with a percent inhibition measured as shown in Table 8 below.

TABLE-US-00016 TABLE 8 Anti-FPN1 Mab Percent (%) Inhibition of Mature Human Hepcidin- induced Internalization and Degradation In Vitro Mab 34A9 Mab 1G9 Mab 3D8 Mab at 600 nM 39.0% 67.9% 66.4% Mab at 200 nM 30.8% 62.4% 63.8% Mab at 67 nM 23.6% 61.8% 55.7% Mab at 22 nM 14.1% 49.9% 50.2% Mab at 7.4 nM 4.8% 24.3% 28.1%

[0172] Data generated in experiments conducted essentially as described in Example 7 support the conclusion that Mabs 1G9, 34A9, and 3D8 greatly inhibit the ability of human hepcidin-25 to cause the internalization and degradation of human FPN1 in vitro.

EXAMPLE 8

Bioactivity of Mab 1G9 Relative to a Control Murine IgG1 following a Single Intravenous Dose to Cynomolgus Monkeys

[0173] The physiological effects of anti-human FPN1 murine Mab 1G9 on serum hepcidin and serum iron levels were investigated by administering the Mab as a single intravenous dose to male Cynomolgus monkeys (Macaca fascicularis; 3-4 kg) and comparing its effects to a control administration of murine IgG1. Following administration blood samples were collected for analysis of serum iron and serum hepcidin. The dose (30 mg/kg) was administered as an injection via a saphenous vein. Immediately after dose administration, but before the needle was removed from the animal, the dose apparatus was flushed with approximately 2 mL of saline.

TABLE-US-00017 TABLE 9 Concentration Volume (mg/mL) (mL/kg) 1 Murine IgG1 Control 9.53 3.15 2 Murine 1G9 12.6 2.38

Sampling for Serum Hepcidin:

[0174] Blood was collected prior to dosing and at 0.5, 1, 3, 6, 10, 24, 48, 72, 96, and 168 hours post-dose. Blood (approximately 0.5 mL) was collected via a femoral vein into tubes containing no anticoagulant. Blood was allowed to clot under ambient conditions prior to centrifugation to obtain serum. Serum samples were placed on dry ice prior to storage at approximately -70° C.

Sampling for Serum Iron:

[0175] Blood was collected prior to dosing and at 1, 3, 6, 24, 48, 72, 96, and 168 hours post-dose. All blood samples were collected, handled, processed, stored, and analyzed in accordance with methods considered acceptable within the medical community. Serum iron levels may be measured by any method known in the art which is generally considered within the medical community to be an acceptable method of measuring total serum iron (Fe). Serum concentrations of hepcidin were determined by liquid chromatography-mass spectrometry essentially as described in Murphy, et al., Blood, 110:1048-54 (2007). Assays for measuring serum iron are well-known in the art (see, for example, Goodwin, J. F., et al., Clinical Chemistry 12: 47-57 (1966), and J. Clin. Path., 24:334-335 (1971)).

[0176] Serum hepcidin concentrations were unaffected by the administration of control murine IgG1 and ranged from 1.5 to 31 ng/mL over the time course studied. Average hepcidin levels in the control animals were 11.5±8.8 ng/mL (mean±SD). After administration of murine Mab 1G9, serum hepcidin levels were elevated from a baseline of 7.6 and 14.7 ng/mL to a peak of 49.7 and 79.1 ng/mL, respectively. The peak in serum hepcidin occurred approximately 10 hours after administration of the murine Mab 1G9 (FIG. 6). The elevation of serum hepcidin is likely due to the interaction of murine Mab 1G9 with its target FPN1, which, upon binding to FPN1 blocks the interaction of FPN1 and hepcidin, thereby slowing FPN1 clearance and/or internalization.

[0177] Serum iron was not elevated in animals treated with control murine IgG and ranged from 64 to 97 μg/dL over the time frame studied. After administration of murine Mab 1G9, serum iron levels were elevated from a baseline of 136 and 144 μg/dL to a peak of 306 and 292 μg/dL, respectively. The peak in serum iron occurred approximately 48 hrs after administration of murine Mab 1G9 (FIG. 7). Serum iron levels gradually returned to baseline by 96 hours after administration, indicating that the elevation of serum iron levels is irreversible.

EXAMPLE 9

Cell-Based Assay for Anti-FPN1 Antibody Inhibition of Hepcidin-25-Induced Internalization and Degradation

[0178] Experiments conducted essentially as described in Example 7 above demonstrate that Mabs Combi-11, 4A10-3, and L2.2-4 inhibit human hepcidin-25 induced internalization and degradation of human FPN1 more effectively in vitro as compared with Mabs 34A9, 3D8, and 1G9 (see Table 10). More specifically, the anti-FPN1 Mabs were tested in a 9-point concentration curve starting at 900 nM and performing 3-fold serial dilutions. The percent (%) inhibition was determined by subtracting the values for 120 nM hepcidin treatment, and then dividing the Mab treated values by the no hepcidin treated value. Relative IC₅₀ were determined in Sigma Plot. Top percent (%) inhibition as well as relative and absolute IC₅₀ are shown in Table 10.

TABLE-US-00018 TABLE 10 Mab Top % Inhibition Relative IC₅₀ (nM) Absolute IC₅₀ (nM) (IgG4) (n = 3) (n = 3) (n = 3) Combi11 92.6 ± 4.5 3.7 ± 0.2 3.8 ± 0.1 4A10-3 85.9 ± 2.2 4.7 ± 1.5 5.7 ± 1.7 L2.2-4 81.6 ± 1.0 4.8 ± 1.2 6.2 ± 1.6 3D8 86.8 ± 7.0 10.2 ± 2.2 13.7 ± 3.4 1G9 69.0 ± 8.0 9.2 ± 3.3 18.8 ± 8.1 34A9 55.2 ± 7.1 58.9 ± 18.6 N.C. N.C.: Fitted top of curve does not reach 50% so absolute IC₅₀ can not be calculated.

EXAMPLE 10

Pharmacodynamic Effect of Humanized Anti-Ferroportin Monoclonal Antibodies 4A10-3 in Cynomolgus Monkeys

[0179] The pharmacodynamics of anti-ferroportin Mabs may be studied after administration of intravenous doses to male Cynomolgus monkeys according to methods known to those skilled in the art. For example, in five independent studies Mab 4A10^-3 was administered to Cynomolgus monkeys as a single intravenous bolus (n=4/group) at doses of 0.3, 1.0, 3.0, 10 and 30 mg/kg. Blood samples (approximately 0.5 mL for iron parameters) were taken prior to the first dose and at 1, 6, 12, 24, 48, 72, 96, 168 and 264 hours post-dose. At higher dose levels, additional blood samples were taken at 360, 456, 552, and 648 hours post-dose. At the time of dosing, the animals weighed between 2 to 3 kg. Blood samples were collected from each animal via a femoral vein into tubes containing no anticoagulant.

[0180] Serum iron concentration-time profiles following intravenous administration of 0.3, 1.0, 3.0, 10 and 30 mg/kg Mab 4A10-3 to male Cynomolgus monkeys was associated with a dose dependent increase in serum iron which peaked at 24 hours after dosing. Peak iron responses (approximately 2-fold increase) and duration of response between the mg/kg and 30 mg/kg doses were similar. In the animals administered 0.3, 1.0, and 3.0 mg/kg doses serum iron returned to baseline values about 48 hours after dosing. In the animals administered 10 mg/kg and 30 mg/kg doses, serum iron returned to baseline values about 72 hours after dosing.

[0181] Furthermore, in a single study, administration of a single subcutaneous injection of Mab 4A10-3 at a dose of 10 mg/kg produced an identical response (n=2; mean±SD) in serum iron, in both intensity and duration, as observed after the equivalent intravenous dose (n=4; mean±SD).

EXAMPLE 11

Pharmacokinetics of Humanized Anti-Ferroportin Monoclonal Antibodies in Rats and Cynomolgus Monkeys

[0182] The pharmacokinetics of anti-ferroportin Mabs may be studied in vivo according methods well-known to those skilled in the art. The pharmacokinetics of anti-FPN1 Mabs 4A10-3 and Combi 11 were investigated after single intravenous doses to male Cynomolgus monkeys and Sprague Dawley rats, for example. At the time of dosing the Cynomolgus monkeys used weighed between 2.2 and 5.5 kg and the Sprague Dawley rats weighed between 240 and 265 g.

[0183] The pharmacokinetic study conducted in Cynomolgus monkeys was performed in three phases, with doses at 1.0 mg/kg, 3.0 mg/kg, and 0.3 mg/kg administered at approximately 2 week intervals. At each phase, either Mab 4A10-3 or Mab Combi11 was administered as a single intravenous bolus (n=4 per group). Blood samples were taken prior to the first dose and at 1, 6, 12, 24, 48, 72, 96, 168, and 264 hours post-dose.

[0184] In rats, Mab 4A10-3 or Mab Combi 11 was administered as a single intravenous bolus dose of 3 mg/kg (n=3 per group). Serial blood samples were taken prior to dose and at 0.08, 1, 4, 8, 24, 48, 72, 120 and 168 hours post-dose.

[0185] Serum concentrations of Mabs 4A10-3 and Combi 11 were determined using a human IgG sandwich ELISA format. The standard curve range was 5 to 400 ng/mL, with a working lower limit of quantitation (LLOQ) defined as 10 ng/mL. Pharmacokinetic parameters were determined using non-compartmental analysis in WinNonlin version 5.2.

[0186] Serum concentration-time profiles following intravenous administration to male Cynomolgus monkeys are plotted in FIG. 10. Mab 4A10-3 was cleared much more slowly (approximately 5-fold) than Mab Combi11 at all doses studied. Differences were apparent at the first time point examined (1 hour) when serum concentrations of Mab Combi11 were approximately 50% of that observed for Mab 4A10-3. At 24 hours post-dose, serum concentrations of Mab 4A10-3 were 20-33% of Cmax compared to only 6-9% for Combi11. Peripheral concentrations of Mab Combi11 were not evident after the 0.3 mg/kg dose. The clearance of Mab 4A10-3 was somewhat faster at the two lower doses compared to the 3 mg/kg dose. The T₁/2 for Mab 4A10-3 ranged from 2 to 3 days. However, the T₁/2 for Mab Combi11 ranged from about 12 to about 27 hours.

[0187] The enhanced clearance of Mab Combi11 relative to Mab 4A10-3 was hypothesized to result from increased non-specific interactions of Mab Combi11 with cell surface proteins which do not occur for Mab 4A10-3. In order to evaluate this hypothesis the pharmacokinetics of Mab 4A10-3 and Mab Combi11 was studied in rats since neither Mab binds effectively to rat ferroportin.

[0188] Serum concentration-time profiles following intravenous administration to male rats are plotted in FIG. 11. Similar to the observation in primates, Mab 4A10-3 cleared more slowly (approximately 5-fold) than Mab Combi11 in rats (data not shown). Again, differences were apparent at the first time point examined (0.08 hours) when serum concentrations of Mab Combi11 were approximately 50% of that observed for Mab 4A10-3. The T₁/2 for Mab 4A10-3 and Mab Combi11 was approximately 4.5 days and 3 days, respectively, in rats (data not shown).

[0189] These data strongly suggest that the more rapid clearance observed for Mab Combi11 was not attributable to target receptor-mediated clearance since neither Mab 4A10-3 nor Mab Combi11 binds rat ferroportin.

EXAMPLE 12

Anti-Human FPN1 Mabs with Delayed Clearance and/or Low Non-Specific (Heparin) Binding

[0190] The pharmacokinetic studies of Mab Combi11 described above in Example 11 suggested that Mab Combi11 was more rapidly cleared from serum as compared to Mab 4A10-3. The data also suggested that the more rapid clearance of Mab Combi11 as compared to Mab 4A10-3 was not attributable to increased target receptor-mediated clearance of Mab Combi11 relative to that of Mab 4A10-3.

[0191] Because multiple arginine residues had been introduced during the engineering of Mab Combi11, it was suspected that the resulting increase in positive charge of Mab Combi11 as compared with Mab 4A10-3, for example, resulted in increased undesirable non-specific binding to negatively-charged membrane surfaces and to heparin. Indeed, modeling of the structure of Mab Combi11 showed a strong positively-charged patch on the surface of Mab Combi11 which was more pronounced in Mab Combi11 than some of the other human engineered anti-FPN1 Mabs, including Mab 4A10-3 and Mab L2.2-4.

[0192] Mabs Combi11, 4A10-3, and L2.2-4 were tested for non-specific heparin binding using a heparin ELISA according to methods known to one skilled in the art. Mabs Combi11, 4A10-3, 3D8, and L2.2-4 were also tested for binding to human FPN1 expressing HEK 293 cells as well as to control HEK 293 cells lacking human FPN1 expressed on the cell surface.

[0193] The heparin ELISA using Mab Combi-11 showed that Mab Combi11 binds strongly to heparin whereas Mabs 4A10-3 and L2.2-4 did not. Furthermore, Mab Combi-11 also bound strongly to both human FPN1 expressing HEK 293 cells and control HEK 293 cells lacking human FPN1 expressed on the cell surface. On the other hand, Mabs 4A10-3, 3D8, and L2.2-4 significantly bound to human FPN1 expressing HEK 293 cells but not to the control HEK 293 cells.

[0194] Mab Com11GY was therefore generated to reduce the non-specific binding observed with Mab Combi11 by replacing the arginine amino acid residue in the HCDR2 with a glycine amino acid residue. In addition, another potentially problematic amino acid residue found in Mab Combi11, the tryptophan amino acid residue in the LCDR2, was substituted with a tyrosine amino acid residue in Mab Com11GY. Preliminary binding data, using supernatants from cells expressing Mabs Com11GY, demonstrated a lack of non-specific binding to control HEK 293 cells, i.e., human FPN1 non-expressing cells, whereas both Mabs 1B7 and 1F8 demonstrated significantly more non-specific binding to the same control cells.

EXAMPLE 13

Assay for the Inhibition of Human Hepcidin-25 Binding to FPN1

[0195] Human engineered, affinity matured anti-human FPN1 antibodies may be assayed for the ability to inhibit human hepcidin-25 binding to human FPN1 expressed in HEK 293 cells. Briefly, transfected FPN/293 cells are plated in poly-D-lysine coated plates on 96 well plates (BD Biosciences, San Jose, Calif.; BD Biocoat plates #35 4640) at 40,000 cells per well in 80 μL of assay medium (DMEM 11965, 10% dialyzed FBS, 20 μM FAC, penicillin-streptomycin), centrifuged for 1 minute at 1000 revolutions per minute, and then incubated 4 hours at 37° C., 10% CO₂. FPN1 expression is induced by adding 20 μL of Doxycycline at 10 nM to the plated cells (2 nM final concentration of doxycycline. Doxycyline induced and un-induced control cells are incubated for 5 hours at 37° C., 10% CO₂. Next, the inducing agent is removed by washing the plate 2× with DMEM. The cells are incubated overnight in 100 μL of assay medium. Next, the assay media is removed and replaced with 40 μL test antibody or an isotype control antibody solution in triplicate and incubated at 37° C., 10% CO₂ for 20 minutes. Next, 20 μL of biotinylated mature human hepcidin is added to the wells to a final concentration of 30 nM per well. The samples are incubated for 1 hour, at 37° C., 10% 10% CO₂ before washing 4 times with 200 μL 2% FBS, D-PBS (Gibco, catalog no. 14040). Next, 65 μL of lysing buffer (0.5% Triton X-100, 10 mM EDTA) are added to all the wells and the plates are shaken for 10 minutes. Next, 50 μL of the solution in each well is transferred to individual wells of a streptavidin coated Greiner microtiter plate (60 μL of 2 μg/mL streptavidin (Sigma, St. Louis, Mo.; catalog no. 54762) in PBS, incubated at 4° C. overnight, washed 2 times (0.1% Tween 20, TBS), blocked with casein/PBS), and then incubated for one hour at room temperature. Next, the wells of the plate are washed 3 times (0.1% Tween 20, TBS) and 50 μL anti-human hepcidin-25 Mab 3.23 at 0.5 μg/mL is added and the samples are incubated one hour at room temperature. The anti-human hepcidin-25 Mab 3.23 is described in PCT International Patent Application Publication WO 2009/058797. Next, the plates are washed three times and 50 μL of goat anti-human IgG-horseradish peroxidase (Southern Biotech catalog no. 2060-05) is added at 1:2000 dilution. After incubating one hour at room temperature, the plate is washed 4× and 50 μL of OPD substrate (Sigma; catalog no. P6912) is added. The reaction is stopped with 100 μL 1 N HCl after 4 minutes. Absorbance at 490 nm (A₄₉₀) is read using an appropriate ELISA plate reader. The assay range is determined by subtracting the A₄₉₀ of un-induced wells from the induced wells receiving control antibody.

[0196] The data shown in Table 11 demonstrate that humanized Mab 3D8 and affinity matured variants thereof significantly inhibit the ability of human hepcidin-25 to bind human FPN1. More specifically, Mab 3D8, a humanized form of the mouse Mab 1G9, having the human heavy chain framework VH1-69 and light chain framework O2, demonstrated an IC₅₀ of about 400 nM as determined in this assay format. Affinity matured Mabs 4A10-3, Combi11, and L2.2-4 demonstrated significantly improved inhibition of binding as determined in this assay format.

TABLE-US-00019 TABLE 11 % Inhibition Antibody concentration Antibody 2000 nM 500 nM 125 nM 31.25 nM 7.8 nM 3D8 Mean 54.7 64.7 21.8 4.0 -2.8 SD 9.0 2.5 14.9 11.5 2.7 4A10-3 Mean 95.3 84.9 59.3 13.4 5.4 SD 2.1 1.7 6.4 4.2 14.4 combi 11 Mean 79.9 81.6 88.4 32.1 18.7 SD 9.0 8.8 4.8 3.1 8.4 L2.2-4 Mean 85.1 99.8 76.5 27.5 -0.1 SD 20.8 6.5 9.3 3.9 2.9 control IgG Mean -2.5 -0.7 1.9 7.9 2.5 SD 5.8 10.3 6.2 6.5 14.1 Note: 75 kg/mole was the molecular weight used to calculate the antibody concentration.

EXAMPLE 14

In Vitro Assay of the Effects of FPN1 Mabs on Cellular Ferritin Levels

[0197] As described in Example 5, Caco-2 cells, a human enterocyte cell line, endogenously expressing FPN1, may be monitored for changes in ferritin. In experiments conducted essentially as described in Example 5, the effect of anti-human FPN1 antibodies on mature hepcidin modulated iron regulation in Caco-2 cells was determined and is expressed as percent inhibition, averaged over a number of independent experiments in Table 12 below.

[0198] The data indicate that the effects of hepcidin on ferritin concentration in the cells can be inhibited by anti-human FPN1 Mabs in a dose-dependent manner. As indicated by the EC50 values, some anti-human Mabs are more potent in inhibiting the effect of hepcidin than others, for example, Combi11≈4A10-3>L2-2-4>3D8.

TABLE-US-00020 TABLE 12 Percentage inhibition (± SEM) by anti-human FPN1 Mabs on mature hepcidin induced increases in cellular ferritin levels in Caco-2 cells in vitro Control human Concentration (M) Combi11 4A10-3 L2-2-4 3D8 IgG₄ 6.67E-7M 75.2 (7.4) 61.9 (6.3) 61.7 (3.9) 29.8 (6.1) 18.7 (3.8) 2.22E-7M 69.3 (6.2) 59.6 (5.4) 53.8 (11.7) 25.2 (4.4) 24.2 (5.2) 7.4E-8M 61.1 (5.6) 45.6 (5.1) 40.6 (10.3) 12.0 (5.6) 21.1 (4.4) 2.47E-8M 45.3 (6.5) 36.2 (8.1) 30.1 (4.4) 3.4 (4.7) 22.1 (3.8) 8.0E-9M 31.6 (9.1) 28.0 (7.5) 39.5 (6.3) 14.9 (3.6) 16.1 (3.7) 2.7E-9M 34.8 (5.7) 24.9 (7.6) 39.6 (7.5) 10.6 (10.6) 22.4 (3.6) 9.0E-10M 23.3 (6.3) 19.5 (4.8) 24.4 (8.2) 8.6 (6.4) 17.5 (3.9) 3.0E-10M 14.2 (5.6) 12.0 (10.6) 24.1 (9.8) 7.3 (6.6) 15.5 (3.2) Number of 6 6 3 4 16 experiments (n) EC50 (nM) 28 37 193 360 N.C. N.C.: Negative control EC₅₀ can not be calculated.

Sequence CWU 1

2141571PRTHomo sapiens 1Met Thr Arg Ala Gly Asp His Asn Arg Gln Arg Gly Cys Cys Gly Ser1 5 10 15Leu Ala Asp Tyr Leu Thr Ser Ala Lys Phe Leu Leu Tyr Leu Gly His 20 25 30Ser Leu Ser Thr Trp Gly Asp Arg Met Trp His Phe Ala Val Ser Val 35 40 45Phe Leu Val Glu Leu Tyr Gly Asn Ser Leu Leu Leu Thr Ala Val Tyr 50 55 60Gly Leu Val Val Ala Gly Ser Val Leu Val Leu Gly Ala Ile Ile Gly65 70 75 80Asp Trp Val Asp Lys Asn Ala Arg Leu Lys Val Ala Gln Thr Ser Leu 85 90 95Val Val Gln Asn Val Ser Val Ile Leu Cys Gly Ile Ile Leu Met Met 100 105 110Val Phe Leu His Lys His Glu Leu Leu Thr Met Tyr His Gly Trp Val 115 120 125Leu Thr Ser Cys Tyr Ile Leu Ile Ile Thr Ile Ala Asn Ile Ala Asn 130 135 140Leu Ala Ser Thr Ala Thr Ala Ile Thr Ile Gln Arg Asp Trp Ile Val145 150 155 160Val Val Ala Gly Glu Asp Arg Ser Lys Leu Ala Asn Met Asn Ala Thr 165 170 175Ile Arg Arg Ile Asp Gln Leu Thr Asn Ile Leu Ala Pro Met Ala Val 180 185 190Gly Gln Ile Met Thr Phe Gly Ser Pro Val Ile Gly Cys Gly Phe Ile 195 200 205Ser Gly Trp Asn Leu Val Ser Met Cys Val Glu Tyr Val Leu Leu Trp 210 215 220Lys Val Tyr Gln Lys Thr Pro Ala Leu Ala Val Lys Ala Gly Leu Lys225 230 235 240Glu Glu Glu Thr Glu Leu Lys Gln Leu Asn Leu His Lys Asp Thr Glu 245 250 255Pro Lys Pro Leu Glu Gly Thr His Leu Met Gly Val Lys Asp Ser Asn 260 265 270Ile His Glu Leu Glu His Glu Gln Glu Pro Thr Cys Ala Ser Gln Met 275 280 285Ala Glu Pro Phe Arg Thr Phe Arg Asp Gly Trp Val Ser Tyr Tyr Asn 290 295 300Gln Pro Val Phe Leu Ala Gly Met Gly Leu Ala Phe Leu Tyr Met Thr305 310 315 320Val Leu Gly Phe Asp Cys Ile Thr Thr Gly Tyr Ala Tyr Thr Gln Gly 325 330 335Leu Ser Gly Ser Ile Leu Ser Ile Leu Met Gly Ala Ser Ala Ile Thr 340 345 350Gly Ile Met Gly Thr Val Ala Phe Thr Trp Leu Arg Arg Lys Cys Gly 355 360 365Leu Val Arg Thr Gly Leu Ile Ser Gly Leu Ala Gln Leu Ser Cys Leu 370 375 380Ile Leu Cys Val Ile Ser Val Phe Met Pro Gly Ser Pro Leu Asp Leu385 390 395 400Ser Val Ser Pro Phe Glu Asp Ile Arg Ser Arg Phe Ile Gln Gly Glu 405 410 415Ser Ile Thr Pro Thr Lys Ile Pro Glu Ile Thr Thr Glu Ile Tyr Met 420 425 430Ser Asn Gly Ser Asn Ser Ala Asn Ile Val Pro Glu Thr Ser Pro Glu 435 440 445Ser Val Pro Ile Ile Ser Val Ser Leu Leu Phe Ala Gly Val Ile Ala 450 455 460Ala Arg Ile Gly Leu Trp Ser Phe Asp Leu Thr Val Thr Gln Leu Leu465 470 475 480Gln Glu Asn Val Ile Glu Ser Glu Arg Gly Ile Ile Asn Gly Val Gln 485 490 495Asn Ser Met Asn Tyr Leu Leu Asp Leu Leu His Phe Ile Met Val Ile 500 505 510Leu Ala Pro Asn Pro Glu Ala Phe Gly Leu Leu Val Leu Ile Ser Val 515 520 525Ser Phe Val Ala Met Gly His Ile Met Tyr Phe Arg Phe Ala Gln Asn 530 535 540Thr Leu Gly Asn Lys Leu Phe Ala Cys Gly Pro Asp Ala Lys Glu Val545 550 555 560Arg Lys Glu Asn Gln Ala Asn Thr Ser Val Val 565 57021716DNAHomo sapiens 2atgaccaggg cgggagatca caaccgccag agaggatgct gtggatcctt ggccgactac 60ctgacctctg caaaattcct tctctacctt ggtcattctc tctctacttg gggagatcgg 120atgtggcact ttgcggtgtc tgtgtttctg gtagagctct atggaaacag cctccttttg 180acagcagtct acgggctggt ggtggcaggg tctgttctgg tcctgggagc catcatcggt 240gactgggtgg acaagaatgc tagacttaaa gtggcccaga cctcgctggt ggtacagaat 300gtttcagtca tcctgtgtgg aatcatcctg atgatggttt tcttacataa acatgagctt 360ctgaccatgt accatggatg ggttctcact tcctgctata tcctgatcat cactattgca 420aatattgcaa atttggccag tactgctact gcaatcacaa tccaaaggga ttggattgtt 480gttgttgcag gagaagacag aagcaaacta gcaaatatga atgccacaat acgaaggatt 540gaccagttaa ccaacatctt agcccccatg gctgttggcc agattatgac atttggctcc 600ccagtcatcg gctgtggctt tatttcggga tggaacttgg tatccatgtg cgtggagtac 660gttctgctct ggaaggttta ccagaaaacc ccagctctag ctgtgaaagc tggtcttaaa 720gaagaggaaa ctgaattgaa acagctgaat ttacacaaag atactgagcc aaaacccctg 780gagggaactc atctaatggg tgtgaaagac tctaacatcc atgagcttga acatgagcaa 840gagcctactt gtgcctccca gatggctgag cccttccgta ccttccgaga tggatgggtc 900tcctactaca accagcctgt gtttctggct ggcatgggtc ttgctttcct ttatatgact 960gtcctgggct ttgactgcat caccacaggg tacgcctaca ctcagggact gagtggttcc 1020atcctcagta ttttgatggg agcatcagct ataactggaa taatgggaac tgtagctttt 1080acttggctac gtcgaaaatg tggtttggtt cggacaggtc tgatctcagg attggcacag 1140ctttcctgtt tgatcttgtg tgtgatctct gtattcatgc ctggaagccc cctggacttg 1200tccgtttctc cttttgaaga tatccgatca aggttcattc aaggagagtc aattacacct 1260accaagatac ctgaaattac aactgaaata tacatgtcta atgggtctaa ttctgctaat 1320attgtcccgg agacaagtcc tgaatctgtg cccataatct ctgtcagtct gctgtttgca 1380ggcgtcattg ctgctagaat cggtctttgg tcctttgatt taactgtgac acagttgctg 1440caagaaaatg taattgaatc tgaaagaggc attataaatg gtgtacagaa ctccatgaac 1500tatcttcttg atcttctgca tttcatcatg gtcatcctgg ctccaaatcc tgaagctttt 1560ggcttgctcg tattgatttc agtctccttt gtggcaatgg gccacattat gtatttccga 1620tttgcccaaa atactctggg aaacaagctc tttgcttgcg gtcctgatgc aaaagaagtt 1680aggaaggaaa atcaagcaaa tacatctgtt gtgtag 17163573PRTMacaca irus 3Met Thr Arg Ala Gly Asp His Asn Arg Gln Arg Gly Cys Cys Gly Ser1 5 10 15Leu Ala Asp Tyr Leu Thr Ser Ala Lys Phe Leu Leu Tyr Leu Gly His 20 25 30Ser Leu Ser Thr Trp Gly Asp Arg Met Trp His Phe Ala Val Ser Val 35 40 45Phe Leu Val Glu Leu Tyr Gly Asn Ser Leu Leu Leu Thr Ala Val Tyr 50 55 60Gly Leu Val Val Ala Gly Ser Val Leu Val Leu Gly Ala Ile Ile Gly65 70 75 80Asp Trp Val Asp Lys Asn Ala Arg Leu Lys Val Ala Gln Thr Ser Leu 85 90 95Val Val Gln Asn Val Ser Val Ile Leu Cys Gly Ile Ile Leu Met Met 100 105 110Val Phe Leu His Lys His Glu Leu Leu Thr Met Tyr His Gly Trp Val 115 120 125Leu Thr Ser Cys Tyr Ile Leu Ile Ile Thr Ile Ala Asn Ile Ala Asn 130 135 140Leu Ala Ser Thr Ala Thr Ala Ile Thr Ile Gln Arg Asp Trp Ile Val145 150 155 160Val Val Ala Gly Glu Asp Arg Ser Lys Leu Ala Asn Met Asn Ala Thr 165 170 175Ile Arg Arg Ile Asp Gln Leu Thr Asn Ile Leu Ala Pro Met Ala Val 180 185 190Gly Gln Ile Met Thr Phe Gly Ser Pro Val Ile Gly Cys Gly Phe Ile 195 200 205Ser Gly Trp Asn Leu Val Ser Met Cys Val Glu Tyr Phe Leu Leu Trp 210 215 220Lys Val Tyr Gln Lys Thr Pro Ala Leu Ala Val Lys Ala Ala Phe Lys225 230 235 240Glu Glu Glu Thr Glu Leu Lys Gln Leu Asn Leu His Lys Asp Thr Glu 245 250 255Pro Lys Pro Leu Glu Gly Thr His Leu Met Gly Val Lys Asp Ser Asn 260 265 270Ile His Glu Leu Glu His Glu Gln Glu Pro Thr Cys Ala Ser Gln Met 275 280 285Ala Glu Pro Phe Arg Thr Phe Arg Asp Gly Trp Val Ser Tyr Tyr Asn 290 295 300Gln Pro Val Phe Leu Ala Gly Met Gly Leu Ala Phe Leu Tyr Met Thr305 310 315 320Val Leu Gly Phe Asp Cys Ile Thr Thr Gly Tyr Ala Tyr Thr Gln Gly 325 330 335Leu Ser Gly Ser Ile Leu Ser Ile Leu Met Gly Ala Ser Ala Ile Thr 340 345 350Gly Ile Met Gly Thr Val Ala Phe Thr Trp Leu Arg Arg Lys Cys Gly 355 360 365Leu Val Arg Thr Gly Leu Ile Ser Gly Leu Ala Gln Leu Ser Cys Leu 370 375 380Ile Leu Cys Val Ile Ser Val Phe Met Pro Gly Ser Pro Leu Asp Leu385 390 395 400Ser Val Ser Pro Phe Glu Asp Ile Arg Ser Arg Phe Ile Gln Gly Glu 405 410 415Ser Ile Thr Pro Thr Lys Ile Pro Glu Thr Ile Ile Thr Thr Glu Ile 420 425 430Tyr Met Ser Asn Gly Ser Asn Ser Ala Asn Ile Val Pro Glu Thr Ser 435 440 445Pro Glu Ser Val Pro Ile Ile Ser Val Ser Leu Leu Phe Ala Gly Val 450 455 460Ile Ala Ala Arg Ile Gly Leu Trp Ser Phe Asp Leu Thr Val Thr Gln465 470 475 480Leu Leu Gln Glu Asn Val Ile Glu Ser Glu Arg Gly Ile Ile Asn Gly 485 490 495Val Gln Asn Ser Met Asn Tyr Leu Leu Asp Leu Leu His Phe Ile Met 500 505 510Val Ile Leu Ala Pro Asn Pro Glu Ala Phe Gly Leu Leu Val Leu Ile 515 520 525Ser Val Ser Phe Val Ala Met Gly His Ile Met Tyr Phe Arg Phe Ala 530 535 540His Asn Thr Leu Gly Asn Lys Leu Phe Ala Cys Gly Pro Asp Ala Lys545 550 555 560Glu Val Arg Lys Glu Asn Gln Ala Asn Thr Ser Val Val 565 57041320DNAArtificialSynthetic 4caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc agtgaaggtt 60tcctgcaagg catctggcta cgccttcact tcgttcttga tagagtgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggaacg agtaatccta ggactgggag gacgaagtat 180aaatccaagt tcaggggcag agtcaccatt accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gcgcgagttt 300tttgactact ggggccaagg aaccacggtc accgtctcct cagcctccac caagggccca 360tcggtcttcc cgctagcgcc ctgctccagg agcacctccg agagcacagc cgccctgggc 420tgcctggtca aggactactt ccccgaaccg gtgacggtgt cgtggaactc aggcgccctg 480accagcggcg tgcacacctt cccggctgtc ctacagtcct caggactcta ctccctcagc 540agcgtggtga ccgtgccctc cagcagcttg ggcacgaaga cctacacctg caacgtagat 600cacaagccca gcaacaccaa ggtggacaag agagttgagt ccaaatatgg tcccccatgc 660ccaccctgcc cagcacctga ggccgccggg ggaccatcag tcttcctgtt ccccccaaaa 720cccaaggaca ctctcatgat ctcccggacc cctgaggtca cgtgcgtggt ggtggacgtg 780agccaggaag accccgaggt ccagttcaac tggtacgtgg atggcgtgga ggtgcataat 840gccaagacaa agccgcggga ggagcagttc aacagcacgt accgtgtggt cagcgtcctc 900accgtcctgc accaggactg gctgaacggc aaggagtaca agtgcaaggt ctccaacaaa 960ggcctcccgt cctccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagagcca 1020caggtgtaca ccctgccccc atcccaggag gagatgacca agaaccaggt cagcctgacc 1080tgcctggtca aaggcttcta ccccagcgac atcgccgtgg agtgggaaag caatgggcag 1140ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1200tacagcaggc taaccgtgga caagagcagg tggcaggagg ggaatgtctt ctcatgctcc 1260gtgatgcatg aggctctgca caaccactac acacagaaga gcctctccct gtctctgggt 13205570PRTRattus sp 5Met Thr Lys Ser Arg Asp Gln Thr His Gln Glu Gly Cys Cys Gly Ser1 5 10 15Leu Ala Asn Tyr Leu Thr Ser Ala Lys Phe Leu Leu Tyr Leu Gly His 20 25 30Ser Leu Ser Thr Trp Gly Asp Arg Met Trp His Phe Ala Val Ser Val 35 40 45Phe Leu Val Glu Leu Tyr Gly Asn Ser Leu Leu Leu Thr Ala Val Tyr 50 55 60Gly Leu Val Val Ala Gly Ser Val Leu Val Leu Gly Ala Ile Ile Gly65 70 75 80Asp Trp Val Asp Lys Asn Ala Arg Leu Lys Val Ala Gln Thr Ser Leu 85 90 95Val Val Gln Asn Val Ser Val Ile Leu Cys Gly Ile Ile Leu Met Met 100 105 110Val Phe Leu His Lys Asn Glu Leu Leu Asn Met Tyr His Gly Trp Val 115 120 125Leu Thr Val Cys Tyr Ile Leu Ile Ile Thr Ile Ala Asn Ile Ala Asn 130 135 140Leu Ala Ser Thr Ala Thr Ala Ile Thr Ile Gln Arg Asp Trp Ile Val145 150 155 160Val Val Ala Gly Glu Asn Arg Ser Arg Leu Ala Asp Met Asn Ala Thr 165 170 175Ile Arg Arg Ile Asp Gln Leu Thr Asn Ile Leu Ala Pro Met Ala Val 180 185 190Gly Gln Ile Met Thr Phe Gly Ser Pro Val Ile Gly Cys Gly Phe Ile 195 200 205Ser Gly Trp Asn Leu Val Ser Met Cys Val Glu Tyr Phe Leu Leu Trp 210 215 220Lys Val Tyr Gln Lys Thr Pro Ala Leu Ala Val Lys Ala Ala Leu Lys225 230 235 240Val Glu Glu Ser Glu Leu Lys Gln Leu Thr Ser Pro Lys Asp Thr Glu 245 250 255Pro Lys Pro Leu Glu Gly Thr His Leu Met Gly Glu Lys Asp Ser Asn 260 265 270Ile Arg Glu Leu Glu Cys Glu Gln Glu Pro Thr Cys Ala Ser Gln Ile 275 280 285Ala Glu Pro Phe Arg Thr Phe Arg Asp Gly Trp Val Ser Tyr Tyr Asn 290 295 300Gln Pro Val Phe Leu Ala Gly Met Gly Leu Ala Phe Leu Tyr Met Thr305 310 315 320Val Leu Gly Phe Asp Cys Ile Thr Thr Gly Tyr Ala Tyr Thr Gln Gly 325 330 335Leu Ser Gly Ser Ile Leu Ser Val Leu Met Gly Ala Ser Ala Ile Thr 340 345 350Gly Ile Met Gly Thr Val Ala Phe Thr Trp Leu Arg Arg Lys Cys Gly 355 360 365Leu Val Arg Thr Gly Leu Phe Ser Gly Leu Ala Gln Leu Ser Cys Leu 370 375 380Ile Leu Cys Val Ile Ser Val Phe Met Pro Gly Ser Pro Leu Asp Leu385 390 395 400Ser Val Ser Pro Phe Glu Asp Ile Arg Ser Arg Phe Ile His Glu Glu 405 410 415Ala Val Ser Ser Thr Thr Lys Ile Pro Glu Thr Glu Met Leu Met Ser 420 425 430Asn Val Ser Asn Val Val Asn Thr Val His Glu Met Ser Thr Lys Ser 435 440 445Val Pro Ile Ile Ser Val Ser Leu Leu Phe Ala Gly Val Ile Ala Ala 450 455 460Arg Ile Gly Leu Trp Ser Phe Asp Leu Thr Val Thr Gln Leu Leu Gln465 470 475 480Glu Asn Val Ile Glu Ser Glu Arg Gly Ile Ile Asn Gly Val Gln Asn 485 490 495Ser Met Asn Tyr Leu Leu Asp Leu Leu His Phe Ile Met Val Ile Leu 500 505 510Ala Pro Asn Pro Glu Ala Phe Gly Leu Leu Val Leu Ile Ser Val Ser 515 520 525Phe Val Ala Met Gly His Leu Met Tyr Phe Arg Phe Ala Gln Lys Thr 530 535 540Leu Gly Asn Gln Ile Phe Val Cys Ala Pro Asp Glu Lys Glu Val Thr545 550 555 560Asp Glu Ser Gln Pro Asn Thr Ser Val Val 565 57061713DNARattus sp 6atgaccaagt caagagatca gacccatcag gaaggatgct gtggatcttt agcaaactac 60ctgacctcag caaaattcct cctctacctt ggccactctc tctccacttg gggggatcgg 120atgtggcact ttgcagtgtc tgtgtttctg gtggaactct acggaaacag cctcctcttg 180acagctgtct acgggttggt ggtggcaggc tctgttctgg tcctgggagc catcattggt 240gactgggtgg ataagaatgc cagacttaaa gtggcccaga cgtccctggt ggttcagaat 300gtatcagtca ttctctgcgg gatcatcctg atgatggttt tcttacacaa gaatgagctt 360ctgaacatgt atcatggatg ggtccttact gtctgctaca tcctgatcat caccattgca 420aacattgcga atttggccag tactgccact gcaattacaa tccaaaggga ctggattgtt 480gtcgtagcag gagaaaacag gagcagatta gcagacatga atgctaccat tagaaggatt 540gaccagctaa ccaacatcct ggcccccatg gctgttggcc agattatgac attcggttcc 600ccagtcattg gctgtggttt catttctggt tggaatttgg tgtccatgtg tgtggagtac 660ttcttgctct ggaaggttta ccagaagacc cctgctctgg ctgtaaaagc tgctctcaag 720gtagaggagt cagaactgaa gcagctgacc tcacctaaag atactgagcc aaaacctttg 780gagggaactc acctaatggg tgagaaagac tctaacatcc gtgaacttga atgtgaacaa 840gaacccacct gtgcctccca gatcgcagaa cccttccgca cttttcgaga tggatgggtc 900tcctactata accagcccgt atttttggct ggcatgggcc tggctttcct ctatatgaca 960gtcctgggct tcgactgtat caccacagga tatgcttaca ctcagggact gagtggttcc 1020atcctcagtg ttttgatggg agcatcagca ataactggaa taatgggaac tgtggccttc 1080acttggctac gtcgaaaatg tggccttgtt cggactggtc tgttctcagg actggctcag 1140ctttcttgtt tgatcttgtg tgtgatctcc gtgttcatgc ctggaagccc cttggacctg 1200tctgtttctc catttgaaga tatccgttct aggtttatac atgaggaggc agtgtcctca 1260actaccaaaa tacctgaaac agaaatgctt atgtctaatg tgtctaatgt tgtcaatacc 1320gtccatgaga tgagtactaa atccgtcccc ataatctccg tcagcctgct gtttgcagga 1380gtcattgctg ctagaatcgg tctttggtcc tttgatttga ctgtgacaca gttgctgcaa 1440gaaaatgtaa

ttgaatcaga aagaggcatt atcaatggtg tgcagaactc catgaactac 1500cttctcgacc ttctgcattt catcatggtc atcttggccc caaatcctga agcttttggc 1560ttgctagtat tgatttcagt ctcctttgtg gcaatgggac atcttatgta tttccgtttt 1620gcccagaaga ctctgggcaa ccagattttt gtttgtgctc ctgatgaaaa ggaagttaca 1680gatgaaagtc agcctaatac atctgttgtg tag 17137570PRTMus sp 7Met Thr Lys Ala Arg Asp Gln Thr His Gln Glu Gly Cys Cys Gly Ser1 5 10 15Leu Ala Asn Tyr Leu Thr Ser Ala Lys Phe Leu Leu Tyr Leu Gly His 20 25 30Ser Leu Ser Thr Trp Gly Asp Arg Met Trp His Phe Ala Val Ser Val 35 40 45Phe Leu Val Glu Leu Tyr Gly Asn Ser Leu Leu Leu Thr Ala Val Tyr 50 55 60Gly Leu Val Val Ala Gly Ser Val Leu Val Leu Gly Ala Ile Ile Gly65 70 75 80Asp Trp Val Asp Lys Asn Ala Arg Leu Lys Val Ala Gln Thr Ser Leu 85 90 95Val Val Gln Asn Val Ser Val Ile Leu Cys Gly Ile Ile Leu Met Met 100 105 110Val Phe Leu His Lys Asn Glu Leu Leu Thr Met Tyr His Gly Trp Val 115 120 125Leu Thr Val Cys Tyr Ile Leu Ile Ile Thr Ile Ala Asn Ile Ala Asn 130 135 140Leu Ala Ser Thr Ala Thr Ala Ile Thr Ile Gln Arg Asp Trp Ile Val145 150 155 160Val Val Ala Gly Glu Asn Arg Ser Arg Leu Ala Asp Met Asn Ala Thr 165 170 175Ile Arg Arg Ile Asp Gln Leu Thr Asn Ile Leu Ala Pro Met Ala Val 180 185 190Gly Gln Ile Met Thr Phe Gly Ser Pro Val Ile Gly Cys Gly Phe Ile 195 200 205Ser Gly Trp Asn Leu Val Ser Met Cys Val Glu Tyr Phe Leu Leu Trp 210 215 220Lys Val Tyr Gln Lys Thr Pro Ala Leu Ala Val Lys Ala Ala Leu Lys225 230 235 240Val Glu Glu Ser Glu Leu Lys Gln Leu Thr Ser Pro Lys Asp Thr Glu 245 250 255Pro Lys Pro Leu Glu Gly Thr His Leu Met Gly Glu Lys Asp Ser Asn 260 265 270Ile Arg Glu Leu Glu Cys Glu Gln Glu Pro Thr Cys Ala Ser Gln Met 275 280 285Ala Glu Pro Phe Arg Thr Phe Arg Asp Gly Trp Val Ser Tyr Tyr Asn 290 295 300Gln Pro Val Phe Leu Ala Gly Met Gly Leu Ala Phe Leu Tyr Met Thr305 310 315 320Val Leu Gly Phe Asp Cys Ile Thr Thr Gly Tyr Ala Tyr Thr Gln Gly 325 330 335Leu Ser Gly Ser Ile Leu Ser Ile Leu Met Gly Ala Ser Ala Ile Thr 340 345 350Gly Ile Met Gly Thr Val Ala Phe Thr Trp Leu Arg Arg Lys Cys Gly 355 360 365Leu Val Arg Thr Gly Leu Phe Ser Gly Leu Ala Gln Leu Ser Cys Leu 370 375 380Ile Leu Cys Val Ile Ser Val Phe Met Pro Gly Ser Pro Leu Asp Leu385 390 395 400Ser Val Ser Pro Phe Glu Asp Ile Arg Ser Arg Phe Val Asn Val Glu 405 410 415Pro Val Ser Pro Thr Thr Lys Ile Pro Glu Thr Val Phe Thr Thr Glu 420 425 430Met His Met Ser Asn Met Ser Asn Val His Glu Met Ser Thr Lys Pro 435 440 445Ile Pro Ile Val Ser Val Ser Leu Leu Phe Ala Gly Val Ile Ala Ala 450 455 460Arg Ile Gly Leu Trp Ser Phe Asp Leu Thr Val Thr Gln Leu Leu Gln465 470 475 480Glu Asn Val Ile Glu Ser Glu Arg Gly Ile Ile Asn Gly Val Gln Asn 485 490 495Ser Met Asn Tyr Leu Leu Asp Leu Leu His Phe Ile Met Val Ile Leu 500 505 510Ala Pro Asn Pro Glu Ala Phe Gly Leu Leu Val Leu Ile Ser Val Ser 515 520 525Phe Val Ala Met Gly His Leu Met Tyr Phe Arg Phe Ala Gln Lys Thr 530 535 540Leu Gly Asn Gln Ile Phe Val Cys Gly Pro Asp Glu Lys Glu Val Thr545 550 555 560Asp Glu Asn Gln Pro Asn Thr Ser Val Val 565 57081713DNAMus sp 8atgaccaagg caagagatca aacccatcag gaaggatgct gtggatcctt agcaaactac 60ctgacctcag caaaattcct cctctacctt ggccactctc tctccacttg gggggatcgg 120atgtggcact ttgcagtgtc tgtgtttctg gtggaactct atggaaacag ccttctcttg 180acagctgtct atggactggt ggtggcaggc tctgttctgg tcctgggagc catcattggt 240gactgggtgg ataagaatgc cagacttaaa gtggcccaga cgtcactagt ggttcagaat 300gtgtccgtca tcctctgcgg aatcatcctg atgatggttt tcctacacaa gaatgagctc 360ctgaccatgt accatggatg ggtccttact gtctgctaca tcctgatcat cactattgca 420aacattgcaa atttggccag tactgccact gcgatcacaa tccaaaggga ctggattgtt 480gttgtggcag gagaaaacag gagcagatta gcagacatga atgctaccat tagaaggatt 540gaccagctaa ccaacatcct ggcccccatg gctgtcggcc agattatgac atttggttct 600ccagtcattg gctgtggttt catttccggt tggaatttgg tgtccatgtg tgtggagtac 660ttcttgctct ggaaggttta ccagaagacc cctgctctgg ctgtaaaagc tgctctcaag 720gtagaggagt cagaactgaa gcagctgacc tcacctaaag atactgagcc aaaacctttg 780gagggaactc atctaatggg tgagaaagac tccaacatcc gtgaacttga atgtgaacaa 840gagcccacct gtgcctccca gatggcagag cccttccgca ctttccgaga tggatgggtc 900tcctactata accagccagt gtttctggct ggcatgggcc tggctttcct ctatatgaca 960gtcctgggct ttgactgtat cactacaggg tacgcctaca ctcaggggct gagtggatcc 1020atccttagta ttttgatggg agcatcagca ataactggaa taatgggaac tgtggccttc 1080acctggctac gtcgaaaatg tggccttgtt cggactggtc tattctcagg actagcccag 1140ctttcctgtt taatcttgtg tgtgatctcc gtattcatgc ctggaagccc cttggacctg 1200tctgtttctc catttgaaga tatccgttct aggtttgtga atgtggagcc agtgtcccca 1260actaccaaaa tacctgagac cgtctttaca acagaaatgc atatgtccaa catgtctaat 1320gtccatgaga tgagtactaa acccatcccc atagtctctg tcagcctgct gtttgcagga 1380gtcattgctg ctagaatcgg tctttggtcc tttgatttga cggtgacaca gttgctgcaa 1440gaaaatgtaa ttgaatctga aagaggcatt atcaatggtg tgcagaactc catgaactac 1500cttcttgacc ttctgcattt catcatggtc atcttggccc caaatcctga agcttttggc 1560ttgctggtat tgatttcagt ctcctttgtg gcaatgggac atcttatgta tttccgattt 1620gcccagaaga ctctgggcaa ccagattttt gtttgtggtc ctgatgaaaa agaagttaca 1680gatgaaaatc aaccgaatac atctgttgta tag 1713956PRTHomo sapiens 9Pro Gly Ser Pro Leu Asp Leu Ser Val Ser Pro Phe Glu Asp Ile Arg1 5 10 15Ser Arg Phe Ile Gln Gly Glu Ser Ile Thr Pro Thr Lys Ile Pro Glu 20 25 30Ile Thr Thr Glu Ile Tyr Met Ser Asn Gly Ser Asn Ser Ala Asn Ile 35 40 45Val Pro Glu Thr Ser Pro Glu Ser 50 5510642DNAArtificialSynthetic 10gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgca gggcgagtaa gagcattagc aaatatacag cctggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gggtccaagc ggcactgggg agtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg caacttacta ttgtcaacaa cataatgaat acccgtacac gttcggcgga 300gggaccaagg tggagatcaa acggactgtg gctgcaccat ctgtcttcat cttcccgcca 360tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gc 6421139PRTArtificialSynthetic 11Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn Glu Gly Pro1 5 10 15Gly Pro Gly Ser Pro Phe Glu Asp Ile Arg Ser Arg Phe Ile Gln Gly 20 25 30Glu Ser Ile Thr Pro Thr Lys 351220PRTHomo sapiens 12Ser Pro Phe Glu Asp Ile Arg Ser Arg Phe Ile Gln Gly Glu Ser Ile1 5 10 15Thr Pro Thr Lys 201314PRTHomo sapiens 13Glu Asp Ile Arg Ser Arg Phe Ile Gln Gly Glu Ser Ile Thr1 5 101414PRTHomo sapiens 14Arg Ser Arg Phe Ile Gln Gly Glu Ser Ile Thr Pro Thr Lys1 5 101513PRTHomo sapiens 15Ser Pro Phe Glu Asp Ile Arg Ser Arg Phe Ile Gln Gly1 5 101611PRTHomo sapiens 16Arg Ser Arg Phe Ile Gln Gly Glu Ser Ile Thr1 5 101710PRTArtificialSynthetic 17Gly Tyr Ala Phe Thr Asn Phe Leu Ile Glu1 5 101817PRTArtificialSynthetic 18Thr Ile Asn Pro Glu Thr Gly Gly Thr Lys Tyr Asn Glu Lys Phe Arg1 5 10 15Gly195PRTArtificialSynthetic 19Glu Phe Phe Asp Tyr1 52011PRTArtificialSynthetic 20Arg Ala Ser Lys Ser Ile Ser Lys Tyr Leu Ala1 5 10217PRTArtificialSynthetic 21Ala Gly Ser Thr Leu His Ser1 5229PRTArtificialSynthetic 22Gln Gln His Asn Glu Tyr Pro Tyr Thr1 52310PRTArtificialSynthetic 23Gly Tyr Ala Phe Thr Ser Phe Leu Ile Glu1 5 102417PRTArtificialSynthetic 24Thr Ile Asn Pro Arg Thr Gly Gly Thr Lys Tyr Asn Glu Lys Phe Arg1 5 10 15Gly2517PRTArtificialSynthetic 25Thr Ile Asn Pro Lys Thr Gly Gly Thr Lys Tyr Asn Glu Lys Phe Arg1 5 10 15Gly2617PRTArtificialSynthetic 26Thr Ile Asn Pro Glu Thr Gly Gly Thr Lys Tyr Asn Ala Lys Phe Arg1 5 10 15Gly277PRTArtificialSynthetic 27Ala Gly Ser Lys Leu His Ser1 5287PRTArtificialSynthetic 28Ala Gly Ser Arg Leu His Ser1 5299PRTArtificialSynthetic 29Phe Gln His Asn Glu Tyr Pro Tyr Thr1 53010PRTArtificialSynthetic 30Gly Tyr Ala Phe Thr Xaa Phe Leu Ile Glu1 5 103117PRTArtificialSynthetic 31Thr Ile Asn Pro Xaa Thr Gly Gly Thr Lys Tyr Asn Xaa Lys Phe Arg1 5 10 15Gly327PRTArtificialSynthetic 32Ala Gly Ser Xaa Leu His Ser1 5339PRTArtificialSynthetic 33Xaa Gln His Asn Glu Tyr Pro Tyr Thr1 53417PRTArtificialSynthetic 34Thr Ile Asn Pro Lys Thr Gly Gly Thr Lys Tyr Asn Ala Lys Phe Arg1 5 10 15Gly3517PRTArtificialSynthetic 35Thr Ser Asn Pro Arg Thr Gly Gly Thr Lys Tyr Asn Glu Lys Phe Arg1 5 10 15Gly3617PRTArtificialSynthetic 36Thr Ile Asn Pro Arg Thr Gly Gly Thr Lys Tyr Lys Glu Lys Phe Arg1 5 10 15Gly3711PRTArtificialSynthetic 37Arg Ala Ser Lys Ser Ile Ser Lys Tyr Thr Ala1 5 103811PRTArtificialSynthetic 38Arg Ala Ser Lys Ser Ile Ser Lys Tyr Ser Ala1 5 103911PRTArtificialSynthetic 39Arg Ala Ser Lys Ser Ile Ser Lys Tyr Ala Ala1 5 10409PRTArtificialSynthetic 40His Gln His Asn Glu Tyr Pro Tyr Thr1 54117PRTArtificialSynthetic 41Thr Ser Asn Pro Arg Thr Gly Gly Thr Lys Tyr Lys Glu Lys Phe Arg1 5 10 15Gly4211PRTArtificialSynthetic 42Arg Ala Ser Lys Ser Ile Ser Lys Tyr Xaa Ala1 5 104317PRTArtificialSynthetic 43Thr Xaa Asn Pro Xaa Thr Gly Gly Thr Lys Tyr Xaa Xaa Lys Phe Arg1 5 10 15Gly44114PRTMus sp 44Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Phe 20 25 30Leu Ile Glu Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Thr Ile Asn Pro Glu Thr Gly Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Asn Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys 85 90 95Val Arg Glu Phe Phe Asp Tyr Trp Gly Gln Gly Thr Ser Leu Thr Val 100 105 110Ser Ser45114PRTArtificialSynthetic 45Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Phe 20 25 30Leu Ile Glu Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Thr Ile Asn Pro Arg Thr Gly Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Asn Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys 85 90 95Val Arg Glu Phe Phe Asp Tyr Trp Gly Gln Gly Thr Ser Leu Thr Val 100 105 110Ser Ser46114PRTArtificialSynthetic 46Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Phe 20 25 30Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Thr Ser Asn Pro Arg Thr Gly Gly Thr Lys Tyr Lys Glu Lys Phe 50 55 60Arg Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110Ser Ser47107PRTMus sp 47Glu Thr Thr Val Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly1 5 10 15Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Leu Ala Trp Phe Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile 35 40 45Tyr Ala Gly Ser Thr Leu His Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys 100 10548107PRTArtificialSynthetic 48Glu Thr Thr Val Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly1 5 10 15Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Leu Ala Trp Phe Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile 35 40 45Tyr Ala Gly Ser Lys Leu His Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Met Tyr Tyr Cys Phe Gln His Asn Glu Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys 100 10549107PRTArtificialSynthetic 49Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Thr Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Gly Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 10550438PRTMus sp 50Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Phe 20 25 30Leu Ile Glu Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Thr Ile Asn Pro Glu Thr Gly Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Asn Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys 85 90 95Val Arg Glu Phe Phe Asp Tyr Trp Gly Gln Gly Thr Ser Leu Thr Val 100 105 110Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly 115 120 125Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys 130 135 140Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu145 150 155 160Ser Ser Gly Val His Thr

Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr 165 170 175Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu 180 185 190Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp 195 200 205Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr 210 215 220Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp225 230 235 240Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp 245 250 255Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp 260 265 270Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn 275 280 285Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp 290 295 300Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro305 310 315 320Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala 325 330 335Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp 340 345 350Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile 355 360 365Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn 370 375 380Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys385 390 395 400Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys 405 410 415Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu 420 425 430Ser His Ser Pro Gly Lys 43551438PRTArtificialSynthetic 51Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Phe 20 25 30Leu Ile Glu Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Thr Ile Asn Pro Arg Thr Gly Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Asn Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys 85 90 95Val Arg Glu Phe Phe Asp Tyr Trp Gly Gln Gly Thr Ser Leu Thr Val 100 105 110Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly 115 120 125Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys 130 135 140Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu145 150 155 160Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr 165 170 175Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu 180 185 190Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp 195 200 205Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr 210 215 220Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp225 230 235 240Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp 245 250 255Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp 260 265 270Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn 275 280 285Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp 290 295 300Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro305 310 315 320Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala 325 330 335Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp 340 345 350Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile 355 360 365Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn 370 375 380Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys385 390 395 400Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys 405 410 415Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu 420 425 430Ser His Ser Pro Gly Lys 43552440PRTArtificialSynthetic 52Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Phe 20 25 30Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Thr Ser Asn Pro Arg Thr Gly Gly Thr Lys Tyr Lys Glu Lys Phe 50 55 60Arg Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys 115 120 125Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys 130 135 140Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu145 150 155 160Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 165 170 175Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 180 185 190Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val 195 200 205Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 210 215 220Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys225 230 235 240Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 245 250 255Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 260 265 270Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 275 280 285Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 290 295 300Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys305 310 315 320Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 325 330 335Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 340 345 350Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 355 360 365Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 370 375 380Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu385 390 395 400Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 405 410 415Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 420 425 430Lys Ser Leu Ser Leu Ser Leu Gly 435 44053214PRTMus sp 53Glu Thr Thr Val Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly1 5 10 15Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Leu Ala Trp Phe Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile 35 40 45Tyr Ala Gly Ser Thr Leu His Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala 100 105 110Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu145 150 155 160Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205Phe Asn Arg Asn Glu Cys 21054214PRTArtificialSynthetic 54Glu Thr Thr Val Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly1 5 10 15Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Leu Ala Trp Phe Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile 35 40 45Tyr Ala Gly Ser Lys Leu His Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Met Tyr Tyr Cys Phe Gln His Asn Glu Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala 100 105 110Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu145 150 155 160Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205Phe Asn Arg Asn Glu Cys 21055214PRTArtificialSynthetic 55Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Thr Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Gly Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 21056114PRTArtificialSynthetic 56Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Xaa Phe Thr Ser Phe 20 25 30Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Thr Ser Asn Pro Arg Thr Gly Xaa Xaa Lys Tyr Lys Xaa Lys Phe 50 55 60Arg Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Phe Phe Xaa Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110Ser Ser57107PRTArtificialSynthetic 57Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Thr Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Gly Ser Lys Xaa Xaa Xaa Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 10558342DNAMus sp 58caggtgcagc tgaagcagtc tggagctgaa ctggtaaggc ctgggacttc agtgaaggtg 60tcctgcaagg cttctggata cgccttcact aatttcttga tagagtggtt aaagcagagg 120cctggacagg gccttgagtg gattggaacg attaatcctg aaactggtgg tactaagtat 180aatgagaagt tcaggggcaa ggcaacactg actgctgaca aatcttccag cactgcctat 240atgcagctca acagcctgac atctgatgac tctgcggtct atttctgtgt cagagagttt 300tttgactact ggggccaagg caccagtctc acagtctcct ca 34259321DNAMus sp 59gaaacaactg tgacccagtc tccatcttat cttgctgcat ctcctggaga aaccattact 60attaattgca gggcgagtaa gagcattagc aaatatttag cctggtttca agagaaacct 120gggaaaacta ataagcttct tatctacgct ggatccactt tgcactctgg aattccatca 180aggttcagtg gcagtggatc cggtacagat ttcactctca ccatcagtag cctggagcct 240gaagattttg caatgtatta ctgtcaacaa cataatgaat acccgtacac gttcggagga 300gggaccaagc tggagctgaa a 32160342DNAArtificialSynthetic 60caggtgcagc tgaagcagtc tggagctgaa ctggtaaggc ctgggacttc agtgaaggtg 60tcctgcaagg cttctggata cgccttcact tcgttcttga tagagtggtt aaagcagagg 120cctggacagg gccttgagtg gattggaacg attaatccta ggactggtgg tactaagtat 180aatgagaagt tcaggggcaa ggcaacactg actgctgaca aatcttccag cactgcctat 240atgcagctca acagcctgac atctgatgac tctgcggtct atttctgtgt cagagagttt 300tttgactact ggggccaagg caccagtctc acagtctcct ca 34261321DNAArtificialSynthetic 61gaaacaactg tgacccagtc tccatcttat cttgctgcat ctcctggaga aaccattact 60attaattgca gggcgagtaa gagcattagc aaatatttag cctggtttca agagaaacct 120gggaaaacta ataagcttct tatctacgct ggatccaagt tgcactctgg aattccatca 180aggttcagtg gcagtggatc cggtacagat ttcactctca ccatcagtag cctggagcct 240gaagattttg caatgtatta ctgtttccaa cataatgaat acccgtacac gttcggagga 300gggaccaagc tggagctgaa a 32162342DNAArtificialSynthetic 62caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc agtgaaggtt 60tcctgcaagg catctggcta cgccttcact tcgttcttga tagagtgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggaacg attaatccta ggactggtgg tactaagtat 180aatgagaagt tcaggggcag agtcaccatt accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gcgcgagttt 300tttgactact ggggccaagg aaccacggtc accgtctcct ca 34263321DNAArtificialSynthetic 63gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgca gggcgagtaa gagcattagc aaatatttag cctggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgct ggatccaagt tgcactctgg agtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg caacttacta ttgtttccaa cataatgaat acccgtacac gttcggcgga 300gggaccaagg tggagatcaa a 32164342DNAArtificialSynthetic 64caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc agtgaaggtt 60tcctgcaagg catctggcta cgccttcact tcgttcttga tagagtgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggaacg agtaatccta ggactggtgg tactaagtat 180aaagagaagt tcaggggcag agtcaccatt accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac

acggccgtgt attactgtgc gcgcgagttt 300tttgactact ggggccaagg aaccacggtc accgtctcct ca 34265321DNAArtificialSynthetic 65gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgca gggcgagtaa gagcattagc aaatatacag cctggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gggtccaagt tgcactctgg agtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg caacttacta ttgtcaacaa cataatgaat acccgtacac gttcggcgga 300gggaccaagg tggagatcaa a 32166329PRTArtificialSynthetic 66Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly 32567329PRTArtificialSynthetic 67Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly 32568325PRTHomo sapiens 68Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly145 150 155 160Met Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn225 230 235 240Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu305 310 315 320Ser Leu Ser Pro Gly 32569326PRTArtificialSynthetic 69Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp145 150 155 160Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230 235 240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser Leu Gly 32570326PRTArtificialSynthetic 70Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp145 150 155 160Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230 235 240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser Leu Gly 32571978DNAArtificialSynthetic 71gcctccacca agggcccatc ggtcttcccg ctagcgccct gctccaggag cacctccgag 60agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 240tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc 300aaatatggtc ccccatgccc accctgccca gcacctgagg ccgccggggg accatcagtc 360ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 420tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 480ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 540cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 600tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 660gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 720aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 780tgggaaagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 840gacggctcct tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg 900aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 960ctctccctgt ctctgggt 97872107PRTHomo sapiens 72Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 10573321DNAHomo sapiens 73cggactgtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300agcttcaaca ggggagagtg c 3217423PRTHomo sapiens 74Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys 207515PRTHomo sapiens 75Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr1 5 10 157632PRTHomo sapiens 76Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr1 5 10 15Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 307710PRTHomo sapiens 77Phe Gly Gly Gly Thr Lys Val Glu Ile Lys1 5 107825PRTHomo sapiens 78Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser 20 257914PRTHomo sapiens 79Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly1 5 108032PRTHomo sapiens 80Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu1 5 10 15Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 308111PRTHomo sapiens 81Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser1 5 108232PRTHomo sapiens 82Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr1 5 10 15Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys 20 25 308325PRTHomo sapiens 83Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser 20 258432PRTHomo sapiens 84Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu1 5 10 15Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 308523PRTHomo sapiens 85Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys 208632PRTHomo sapiens 86Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr1 5 10 15Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys 20 25 308732PRTHomo sapiens 87Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu1 5 10 15Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 3088193PRTHomo sapiens 88Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu1 5 10

15Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 25 30Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg65 70 75 80Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu 85 90 95Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser 100 105 110Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly 115 120 125Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu 130 135 140Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile145 150 155 160Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190Arg8984PRTHomo sapiens 89Met Ala Leu Ser Ser Gln Ile Trp Ala Ala Cys Leu Leu Leu Leu Leu1 5 10 15Leu Leu Ala Ser Leu Thr Ser Gly Ser Val Phe Pro Gln Gln Thr Gly 20 25 30Gln Leu Ala Glu Leu Gln Pro Gln Asp Arg Ala Gly Ala Arg Ala Ser 35 40 45Trp Met Pro Met Phe Gln Arg Arg Arg Arg Arg Asp Thr His Phe Pro 50 55 60Ile Cys Ile Phe Cys Cys Gly Cys Cys His Arg Ser Lys Cys Gly Met65 70 75 80Cys Cys Lys Thr9061PRTHomo sapiens 90Gly Ser Val Phe Pro Gln Gln Thr Gly Gln Leu Ala Glu Leu Gln Pro1 5 10 15Gln Asp Arg Ala Gly Ala Arg Ala Ser Trp Met Pro Met Phe Gln Arg 20 25 30Arg Arg Arg Arg Asp Thr His Phe Pro Ile Cys Ile Phe Cys Cys Gly 35 40 45Cys Cys His Arg Ser Lys Cys Gly Met Cys Cys Lys Thr 50 55 609125PRTHOMO SAPIENS 91Asp Thr His Phe Pro Ile Cys Ile Phe Cys Cys Gly Cys Cys His Arg1 5 10 15Ser Lys Cys Gly Met Cys Cys Lys Thr 20 259225PRTMONKEY 92Asp Thr His Phe Pro Ile Cys Ile Phe Cys Cys Gly Cys Cys His Arg1 5 10 15Ser Lys Cys Gly Met Cys Cys Arg Thr 20 259325PRTRATTUS SP 93Asp Thr Asn Phe Pro Ile Cys Leu Phe Cys Cys Lys Cys Cys Lys Asn1 5 10 15Ser Ser Cys Gly Leu Cys Cys Ile Thr 20 259425PRTMUS SP 94Asp Thr Asn Phe Pro Ile Cys Ile Phe Cys Cys Lys Cys Cys Asn Asn1 5 10 15Ser Gln Cys Gly Ile Cys Cys Lys Thr 20 25957PRTHomo sapiens 95Arg Ser Arg Phe Ile Gln Gly1 59624PRTArtificialSynthetic 96Gly Gly Ser Pro Phe Glu Asp Ile Arg Ser Arg Phe Ile Gln Gly Glu1 5 10 15Ser Ile Thr Pro Thr Lys Gly Cys 209716PRTArtificialSynthetic 97Gly Gly Ser Pro Phe Glu Asp Ile Arg Ser Arg Phe Ile Gln Gly Cys1 5 10 159821PRTArtificialSynthetic 98Gly Gly Ile Gln Gly Glu Ser Ile Thr Pro Thr Lys Ile Pro Glu Ile1 5 10 15Thr Thr Glu Gly Cys 209919PRTArtificialSynthetic 99Gly Gly Met Pro Gly Ser Pro Leu Asp Leu Ser Val Ser Pro Phe Glu1 5 10 15Asp Gly Cys10019PRTArtificialSynthetic 100Gly Gly Ser Pro Leu Asp Leu Ser Val Ser Pro Phe Glu Asp Ile Arg1 5 10 15Ser Gly Cys10118PRTArtificialSynthetic 101Gly Gly Glu Asp Ile Arg Ser Arg Phe Ile Gln Gly Glu Ser Ile Thr1 5 10 15Gly Cys10218PRTArtificialSynthetic 102Gly Gly Arg Ser Arg Phe Ile Gln Gly Glu Ser Ile Thr Pro Thr Lys1 5 10 15Gly Cys10310PRTArtificialSynthetic 103Gly Arg Ala Phe Thr Ser Phe Leu Ile Glu1 5 1010410PRTArtificialSynthetic 104Gly Lys Ala Phe Thr Ser Phe Leu Ile Glu1 5 1010510PRTArtificialSynthetic 105Gly Tyr Arg Phe Thr Ser Phe Leu Ile Glu1 5 1010610PRTArtificialSynthetic 106Gly Tyr Ala Phe Arg Ser Phe Leu Ile Glu1 5 1010710PRTArtificialSynthetic 107Gly Xaa Xaa Phe Xaa Ser Phe Leu Ile Glu1 5 1010817PRTArtificialSynthetic 108Thr Ser Asn Pro Arg Thr Arg Gly Thr Lys Tyr Lys Glu Lys Phe Arg1 5 10 15Gly10917PRTArtificialSynthetic 109Thr Ser Asn Pro Arg Thr Gly Arg Thr Lys Tyr Lys Glu Lys Phe Arg1 5 10 15Gly11017PRTArtificialSynthetic 110Thr Ser Asn Pro Arg Thr Gly Gly Arg Lys Tyr Lys Glu Lys Phe Arg1 5 10 15Gly11117PRTArtificialSynthetic 111Thr Ser Asn Pro Arg Thr Gly Gly Thr Lys Tyr Lys Thr Lys Phe Arg1 5 10 15Gly11217PRTArtificialSynthetic 112Thr Ser Asn Pro Arg Thr Gly Gly Thr Lys Tyr Lys Ser Lys Phe Arg1 5 10 15Gly11317PRTArtificialSynthetic 113Thr Ser Asn Pro Arg Thr Gly Gly Thr Lys Tyr Lys Trp Lys Phe Arg1 5 10 15Gly11417PRTArtificialSynthetic 114Thr Ser Asn Pro Arg Thr Gly Gly Thr Lys Tyr Lys Glu Val Phe Arg1 5 10 15Gly11517PRTArtificialSynthetic 115Thr Ser Asn Pro Arg Thr Gly Gly Thr Lys Tyr Lys Glu Lys Phe Arg1 5 10 15Arg11617PRTArtificialSynthetic 116Thr Ser Asn Pro Arg Thr Gly Arg Thr Lys Tyr Lys Ser Lys Phe Arg1 5 10 15Gly11717PRTArtificialSynthetic 117Thr Ser Asn Pro Arg Thr Gly Arg Thr Lys Tyr Lys Thr Lys Phe Arg1 5 10 15Gly11817PRTArtificialSynthetic 118Thr Ser Asn Pro Arg Thr Xaa Xaa Xaa Lys Tyr Lys Xaa Xaa Phe Arg1 5 10 15Xaa1195PRTArtificialSynthetic 119Glu Phe Phe Val Tyr1 51205PRTArtificialSynthetic 120Glu Phe Phe Xaa Tyr1 51217PRTArtificialSynthetic 121Ala Gly Ser Lys Arg His Ser1 51227PRTArtificialSynthetic 122Ala Gly Ser Lys Leu Arg Ser1 51237PRTArtificialSynthetic 123Ala Gly Ser Lys Leu Val Ser1 51247PRTArtificialSynthetic 124Ala Gly Ser Lys Leu Tyr Ser1 51257PRTArtificialSynthetic 125Ala Gly Ser Lys Leu His Trp1 51267PRTArtificialSynthetic 126Ala Gly Ser Lys Leu His Tyr1 51277PRTArtificialSynthetic 127Ala Gly Ser Lys Arg His Trp1 51287PRTArtificialSynthetic 128Ala Gly Ser Lys Arg Tyr Tyr1 51297PRTArtificialSynthetic 129Ala Gly Ser Lys Xaa Xaa Xaa1 5130114PRTArtificialSynthetic 130Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Phe 20 25 30Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Thr Ser Asn Pro Arg Thr Gly Arg Thr Lys Tyr Lys Ser Lys Phe 50 55 60Arg Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110Ser Ser131342DNAArtificialSynthetic 131caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc agtgaaggtt 60tcctgcaagg catctggcta cgccttcact tcgttcttga tagagtgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggaacg agtaatccta ggactgggag gacgaagtat 180aaatccaagt tcaggggcag agtcaccatt accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gcgcgagttt 300tttgactact ggggccaagg aaccacggtc accgtctcct ca 342132107PRTArtificialSynthetic 132Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Thr Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Gly Ser Lys Arg His Trp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105133321DNAArtificialSynthetic 133gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgca gggcgagtaa gagcattagc aaatatacag cctggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gggtccaagc ggcactgggg agtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg caacttacta ttgtcaacaa cataatgaat acccgtacac gttcggcgga 300gggaccaagg tggagatcaa a 321134114PRTArtificialSynthetic 134Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Phe 20 25 30Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Thr Ser Asn Pro Arg Thr Gly Gly Arg Lys Tyr Lys Glu Lys Phe 50 55 60Arg Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110Ser Ser135342DNAArtificialSynthetic 135caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc agtgaaggtt 60tcctgcaagg catctggcta cgccttcact tcgttcttga tagagtgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggaacg agtaatccta ggactggtgg tcggaagtat 180aaagagaagt tcaggggcag agtcaccatt accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gcgcgagttt 300tttgactact ggggccaagg aaccacggtc accgtctcct ca 342136107PRTArtificialSynthetic 136Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Thr Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Gly Ser Lys Leu His Trp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105137321DNAArtificialSynthetic 137gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgca gggcgagtaa gagcattagc aaatatacag cctggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gggtccaagc tgcactgggg agtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg caacttacta ttgtcaacaa cataatgaat acccgtacac gttcggcgga 300gggaccaagg tggagatcaa a 321138114PRTArtificialSynthetic 138Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Phe 20 25 30Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Thr Ser Asn Pro Arg Thr Gly Gly Arg Lys Tyr Lys Glu Lys Phe 50 55 60Arg Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110Ser Ser139342DNAArtificialSynthetic 139caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc agtgaaggtt 60tcctgcaagg catctggcta cgccttcact tcgttcttga tagagtgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggaacg agtaatccta ggactggggg gaggaagtat 180aaagagaagt tcagggggag agtcaccatt accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gcgcgagttt 300tttgactact ggggccaagg aaccacggtc accgtctcct ca 342140107PRTArtificialSynthetic 140Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Thr Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Gly Ser Lys Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105141321DNAArtificialSynthetic 141gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgca gggcgagtaa gagcattagc aaatatacag cctggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gggtccaagt tgcggtctgg agtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg caacttacta ttgtcaacaa cataatgaat acccgtacac gttcggcgga 300gggaccaagg tggagatcaa a 321142114PRTArtificialSynthetic 142Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Ser Phe 20 25 30Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Thr Ser Asn Pro Arg Thr Gly Arg Thr Lys Tyr Lys Thr Lys Phe 50 55 60Arg Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110Ser Ser143342DNAArtificialSynthetic 143caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc agtgaaggtt 60tcctgcaagg catctggcta ccgcttcact tcgttcttga tagagtgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggaacg agtaatccta ggactggtag gacaaagtat 180aaaaccaagt tcaggggcag agtcaccatt accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gcgcgagttt 300tttgactact ggggccaagg aaccacggtc accgtctcct ca 342144107PRTArtificialSynthetic 144Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Thr Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Gly Ser Lys Arg Tyr Tyr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105145321DNAArtificialSynthetic 145gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgca gggcgagtaa gagcattagc aaatatacag cctggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gggtccaagc ggtactacgg agtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg caacttacta ttgtcaacaa cataatgaat acccgtacac gttcggcgga 300gggaccaagg tggagatcaa a 321146114PRTArtificialSynthetic 146Gln

Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Ser Phe 20 25 30Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Thr Ser Asn Pro Arg Thr Gly Gly Thr Lys Tyr Lys Glu Lys Phe 50 55 60Arg Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Phe Phe Val Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110Ser Ser147342DNAArtificialSynthetic 147caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc agtgaaggtt 60tcctgcaagg catctggcta ccgcttcact tcgttcttga tagagtgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggaacg agtaatccta ggactggtgg gacaaagtat 180aaagagaagt tcaggggcag agtcaccatt accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gcgcgagttt 300tttgtctact ggggccaagg aaccacggtc accgtctcct ca 342148107PRTArtificialSynthetic 148Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Thr Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Gly Ser Lys Arg Tyr Tyr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105149321DNAArtificialSynthetic 149gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgca gggcgagtaa gagcattagc aaatatacag cctggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gggtccaagc ggtactacgg agtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg caacttacta ttgtcaacaa cataatgaat acccgtacac gttcggcgga 300gggaccaagg tggagatcaa a 321150440PRTArtificialSynthetic 150Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Phe 20 25 30Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Thr Ser Asn Pro Arg Thr Gly Arg Thr Lys Tyr Lys Ser Lys Phe 50 55 60Arg Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys 115 120 125Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys 130 135 140Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu145 150 155 160Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 165 170 175Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 180 185 190Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val 195 200 205Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 210 215 220Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys225 230 235 240Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 245 250 255Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 260 265 270Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 275 280 285Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 290 295 300Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys305 310 315 320Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 325 330 335Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 340 345 350Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 355 360 365Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 370 375 380Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu385 390 395 400Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 405 410 415Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 420 425 430Lys Ser Leu Ser Leu Ser Leu Gly 435 440151214PRTArtificialSynthetic 151Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Thr Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Gly Ser Lys Arg His Trp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 210152440PRTArtificialSynthetic 152Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Phe 20 25 30Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Thr Ser Asn Pro Arg Thr Gly Gly Arg Lys Tyr Lys Glu Lys Phe 50 55 60Arg Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys 115 120 125Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys 130 135 140Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu145 150 155 160Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 165 170 175Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 180 185 190Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val 195 200 205Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 210 215 220Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys225 230 235 240Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 245 250 255Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 260 265 270Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 275 280 285Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 290 295 300Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys305 310 315 320Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 325 330 335Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 340 345 350Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 355 360 365Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 370 375 380Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu385 390 395 400Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 405 410 415Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 420 425 430Lys Ser Leu Ser Leu Ser Leu Gly 435 4401531320DNAArtificialSynthetic 153caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc agtgaaggtt 60tcctgcaagg catctggcta cgccttcact tcgttcttga tagagtgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggaacg agtaatccta ggactggtgg tcggaagtat 180aaagagaagt tcaggggcag agtcaccatt accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gcgcgagttt 300tttgactact ggggccaagg aaccacggtc accgtctcct cagcctccac caagggccca 360tcggtcttcc cgctagcgcc ctgctccagg agcacctccg agagcacagc cgccctgggc 420tgcctggtca aggactactt ccccgaaccg gtgacggtgt cgtggaactc aggcgccctg 480accagcggcg tgcacacctt cccggctgtc ctacagtcct caggactcta ctccctcagc 540agcgtggtga ccgtgccctc cagcagcttg ggcacgaaga cctacacctg caacgtagat 600cacaagccca gcaacaccaa ggtggacaag agagttgagt ccaaatatgg tcccccatgc 660ccaccctgcc cagcacctga ggccgccggg ggaccatcag tcttcctgtt ccccccaaaa 720cccaaggaca ctctcatgat ctcccggacc cctgaggtca cgtgcgtggt ggtggacgtg 780agccaggaag accccgaggt ccagttcaac tggtacgtgg atggcgtgga ggtgcataat 840gccaagacaa agccgcggga ggagcagttc aacagcacgt accgtgtggt cagcgtcctc 900accgtcctgc accaggactg gctgaacggc aaggagtaca agtgcaaggt ctccaacaaa 960ggcctcccgt cctccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagagcca 1020caggtgtaca ccctgccccc atcccaggag gagatgacca agaaccaggt cagcctgacc 1080tgcctggtca aaggcttcta ccccagcgac atcgccgtgg agtgggaaag caatgggcag 1140ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1200tacagcaggc taaccgtgga caagagcagg tggcaggagg ggaatgtctt ctcatgctcc 1260gtgatgcatg aggctctgca caaccactac acacagaaga gcctctccct gtctctgggt 1320154214PRTArtificialSynthetic 154Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Thr Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Gly Ser Lys Leu His Trp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 210155642DNAArtificialSynthetic 155gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgca gggcgagtaa gagcattagc aaatatacag cctggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gggtccaagc tgcactgggg agtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg caacttacta ttgtcaacaa cataatgaat acccgtacac gttcggcgga 300gggaccaagg tggagatcaa acggactgtg gctgcaccat ctgtcttcat cttcccgcca 360tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gc 642156440PRTArtificialSynthetic 156Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Phe 20 25 30Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Thr Ser Asn Pro Arg Thr Gly Gly Arg Lys Tyr Lys Glu Lys Phe 50 55 60Arg Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys 115 120 125Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys 130 135 140Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu145 150 155 160Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 165 170 175Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 180 185 190Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val 195 200 205Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 210 215 220Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys225 230 235 240Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 245 250 255Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 260 265 270Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 275 280 285Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 290 295 300Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys305 310 315 320Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 325 330 335Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 340 345 350Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 355 360 365Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 370 375 380Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu385 390 395 400Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 405 410 415Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 420 425 430Lys Ser Leu Ser Leu Ser Leu Gly 435 4401571320DNAArtificialSynthetic 157caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc agtgaaggtt

60tcctgcaagg catctggcta cgccttcact tcgttcttga tagagtgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggaacg agtaatccta ggactggggg gaggaagtat 180aaagagaagt tcagggggag agtcaccatt accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gcgcgagttt 300tttgactact ggggccaagg aaccacggtc accgtctcct cagcctccac caagggccca 360tcggtcttcc cgctagcgcc ctgctccagg agcacctccg agagcacagc cgccctgggc 420tgcctggtca aggactactt ccccgaaccg gtgacggtgt cgtggaactc aggcgccctg 480accagcggcg tgcacacctt cccggctgtc ctacagtcct caggactcta ctccctcagc 540agcgtggtga ccgtgccctc cagcagcttg ggcacgaaga cctacacctg caacgtagat 600cacaagccca gcaacaccaa ggtggacaag agagttgagt ccaaatatgg tcccccatgc 660ccaccctgcc cagcacctga ggccgccggg ggaccatcag tcttcctgtt ccccccaaaa 720cccaaggaca ctctcatgat ctcccggacc cctgaggtca cgtgcgtggt ggtggacgtg 780agccaggaag accccgaggt ccagttcaac tggtacgtgg atggcgtgga ggtgcataat 840gccaagacaa agccgcggga ggagcagttc aacagcacgt accgtgtggt cagcgtcctc 900accgtcctgc accaggactg gctgaacggc aaggagtaca agtgcaaggt ctccaacaaa 960ggcctcccgt cctccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagagcca 1020caggtgtaca ccctgccccc atcccaggag gagatgacca agaaccaggt cagcctgacc 1080tgcctggtca aaggcttcta ccccagcgac atcgccgtgg agtgggaaag caatgggcag 1140ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1200tacagcaggc taaccgtgga caagagcagg tggcaggagg ggaatgtctt ctcatgctcc 1260gtgatgcatg aggctctgca caaccactac acacagaaga gcctctccct gtctctgggt 1320158214PRTArtificialSynthetic 158Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Thr Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Gly Ser Lys Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 210159642DNAArtificialSynthetic 159gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgca gggcgagtaa gagcattagc aaatatacag cctggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gggtccaagt tgcggtctgg agtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg caacttacta ttgtcaacaa cataatgaat acccgtacac gttcggcgga 300gggaccaagg tggagatcaa acggactgtg gctgcaccat ctgtcttcat cttcccgcca 360tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gc 642160440PRTArtificialSynthetic 160Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Ser Phe 20 25 30Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Thr Ser Asn Pro Arg Thr Gly Gly Thr Lys Tyr Lys Glu Lys Phe 50 55 60Arg Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Phe Phe Val Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys 115 120 125Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys 130 135 140Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu145 150 155 160Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 165 170 175Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 180 185 190Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val 195 200 205Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 210 215 220Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys225 230 235 240Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 245 250 255Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 260 265 270Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 275 280 285Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 290 295 300Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys305 310 315 320Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 325 330 335Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 340 345 350Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 355 360 365Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 370 375 380Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu385 390 395 400Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 405 410 415Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 420 425 430Lys Ser Leu Ser Leu Ser Leu Gly 435 4401611320DNAArtificialSynthetic 161caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc agtgaaggtt 60tcctgcaagg catctggcta ccgcttcact tcgttcttga tagagtgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggaacg agtaatccta ggactggtgg gacaaagtat 180aaagagaagt tcaggggcag agtcaccatt accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gcgcgagttt 300tttgtctact ggggccaagg aaccacggtc accgtctcct cagcctccac caagggccca 360tcggtcttcc cgctagcgcc ctgctccagg agcacctccg agagcacagc cgccctgggc 420tgcctggtca aggactactt ccccgaaccg gtgacggtgt cgtggaactc aggcgccctg 480accagcggcg tgcacacctt cccggctgtc ctacagtcct caggactcta ctccctcagc 540agcgtggtga ccgtgccctc cagcagcttg ggcacgaaga cctacacctg caacgtagat 600cacaagccca gcaacaccaa ggtggacaag agagttgagt ccaaatatgg tcccccatgc 660ccaccctgcc cagcacctga ggccgccggg ggaccatcag tcttcctgtt ccccccaaaa 720cccaaggaca ctctcatgat ctcccggacc cctgaggtca cgtgcgtggt ggtggacgtg 780agccaggaag accccgaggt ccagttcaac tggtacgtgg atggcgtgga ggtgcataat 840gccaagacaa agccgcggga ggagcagttc aacagcacgt accgtgtggt cagcgtcctc 900accgtcctgc accaggactg gctgaacggc aaggagtaca agtgcaaggt ctccaacaaa 960ggcctcccgt cctccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagagcca 1020caggtgtaca ccctgccccc atcccaggag gagatgacca agaaccaggt cagcctgacc 1080tgcctggtca aaggcttcta ccccagcgac atcgccgtgg agtgggaaag caatgggcag 1140ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1200tacagcaggc taaccgtgga caagagcagg tggcaggagg ggaatgtctt ctcatgctcc 1260gtgatgcatg aggctctgca caaccactac acacagaaga gcctctccct gtctctgggt 1320162214PRTArtificialSynthetic 162Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Thr Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Gly Ser Lys Arg Tyr Tyr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 210163642DNAArtificialSynthetic 163gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgca gggcgagtaa gagcattagc aaatatacag cctggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gggtccaagc ggtactacgg agtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg caacttacta ttgtcaacaa cataatgaat acccgtacac gttcggcgga 300gggaccaagg tggagatcaa acggactgtg gctgcaccat ctgtcttcat cttcccgcca 360tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gc 642164440PRTArtificialSynthetic 164Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Ser Phe 20 25 30Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Thr Ser Asn Pro Arg Thr Gly Arg Thr Lys Tyr Lys Thr Lys Phe 50 55 60Arg Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys 115 120 125Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys 130 135 140Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu145 150 155 160Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 165 170 175Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 180 185 190Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val 195 200 205Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 210 215 220Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys225 230 235 240Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 245 250 255Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 260 265 270Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 275 280 285Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 290 295 300Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys305 310 315 320Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 325 330 335Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 340 345 350Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 355 360 365Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 370 375 380Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu385 390 395 400Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 405 410 415Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 420 425 430Lys Ser Leu Ser Leu Ser Leu Gly 435 4401651320DNAArtificialSynthetic 165caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc agtgaaggtt 60tcctgcaagg catctggcta ccgcttcact tcgttcttga tagagtgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggaacg agtaatccta ggactggtag gacaaagtat 180aaaaccaagt tcaggggcag agtcaccatt accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gcgcgagttt 300tttgactact ggggccaagg aaccacggtc accgtctcct cagcctccac caagggccca 360tcggtcttcc cgctagcgcc ctgctccagg agcacctccg agagcacagc cgccctgggc 420tgcctggtca aggactactt ccccgaaccg gtgacggtgt cgtggaactc aggcgccctg 480accagcggcg tgcacacctt cccggctgtc ctacagtcct caggactcta ctccctcagc 540agcgtggtga ccgtgccctc cagcagcttg ggcacgaaga cctacacctg caacgtagat 600cacaagccca gcaacaccaa ggtggacaag agagttgagt ccaaatatgg tcccccatgc 660ccaccctgcc cagcacctga ggccgccggg ggaccatcag tcttcctgtt ccccccaaaa 720cccaaggaca ctctcatgat ctcccggacc cctgaggtca cgtgcgtggt ggtggacgtg 780agccaggaag accccgaggt ccagttcaac tggtacgtgg atggcgtgga ggtgcataat 840gccaagacaa agccgcggga ggagcagttc aacagcacgt accgtgtggt cagcgtcctc 900accgtcctgc accaggactg gctgaacggc aaggagtaca agtgcaaggt ctccaacaaa 960ggcctcccgt cctccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagagcca 1020caggtgtaca ccctgccccc atcccaggag gagatgacca agaaccaggt cagcctgacc 1080tgcctggtca aaggcttcta ccccagcgac atcgccgtgg agtgggaaag caatgggcag 1140ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1200tacagcaggc taaccgtgga caagagcagg tggcaggagg ggaatgtctt ctcatgctcc 1260gtgatgcatg aggctctgca caaccactac acacagaaga gcctctccct gtctctgggt 1320166214PRTArtificialSynthetic 166Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Thr Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Gly Ser Lys Arg Tyr Tyr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 210167642DNAArtificialSynthetic 167gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgca gggcgagtaa gagcattagc aaatatacag cctggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gggtccaagc ggtactacgg agtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg caacttacta ttgtcaacaa cataatgaat acccgtacac gttcggcgga 300gggaccaagg tggagatcaa acggactgtg

gctgcaccat ctgtcttcat cttcccgcca 360tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gc 64216814PRTHomo sapiens 168Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile1 5 1016910PRTHomo sapiens 169Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys1 5 1017011PRTArtificialSynthetic 170Arg Ala Ser Lys Ser Ile Ser Lys Tyr Xaa Ala1 5 101717PRTArtificialSynthetic 171Ala Gly Ser Lys Xaa His Xaa1 51729PRTArtificialSynthetic 172Xaa Gln His Asn Glu Tyr Pro Tyr Thr1 517317PRTArtificialSynthetic 173Thr Xaa Asn Pro Arg Thr Gly Xaa Thr Lys Tyr Xaa Xaa Lys Phe Arg1 5 10 15Gly1747PRTArtificialSynthetic 174Ala Gly Ser Lys Xaa Xaa Xaa1 517510PRTArtificialSynthetic 175Gly Tyr Xaa Phe Thr Ser Phe Leu Ile Glu1 5 1017617PRTArtificialSynthetic 176Thr Ser Asn Pro Arg Thr Gly Xaa Xaa Lys Tyr Lys Xaa Lys Phe Arg1 5 10 15Gly1777PRTArtificialSynthetic 177Ala Gly Ser Lys Arg His Tyr1 5178114PRTArtificialSynthetic 178Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Phe 20 25 30Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Thr Ser Asn Pro Arg Thr Gly Gly Thr Lys Tyr Lys Ser Lys Phe 50 55 60Arg Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110Ser Ser179440PRTArtificialSynthetic 179Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Phe 20 25 30Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Thr Ser Asn Pro Arg Thr Gly Gly Thr Lys Tyr Lys Ser Lys Phe 50 55 60Arg Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys 115 120 125Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys 130 135 140Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu145 150 155 160Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 165 170 175Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 180 185 190Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val 195 200 205Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 210 215 220Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys225 230 235 240Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 245 250 255Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 260 265 270Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 275 280 285Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 290 295 300Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys305 310 315 320Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 325 330 335Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 340 345 350Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 355 360 365Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 370 375 380Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu385 390 395 400Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 405 410 415Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 420 425 430Lys Ser Leu Ser Leu Ser Leu Gly 435 440180107PRTArtificialSynthetic 180Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Thr Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Gly Ser Lys Arg His Tyr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105181214PRTArtificialSynthetic 181Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Thr Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Gly Ser Lys Arg His Tyr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 21018210PRTArtificialSynthetic 182Gly Tyr Xaa Phe Thr Ser Phe Leu Ile Glu1 5 1018311PRTHomo sapiens 183Ala Asn Ile Val Pro Glu Thr Ser Pro Glu Ser1 5 1018410PRTHomo sapiens 184Ile Val Pro Glu Thr Ser Pro Glu Ser Val1 5 1018510PRTHomo sapiens 185Asn Ile Val Pro Glu Thr Ser Pro Glu Ser1 5 101868PRTHomo sapiens 186Val Pro Glu Thr Ser Pro Ser Val1 518710PRTHomo sapiens 187Pro Glu Thr Ser Pro Glu Ser Val Pro Ile1 5 1018810PRTHomo sapiens 188Thr Ser Pro Glu Ser Val Pro Ile Ile Ser1 5 101899PRTHomo sapiens 189Ala Asn Ile Val Pro Glu Thr Ser Pro1 51909PRTHomo sapiens 190Ile Val Pro Glu Thr Ser Pro Glu Ser1 51918PRTHomo sapiens 191Ala Asn Ile Val Pro Glu Thr Ser1 51928PRTHomo sapiens 192Ile Val Pro Glu Thr Ser Pro Glu1 51937PRTHomo sapiens 193Ile Val Pro Glu Thr Ser Pro1 51945PRTHomo sapiens 194Ile Gln Gly Glu Ser1 519510PRTHomo sapiens 195Ile Thr Pro Thr Lys Ile Pro Glu Ile Thr1 5 1019610PRTHomo sapiens 196Ile Gln Gly Glu Ser Ile Thr Pro Thr Lys1 5 1019710PRTHomo sapiens 197Gln Gly Glu Ser Ile Thr Pro Thr Lys Ile1 5 1019810PRTHomo sapiens 198Gly Glu Ser Ile Thr Pro Thr Lys Ile Pro1 5 1019910PRTHomo sapiens 199Glu Ser Ile Thr Pro Thr Lys Ile Pro Glu1 5 1020010PRTHomo sapiens 200Ser Ile Thr Pro Thr Lys Ile Pro Glu Ile1 5 1020110PRTHomo sapiens 201Ile Thr Pro Thr Lys Ile Pro Glu Ile Thr1 5 1020213PRTHomo sapiens 202Ile Thr Thr Glu Ile Tyr Met Ser Asn Gly Ser Asn Ser1 5 1020312PRTHomo sapiens 203Thr Glu Ile Tyr Met Ser Asn Gly Ser Asn Ser Ala1 5 1020410PRTHomo sapiens 204Ile Thr Thr Glu Ile Tyr Met Ser Asn Gly1 5 1020510PRTHomo sapiens 205Thr Thr Glu Ile Tyr Met Ser Asn Gly Ser1 5 1020610PRTHomo sapiens 206Thr Glu Ile Tyr Met Ser Asn Gly Ser Asn1 5 1020710PRTHomo sapiens 207Glu Ile Tyr Met Ser Asn Gly Ser Asn Ser1 5 1020810PRTHomo sapiens 208Ile Tyr Met Ser Asn Gly Ser Asn Ser Ala1 5 1020910PRTHomo sapiens 209Arg Asp Gly Trp Val Ser Tyr Tyr Asn Gln1 5 102107PRTHomo sapiens 210Glu Ile Tyr Met Ser Asn Gly1 52118PRTHomo sapiens 211Ile Tyr Met Ser Asn Gly Ser Asn1 52125PRTHomo sapiens 212Ile Thr Pro Thr Lys1 521310PRTHomo sapiens 213Ser Ile Thr Pro Thr Lys Ile Pro Glu Ile1 5 1021410PRTHomo sapiens 214Glu Ile Tyr Met Ser Asn Gly Ser Asn Ser1 5 10

Claims

1. A monoclonal antibody comprising: a. a light chain and a heavy chain as shown in SEQ ID NO: 154 and SEQ ID NO: 152, respectively; b. a light chain and a heavy chain as shown in SEQ ID NO: 181 and SEQ ID NO: 179, respectively; or c. a light chain and a heavy chain as shown in SEQ ID NO: 158 and SEQ ID NO: 156, respectively.

2. The monoclonal antibody of claim 1 comprising two light chain polypeptides and two heavy chain polypeptides, and wherein each of the light chain polypeptides have the amino acid sequence as shown in SEQ ID NO: 154 and each of the heavy chain polypeptides have the amino acid sequence as shown in SEQ ID NO: 152.

3. The monoclonal antibody of claim 1 comprising two light chain polypeptides and two heavy chain polypeptides, and wherein each of the light chain polypeptides have the amino acid sequence as shown in SEQ ID NO: 181 and each of the heavy chain polypeptides have the amino acid sequence as shown in SEQ ID NO: 179.

4. The monoclonal antibody of claim 1 comprising two light chain polypeptides and two heavy chain polypeptides, and wherein each of the light chain polypeptides have the amino acid sequence as shown in SEQ ID NO: 158 and each of the heavy chain polypeptides have the amino acid sequence as shown in SEQ ID NO: 156.

5. A polynucleotide comprising a nucleotide sequence encoding a light chain polypeptide as shown in SEQ ID NO: 154 or a heavy chain polypeptide as shown in SEQ ID NO: 152.

6. A recombinant expression vector comprising the polynucleotide of claim 5.

7. A host cell which has been transformed by an expression vector of claim 6.

8. The host cell according to claim 7 wherein said cell is a Chinese hamster ovary (CHO), NS0 myeloma, COS, or SP2/0 cell.

9. A method of increasing serum iron levels, reticulocyte count, red blood cell count, hemoglobin, and/or hematocrit comprising administering to a subject an effective amount of a monoclonal antibody comprising: a. a light chain and a heavy chain as shown in SEQ ID NO: 154 and SEQ ID NO: 152, respectively; b. a light chain and a heavy chain as shown in SEQ ID NO: 181 and SEQ ID NO: 179, respectively; or c. a light chain and a heavy chain as shown in SEQ ID NO: 158 and SEQ ID NO: 156, respectively.

10. A method of treating anemia, anemia of cancer, or anemia of cancer associated with elevated levels of hepcidin in a subject, comprising administering to the subject an effective amount of a monoclonal antibody comprising: a. a light chain and a heavy chain as shown in SEQ ID NO: 154 and SEQ ID NO: 152, respectively; b. a light chain and a heavy chain as shown in SEQ ID NO: 181 and SEQ ID NO: 179, respectively; or c. a light chain and a heavy chain as shown in SEQ ID NO: 158 and SEQ ID NO: 156, respectively.

11. The method of claim 9, further comprising administering to said human patient an ESA, or other therapeutic agent or therapeutic treatment conventionally employed to increase serum iron levels, reticulocyte count, red blood cell count, hemoglobin, and/or hematocrit in a human.

12. The method of claim 10, further comprising administering to said human patient an ESA, or other therapeutic agent or therapeutic treatment conventionally employed to increase serum iron levels, reticulocyte count, red blood cell count, hemoglobin, and/or hematocrit in a human.

13. A pharmaceutical composition, comprising the monoclonal antibody of claim 1, and a pharmaceutically acceptable carrier, diluent, or excipient.

14. A process for producing a monoclonal antibody comprising the steps of: (i) culturing a host cell of claim 8 under conditions suitable to allow expression of the monoclonal antibody; and (ii) recovering the expressed monoclonal antibody.

15. The monoclonal antibody obtained by the process of claim 14.

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