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AU2013227987A1 - Anti-hepcidin antibodies and methods of use - Google Patents

Anti-hepcidin antibodies and methods of use Download PDF

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AU2013227987A1
AU2013227987A1 AU2013227987A AU2013227987A AU2013227987A1 AU 2013227987 A1 AU2013227987 A1 AU 2013227987A1 AU 2013227987 A AU2013227987 A AU 2013227987A AU 2013227987 A AU2013227987 A AU 2013227987A AU 2013227987 A1 AU2013227987 A1 AU 2013227987A1
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Australia
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antibody
seq
amino acid
hepcidin
nos
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AU2013227987A
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Keegan Cooke
Ian Foltz
Michael Gallo
Randal R. Ketchem
Christopher Mehlin
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Amgen Inc
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Amgen Inc
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Priority claimed from AU2009246946A external-priority patent/AU2009246946B2/en
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Priority to AU2013227987A priority Critical patent/AU2013227987A1/en
Publication of AU2013227987A1 publication Critical patent/AU2013227987A1/en
Abandoned legal-status Critical Current

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Abstract

The invention relates to monoclonal antibodies that bind hepcidin and methods of making and using such antibodies. Also provided are methods of treating hepcidin-related disorders.

Description

ANTI-HEPCIDIN ANTIBODIES AND METHODS OF USE CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application is a divisional application of Australian Application No. 2009246946, which is incorporated in its entirety herein by reference. [0001a] This application claims the benefit of U.S. Provisional Application No. 61/049,687, filed 01 May 2008, which is hereby incorporated by reference. FIELD OF THE INVENTION [0002] The invention relates to hepcidin, hepcidin antagonists (including antibodies that bind hepcidin) and their ability to modulate hepcidin activity. BACKGROUND OF THE INVENTION [0002a ] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field. [0003] Iron is an essential trace element required for growth and development of all living organisms. Iron content in mammals is regulated by controlling iron absorption, iron recycling, and release of iron from the cells in which it is stored. Iron is absorbed predominantly in the duodenum and upper jejunum by enterocytes. A feedback mechanism exists that enhances iron absorption in individuals who are iron deficient, and that reduces iron absorption in individuals with iron overload (Andrews, Ann. Rev. Genomics Hum. Genet., 1:75 (2000); Philpott, Hepatology, 35:993 (2002); Beutler et al., Drug-Metab. Dispos., 29:495 (2001)). Iron is recycled from degraded red cells by reticuloendothelial macrophages in bone marrow, hepatic Kupffer cells and spleen. Iron release is controlled by ferroportin, a major iron export protein located on the cell surface of enterocytes, macrophages and hepatocytes, the main cells capable of releasing iron into plasma. Hepcidin binds to ferroportin and decreases its functional activity by causing it to be internalized from the cell surface and degraded. (Nemeth et al., Science, 306:2090-3, 2004; De Domenico et al., Mol. Biol. Cell., 18:2569-2578, 2007). [0004] Hepcidin is an important regulator of iron homeostasis (Philpott, Hepatology, 35:993 (2002); Nicolas et al., Proc. Natl. Acad. Sci. USA, 99:4396 (2002)). High levels of human hepcidin result in reduced iron levels, and vice versa. Mutations in the hepcidin gene which result in lack of hepcidin activity are associated with juvenile hemochromatosis, a severe iron overload disease (Roetto et al., Nat. Genet., 33:21-22, 2003). Studies in mice have demonstrated a role of hepcidin in control of normal iron homeostasis (Nicolas et al., Nat. - 1 trene, 34:7-101. 2003; Nicolas et a, Proc, Ad, Acad. Sci USA, 99:4596-460 1 2002; Nicolas et at, Proc. Nait Acad, Sct Al, 98:870-8785, 2001. U005) In addition, data. is accrnulating imupicating hepcidin in iron sequestration during inflammation (See, e~g, Weinstein et at, Blood, 100:3776-3678 1 2002; Kemna et al, Blood, 106:1864-866, 2005; Nicolas et aJ Clin. Invest 1 10:1037~1044., 2002; Nemeth et at,. 1. Clin. Tnvest, 11311-1276, 2004; Neneth et at, Blood, 101:2461-2463, 2003 and Rivera et at, Blood, 105:1 797 1802, 2005), Hepcidin gene expression has been observed to be robusty upreglated after inflammatory stimuli, such as infetions, which induce the acute phase response of the innate immune systems of vertebrates. In mice, hepcidin gene expression was shown to be upreguated by lipopolysaccharide (LPS) turpentine, Freund's complete adjuvant, and adenoviral infections. Hepcdin expression is induced by the intammatory cytokine interleukin-6 (IL-6), A strong correlation between hepcidin expression and anemia of inflammation was also found in patients with chromi inflammatory diseases, including bacterial, fuingai, and viral inftaions, 10006) Human hepcidin, a 25 amino aczd peptide with anti-microbial and iron regulating activty, was discovered independently by two groups investigatming novel anti microbial peptide. (Krause et at, FEBS Len, 48(V 147 (2000); Park et aL, J.Biol3 Chem. 276:7806 (2001)) It hs also been referred to as LEAPM (liverexpressed antimicrobial peptide) A hepcidin DNA encoding an 83 amino acid pre-popeptide in mice and an 84 amio acid pre-propeptide in rat and human were subsequently identified in a search for liver specific genes that were regulated by iron (Pigeon et a, j Bi/ Ch0ema, 276:7811 (20014)) 'The 2.4 residue N-terminal signal peptide is first cleaved to produce pro-hepeidin, which is then further processed to produce mature hepeidin, found in both blood and urine. In human uine, the predominant fbrm contains 25 amino acids, although shorter 22 and .20 amin acid peptides are also present. 100071 The mature peptide is notable for containing eigh cysteine residues linked as four disulfide bridges. The structure of hepcidin was studied by Hunter et al, J Biot Che,, 27737597-37603 (2002), byNMR using clenicl synthesized hepcidin with an identical PLC retention time to that of native hepeidn purified from urine, Hunter et al reported their determination that hepcidin. folded into a hairpin loop structure containing a vicinal disulide bond (C-C8, C2-C7, (3-C6, (24-CS) See also Lauth et at, . Riot Chea, 280:272-9282 (2005) However, as discovered and disclosed in copending U.S. Patent Application No. 12/022,515, incorporated by reference herein i ts entirety, the structure of hepoidin was determined to have a disuitide bond connectivity different than noted above, U.S. Patent Application Publication Nos. 2003/0187228, 2004/0096987, 2004/0096990, 2005/0148025, 2006/0019339, 2005/0037971 and 2007/0224186; .S, Pten Nos. 7,232892 and 7,294,690 and international Publication No, WO 02/98444 discuss hepcidin antibodies, SUMMARY OF THE MENTION 1000 9 1 Various embodiments of the invention provide antibodies, including monoclonal antibodies hat specifically bind human hepcidin, methods ofproducig such antibodies, methods of using uch antibodies fir detectog hepcidin, pharnaceutiea formulations including such antibodies, methods of preparing the [harmaceutical tormulanons, and methods of treating patients with the pharaceutical formulations, including combination therapy with erythropoiests stimulators as described below, Nucleic acids encoding such antibodies, vectors and recombmant host cells comprising such nuclei acids, and methods of producing such antibodies are also provided, 100 101 in some embodiments, an isolated anybody is provided that binds to human hepcidin of SEQ ID NO: 9 with an affinity Ko of less than about 10oM that exhibits at least one of the properties selected from the group consisting of (a) at least about a 50~oid higher KQ at a pHl of about 5.5 or about 6 compared to its Ko for said hepcidin at a piH of about 7A.; (b) at least about a 5-old faster clearance of said hepcidin compared to antibody 1 Sand (c) an off rate of about 6x 10 ' or higher at about pH 5.5 Or about pH 6. Av or in addition to one or more of the foregoing properties, the antibody exhibits at least one of the properties selected rom the group consisting of: (a) reduces the level of total human hepoidin in serum by at east about 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% in a C57M6 mouse about 2.4 hours after the administration to said mouse of (i) a I mg doses of said antibody and (ii) a pre-compiexed single dose of 3.7 pg of human hepcidin with a 1 tug dose of said antibody; (b) reduces the level of total human hepcidin in serum in a mouse by at least about 20%, 30%, 40%, 50% 60%, 70%, 80% or 90% about 24 hours after said mouse is adminstered a single dose of 3.7 pg of human hepcidin, wherein said hepcidin is administered three days after said mouse is predosed with said antibody; (c) results in a greater than about 50% reduction in overall accumulation of total serum hepcidin in mice treated with said antibody compared to antibody PSI; and (d) results in at least about a 2-fold higher intracellular accumulation of heptidin in FeRn transfected HEK293 cells incubated with said antibody compared to antibody 1 SL !null! in some embodiments, an isolated antibody is provided that binds to human hepcidin of SEQ ID NO: 9 with an affinity K of less than about 10'M, wherein said antibody increases circulating iron level or Tsar in a mouse overexpressing human hepcidin for at least I day, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 3, at least 9. at least 10, at least 11 days or more after a single dose of antibody, |00 12 In some embodiments an isolated antibody is provided that binds to human hepcidin of SEQ ID NO; 9, with an affinity K 0 of at least i 0M wherein said antibody is obtained by: (a) replacing an amino acid in the heavy or light chain of said antibody with a histidine; (b) screening the antibody obtained in (a) for differential pH binding; (c) reacng another amino acid in the heavy or light chain of said antibody with a histidme; and (d) screenng said antibody fOr having at least one of the poperties selected from the group consistig of: (i) at least about 50-1000 fold higher K1 at about pH 5" or about p-I 6 compared to its K 0 for said hepcidin at about pH- 7.4; and (ii) an off rate of about 6x 100 s or higher at about pH 5.5 or about pH 6, 0013 In some enbodiments, an antibody described herein deceases iron in ferroportin expressing cells stimulated with 50 ng/mL hepcidin at an EC50 of about 20 nM or less: and/or increases the level in a subject of one of at least hemoglobin or hematocrit. or both; and/or increase in a subject one of at least the red blood cell count., the red blood cell hemoglobin content or the red blood cell mean cell volume of red blood cell count, or any combinations thereof; and/or increases in a subject one of at least the reticulocyte count, the resculoce henoglobinn content or the reticulocyte mean cell volume of reticulocyte count, or any combmations thereof; and/or inhibits the iron-regulatig activity of hepcidin. 100141 in some embodiments, the antibody comprises an amino acid sequence at least 90% identical to SEQ ID NO: 170 or to SEQ ID NO: l68, said polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID Ntis: 171- 1 76, and any sequences comprising at least one amino change to any of SEQ ID NOs: 171-176, in one aspect, the antibody comnprises SEQ i) NOs: 71 173. in another aspect, the antibody comprises SEQ lD N(is: 1 74-176, [00 15) In some embodiments, an antibody described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO: 333 or to SEQ D NO: 331, said polypeptide comparing at least one ammo acid sequence selected from the group consisting of SEQ iD NOs 334-349, and any sequences comprising at least one amino acid change to any of SEQ D N~s:334-349. In one aspect, antibody described herein conpriss SEQ IHDN4s: 334 346. i another aspect, a antibody described herein comprises SEQ 1D NOs: 347-349, '-4jotI6j In some embodiments, an antibody described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO: 343 or to SEQ ID NO: 34I , said polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 344-349, and any sequences comprising at least one amno acid change to any of SEQ 1D NOs: 344~49, In one aspect, an antibody described herein comprises SEQ ID NOs: 344 346. in another aspect, an antibody described herein comprises SEQ ID NOs: 347-349. 100171 In some embodiment, an antibody described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO: 353or to SEQ iD NO: 351, said polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ 1 IOs: 354159 and any sequences comprising at least one amino acid change to any of SEQ ID Os: 354-359. In one aspect, an antibody described herein comprises SEQ ID NOs: 334 356. In another aspect, an antibody described herein comprises SEQ lD iOs: 3574359, 10018 in some embodiments, an antibody described heroin comprises an amino acid sequence at least 90% identical to SEQ ID NO: 363 or to SEQ 1) NO; 361, said polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ tD NOs: 364-369, and any sequences comprising at least one amino acid change to any of SEQ ID NOs: 364.369, in one aspect, an anybody described herein comprises SEQ I) NOs- 364 366. in another aspect, an antibody described herein comprises SEQ ID NOs: 367-369, |0(191 in some embodiments. an antibody described herein comprises an amino acid sequence at least 90% identical to SEQ ID NO: 373 or to SEQ ILD NO: 37, said pol peptide comprising at least one amino acid sequence selected from the group consistI of SEQ HD NiOs; 374-379, and any sequences composing at least one amino acid change to any ofLSEQ ID BOs: 374-379. in one aspect, an antibody described here comprises SEQ ID NOs: 374 376 in another aspect, an antibody described herein comprises SEQ ID NOs: 377-379, 100201 in some embodiments, an antibody described herein comprises n amino acid sequence at least 90% identical to SEQ ID NO: 383 or to SEQ ID NO: 381, said poypeptide comprising at least one amino acid sequence selected &om the group consisting of SEQ ID NOs: 384-389., and any sequences comprsMg at least one amino acid change to any of SEQ ID NOs: 384-389, in one aspect, an antibody described herein comprises SEQ ID NOs: 384 86. In another aspect, an antibody described herein conpises comprising S EQ D NOs: 38 895 j002 1 in some embodiments, an antibody described herein comprises an amino acid sequence at least 90% .dentical to SEQ I) NO: 393 or to SEQ ID NO: 391, said polypeptide comprising at least one amio acid sequence selected frn the group consisting of SEQ ID TVs: 394-399, and any sequences comprising at least one amino acid change to any of SEQ ID Nts: 3941 99, In one aspect, an antibody described herein comprises SEQ ID NOs: 394 396 in another aspect, an antibody described herein eomprises compring SEQ ID NOs: 397199. [00 221 In somn embodimients, an antibody described here comprises an ammo acid sequence of SEQ )D NO: 170 wherein at least one, two, three or all four of the amino acids at positions 52, 57, 99 and 107 of said amino acid sequence are replaced with a histidine, Such an antibody may fhrter comprise S EQ ID NO: 168. In other embodiments, the antibody comprises an amino acid sequence of SEQ ID N O: 168 wherein at east one or both of the amina acids at positions 27 and 89 of said amino acid sequence are replaced with a histidme, Such an antibody may further comprise SEQ ID NO: )70: Optionally, any of the foregoing modified SEQ ID NO: 1.70 and any of the frging m1D NO: 68a be combined in an antibody, in one embodiment, the amino acxds at positions 57 and 107 of SEQ 1D NO: 170 are both replaced with a histidine. In another embodiment, the amino acid at position 107 of SEQ iD NO: 170 and the amino acid at position 27 of SEQ ID NO: 168 are both replaced with a histidine, in another embodiment, the amino acid at position 107 of S EQ ID NO: 170 and the amno acid at position 89 of SEQ ID NO: 168 ae both replaced with a histidine. in yet another embodiment. the amino acids at positions 99 and 107 of SEQ ID NO: 370 are both replaced with a histidine. |00231 Any of the foregoing antibodies may be a monoeional antibody, or a chimeric, humanized, or human antibody. In some embodiments, the antibody is an IgO isotype. such as an igGG, g32. 1gG3 or 1gG4 isotype. 100241 in another aspect, embodiments of the invention include an isolated nucleic acid molecule comprising a nucleotide sequence that encodes any of the foregoing antibodies, an expression vector comprising any of the isolated nucleic acid molecules, operably linked to areuatory control sequence, host cells comprising sUc isolated eacid e e vectors, and methods of using such host cells to produce an antibody, Such production methods compose culturing the host cell under suitable conditions such that the nucleic acid is expressed to produce the antibody, and optionally recovering the antibody from the host cell or culture medium, in a related embodiment, an isolated atibody or agent produced by the afOrementioned method is provided 100251 Ermbodinments described herein include a composition that contains any of the foregoing antibodies, e'g in a therapeutically effective amour, and a pharmaceuticaly acceptable carrier, diuent or excipient. In a related aspect, embodiments of the invention 6include a method of treating a disorder of iron homeostasis in a subject in need thereof by administering any of the foregoing antibodies or compositions, e g in a therapticaily effective amount, Exemplary disorders of iron homeostasis include anemia, sepsis, anemia of inflammation, anemia of cancer, chemotherapy induced anemia, chronic infl.anmatory anemia, congestive heart flu re, end stage renal disorder, chronic kidney disease (stage I, 1I, HIL IV or ), iron dency anu, a disorder of iron homeostasis, ferroportin disease, hemochromatosis, diabetes, inflammation. rheumatoid arthritis, arteriosc erosis, tumors, vascultis, systemic, heroglobnopathies, and red blood cell disorders In related aspects, embodtments of the invention provide mehods of treating a human with an elevated level of hepeidin, or methods of treating a human with anemia, by administering any of the foroegoingati s ionseg.in a teauic five am Ahus provided are uses of any of the foregoing antibodies in preparation of a medicament for treating any of the foregoing subjects or conditions. 00 261 It is understood that co-administration methods involving administration of antibodies with a second therapeutic agent, as described herein, encompass not only the use of the antibody in preparation ot a rnedicamnent for co-administration with the second therapeutic agent, but also the use of the second therapeutic agent in preparation. of a medicament for co adrmmnistrati on with the antibody, I I27 in some embodimemts, the mnanunal is a human suf Tering from a condition selected fom the group consisting of A frican iron overload, alpha thalassemia, Azheimer's disease. anemia, anemia of cancer, anemia ofchronic disease, anemia of inflammation, arteriosclerosis or atherosclerosis (including coronary artery disease, cerebrovasculwa disease or peripheral occlusive arterial disease), ataxias, ataxias related to iron, arrnsfernemia, cancer, ceruloplasmin deficiency, chemotherapy-induced anemia. chronic renal/kidney disease (stage LI1L Ill, IV or V), including end stage renal disease or chronic renal/kidney failure, cirrhosis of liver, classic hemochromatosis, collagen-induced arthritis (CIA), conditions with hepcidin excess (elevated hepcidin), congenital dyserythropoietic anemia, congestive heart failure, Crohn's disease, diabetes, disorders of iron biodistribution, disorders of iron homeostasis, disorders of iron metabolism, ferroportin disease, ferroportin mutation hemochromatosis, folate deficiency, Friedrich's ataxia, funicular myelosis, gracile syndrome. H pye/ori ifection or other bacterial infections, Hallervordan Spatz disease, hemnochromnatosis, hemochromatosis resulting from mutations in transferrin receptor 2, hemoglobi nopathies, hepatitis, hepatitis (Brockt h epatitis C, hepatocellul ar carcinoma, hereditary hemochromtosis. HIV or other viral illnesses, Huntintrn's disease, hyperterritinemia, hyohoi mi~crocy tic anemia, hypofbenma, insulin resistance, iron defleiency anemia, iron deficiency disorders, iron overload disorders, iron-deficiency conditions with hepcidon excess, juvenile hemnochronmatosis (HF E2), multi ple sclerosis, mutation in transferrin receptor 2, NFE, hemojuvelin, fZrroportin or other genes of iron metaosmn, neonatal hemochromatosis, neurodegeerative diseases related to iron, osteopenia, osteoporosis pancreatitis. Pantothenate kinase-associatsed neurodeeneaton, Parkinson's disease, pellagra, pica, porphyria, porphyria cutanea tarda, pseudoencephaliti s, pahronary hem~osiderosis, red blood cell disorders, rheumatoid arthritis, sepsis, siderohhanti anenia, systeni c lupus erythematosus, thalassemia, tha aseria interrmedia, trans fusional iron overkuad, rumors, vasculitis, vitamin B6 deficiency, vitamin t312 deficiency~ and/or Wilson's disease. 1002 S in some embodments, methods of treating anemia are provided, in which a human administered any of the foregoing antibodies or compositions and an erytropoesis stimulator. Exemplary erythropoiesis stimulators include erythropoietin, erythropoietin variants and peptdes or antibodies that bind and activate erythropoietin receptor. Other exemplary ervthropoiesis stinulators include human erythropoietin of SEQ IfD NO; 72 or darbepoetin alifai of S EQ D NO; 71 Exemplary forms of aneoia that may be treated accordmig to such methods include anemia of in flanmmation, anemia of cancer; chemotherapy induced anemia, iron deficiency anemia, a disorder of on n homeostasis, ierroportin disease, or anemia resulting from kidney disease Also provided are methods of treating a mammal with anemia that is hypo-responsive, or even to therapy with an erythropoiesis stimulator comprising st aically t mount of an antibody that specifically qnnm:'admiitrna hea u al binds human hepoidin. Any of the foreging methods may also include administering iron to thet subject. oP 291 The foregoing summary is not intended to define every aspect of the invention and aitiona aspects are described in other sections. such as the Detailed Description, The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of feature esscribed herein may be contemplated, even if the combination of features are not found together in the same sentence, or paragraph, or section of this doceumrent, [00301 In addition to the foregoing, the ivenion can inclade, as an additional aspect., all embodiments of the invention narrower in scope in any way than the variations defined by specific paragraphs herein, For example, certain aspects of the invenion that are described as a genus, and it should be understood that every member of a genus is, individually, an aspect of the mventon Also, aspects described as a genus or selecting a member of a genus, should be understood to embrace combinations of two or more members of the genus. [0031 it should he understood that while various embodiments in the specification are presented using "comprising" language, under various circumstances, a related embodiment may also be described using "consisting o" or "consisting essentially o language. t is to be noted that the term "a" or "an". refers to one or more, for example, "an immunoglobulin molecule) is understood to represent one or more immmnogiobulin molecules. As such, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein, [0032) should also be understood that when describing r , the characteristic being described could be an individual value found withi the range, For example, "a pH from about pH 4 to about phi 6," could be, but is not lined to, p4 42, 4 5.1 5.5 etc and any value in between such values. Additionally, a pH rom about pH 4 to about pH 6 "should not be construed to mean that the pH of a formulation in question varies 2 pH units in the range from pH 4 to pH 6 during storage, but rather a value may be picked in that rane for the pH of the solution, and the ph remains bufiered at about that pH, in some embod iments. when the tern "abou" is used, it means the recited number plhs or minus 5%, 10%, 15% or more of that recited number, The actual variation intended is determinable from the context Although the applicants) invented the full scope of the invention described herein, the applicants do not mitend to claim subject matter descried in the prior art work of others. Therefore, in the event that statutory prior art withm the scope ot a claim is brought to the attention of the applicants by a Patent Office or other entity or individual, the applicants) reserve the right to exercise amendment rights under applicable patent laws to redefine the subject matter of such a claim to specifically exclude such statutory prior art or obvious v s of statutory prior art&fr the scope of such a claim. Variations of the invention defined by such amended claims also are intended as aspects of the invention, 13RI WF DECSCIPTON OFH EM I R U R ES 0033 Figur I shows the off-rate' for antibodes I, 1Ss3 2.7,01 P23FII and 0034 Figure 2 hows rnorine anthepcidin atibody 24, functional ablity to drive down inrmrliratons in a beta4actamase iron-response aay. 19- 35 Fu3hwhability of human anti-hepcidin antibodies 18811- 23F1 I and 24E4o dnve down intraceShuar iron concentrations in a beta dactamnase ro-response assay 10036 igure 4 demonstrates tat an anhepcidinanibody eutrzhashman hepeidi n injected into mice. 00371 Figures 5AE demonstrate that antibody neutralization of human bepcidin vi rally expressed mice restores normal early red cell characteristics> 0038) Figures SA-B demonstrate that treatment with antibody 13211 restored norma& early red cell characteristics, [01039) Figures 7A - demonstrate that treatment with antibody 18811 leads to significant reduction in total hepcidin levels. 10040 Figure 8 shows a titration of adenovirsassociated virus (AAV)-mediated hepcidin expression and resulting serum iron concentrtons, 004 1 Figure 9 shows that viral overexpression of hepcidin causes hypo responsi veness to erythropoietin. {)0421 Figures 10(IA-E demonstrate that an anti-hepidin antibody restores responsiveness to erythropoietin in. ie virally over-expressing hepcidin, 10043 Figures 1 A-C shows that neutralization of hepcidin by antihepeidin antibody treatment restores rcsponsiveness to erythropoietin in human hepcidin knock-in mice with anemia of intlarmmation, [00441 Figure 12 demonstrates that hepcidin levels are elevated in anemia of cancer patients (AoC) and not in normal patients, 1004 Figure 13 demonstrates that hepcidin levels correlate with diagnosis of inflammatory anemia and not iron deficiency anema 100461 Figure 14A shows a decision tree of iron indices and disease states for assessment of a patient, in the absence of hepcidin measurement [00471 Figure 148 shows a theoretical decision tree fbr assessment of a patient usna measurement of hepcin levels. 0048 Figure 15 shows prohepeidin concentration measured by a sandwich imm unassa y, demonstrating that prhepcidin is not detectable in serum. 100491 Figure 1 shows results of a Biacore experiment demonstrating that two monoclonal antibodies can bind to hepcidin at once. (005$0 Figure 17 demonstrates that a sandwich ELISA can be constructed with mnonocional antibodies raised against mature hepcid.
00 53 Figure 18 shows the concentration of hepCidin present in buffer, rabbit serum and pooled human serum as determined by a competitive binding assay. 1(0521 Figure 19 shows the measurement of hepidin in huna se. [003 Figure 20 shows the concentration of hepcidin present in normal human sera usina conpetitive bindingassay [0054 Figure 21 shows the serum antibody concentration of antibodies I51 and I8R1) after administration cf antibody-hpcidi complexes at various imepoints, |00$$ Figure 22 shows the serum hepcidin concentration affer administration of anthody'-antigen complexes at varioustimenpomts. 00 56) Figure 23 shows the total urine hepcidin concentration miuce pre-dosed with antibody IS S or 8 5,I I at various time points, (00 57) Figure 24 shows the serum hepcidin concentration after administration ot antibodies IBI1 and 1) at various timepoints, 100581 Figure 25 shows the serum bepoidin concentration in mice pre-dosed with antibody I $I and I 8BI 1at various timepoints. 00591 Figure 26 demonstrates that antibody 1 81I causes an accumulation of intracelluar hepecidin. DETAILED DESCRIPIION OF THE INVENTION 006;- Described herein se antibodies that exhibit one or mor properties that are associated wih enhanced target antigen clearance omn the circulation. Normally, antibodies are imanrnaid into cells and then recycled back into circulation via a pathway involving the receptor Fen (SEQ ID NO: 400). See, e.g, Prabhat et a, Proc Na 'V A cad, Set, 104(14358I594 (2007). Antibodies (either alone or complexed with antigen) are internalized into the acidified endosomes of the cells. Some of these antibodies in the acidiled endosomes then bind to FcRn, which then recycles the antibodies and any associated antigen back out of the cell Antibodies and/or antigen which did not bind to FQRn are transported to the lysosoimes where they are degraded. 10061 Antibodies are provided herein that exhibit differential pH binding to an antgen at a pH below about 74, as well as improved methods of treatment using such antibodies. For example, in some embodiments, such antibodies: bind to antigen with at least about 50-f11d to M000-fod or more reduced binding affinity at a pH of about 5,5 or about 6 compared to a pH of about 7.4 (as measured by a 50,fold to l 000- fod or higher relative K at 1 1 pH of about 5 5 or about 6 compared to at a pH of about 7.4). in some embodiments, the antibodies exhibit rapid ofrate for antigen of about 6 x f0' & or higher, or about I x J0' s' or higher. Such antibodies are expected to bind amigen in circulation but tend to release the antigen in acidified endosomes at a ph of about 5.5 or about 6. 'The greater release of antigen in acidified lysosomes is associated with greater degradation of the target antigen and enhanced clearance of antigen, Another property may be greater recycling of free antibodies (unbound to antigen) into circulation to hind to additional antigen. In contrast, antibodies that do not release their antigen are more frequently recycled nto circulation as an antibody antigen complex, resultng in the inabihty of the antibody to bind to and ultimately clear additional antigen from circulation. [0062) Also provided are antibodies that produce increased, e.g at least I .5fold or 2 fold, intracellular accumulation of target antigen and/or enhanced clearance of antigen from circulation and/or reduced accumulation of circulating antigen, as well as improved methods of treatment using such antibodies. Other properties of such antibodies may include prevention of build-up of antibody-antigen complexes in circulation, making more recycled &ee antibody available to bind antigen than conventional antibodies, better potency a reduced dose and/or &rquency of administration to achieve therapeutic effectiveness. 100631 Target antigens can include soluble antigens that have a relatiely high leve of production and/or a short halflife in circulation of about 24 hours or less. or about 18, 12, 8, 4. 3, 2. or 1 hour or less, or about 45, 30, or 15 minutes or less.Antibodies will generally bind to the target antigen with a KC in the range of1 X 104 M or l orto 104* M or lower, (eg. about 10ab i 0', about 100, about 10 about 10.", about 10"> about 10' about 10' about i0 about 10 about 101 or less), where lower K 0 indicates better affinity. 10064) Also provided are methods of screening tbr antibodies with desired properties comprising dNyg an antibody that exhibits differential ph binding to a antigen at a pH below about 7.4, and optionally denmonstrating that the antibody exhibits enhanced target armigen clearance relative to an antibody of similar or better binding affity that does not. exhibit difflerential pH binding, and/or optionally demonstrating that the antibody exhibits increased intracellular accumulation of target antigen and/or reduced accumulation of circulating antigen relative to an antibody of similar or better binding affimty that does not exhibit d ifferential pH' binding. 100651 in another aspect, methods of treatment are provided that involve administering therapeutically effective amounts of antibodies with the above-described properties, optionally S12 also invoking detecting circulating blood level of a tar antigen before or concurrent with said administration, and detectng circulating blood level of said target antigen after said administration, e~g. abom 24 hours, 2 days, 3, 4, 5, 6, 7 days or 2 weeks after said administration. 100661 Hepeidin is a good taget antigen for antibodies that exhibit the properties desired herein. Hepcidin has a relatively short half-ife (Rivera e0 al, Blood, 106:2196 2199, 2005). The human hepoidin gene encodes an 84 residue preprpeptide (SEQ ID N0: 8), The corresponding cDNA and genomi sequences are set torth in SEQ HD NOs: 7 and 100, respectively. The 24-esidue N-terminal signal peptide (residues 1-24 of SEQ ID NO: 8) is Brst cleaved to produce pro-hepcidin, which is then further processed by cleavage of the prodomain (residues 25-9 of SEQ 0D Nil 8) to produce the 25-residue mature hepeidin (residues 60T4 of SEQ NO: 8, set forth in SEQ ID NO: 9), in addition to the primary 2 amino acid form, further N-teminaliy truncated forms that are 20 or 22 amino acids in length can be identified in wine (20 amjno acids, SEQ fD NC: 96; and 22 amino acids, SF0 1D) NO 98) Mature human hepcidin contains eight cysteine residues, which are referred to herein sequentially as C! through CS (numbered from the Nterminus to the C-crminus) }0067] in some embodiments, the antibodies described herein bind to mature. correctly folded, bioactive human hepcidin in which disuliide bonds are formed between Ci -C8, C2 CA, C3C6 and C54C7, with the desired. afnity. in some embodiment the antibodies inhibit the non-regulating activity of hepcidin. in some embodiments the monoclonal antibody decreases intracellular iron concentration and/or increases circulating iron concentration at an ECs of about I0) Mv or less, or about 20nM or less. in some embodiments, the antibody exhibiits the property in mammals of increasing red blood cell count numberr) or hemoglobin or hemnatocrit levels, and/or normalizing rediculocyte count, reticulocyte mean cell volume and/or reticul ocyte hemoglobin content, increases circulating iron level or Tsat in a mouse overexpressing human hepcidi~n tbr at least 1, 2, 3, 4,8S, 6, 7, 8, 9, 10 or 11i days or longer after a single dose of the antibody. A NTl-HEPCtiN ANTIEBODIFS AND SPECIFIC BINDING AGENTS [0068|j The term "antibody" is used in the broadest sense and includes fully assembled antibodies, mronocl onal. antibodies, polyclonal antibodies mul'tispecifi c antibodies (including bispecifieantibodies), antibody fragments that can bind an) amigenr (ildmig, Fab' E'(abYs Pv, single e chain antibodies> diabodies), and recombnant peptides comprising the foregomng as long as they exhibit the desired biological activity, NMutimers or aggregates of' intact 13mc' v,,m molecules and/or fragments, including chem'icall derinvatized antibodies, are contemplated. Antibodies of any isotype class or subclass, including IgG, 1gM. lgD. IgA, anid igE, Ig I 1, 1g02, gG3, IgG 4 , igAl and IgA2, or any aliotype, are contemplated. Different isotypes have difrenrit effector functions; for example, Ig1 and gG3 isotypes nave antiody-dependent cel lul ar cytotoxi city (ADCC) activity. 10069) In some e o n, the antibodies described herein exhibit d.iffTrenia pH binding to an antigen The term "kdifrentiaI pH binding" as used herein refers to an antibody that binds to its antigen with high affinity (lower K) at a pH of about 7.4 but binds to the antigen with a lower affinity (higher K) at a lower pH. An antibody that exibits a K 0 that is at least 50, at least 60, at least 70, at least S0, at least 90, at least 100, at least 1, at least 200, at least 250, at least 300, at least 350 at least 400, at least 450, at least W0, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800. at least 850 at least 90, at least 950, at least I 000-fid or more higher for its antigen at apH more acidic than a pH of about 7A (e.g, a pH of about 7.0 about 6,5, about 6,0, about 5,5, about 5.0 or about 4.5) is specifically con temp lated, 100701 The ternm indiig affity" or
M
affnity" as used herein refers to the equirium dissociation constant (Ka) associated with each antigen-antibody interaction In some embodiments tWh antibodies descrbed herein exhibit desirable propenies such as binding affinity as measured by Ko for hcpcidin in the range ofl x 10' NI or less, or rangin down to 10 M or lower, (eimg about 10" 10 0 i 103 1 y0 v 101, la", 110, 10" M or less) at about pH 7.4, where lower K 0 indicates better affinity, Optionalythe antibody further exhibits a KAor hepcidin at least 50 1000 fbd higher (less binding affinity) at about pH 5.5 or about pH 6 compared to at a pH1 of about 7,4. The eqluilibrium~ dissociation constant can be determined in solution equilbrium assay using BIAcore and/or KinEx A, such as described. in Examples 3 and 4. 1(071 l} The binding affinity is directly related to the ratio of the kinetic off-rate (generally reported in units of inverse time, eg. seconds') divided by the kinetic on-rate (generally reported in units of concentration per unit tine, e.g, Ms). Offrate analysis can estmate the interaction that occurs in viv, since a slow off rate would predict a greater degree of interacflan over long period of time. In sonic embodiments, the antibodies desenbed herein exhibit an off-rate of about 6x 1 0OX' or higher, or about 1 x 10 44 or higher (faster o rate) at about pH 5.5 or about pH 6. Optionally, the antibody also exhibits an off rate 1f ixl0 4 or less (slower off-rate) at about pH 7A. In other embodiments, the antibodies described herein exhibit an offrate measuredd in. &^5 that is at least about 10-fold. -14- 2Q0 30, 400, 60, 70, 80t 0 or 00-Fold higher at about pH 5.5 or about pH 6Hompared t thre af-rare at about pH 74, 100 721 In other embodiments the antibodies described herein exhibit specificity for or specify Baly bind to human hepcidin, As used herein, an antibody is "specific for" or "specifcal binds human hepeidim when it has a significantly hiher birding affinity for;, and consequently is capable of distinguishig, human hepcidin compared to other unrelated proteins in different families. in some embodiments, such antibodies may also crossreact with hepcidin of other species, such as urine, rat, or primate hepcidin; while in other embodiments, the antiodies bind only to human or primate hepcidin and not significantly to rodent hepcidin In some emnodiments, antibodies hind to human and cynomologaus monkey hepcidin but not significantly to rodent hepcidin, in some embodimts, antibodies specific for hepeidin cross-react with other n the same fanly, while in other embodiments, the antibodies distinguish hepeidin from other related family members, including defensins or mouse hepc2 [00731 in some embodiments, the antibodies exhibit "enhanced target antigen clearance> meaning they produce a faster r greater reduction in circulating blood levels of total target antigen. For eaxampite, cnhanced antigen clearance compared to an antibody that does not exhibit differential pH binding can be measured by comparing blood levels of target anigen at a certain time point, .g. about 12, 24, 36, 48, or 72 hours after administration of antibody. Enhanced antigen clearance will result A greater reduction in blood level at the same time point, tA ively, for example, enhanced antigen clearance Can be measuredby comparing the time period required to reduce target antigen to, e.g., 25%, 50%, 75% or 90% of its blood level prior to administration of antibody. Enhanced antigen clearance will result in a shorter tine period to ahe such redction. As yet another aternative, enhanced antigen clearance is indicated by greater internalization of target antigens into ells expressing FcRn., as measured by intracellular accumulation of target antigen, 100741 in yet other embodiments, the monoclonal antibodies inhibit (or neutralze) hepcidin iron-egatning activity, in vitro and or in vivo. Such hepcidin-neutraizing antibodies are therapeutically useful for hepcidin-related disorders or disorders of iron oiepcidin neutralaing activity neasurd through a number of markers, or example, ferritin/iron levels, red blood cell count, red blod cell characteristic hemoglobinn content and/or cell volume), early red blood cell characteristics (reticulocyte nurnbers, hemoglobin contend or cell volume) (Clinical Hematology, third edition, Lippincott, Williams and Wilkins; editor Mary L Turgeon, 1999) ferroportin internaliation, or iron - 15transport In one embodiment, the monoclonal antibody decreases intracellular iron concentration at an EC, of about 100 N or less and/or increases circulating iron carnccratamn 10075J in some embodimnnts, a monoclonal antibOdy as described herein antagonizes the effect of human hepcidin or inhibits hepeidin iron-regulating activity, in some embodiments, a monoclonal antibody as described herein exerts an effect at an Cs of about Ix10 N or less, or about ixI0' M or less, For example, an antibody may decrease the intracellular iron level in a cell at an Eso of about [x 10 A M or less, or may reduce ii expression at an E0s0 of about fx104 M or less, as determined by a ferriti n assay. In other embodiments, a monoclonal antibody as described herein may reduce &ree seun hepcidin levels by at least about 20%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 60%, by at least about 70%, by at least about 80%, or by at least about 90%. In other embodiments, a monoclonal antibody as described herein may increase red blood cell count (number), red blood cell mean cell volume or red blood celi hemoglobin content, increase hemoglobin, increase bematocrit, increase Tsat, increase circulating (or serum) iron levels, and/or increase or nornalize reticulocte count. reticuloevte mean cell volume, reticulocyte hemoglobin content or reticulocyte numbers. 10076 1 In some embodiments, the invention contemplates: 1) a nonoceonial antibody that retains any one, two, three, four, five, or six of CDRH1I, CDRH2, CDRH3, CDR L, CDRW L or C.DR L3 of any of antibody Ab43, 27,2,41, RN 1C9, 1S, 1S2, 1S3, 1S4, 135, 3113; 4E, 7A3, 9102, 1259, 15E, 181311, 1808, 19B8, 19C1, 19D12, 1916, 20E12, 22F12, 22H 10, 23A1i, 23F11, 24E4 and 26F11, optionally including one or two mutations in such CDR(s), wherein the antibody exhibits differential pH binding; and/or rapid off rate (e.g, 6x V2 s or higher) ata pH. of about 5.5 or about 6, and/or enhanced hepcidin clearance; 2) a monoclonal antibody that retains all of CDRH 1, CDRH2, CDRH3, or the heavy chain variable region of any of antibody Ab43, 2,7, 2.A, R9, 109, 1, 12, S3, 1 S4, 135, 31B3; 4E1, 7A3, 9D12, l2B9, 15E1, 18 It1, 18D8, 1988, 1901, 19D12, 1916, 20E12, 22F12, 22110, 23A I, 23F1 1, 2454 and 26F 1, optionally including one or two mutations in such COR(s), wherein the antibody exhibits differential p4 binding, and/or rapid off rate (eg, 6x U 2 s or higher) at a pH of about 5.5 or ab out 6, and/or enhanced hepcidin clearance; 3) monoclonal antibody that retains all of CDRL , CR L2, CDREL, or the light chain variable region of any of antibody Ab43 2., 2.41, R9, 1C9 1S1, IS2, 1T, 1S4, 1 S5, 33; 4E1 73 9D12 12B9, 15E1, 18B1, I18D8, 198, 19C1, 19D12, 1916, 20E12, 22F12, 22410, 23A1 , 23F11, 24E4 and 26F11L optionally including one or two mutations in such CD(s), wherein the antibody 16exhibTs differential ph binding, and/or rapid off rate (eg, 6x 1{0 s0 or igher) at a pH of about 5.5 or about 6, and/or enhanced hepcidin clearance; 4) a monoclonal antibody that binds o the same epitope of mature human hepcidin as antibody Ab43, 2,7, 2,41,R9, 1C9, 1S1, S2,1S3, AS4 IS5,3B3;4EA3O9DI121B9, 15E1, 18B 11, 18138,19O8, I9C,19D 12, 1916, 20E12, 22F12, 22H 10, 23A1W, 2311, 24E4 and 26F11, eg. as determined through X ray crystallography, or a conformaonal epiope comprsmng an amino acid within amino acids -5 of SEQ l) NO: 9 and/or ar amino acid within a loop formed by amino acids 1 0-1 3 of SEQ LD NO: 9 andJor an amino acid within a loop formed by amino acids 14-22 of SEQ ID NO: 9, wherein the antibody exhibits deferential pH binding, and/or rapid off rate (e.g., 6xl0Q sa or higher) at a pH of about 5 5 or about 6, and/or enhanced hepidiin clearance; 5) a monoclonal antibody that competes with antibody Ab43,* 2.7, 2,41-9, .R9C 181, 1S2, i 3, 154, S 5, 3B3; 41, 7A3, 9D2 12B9,151, 18311, 188 , 198, 190, 19D] 2, 1916, 20E12, 22F12,221Hi0, 23A11, 23F11, 24E4 and 26F11 tbr binding to mature human hepeidin by more than about 75%, more than about 80%, or more than about 81%, 82%, 83%, 84%, 85%, 86%, 87%. 88%, 89%, 90%, 91%, 92%, 9% 94% or 95% eg, assessed by competitive ELISA or BMacore or by other methods known in the art), wherein the antibody exhibits differential pH bmding, and/or rapid off rate (e.g.g, x 10 or higher) at a ph of about 5.5 or about 6, and/or enhanced hepcidin clearance; 6) a monoclonal antibody that specifically binds to human hepeidin of SEQ ID NO: 9 with an affinity Ko (equilibr dissociation constant) for hepcidin in the range of I x 10' M or less, or ranging down to I0 M1 or lower, (e.g, about 10> ", 10^0 10' 10' 10Y io 10: 10 ( 10' M&N or less) as measured by BlAcore or KinExA and that exhibits at least one, two, three or more of the properties selected &om the group consisting of: i) differential pH bmdicg as shown by at least about 50 tol000 fd lower affinity (or higher Ko) at a pH of about 5,5 or about 6 compared to at about pH 7.4; i) at least about 5, 6, 7, 8, 9, or M0-fold faster clearance of said hepcidin compared to antibody 1; iii) a rapid off rate as measured by, e.g, an off rate of about 6x j04 s or higher at about ph 5,5 or about pH 6, or an off-rate of about i x 10' s' or higher at about pH 5.5 or about pH 6, or an off rate ofat least about 10bId, 20, 30, 40, 50, 60, 70, 80, 90 or 100-fOld higher at about ph 5.5 or about 6, compared to the off-rate at about pH 7,4; iv) reduces the level of total human hepcidin in serum by at least about 90% m I C57BLM6 mouse about 24 hours after the administration to said mouse of(i) a i g dose of said antibody and (ii) a pre-complexed single dose of 3.7 pg of human hepcidin with a I ng dose of said antibody; v) reduces th level of total human hep.idin in serum in a mouse by at least about 90% about 24 hours afer said mouse is administered a single dose of 3,7 tpg of - 17 hnan hcpci where said hepddin is administered three days after said mouse a pre dosed wit said antbody; vi) produce at least aboutI1fod or2>fold higher intracelfutar accundation of human hepeidin in FRn-transected H EK293 cells compared to antibody S1; vii) results in. a greater tAn about 50% reduction in overall accumulation of total serum hepcidin in mice treated with said antibody compared to antibody 1 Si e, , at about 24 hours; and/or viii) increases circulating iron level or Tsat in a mouse expressing hepcidn for at least about I day. at last 2, asi at least 4, at least 5, at least 6, at least 7 at leas 8, at least 9, at least 10, at least I I days or more after a single dose of the antibody, 10077j in some embodiments, an antibody described herein exhibits differential pH binding as shown by at least about 50- 1000 fold lower af5inity (higher Ko) at a pH of about 55 or about 6 compared to at about pH 7.4 and also exhibits (1) at least about 5, 6, 7 89, 9, or 10 fold faster clearance of said hepeidin compared to antibody 11; and/or (2) a rapid off rate of, eg, about 6 x 10 s'' or higher at about pH 5. S or about pH 6; and/or (3) reduces the level of total human hepcidin i serum by at least about 90% in a CS7BL/6 mouse about 24 hours after the administration to said mouse of (i) a I m g dose of said antibody and (ii) a pre complexed single dose of 3, 7 pg of human h nwith a I mg dose of said antibody; and/or (4) reduces the level of total human hepcidin in serum in a mouse by at least about 90% about 24 hours after said mouse is adminstered a single dose of 3.7 pg of human hepcidin, wherein said hepeidm is administered three days after said mouse is pr-dosed with said antibody; and/or (5) furher produces at least about LSfoid or 2-fold higher intracellular accumulation of human hepeidin in FcRn-transfetemd HEK293 cells compared to antibody 181; and/or (6) results in a greater than about 50% reduction in overall accumulation of total serum hepcidin in mice treated with said antibody compared to aib ; and/or (7) increases circulating aron level or Tsat in a mouse expressing hepcidin for at least about I day, at est 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least Il days or more after a single dose of the antibody. |00781 in some embodiments, an antibody described herein exhibits at least about 5. 6, 7. 8, 9, or 10-f6ld faster clearance of said hepcidin compared to antibody iSI and also (1) reduces the level of total human hepcidin in serun by at least about 90% in a C57fBL/6 mouse about 24 hours after the administration to said mouse of(i) a i mg dose of said antibody and (ii) a pre-complexed single dose of 37 pg of human hepcidin with a 1 mg dose of said antibody; and/or (2r humn in serum in a mouse by at least about 90% about 24 hours after said mouse is administered a smgle dose of 3.7 pg of human hepcidin wherein said hepeidin is administered three days after said mouse is predosd with 8~IS said antibody; and/or (3) produces at least about 1.5 ld or 2foid higher intracellular accumulation of human bepeidin in FeRn-transfected - EK293 cells compared to anibody S ; and/or (4) results in greater than about 50% reduction in overall accumulation of total serum hepcidin in mice treated with said antibody compared to antibody Si and/or (.) increases circulating iron level or Tsat in a mouse expressing hepeidin for at least about I day, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 days or more after a single dose oftthe antibody. 10079 1 n some embodiments, the anybody exhibits a rapid oft.rate, e,g., about 6x10' T or higher at about phi 5.5r about pH 6 and also (1) reduces the level of total human hepcidin in serwn by at least about 90% in a C57BL/6 mouse about 24 hours after the administration to said mouse of () a I mg dose of said antibody and (ii) a precomplexed single dose of 3,7 pg of human hepcidin with a I mg dose of said antibody; andor (2) reduces the level of total human hepcidin in serum in a mouse by at least about 90% about 24 hours after said mouse is administered a single dose of 3,7 pg of human hepcidin, wherein said hepcidin is administered three days after said mouse is pre-dosed with said antibody; and/or (3) produces at least about 1S-6d or 2-fold higher intracellular accumulation of human hepeidin in FcRn-transfected H1EK293 cells cmpared. to antibody 1 S ; and/or (4) results in a greater than about 50% reduction in overall accumulation of total serum hepcidin in nice treated with said antibody compared to antibody iSi; and/or (5) increases circulating ion level or Tsat in a mouse expressing hepcidin for at least about I day,a 2, least That least 3, at least 4, at least 5, at least 6 at least 7, at least 8, at least 9, at least 10, at least i days or more after a single dose of the antibody, 0080 In some embodiments, an antibody described herein reduces the level of total human hepcidin in serum in a mouse by at least about 90% about 24 hours after said mnouse is administered a single dose of 3.7 pg of human hepcidin, wherein said hepcidin is administered three days after said mouse is predosed, also ) produces at least about L5-fol~d or 2-fbid higher intracellular accumulation of human bepeidin in FeRn tr'ansfected EK293 cells compared to antibody IS I; and/or (2) results i~n a greater than about 50% reduAction in overall accumulation of total serum bepcidin in mice treated with said antibod y compared to antibody I S; anid/or (3) increases circulating iron level or Tsat in a mouse expressing hepcidin for at least about . day, at least 2, at least 3, at least 4. at least 5, at least 6 at least 7, at least 8, at least 9, at least 10, at least I I days or moore after a single dose of the antibody. 19 110si in some embodiments, an antibody described herein produces at least about l.5i-fold or 2-fold higher intraclilular accumulation of human hepeidin in Fokn-transfected HEK293 cells compared to antibody I .and also results in a greater than about 50% reduction in overall accumulation of total serum hepcidin in mice treated with said antibody compared to antibody IS ; and/or increases circulating iron level or Tsat in a mouse expressing bepeidin for at least about 1 day, at least 2, at least 3, at least 4, at least 5, at least 6 at least 7, at least 8, at least 9, at least I10, at least I days or more after a single dose of the antibody. 10082 In another aspect, methods are provided for modifyina antibodies that lack properties such as differential pH binding and/or enhanced target antigen clearance) to produce antibodies that exhibit such properties The antibody can be an anti-hepcidin antibody produced by such methods. I som embodiments, residues in the CDRs and/or residues that according to three-dimensional mnolng are predicted to be most afiTeted by introduction of an amino acid with a pKa in the range of pH of about 5.5 to about 7A are mutated by the introduction of such an amino acid, e.g, histidine. Histidine is an amino acid that is senstve to pH shifts Tom 74 to 6,0, as the imidazole side chain of histidine has a pKa just Over 6, which may vary slightly higher or lower depending on the environment of the amino acid, Upon a change in pH from about 7.4 to a lower ph of about 6.0 or 5.1 for example, the mutated antibody may undergo an allosteric conformational change that would disrupt antigen-antibody interaction, 100831 Candidate residues for iutation include residues that are directed contact sites with antigen or sites that contribute to the formation of charge-charge interactions along the antibody-antigen binding interface. Other candidate residues include residues withm conserved regions of the antibody, Yet other candidate residues include framework residues that are at least 10% surface exposed and within 4,5 A of a CDR residue. Additional candidate residues include those selected by visual inspection of a 3-dimensional structural model for amino acids in proximity to the CDRs or selected framework residues. Histidine or other desired amino acids can be mutated at at single or multiple positions within the amno acid sequence, For example, mutations which produce some differential pH binding effect as single mutations can be combined as double, triple or more muhiple mutations. Antibodies that have been mutated in such a manner are then screened for differential pH bindmg and then can be further screened fur other properties. 100841 in one aspect, at least one, two, three, four, five, six or more residues in the heavy chai variable region of said antibody are deleted and replaced with a histidme residue. -20- In another aspect, at least one, two, three, four, five, six or more residues in the light chain variable region of said antibody are deleted and replaced with a histidine residue, in some aspects, at least one residue from the light chain variable reion of said anibody and at least one residue from the heavy chan variable region of said antibody is replaced with a histidne residue, in one one embodiment, at least one residue in the heavy chain variable region at position selected from the group consisting of 52, 57, 99 and 107 of SEQ ID NO: 170 is replaced with a histdine residue, in another e at eat one reside in the hght chain variable region at a position selected from the group consisting of 27 and 89 of SEQ IDl NO: 168 is replaced with a histidine residue, In another embodiment, the amino acids at positions 57 and 107 of the heavy chain variable region of SEQ ID NO: 170 are replaced with a histidine residue. In another embodiment, the amino acids at position 1 07 of the heavy chain variable region of SEQ ID NO: 170 and position 27 of the hght chain variable region of SEQ iD NO: 168 are replaced with a hishdine. In another embodiment, the amino acid at poston 107 of the heavy cA aable region of SEQ ID NO: 170 and the amino acid at position 89 of the light chain variable reCgion of S EQ ID NO : 16 is replaced with a histidine. In another embodiment, the amino acid at positions 99 and 107 of the heavy chain variable region of S EQ ID NO: 170 are replaced with a histidine, [ 85 in one embodiment, the antibody comprises at least one, two, three, four, tve or all of the amino acid sequences selected from the group consisting of SEQ ID NOs: 6-21 (Ab 43), In another embodiment, the antibody comprises at least one, two, three, fbur, five or alu of the amino acid sequences selected from the group consisting of SEfQ I) N8Os: 2--3 (2; CDR s). Tn another embodiment, the antibody comprises at least one, two, three, hut Byek or all of the amino acid sequences selected from te group consistig of SEQ ID NOs: 40-45 (241 CD~s). In yet another embodiment, the antibody comprises at least one, two three, four. Sive or a) of the amino acid sequences selected from the group consisting of SEQ ID NOs:52-37 (R9 CDIs), in another embodiment, the antibody comprises at least one, two, three, four, five or all of the amino acid sequences selected from the group consisting of SEQ ID N Os: 1 I -- 1 16 (1C9 CDRs), In another embodiment, the antibody comprises at least one, two, three, four, five or all of the amino acid sequences selected from the group consisting of SEQ ID N40s: 121-1 26 (3133 C)1(s). In yet another embodiment, the antibody comprises at least one, two, three, four, five or all of the amino acid sequences selected from the group consisting of SEQ ID NOs: 13 1136 (4E31 CDRs). n another embodiment, the antibody compises at least one, two, three, four, five or al of the amino acid sequences selected from the group consisting of SEQ ID NOs: 141-446 (7A3 CDRs). In another embodiment, the antbioy comprises at least one, two, three, fur five or all of the amino acid sequences selected from the group consisting of SEQ UD NOs; 151-A 56 (9D12. CDRs, In another embodiment, the antibody comprises at least one, two, three, fur) five or all of the amino acid sequences selected from the group consisting of SEQ ID Nis: 161-166 21289 CDRs in yet another embodimnt , the antibody comprises at least one, two, three, four, five or all of the amino acid sequences selected from the group consisting of SEQ ID NOs: 171-176 (15E CDRs). In another embodiment, the antibody comprises at least one, two, three, four, five or all of the amino acid sequences selected from the group consisting of SQ ID NOs: 334339 (I 81811 CDRS). in another embodiment, the antibody comprises at least one, two, three, four, tve or all of the amino acid sequences selected frm the group consisting of SEQ ID)NOs: 314-319 (182)08 CDRs. in another embodiment, the antibody comprises at least one, two, three, four, five or all of the amino acid sequences selected from the group consistng of SEQ ID Ns: 344-349 (198 C)Rs). n another embodiment, the antibody comprises at least one two, three, four, five or all of the amino acid sequences selected from the group consisting of SEQ Lf) Ntis: 324-329 (19C CDRs). ain yet another embodiment, the antibody composes at least one, two, three, four, five or all of the amino acid sequences selected from the group consisting of SEQ I) NOs: 294-299 (19012 CDRs), In another embodiment, the antibody comprises at least one, two, three, four, live or all of the anmmo acid sequences selected from the group consisting of SEQ iD Nis: 304-309 (19H6 CDRs). in another embodiment, the antibody comprises at least one, two. three, four, five or all of the amino acid sequences selected from the group consisting of SEQ ) NIs: 354-359 (2QE12 CDRs). In other embodiment, the antibody comprises at least one, two, three, four, five or all of the amino acid sequences selected from the group consisting of S EQ 11D2 Ntis: 364-369 (22F12 CDR s), In another embodiment, the antibody conmpses at least one, two, three, fbur, five or all of the amino acid sequences selected from the group consisting of SEQ ID Nts: 374,379 (22H11 0 CDRs), in another embodiment, the antibody composes at least one, two, three, four, five or all of the amino acid sequences selected from the group consisting of SEQ 112 Ntis: 384-389 (23A ii CDRs) In yet another embodiment, the antibody comprises at least one, two, three, four, five or all of the amino acid sequences selected from the group consisting of S EQ ID Ns: 181-186 (23711 CDRs) In another embodiment, the antibody comprises at least one, two, three, fdur, five or all of the amino acid sequences selected from the group consisting of SEQ ID NTs: 394-399 (24E4 CDts), in another embodiment, the antibody comprises at least one, two, three, four, five or all of the amino acid sequences selected from the group consistng of SEQ N)Ns: 9196 (26K1 Ms). In another mbodimen the antibody comprnses at least one, two, three, four Ave or al of the amino acid sequences selected from the group consisting of SEQ ID NOs: 203~205 and 131-33 (181 CDRs). in another embodiment, the antibody comprises at least one two, three, tour, five or all of the amino acid sequences selected frot the group consisting of SEQ ID NOs: 214-216 and 144-146 (152 CORs) in yet another embodunent, the antibody comprises at least one. two, three, four, fiv or all of the ammo acid sequences selected hom the group consisting of SEQ lDNs: 225 22.7 and 164-166 (1S8 CDRs). in another embodiment, the antibody comprises at least one, two, three, four, five or all of the amino acid sequences selected from the group consisting of SEQ D) Nos. 236238 and 174-176 (1S4 CDRsI. In another embodiment, the antibody comprises at least one. two, three, tour, lye or all of the amino acid sequences selected fron the group consisting of SEQ ID NOs: 247249 and 184-186 (1 S5 CDRs), 100861 In some embodiments. the antibdy comprises all three light chain CDRs, all three heavy chain Clts, or all six CDs, in some. embodiments, two light chain CIDs from an antibody may be combined with a turd ight chain CDR from a different antibody. Atemativel y, a CDRL1 fom one antibody can be combined with a CDRL2 from a different antibody and a CDRL3 rom yet another antibody. particularly where the CDRs are highly homologous. Similarly, two heavy chain CDlts horn an antibody may he combined with a third heavy chain CDR front a different antibody; or a CDRHO from one antibody can be combined with a CDR H2 fro a different antibody and a CUTH3 from vet another antibody, particularly where the CDRs are highly homologous, Consensus CDRs may also be used. In one embodiment the anibody comprises one or more of the amino acid sequences set forth in SEQ ID NO: 74 (XASNLES), SEQ 1D NO: 75 (XQSNEE) and SEQ ID NO: 76 (QQXNEX), SEQ ID NO: 28 (RASESVDSYGNSFMH) SEQ ID NO: 77 (WINTXSGVPTYADDFXO), SEQ D NC): 7 (XXYYGX*A*Y), SEQ ID NO: 19 (TYGMS> SEQ ID NO: 284 (VIXYXXSNCYYADSVKC), SEQ HD NO: 285 (WXAXNGXXXXAXXXQX), SEQ ID NO; 286 (AQEGXAPDAFP1) SEQ ID NO, 27 (QAWYSSTNV.), SEQ ID NO: 288 (QA WDSSTAXX), SEQ JD NO: 289 (QSDYSSXXX) wherein X is any amino acid and * can be absent or any amino acid. 0088) In yet another embodiment, the antibody comprises the light and/or heavy chain variable region of an antibody, e.g. SEQ ID NO: 15 (Ab43 heavy chain variable region), and/or SEQ ID NO: 13 (Ab43 light chain variable region): SEQ ID NO: 27 (2.7 heavy chain variable region), and/or SEQ 1D NO: 25 (2,7 light chain variable region); SEQ VD NO: 39(2A1 heavy chain variable region), and/or SEQ ID NO: 37 (2.41 light chain variable 23 region); or SEQ ID NO: 51 (R9 heavy chain vana reion, and/or SEQ 1D NO: 49 (R9 light charm variable region) SEQ ID NO: I 10 (IC9 heavy chain vanable region) and/or SEQ ID NO: 108(1C9 light chain Varible region); or SEQ ID NO: 120 (333 heavy chain variable region) and/or SE0DN: 118 (383Uh hmvral ein;SQI NO: 130 (4E1 SEQ10N . 3 igt han variable wegionV) SFEQ 0 heavy chain vanable region) and/or SEQ ID NO: 128 (4E1 ,.h chain variable region); or SEQ ID NO: 140 (7A heavy chamn variable region) and/or SEQ ID N0:138 (7A3 light chain variable region); or SEQ I) NO:. 150 (9D12 heavy chain variable region) and/or SEQ 1D NO: 148 (912 light chain variable region); SEQ 1D NO: 160 (1239 heavy chain variable region), and/or SEQ MD NO: 158 (12B9 light chain vanable region); SEQ ID NO: 170 (1531 heavy chain vanable region) and/or SEQ ID NO: 168 (15 E1 light chain variable region); SEQ ID NO: 333 (1811 heavy chain variable region) and/or SEQ 1D NO 331 (18311 ligh chain variable region); SEQ ID NO: 313 (18D8 heavy chain vanable region) and/or SEQ 1D NO: 311 (18DS light chain variable region); SEQ ID NO: 343 (1938 heavv chain variable region) and/or SEQ iD NO: 341 (1988 light chain variable region); SEQ 1D NO: 323 (19C1 heavy chain variable region) and/or SEQ ID NO: 321 (1901 light chain variable region); SEQ ID NO: 293 (19D12 heavy chain variable region) and/or SEQ ID NO: 291 (19D12 light chain variable region); SEQ 1) NO: 303 (196 heavy chain variable region) and/or SEQ IL) NO: 301 (196 ligh cham variable reg 3 2. heavy chain variable region) and/or S EQ D NO: 351 (2012 lightchain variable region); SEQ ID N: 363 (22F12 heavy chain variable region) and/or SEQ D NO: 361 (22P2 light ohan variable region); SEQ 11D NO: 373 (22H10 hea vy chain variable region) and/or S EQ ID NO: 371 (2210 light chain Region SQ ID NO: 383 (23 A I j heavy chain variable region) aDNO 381 (23Al1 light chai va riabl region); SEQ ID NO: 180 (23F11 heavy chain variable region) and/or SE ID NO: 178 (23311 light chain variable region); 393 (244 heavy chain variable region) and/or SEQ D N\O: 391 (24E4 light chain variable region); SEQ ID NO: 90 (26F1 heavy chain variable region) andw/r SEQ ID NO: 188 (26F11 light chain variable region); or SEQ ID NO. 20 I1NI heavy chain variable region) and/or SEQ ID NO: 1 28 (11 light chain variable region),; SEQ ID NO: 213 (1S2 light chain variable region) and/or SEQ ID NO: 140 (1S2 heavy chain variable region); S EQ ID NO: 224 (1 S3 light chain variable region) andmor SEQ ID NO:. 160 (1 S3 heavy chain variable region); SEQ ID NO: 235 (1,4 light chain variable region) and/or SEQ ID NO: 170 (1I4 heavy chain variable region; or S EQ ID ND: 246 (I S5 light chain variable region) andor SEQ ID ND : 190 (15 heavy chain variable region).
1089 j in some embodiments, an antibody is provided that comprises a polypeptide having an amino acid sequence at least about 65% 0%,75%, 80%, 81%, 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% .97%, 98%, 99% or rore identical to an arnino acid sequence selected from the group consisting of SEQ I) NOs: 15 (Ab43 heavy chain variable region), 27 (27 heavy chain variable region), 39 (2.41 heavy chain variable region), 51 (R9 heavy chain vanable rego) 10() C9 heavy chain variable region), 120 (31B3 heavy chain variable region), 130 (41 heavy chain variable region), 140 (7A3 heavy chain variable regn) 150 (912 heavy chain variable region), 160(12B9 heavy chain variable region), 170 (15E1 heavy chain vanable region), 332 (18B2 1 heavy chain varable region), 313 (1818 heavy chain variable region), 343 (1918 heavy chain variable region , 323 (19C1 heavy chain variable region), 293 (19D12 heavy chain variable regin), 303 (1696 heavy chain variable region), 353 (20E12 heavy chain variable region), 363 (22F12 heavy chain variable regin), 373 (22 110 heavy chain variable region), 383 (23A 11 heavy chain variable regint 180 (23F I i heavy chain variable region), 393 (24E4 heavy cham variable region), 190 (26F11 heavy chain variable region), 202 (1S1 heavy chain vanable region), 13 (Ab4 i cnain variable region), 25 (2.7 light chain variable region), 37 (2,4 light chain variable region), 49 (R9 light cham variable region), 108 (1C9 light chain variable region 118 (3133 light chain variable region), 128 (4E1 light cham variable region), 138 (7A3 light chain variable region), 148 (912 light chain variable region), 158 (1219 light chain variable region), 168 (15121 light chain variable region, 331 (181311 light chain variable region), 311 (18D8 eight chain variable region), 341 (1918 light chain variable region ), 321 (19C1 light chain variable region), 291 (19121 eight chain variable region), 301 (19H6 jight chain variable regon) 351 (20E12 light chain variable region), 361 (22F12 light chain varable region), 371 (22 H10 light chain variable region ), 381 (23A 11 light chain variable region, 17 (2 3 F11 light chain variable region), 391 (24E4 light chain variable region), 188 (26F11 lit chainK variable region) 213 (1 S2 light chain variable region), 224 (133 light chain varible region), 235 (134 light chain variable region), 246 (155 light chain variable region), the polypeptide rther comprising at least one or more of the amino acid sequences set fort in SEQ I) N0s, 16-21 (Ab43 CDs). 28-33 (2.7CD ), 4045 (2,41 CDRs), 52-57 (R9 CDRs), 111- 16 (1C9 CDRs), 121126 (313 CDRs), 131-136 (4E CD1s), 141-146 (7A3 CDs), 151-456 (9D12 CDRs), i61-166 (1219 CDs), 171-)76 (15E1 CDRsh 334-239 (18211 CD Ys), 314-319 (18D8 CDRs), 344-349 (1988 CDR), 324-329 (1£ 9 1MDs), 294 299 (19201 CDs), 304,309 (19H6 CDRs), 354-359 (20E312 CD1s), 36-369 (221F12 CDRs) 374379 (2210DRs) 384-89 123M I SRs - 18 1(23F13 CDRso 394 , 99(2434 -25 - CDRs), 1196 (2611 CURs), 2A3-205 (181 light chain CDRs) and 131133 (131 heavy chain CRs), 214-216 (1S2 heavy chain CDRs) and 144- 46 (IS2 light chain CDRs) 225 227 (1S3 heavy champ CDRs) and 164-166 (133 light chain CDRs), 236-238 (1 S4 heavy chain CURs) and 174176 (184 light chain CURs), 247-249 (18S heavy chain CURs) and 184- 86 (1SS light chain CURs> in any of the foregoing embodiments, the polypeptide includes a sequence comprising one or two modifications to any of the amino acid sequences set forth in SEQ 1D NOs: 16-21 (Ab43 CDRs), 28-33 (2,7CDRs, 40-45 (241 CURs), 5257 (R9 CDRs) 11 V -16 (M CRs), 121-126 (333 CDRs), 131-136 (4E1 CDRs) 141146 (7A3 CDRs, 151d56(9D12 CDRs), 161-166 (1219 CDRs). 171-176 (15E1 CDRs) 334-339 (18811 CDRs), 314-319 (1808 CDRs), 343-349 (1918 CDRS> 324-329(19C1 CDRs), 294-299 (191D12 CUR> s)304-309 (191-6 CDRs), 354-359 (20E2 CDRs), 364-369 (22F12 CDRs), 374-379 (22H10 CDRs> 34-389 (23A1 1 CDRs), 181-186 (23F)1 CDRs), 394-399(244 CDR s), 191-196(2611 CURs), 203-205 (1S1 light chain CDRs) and 131-133 (1Sl heavy chain CDRs), 214~216 (1S2 heavy chain CDRs) and 144-146 (1S2 light chain CDRs, 225 227 (183 heavy chain CDRs) and 164-166 (1S3 light chain CRs), 236-238 (1S4 heavy chain CUR s) and 174-176 (1 4 light chain R0s), 247-249 (185 heavy chain CDRNs) and 184-186 (1 light chain CRs) 10090 in sone tmbodme nts, the antibody comprises the heavy chain variable region of any of antibodies Ab43, 2.7,2.4iR9, 1C9, 181, 1S2, 13 1S4, 185, 383; 481, 7A3, 912)*12. 1289, I1, 18311, 18D8, 19138, 1901, 19D)2, 196, 2.012, 22F12, 22H 10, 23A11, 23F 1, 24E4 and 26F11 and optionally comprises a constant region selected from the group consisting of a human IgG 1 heavy chain constant region (S EQ 1D NOs: 401-402) and a human 1g02 heavy chain constant region (SEQ ID Ns; 403-404) in some embodiments, the antibody comprises the light chain variable region of any of antibodies Ab43 2 7,4241, R9, 109, 13 1, S2, i S3, I S4, 1S5. 3B3 4E.1, 7A3,912, 1289, 15 i, 181311, 18D8, 1988, 19C1, 19102, 19H6, 20E12, 22F12, 22H 10, 23A 11, 2311, 2484, and 26F 1 1 and optionally composes a human kappa light chain constant region (SEQ i) NOs: 405~406) In another embodimnent, the antibody comprises the light chain variable region of any of antibodies Ab43, 2,7 2 41, R9, 1C9 S1, 1S2, 1S3, 1S4, 1S, 313; 4 1, 73, 9012, 12189, 15 1, 18811, 18)8, 19B8, 199, 19102, 196, 20E1 2, 221 2,2HL10, 23A 11, 23F11, 248E4 and 26F11 and optionally comprises a constant region selected frorn the group consisting ofa human lambda light chain constant region type C1 (SEQ ID TOs: 407-408), a human lambda igh, chain constant region type C2 (SEQ U.) NOs: 409-41I), a human lambda light chain constant region type C3 (SEQ ID NOs: 41 -412> a human lambda light chain constant region 2 6 typ C6 (SEQ !D NOs: 413-414) and a human lanbda light chain constant region type C7 (SEQ I D NO: 415-416), { 0091 The eDNA and amino acid sequences for the full length light and heavy chains of each of antibodies 1 C9,3B3, 4E1, 7A3, 9D12, 1239, 15E, 23F11 and 26F11 are so provided. The cDNA sequences encodn the full length light chain of antibodies RC9, 3B3 4El ,7A3,92 12B9, 15E!, 123F1, 26F l, 1S2, IS3, 1 S4 and IS5, iNludg the constant regon. are set frt in SEQ ID s: 197, 208, 219, 230, 241, 252, 256, 260, 264, 217, 228, 239 and 250, respectively. The ano acu sequences of the full length ight chain of antibodies 1C9, 3B3, 4E1, 7A3, 9D12, 1239, 15E, 23Fii, 2.6FII, 132, 133, i 4 and 155 including the constant region, are set forth in SEQ ID NOs: 198 (of which residues I,20 correspond to the signal peptide and the reminder is the mature poiypptide), 209 (of which residues 1-19 correspond to the signal peptide and the remainder is the mature polypeptide), 220 (of which residues 1-20 correspond to the signal peptide and the remainder is the mature polypeptide), 21 (of whch residues 1-20 correspond to the signal peptide and the remainder is the mature polypepide), 242 (of whch residues 1-19 correspond to the signal peptide and the remainder is the anture polypeptide, 253 (of which residues -2N0 correspond to the signal peptide and the remainder is the mature polypeptide), 257 (of which residues I 20 correspond to the signal peptide and the remainder is the mature polypeptide 261 (of whih rosdues 19 correspond to the signal peptide and the remainder is the mature polypeptide) 265 (o f which residues 1-1 correspond to the signal peptide and the reminder is the mature poypeptide), 2.18 (of which residues 1-22 correspond to the signal peptide and the remainder is the mature polypeptide), 229 (of which residues I ,22 correspond to the signal peptide and the remainder is the mature poiypeptide), 240 (of which residues 1-22 correspond to the signal peptide and the remainder is the mature polypeptide) and 251 (of which residues 122 correspond to the signal peptide and the remainder is the mature polypeptide) respectively, 10092 The cDNA sequences encoding the full length heavy chain af antibodies I C9, 333, 4E1, 7A3, 9D12, 1239, 1511, 231 1, 26F11 and 181, including the constant region, are set forth in SEQ ID NOs: 199, 210, 221, 232, 243,254, 258, 262,266 and 206, r 'The amino acd sequences of the full length heavy chain of antibodies 1C9 ,3B3, 4El, 7A3, 9012, 12F39, 150E1, 23F11, 26F Il and 1S!, including the constant region, are set forth in SEQ ID 4s: 200 (of which residues 1-1 9 correspond to the signal peptide and the remainder is the mature polypeptide) 2 11 (of which residues 1-19 correspond to the signal peptide and the remainder is the mature polypeptide) 222 (of which residues 1-19 correspond to the signal peptide and the remainder is the mature polypephide). 233 (of which residues 1-19 correspond -27 to the signal peptide and the remainder is the mature polypeptide 244 (no signal peptide , 255 (of which residues j -19 correspond to the signal peptide and the remainder is the mature polypeptide), 259 (of which residues 1-19 corrspond to the signal peptide and the remainder is the nmture polypeptide), 263 (of which residues 1-20 correspond to the signal peptide and the remainder is the mature polypeptide), 267 (of which residues 119 correspond to the signa peptide and the remainder is the mature polyeptide) and 207 (of which residues 1-1 9 correspond to the signal peptide and the remainder is the mature polypeptide), respectively, 10093 in some embodiments of the invention,abodisomprise amno acids 20 467 of SEQ ID NO: 207 (AS1 heavy champ) and amino acids 21-234 of SEQ ID NO: 220 (1S light chain); or amino acids 20-466 of SEQ ID NO: 233 (1S2 heavy chain) and amino acids 23234 of SEQ f.D NO: 218 (1S2 light chain; or amino acids 20466 of S EQ ID NO: 255 (1 S3 heavy chain) and amino acids 23-234 of S EQ D NO: 229 (1.3 light chain); or amino acids 20~466 of SEQ ID NO: 259 (114 heavy cham) and wherein amino acids 23-234 of SEQ ID NO: 240 (1 S4 light champ ; or anino acids 20-466 of SEQ ID NO: 267 (18S heavy chain) and ammo acids 23234 of SEQ ID NO: t chain) 0094) 1 The term monoclonall antibody' as used herem refers to an antibody; as that term is defined herein, obtained from a population of substantially homogeneous antibodies, ie. the individual antibodis comiising the population are identical except fOr possible naturally occurring mutations or alternative post-ranslational modifications that may be present in minor amounts, whether produced from hybridomas or recombinant DNA techniques. Nonlimiting examples of monoclonal antibodies include nmrin rabbit, rat chicken, chimeric, humanized, or human antibodies, fully assembled antibodies, multispecific antibodies includingg bispecific antibodies), antibody fragments that can bind an antigen (including, Fabt F'(ab) Fv. single cAwin antibodies, diabodies), maxibodies, nanobodies and recomhinant peptides comprising the foregomg as long as they exhibit the desired bioiogical activity, or variants or derives theeof Humaningbody sequence to be more human-like is described in, eg, Jones et al Nu' 321:522. 525 (1 986); Morrison et al, Proc. ~Nt .cad Sc, US A, 81:6851 6855 (1984); Morrison and Ui, Adt Jnmunol, 14:65 92 (1988); Verhoeyeret al, Science 239:1534 1536 (198) Padan, Molec. hmmun, 28:489 498 (1991); Padlan, Mole. nmiunaL, 31(3169 217(99 and Kettleborough, CA. et aL Protein Engineering, 4(7):773 83 (1991); Co, M. S., et al. (1994), I bimuno/ 152, 2968 2976); Studnicka et Al v Protein Engiwering '7: 805-814 (1994) each of which is incorporated herein by reference in its entirety, One method for isolating human monoclonal amibodies is the use of phage display technology. Phage display is described in e.gA Dower e al, V -2S 91/17271, McCafferty et at, WO 92/01047, and Caton and Koprowski, Proc, MNk A cad, Si, USA, 87-6450-6454 (1990), each of which is incorporated herein by reference in its entirety. Another method for isolating human monoclonal antibodies uses tansgcn animals that have no endogenous imunoglobulin production and are engineered to contain human immunoglobulin oci See, e.g, jakobovits et at Proc Nat. Ac, Si UA, 90:2551 (1993 jakobovits et at ature, 362:255,258 (1993); Bmggermann et aL, Year in bwmuna, 7:33 (1993); WO 91/10741, WO 96/34096, WO 98/24893, or US. Patent Application Publication Nos 2003/0194404, 2003/0031667 or 2002/01992i3; each incorporated herein by reference in itsenrty 100951 An isolatedd " antibody refers to an antibody, as thaI term is defined herein th' has been identified and separated from a component of ts natural environment Conaminan components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other protemnaccous or nonproteinaceous solutes. In certain embodiments, the antibody will be pulled (1) to greater than 95% by weight of antibody, or more than 99% by weight, (2) to a degree sufficient to obtain at least '5 residues of N-terminal or amino acid sequence, or (3) to homogeneitv by SDSPAGE under reducing or CJoonmasic blue or silver stain. isolated naturally occurring antibody includes the antibody in sutu within recombinant cells since at least one component of the antibody's natural wilno be present. Ordinarily, however, isolated antibody will bpreparedb at least one purifcation step. 100961 An "immunogobulin" or "native antibody" is a tetrameric glycoprotein. In a naturally-occurrin~g immunoglobu.Iin, each retramer is compnosed of two identical pairs of polypeptide chams, each pair having one "light" (about 25 kia) and one "heavy" chain (about 5070 kDa). The aminoterminal portion of each chaincludes a "variable" ("V") region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-erminal portion of emchaindeis a cntant region primarily responsible fr effector fonction. immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chains. Heavy chains are classified as mu(p) delta (A), gamma ('Y alpha (a) and epsilon (4) and define the antibody's isotype as IgM, IgD .. gG. IgA, and IgE, respectively. Several of these may be further divided into subclasses or isotopes, e.g, igG , igU2, igG3, 1gM4 1gM and igA2. Different isotopes have different effector ftuntions; for example igGI and 1gG3 isotopes have antibody dependent cellular cvtotoxicity (A DCC) activhy. Human light chains are classified as kappa (x) and larbda (W) light chains. Within light and heavy chains, the variable and constant regions are joined by a " region of about 12 or more ammo acids, with the heavy chain also including a "D" region of about 10 more amno acids. See generally) Fundamental immunology, Ch, 7 (Paul, W, ed., 2nd ed, Raven Press, NY, (1989)), 00971 Allotypes are variations in antibody sequence, often in the constant region, that can be inmunogenic and are encoded by specific alleles in. humans. Alotypes have been identifined for five of the human GHC genes, the IGHG1, 1G1HG2, 1GH43, 1GHA2 and 1I genes, and are designated as Gin, G2m, (3m, A2m, and Em alloypes, repetvey Ae 18 Gm allotypes are known: ni m(rQ nG! m(2), G1m (1, 2, 3, 17) or 1m (a , z) Om (23) or C2m (n 3mW (5, 6, 10, I 1, 13, 14, 15, 16, 21, 24, 26, 27, 28) or 11m (hi, c3, b5, bO b3,4, sA , 1,1gi, c5, u, V, g). There are two A2mn allotypes A2(1) and A2m(2). (10998 For a detailed description of the structure and generation of antibodies, see Roth. D. and Craig, N.'L, Cel, 94:41 -414 (1998), herein incorporated by rerence in its entirety, Bri efly, the process 1'r generating DNA encoding the heavy and light chain immunoglobuiin sequences occurs primarily in developing B-cells, Pnor to the rearranging and joining of various imninoglobuin gene segments. the V, D, and constant (C) gene segments are found generally in relatively close proximity on a single chromosome. During B-cel-differentiation, one of each of the appropriate family members of the V , (or only V and J in the case of light chain genes} gene segments are recombined to form functionally rearranged vaalnein fte ev n ih munoglobulin genes. This gene segment vaible regions of the heavy and light in -'og 1 ne rearrangement process appears to be sequential First. heavy chain D-t-J joints are made, followed by heavy chain V-to-DJ jints and hght chain V-o-i jomts. In addition to the rearrangement of V, D and j segments, further diversity is generated in the primary repertoire of iimunogiobulin heavy and light chains by way of variable recombination at the locations where the V and S segments in the light chain are joined and where the D and S segments ot the heavy chain are joined. Such vacation the light chain typically occurs within the last codon of the V gene segment and the frst codon of the j segment Similar imprecision in joining occurs on the heavy chain chromosome between the 1 and b, segments and may extend over as many as 10 nucleotides. Furthermore, several nucleotides may be inserted between the D and jN and between the VA and D gene segments which are not encoded by genomic DN A The addition of these nucleotides is known as N-region diversity. The net effct of' such rearrangements in the variable region gene segments and the variable recombination which may occur during such joining is the production of a primary antibody repertoire.
*)99q The term "hypervariable" region refers to amino acid residues from a compiementarity determining region or CD(. residues 24,34 (LM) 50-56 (L) and 89-97 (L) in the light chain variable domain and 31-35 (HI) I0-65 (H2) and 95-102 (H3) in the heavy chain variable domam as described by Kabat et at. Sequences of Proteins of hmmunologicai Interest, 5" Ed. Pubic Health Service, National Instites of Health, Bethesda, Nd. (191)). Even a single CDR may recognized an id antigen, although with a lower affinity than the entire antigen binding site containing all of the CDRs, 00111 An alternative defimtion of residues from a bypervariable "loop' is described by Chothia eaL, I Mo iJio 196: 901-917 (1987) as residues 26-32 (L), 50-52 (2) and 91-96 (L3) in the Lght chain variabe domain and 26-32 (Wi), 53-5S (H2) and 96-101 (H3) n the heavy chain variable domain . 410 101i "Framework" or FR residues are those variable region residues other than the hypervariable region residues. 1001 012 "Antibody fragments" comprise a portion of an intact immunogiobuin, e.g, an antigen binding or variable region of the intact antibody, and include multispecific (bispecitec trispecific, etc.) antibodies formed from antibody fragments, Fragments of immunoglobulin may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies, {0 1031 Nonlimniting examples of antibody fragments include Fab, Pab F(abt Fv (variable region) domain antibodies (dAb, containing a VIH domain) (Ward et aL, Nature, 341:544546, 1989) complementarity determining region (CDR) agentst, single-chain anibodies (scFv, containing VH and VL domains on a single polypeptide chain) (Bird t al, Science, 242:423-426, 1988, and Huston et alt Proc Nat Acad Si USA 85:5879-5883, I98, optionally including a poypeptide linker; and optionally mutispecifc, uber et al, J, Immunol. 152: 5368 (1994)), single chain antibody fragments, diabodies (VH and VL domains on a single poy)eptide chain that pair with complementary VL. and VtH domains of another chain) (PP 404,97; WO 93/11 161; and Holiger et al Prae Natc Aca. Sc. USA 90:6444-6448 ( 99 3)), triabodies, tetrabodies, minibodies (scFv fused to CH3 via a peptide linker hingelesss) or via an igG hinge) (Olafsen, et a. Protein Eng Des Selt 2004 Apr;17(4)315-23), linear antibodies (tandem Fd segments (V i CH I - C -H 1) (Zapata et at, Protein Eng., 8(10):1057-1062 (1995)): cheating recombinant antibodies (crAb, which can bind to two adjacent. epitopes on the sane antigen) Neri et al., Ma Biol. 246:367~73, 1995), bibodie s (bispecific Fab--scFv) or tribodies (trispecific Fab-(sc~v)(2)) (Seboonjans et al, J Immunol. 165:7050-57, 2000; Wilens et aR, Chromatogr B. Analyt Technol -31 Biome4 ife Sc.. 786:1 6, 20't03), intrabodies (biocca, etLatEMOJ, 9:101-_08) 1990; Colby et al, Proc. NaJlAcd Sci. USA, 1 01-17616-21, 2004) which may also comprse cell signa Isequences which retain or direct the antibody intracelularly (Mhashilkar et al EMBO J. 14:1542151, 1995; Wheeler et a lSE B 1, 1717335, 2003), transbodies (cell-permneable antibodies contaminIg a protein transduction domain (P1) fused to scev (Heng et al, Rtpoiheses, 64:1105-8, 2005), nanobodies (approximately 5kDa variabtle domain ofth heavy chaMin) (Cortez-Retaoo et al!, Cancer Research 642853-57 2004), small modular immunopharmaceuticals (SM IPs) (US. Patent Application Piublication 2003/01 33939 and US Patent Applheation Publication 2003/018592) an antigen-binding-domam itanoglobubi fusion protein, a camelized antibody (in which VH recombies with a constant region that contais hinge, CH 1, CH2 and CH3 domisins) (Desmyter et at, . io. Chem. 276:26285-90 2001: Ewert et at, Rochemisrv 41 :3628-36, 2002; US. Patent Application Publication Nos. 2005/01 36049 and 2005/0037421), a VHH containing antibody, heavy chain antibodies (HOAb bhomodiners of two heavy chains having the structure H2L2), orvarants or derivatives thereof, and polypeptidos that contain at least a portion of an immunoglobulin that is sufcient to confer specific anti gen binding to the polypeptide, such as a CDR sequence, as long as the antibody retains the doted biological activity. 100 1041 The ter "variant" refers to a polypeptide sequence of an antibody that contains at least one amino acid substitution, deletion, or insertion in the variable region or the portion cqn talent to the variable region, provided that the variant retains the desired binding biiological activity. I affinity or bilgclatvti ddition, the antibodies as described herein may have amitno acid modifications in the constant region to modify effector function of the antibody, including half-ife or clearance, ADCC and/or CDC activity. Such modifications can enhance pharmacokinetica or enhance the effectiveness or the antibody in treating cancer, or example. See Shields et at, J Rio. Chem 276(9)6591-6604 (2001), incorporated by reference herein in is entirety. In the case of igG1, modifications to the constant region., particular the hinge or CR2 region, may increase or decrease effector fimtion, including ADCC and/or CDC activity In other embodiments, an g02 constant region is modified to decrease antibody antigen aggregate formation, In the case of tgG4, modifications to the constant region, particularly the binge region, may reduce the formation of half-antibodies. 1001051 The term "modification" includes but is not limited to, one or more amino acd change (including substitutions, msertions or deletions); chemical modifications that do not interfere with hepci din-bnding activity; covalent mnodification by conjugation to therapeutic or diagnostic agents; labeling (eg, with radionuclides or various enzymes) covalent polymer -32 attachment such as pegylation (denvazation with polyethylene glycol) and insertion or substitution by chemical synthesis of non-natural amino acids. In some embodiments, modified polypeptides (including antibodies) will retin the binding properties of unmodified molecules 001061 The term "derivative" refers to antibodies or polypeptides that are covalently modified by conjugation to therapeutic or diagnostic agents, labehng (e.g. with radiouchdes or vanous enzyme), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of non-natal amino acids. in some embodiments, derivatives will retain the binding properties of underivatized molecules 00 1071 Methods for making bispecific or other multispecific antibodies are known in the art and include chemical crosslinking use of leucine zippers (Kostelny ct at, J Pmuni 148 1 5471 553, 1992); diabody technology (Hollinger et at, Poc NadtAad, Sci USA, 90:6444-48, 1993); scfv diners (Guber et at. J! /Mmunot, 152: 5368, 1994), linear antibodies (Zapata et alM rmn 8:105T62, 1995); and chelatng recombinant antibodies (Neri eN al. I MoL Biot, 246, 36M73, 1995). (00108) Thus, a variety of compositions comprising one, two, and/or three CDs of a heavy chain variable region or a hght chain variable region of an antibody may be generated by techniques known in the art. RECONM[NAN PRODUCTION OF ANTIMOAS 1001091 isolated nucleic acids encoding monoclonal antibodies described herein are also provided, optional operable linked to control sequences recognized by a host cell, vectors and host cells comprising the nucleic acids, and recombinant techniques for the production of the antibodies, which may comprise culturing the host cell so that th nucleic acid is expressed and, optionally, recovering the antibody from the host cell culture or culture mediunt [00 1101 Relevant amino acid sequence from an immunoglobulin of interest may be determined by direct protein sequencing, and suitable encoding nucleotide sequences can be designed according to a universal codon table, Alternatively, genomic or cDNA encoding the monoclonal antibodies may be isolated and sequenced from cells producing such antibodies using conventional procedures (e~g. by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal '33 00 4 I Cloning vs carried out using standard techmques (see, eg, Sambrook et al (989) Molecular Cloning: A Laboratory Guide, Voai 1-, Cold Spring Harbor Press, which is incorporated herein by reference). For example, a DNA library may be contracted by reverse transcription of polyA+ mRNA, eg membraneassociated mRNA, and the library screened using probes specific for human immunoglobulin polypeptide gene sequences, In one embodiment, however, the polymerase chain reaction (PCR) is used to amplify eDNAs (or portions of fuldength cDN As) encoding an umnunogiobulin gene segment of mterest (eg, a light or heavy chain vanable segment). The amplified sequences can be readily cloned into any suitable vector, eg, expression vectors, nigene vectors, or phage display vectors, It will be appreciated that the particular method of cloning used is rot cntical, so long as it is possible to determine the sequence of some portion of the immunoglobulin polypeptide of interest. {0) 1121 One source for antibody nucleic acids is a hybrildoma produced by obtaining a W cell &om an animal imnunized with the antUgen of interest and fusing it to an inmortal cell Alternatively, nucleic acid can be isolated from B eels (or whole spleen) of the immunized animat. Yet another source of nucleic acids encnsoding antibodies is a library of such nuclei acids generated, for example, through phage d isplay technology. Polynucleotides encoding peptides of interest, e.g, vanable region pepides with desired binding characteristics, can be identified by standard techniques such as pning, 4)00 113 The sequence encoding an entire variable region of the immnunoglobulin polypeptide may be determined; however, it wil sometimes be adequate to sequence only a portion of a variable region, for example, the CDRtencoding portion. Sequencing is carried out using standard techniques (see, e.g, Sambrook et at (I989) Molecular Cloning: A Laboratory Guide, Vols 14, Cold Spring Harbor Press, and Sanger, F. et at, (1977) Proc, Nd.Aca d. Sci USA, 74: 5463-5467, which is incorporated herein by reference)' By comparing the sequence of the cloned nucleic acid with published sequences of human immu;al ll genes and cDNAs, one of skill wl readily be able to determine, depending sequenced, (i) the germline segment usage of the hybridoma immunoglobulin polypeptide (including the isotype of the heavy chain) and (ii) the sequence of the heavy and light chain variable regions, including sequences resulting from N-region addition and the process of somatic mutation, One source of immunoglobulin gene sequence information is the National Center for Biotechnology Information, National Library of Medicine, National institutes of Health, Bethesda, Md, .34t(0 114| As used herein an isolated" nucleic acid molecule or "isolated" nucleic acid sequence is a nucleic acid molecule that is either (1) identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the nucleic acid or (2) cloned, ampli fled, tagged, or otherwise distinguished from background nucleic acids such that the sequence of the nucleic acid of interest can be determied An isolated nucleic acid molecule is other than in the form or seeing in which it is found in nature. However, an isolated nucleic acid molecule incIludes a nucleic acid moecule contained in cells that ordinarily express the antibody where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells. 1001151 Once isolated, the DNA nA be operably linked to expression control sequences or placed into expression vectors, which are then transfected into host cells that do not otherwise produce immunoglobulin protein, to direct the synthesis of monoclonal antibodies in the recombinant host cells. Recombinant production of antibodies is well known in the art {00116) Expression control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism, The control sequences that are suitable for prokaryotes, tor examplec, include a promoter, optionallty an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, pladenytion and enhances. 001 17 Nucleic acid is operably linked when it is Placed into a functional relationship with another nucleic acid sequence. For example, DN A tor a presequence or secretor y leader is operable linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operable linked to a coding sequence ifit affets the transcription ofWthe sequence; or a ribosome binding site is operably linked to a codng sequence if it is positioned so as to facilitate translation. Generally, operably linked means that the DNA sequence being linked are contiguous, and, in the case of a secretary leaders contiguous and in reading phase Howeveri enhancers do not have to be contiguous. Linking is accomplished by ligation at conveniem restriction sites. If such sites do not exist, the synthetic oligoncleotide adaptors or blinkers are used in accordance with conventional practice, 10 1181 Many vectors are known in the art. Vector components may include one or more of the following: asignal sequence (that may, for example, direct secretion of the antibody), an orgin of replication, one or more selective marker genes (that may, for example confer antibiotic or other drug resistance, complement auxotrophic deficiencies, or supply - 35 critical nutrients not available in the media), an enhancer element, a prom other, and a transcription emirmnation sequence, all of which are well known in the ad. 00 1 19 Cell, cell line, and cell culture are oten used interchaneab-ly and all such designations heremn include progeny. Transformants and transformed eellIs include the primary subject cell and cultures derived therefrom without regard for the number ofransfers. it is also understood that all progeny may not be precisely identical i DNA content, due to deliberate or inadvertent mutations. Mutat progeny that have the same fInction or biological activity as screened for in the originally transformed cell are included, 100 1201 Exemplary host cells include prokaryote, yeast, or higher eukaryote cells (Les a nmultcellular organism), Prokaryotic host cells include eubacteria, such as Gram-negative or Grarn-posiive orgasms, for example, Enercbcweriaceae such as Escwrichia, e.g. E cali, Enterobacter. Ermmau, Klebstela, Proteus, SalonellaU e4g, Salmownella ryphimnurn, Serau, e.g, Serratia marcescans, and SKigella, as well as aci i such as 8. suNtiis and B. i cni/rmis, Psreudomonas, and Streptmyces, Eukaryod microbes such as flamentous fungi or yeast are stable cloning or expression hosts for recombinant polypcptides or antibodies, Saccharomyces cerevsae, or common bakers yeas is the most commonly used among lower eukaryotic host microorganisms, However, a number of other genera, species, and strains are commonly available and useful herein, such as PScha, sig. P pasoris, Schizcsaccharamyces pombe; Kluyvermyce Yarrowia; Candsdd; Trichoderma reesia; Neurospoira craissa; Schwar niomyces such as Schwann~mcesr occldentalis; and tilamentous fnigi such as, e. Neurospora, Penicilium, Ty ad, and Aspergdius hosts such as A nidians and A. niger. t00 1211 Host cells for the expression of glycosylated polypeptide or antibody can be derived from multicellular organisms, Exanples cf invertebrate cells include plant and insect cells, Numerous baculoviral strains and variants and corresponding permissive inrsect host cells from. hosts such as Spadopterafrugiperda (caterpillar), Aedes aegypt mosquitoto, AMdes abtopitu (msut) Drosophila mela ngascer (fruitfly), and Bombyxv maert have been identity. A variety of viral strains for transfection of such cells are publicly available, e.g the L, variant of Autographa califbmica NPV and the Bm- strain of Bombyx mori NPV/ 100122j Vertebrate host cells are also suitable hosts, and recombine production of polypeptide or antibody from such cels has become routine procedure. Examples of useful mammalian host cell lines are Chinese hamster ovary cells, including CH OK 1 cells (A TCC CCL6I), DXB>- I, D-44, and Chinese hamster ovary cells/-DIFR (CH, Urlaub et Wt, PtrocNi,/ Acad. Sci USA, 77: 4216 (198)); monkey kdney CV I lne transformed by SV40 (CCS-7, A TCC C.RL 16$1); human embryonic kidney line (293 or 293 eellIs subeloned for growth in suspension culture, [Graham et at J Gen vrot 36: $9 (1977)]; baby hamster kidney cells (BHK, AwC CCL 10); mouse sertoli cells (TM4, Mather, Bitt Reprod, 23: 243-251 (1980)); monkey kidney cells (CV ATCC CCL 70); African green monkey kidney cells (VER-76, ATCC CRL 1587); human cervical carcinoa cells (H ELA, ATCC CCL 2); canine kidney cels (MDCK ATCC CCL 34); buffalo rat hver cells (BRL 3A. ATCC CRL 1442); human lung cells (W 138 ATCC CCL 75); human hepatona cells (Hep G2, kil 8065); mouse mammary tumor (MMT 0656,ATCC CCL5 1); TRI cells (Mather et al Anna/s k Yl A cad Sci, 383: 44-68 (1982)); MRC 5 cells or FS4 cells; or mammalian myeloma cells. 001 23] Host cells are transforned or transfected with the above-described nuclei acids or vectors for antibody production and cultured in conventional nutrient nWdia niodified as appropriate fr inducing promoters, selecting transform~ants, or amplifying die genes encoding the desired sequences. in addition, novel vectors and transftcted cell lines with muiple copies of transcription units separated by a selective marker are particularly useful for the expression of antibodies. 100 1241 The host ell s used to produce an antibody described herein may be cultured in a variety of media, Commercially available media such as Ham's Fl0 (Sigma), Minimal Essential MEdiurm ((MM), (Sigma) RPMi-1640 (Sigma), and Dulbecco's Modified iagle's Medium ((DM EM), Sigma) are suitable for cuiuring the host cells, In addition, any of the media described in -lam et al, MTh. Ent, 58: 44 (1979), Barnes et al AnaL Biochem, 102: 255 ( O980), U.S. Patent Ns, 4,767,704; 4A57,865; 4,927,762; 4,560,655; or 5,122169; WO 90/03430; WOQ 87/00195; or U.S. Patent Re. No. 30,985 may be used as culture media for the host cells, Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferring, or epidermal growth factor), salts (such as sodium chloride, calcium, and phosphate), buffers (such as HiHPES)t nucleotids (such as adenosine and thymidine), antibiotics (such as GentamycintM drug), trace elements (defined as inorganic compounds usually present at final concenrations in the micromolar range), and glucose or an equivalent energy sourMe Any other necessary supplements may also he included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression and will be apparent to the ordmanly skilled artisan. 1001251 Upon culturing the host cells, the antibody can be produced intraceliuiarly, in the periplasmic space, or directly secreted into the medium if the antibody is produced 37 intracelarly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugat6ion or ultrafiltration. 1001261 The antibody can be punifed using, for example, hydroxylapatite chrmmatography, caton or aMon exchange chromatography, or affinity chromatography, using the antigen of interest or protein A or protein G as an affinity ligand. Protein A can be used to purify antibodies that are based on human yI, 72, or 74 heavy chains (Lindmnark et a, JImuAtl M&e. 62: 1- 3 (1983)), Protein G is recommended for all mouse isotypes and for human y3 (Guss et al, EMOW 5: 15671575 (1986)). The matrix to which the affnity ligand is attached is mot often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyllhenzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a Ci 3 domain, the Bakerbond ABX" 2 resin (J, T. Raker, Phiilipsrg, NJ,) is useMti for purification. Other techniques for protein punifiaion such as ethanol precipitation, Reverse Phase HIPLC, chronatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also possible depending on the antibody to be recovered. Chimeric and H umanzed antibodies [001271 Because chimeric or humanized antibodies are less immunogenic in humans than the parental rodent monoclonal antibodies, they can be used for the treatmtnent of humans with for less ask of anaphylaxis. Thus, these antibodies are contemplated in therapeutic applications that involve i vivo administration to a human. 00 1281 For example, a marine antibody on repeated in vivo administration in man either alone or as a conjugate will bring about an immune response in the recipient, sometimes called a H AMA response (Human Anti Mouse Antibody). The HAMA response may limit the effectiveness of the pharmaceutical if repeated dosing is required, The immunogenicity of the antibody may be reduced by chemical modification of the antibody with a hydrophihe polymer such as polyethylene glycol or by using the methods of genetic engineering to make the antibody binding strue more human like 001t 29' The phrase "chimeri antibody;' as used herein, refers to an antibody containg sequence derived from two different antibodies which typily originate from different species, Most typically, chimeric antibodies comprise variable Ig domains of a rodent monoclonal antibody fused to human constant g domains. Such antibodies can be generated using standard procedures known in the art (See Mormison, S, ,et a (1984) Chimer> Humran Antibody Molecules; Mouse Antigen Binding Domains with H human .38, Constant Region Domains" Proc, N d, A ad, Sci. USA, 81, 6841-6855; and. Boulianne, CO L et al, Naure 312 643-646, (1984)), Although sone chimeric monoclonal antibodies have proved less immunogenic in humans, the rodent variable Ig domains can still lead to a significant human anti-rodent response 00 30| The phrase "humnized antibody' refers to an antibody derived from a non human antibody, typically a rodent monoclonal antibody. Alternatively, a hunmanized antibody may be derived from a chineric antibody, 001t 3 1.i Humanized antibodies may be achieved by a variety of nthods including, for e>:ample: (1) grafRing thea nonu.man complementanty deterunnng regions (CDRs) orto a human framework and constant region (a process referred to in the an as humanizing through "CDR grafting"), or, alernatively, (2) transplanting the entire non-uman variable domains, bt cloakingg" them with a humran-like surface by replacement of surface residues (a process referred to in the art as "veneering"), These rethods are disclosed i e Jones et at, Nature 321:522 525 ( 986); Morrison et al.. Proc, Nd, A cad, Sc, USA, 81: 683 6855( Mornson and i, Adv. Inuzot, 44:65 92 (1988); Verhoeyeret al, Science 2391534 1536 (1988); Padtan, Aolec. Imun 28:489 498 (1991); Padan, Aolc immunol 31(3):169 217 (1994); and Ketleborough, C Ae al., Protein Eng, 4(7:773 83 (1991) each of which is incorporated herein by reference in its entirety, 100 132j CUR grading involves introducing one or nore of the sx. CRs from the mouse heavy and. light chain variable ig domains into the appropriate framework regions of a human variable Ig domain, This technique (Riechmann, L3 et at, Nate 332, (1988)) utilizes the conserved framework regions (FR l-R4) as a scaffold to support the CDR loops which are the primary contacts with antgen. A significant disadvantage of CDR grating, however, is that it can result in a humanized antibody that has a substantially lower binding afhmity than the origmal mouse antibody, because amino acids of the framework regions can contribute to antigen binding, and because amino acids of the CDR loops can influence the association of the two variable ig domains, To maintain the affinity of the humanized monoclonal antibody, the CDR grafting technique can be improved by choosing human fram'ework regions that most closely resemble the framework regions of the original mouse antibody, and by site-directed mutagenesis of single amino acids within the framework or CDRs aided by computer modeling of the antigen binding site (egg Co, N, S, et al. (1994),). Lmmuanot 152. 2.968-2976). 1001331 One method of 'anizing antibodies comprisesigning the nortuman heavy and light chain sequences to human heavy and h chain sequences, selectng and repling the nonhuman framework with a human framework based on such alignment, molecular modeling to predict the conformation of the humanized sequence and comparng to the confmation of the parent antibody, Thus proves is followed by repeated back mutation of residues in the CDR region which disturb the structure of the CDRs until the predicted conformation of the humanized sequence model closely approximates the conformation of the non-nuan CDRs of the parent nonhuman antibody. Such humanized antbodes may be father derivatized to facilitate uptake and clearance, e.g, via Ashwell receptors (See, eg, U.S, Patent Nos. 5,530,101 and 5585089), |00134 A number of humamiations of mouse monoclonal antibodies by rational design have been reported (See, for example, U.S. Patent Applcation P ubhcation No, 2002/009l240 published July 11, 2002, WO 92/11018 and U.S. Paten. N 5,693762, U,. Patent No. 3766,86 E-unian Eingineere nid 100135 The phrase "Hiuman EnineercdM antibody refers to an antibody derived from a nonihuman anybody typically a rodent rmonoconal antibody or possibly a chimeric antibody. Human Engineeringd M of antibody variable domains has been described by Studnicka [See, eg udnicka et a. US Patent No 5,766,86; Studnicka et at Protein Engineering, 7: 803-8 14 (1.9941] as a method for reducing imnmunogenicity while nmantainng biting activity of antibody molecules . According to the method, each variable region amino acid has been assigned a risk of substitution. Amino acid substtutions are distinguished by one of three risa eisk changes are those that have the greatest potential for reducing immiunogenicity with the least chance of disrupting antigen binding; (2) moderate risk changes are those that would further reduce immunogenicity, but have a greater chance of affecting antigen binding or protein foldmg; (3) high risk residues are those that are important for binding or for maintaining antibody structure and carry the highest risk that antigen binding or protein folding will be affected, Due to the three-imensional structural role of prolines, nodif'ations at prolines are generally considered to be at least moderate risk changes, even f the position is typically a low risk position. S00 136) Variable regions of the light and heavy chains of a rodem antibody can be Human EngineeredI by subsituting human amino acids at positions determined to be urdikely to adversely effect either antigen binding or protein folding, but likely to reduce mmunogenicity in a human environmentJ Ahaugh any human variable region carn be used, including an. individual VH or VL sequence or a human consensus VH or VL sequence or an 40 dividual or consensus human germline sequence, generally a human sequence with highest identity or homology to the rodent sequence is used to minmize the number of substitutions, The anino acid residues at any number of the low risk positions, or at all of the low risk positions, can be changed. For example, at each low risk position where the abgned marine and human amino acid residues differ, an amino acid modification is introduced that replaces the rodent residue with the human residue. In addition, the amino acid residues at any number or all of the moderate risk positions can be changed. in some embodiments, al of the low and moderate risk positions are changed from roden to human sequence. 100137)Synthetic genes containing modifled heavy and/or ight chain variable regions are constructed and linkedd to human Y heavy chain and/or kappa light chain, constant regions Any human heavy chainand light chain constant regions of any class or subclass may be used in combination with the Human Engineered: antibody vahle. regions Antibodies From Transgenic Animal: Engineered To Comain Human immunoglobulin Loci 1001381 Antibodies to hepcidin can also be produced using transgenic animals that have no endogenous imrunogiobulin production and are engineered to contain human immunoglobulin ioci. For example, WO) 98/24893 hdioses transgenic animals having a human Ig locus wherein the animals do not produce ftmetional endogenous mnunogiobulins due to the inactivation of endogenous heavy and light chain loci. Transgenic non-pimate mammal ian hosts capable of mounting an immune response to an imnmutnogen,. wherein the antibodies have primate constant and/or variable regions, and wherein the endogenous immunoglobulin encoding loci arc substituted or inactivated have also been discussed. WEO 96/30498 discloses the use of the Crc/Lox system to modify the immunoglobulin locus m a mammal, such as to replace all or a portion of the constant or variable region to form a modified antibody molecue, WO 94/02602 discloses non-human mammalian hosts having inactivated endogenoIs Ig lici and functional human 1g loci, U.S. Patent No, 5,939,598 discloses methods of making transgenic mice in which the mice lack endogenous heavy chains, and express an exogenous immunogiobulin locus comprising one or more xenogeneic constant regions,. 1001391 Using a transgenic animal described above, an immune response can be produced to a selected antigenic molecule, and antibody producing cells can be removed from the animal and used to produce hybridomas that secrete humanderived monoconal antibodies. Inmunization protocols, adj uvants, and the like are known in the art, and are used in mmunization of for example, a tranagenic mouse as described in W) 96/33735, The monoclonal antibodies can be tested for the ability to inhibit or neutralize the biological activity or physiological effet of the corresponding protein, |00 14 0 See also Jakobovits et at, Proc, Nat, A cad. S. USA, 90:255l (1993); Jakobovis et a, Nature, 362:255-258 (1993); Bruggermann ct aL, Year inhimmuno, 7:33 ()993; and US, Pat. No. 5,591 ,669, U.S. Patent No. 5,589,369, U.S Patent No, 5,5,807; and US Patent Application Publication No, 2002/0199213. US. Patent Appiication Publicaton No. 2003/0092125 describes methods for biasing the immune response of an animal to the desired epitope, Human antibodies may also be generated by in vra t cells (see US, Pat binos. 5,67110 and 5229,275). Antibody production by phage display techniques 100 14 1 The development of technologies for making repertoires of recombinant human antibody genes, and the display of the encoded antibody fragments on the surface of filamentous bacteriphage, has provided another means for generating human-derived antibodies. Phage display is described in e.g, Dower at a, W/O 91/17271, McCatferty et at, WO 92/01047 and Caton and Koprowski, Proc. Nad. Ac Set USA, 87:6450-6454 (1990), each of which is incorporatedh ererence n its entirety. The antibodies produced by phage technology are usualIy produced as antigen binding fragments, e-g. Fv or Lab fragroents, in bacteria and thus lack effector functions. Effector functions can be introduced by one of two strategies: The fragments can be engineered either into complete antibodies fb in mammalian cels, or into bispecific antibody fragments with a second binding site capable of triggering an effector function. 10 142] Typically, the Fd fragnnt (V110) and light chain (Vte) of antibodies are separately Cloned by PCR and recombined randomly in combinatorial phage display libraries, which can then be selected for binding to a particular antigen. The antibody fragments are expressed on the phage surface, and selection of v or Fab (and therefore the phage containing the DNA encoding the antibody fragment) by antigen binding is accomplished through several rounds of antigen binding and re-ampaieation, a procedure termed panning, Antibody fragments specific for the antigen are enriched and finally isolated, |00 143 Phage display techniques can also be used in an approach for the humanization of rodent monoclonal antibodies, called "guided selection" (see Jespers, U S, e al., iofTechnology 12, 899-903 (1994)). For this the Fd fagment of the mouse monoclonal antibody can be displayed in combination with a human light chain library, and the resulting hybrid Lab library may then be selected wit antigen, The mouse Fd fragment thereby -42., provides a template to guide the selecton, Subsequenly, the selected human ih chains are combined with a human Fd fragment library Selection of the resulting library yields enirel y human Fab 10t 144) A variety of procedures have been described for deriving human antibodies front phage-display libranes (See, for example, 22oogenboomr et at, a.ol 22-81 (i 991); Marks at al. 4L MoBialt 2 2
,
2 :58,-597 (1991Y US. Pa, Nos 5,565,332 and 5373,905; Clackson, T" and Wells, 3. A , T//3TEC/J, 12, 173-84 (1994)), In particular, i vitro selection and evolution of amibodies derived fron phage display libraries has become a powerful tool (See Burton, D, R, and Barbas iHL C. f, Ad immww.S 57, 191-280 (1994); and, Winter, G, et ak Annu, Rev. Imunol, 12, 433-45S (1994); ,, Patent Appication Pubication No., 2002/00042 15 and WO9G20 1047; US. Patent Application Publication No, 2003/0190317 published October 9, 2003 amd U.S. Patent No. 6,54,287; U.S, Patent No, SV77293. [00145 Watkns, "Screening of Phage~Expressed Antibody Libraries by Capture Lif Methods in A-olecu/ar iology, Antibody Piage isplay: Metods and Protocols, 178 1 87 193, and U.S. Patent Application Publication No. 2003/0044772 published March 6, 2003 describes methods for screening phage-expressed antibody libraries or other binding molecules by capture lift, a method involving immobilizaion of the candidate binding molecules on a solid support 100 1461 As noted above, antibody fragments comprise a ponion of an intact full length antibody, or an antigen binding or variable region of the intact antibody, and include linear antibodies and nmltispecific antibodies formed from antibody fragments. Nonlimiting examples of antibody fragments include Fab, Fab, F(ab)2., Ev, Ed, domain antibody (dAb), complenmentarityv determnin reio (CDRt) &ragmenms, single-chain antibodies (sc~v), single chain antibody &agmnensts, diabodies, triabodies, tetrabodies, minibodies, linear antibodies, chelating recombinant antibodies. tribodies or bibodies, intrabodies, nanobodies, small modular immunopharnaceuticals (SMl. Pa), an antigen-binding-domam immunoglobulin fusion protein, a canmelized antibody, a VH-H coining antibody or muteins or derivatves thereoL and polypeptides that contain at least a portion of an imrunogobulin that is sufficiem to confer specific antigen binding to the poypeptide, such as a CT sequence, as long as the antibody retains the desired biological activity Such antigen fragments may be produced by -43 the modification of whole antibodies or synthesized de novo using recombinant DNA technologies or peptide synthesisa 10 W1 "I antibody t'~ne t 1001 47 The term "diabOdies' refers to srnall atbdfrgeswith two antigen binding sites, which fragments comprise a heavy-ehain variable domain (V H) connected to a ighI-chain variable domain (V L) in the same polypeptide chain (V1 VL) By using a linked that is too short to adow pairing between the two domains on the same chain the domains are forced to pair with the complementary domains of another chain and create two antigaen binding sites. Diabodies are described more fully in, for ample EP 404,097; WO 93/1 1161; and Hollinger et at. Proc. Nad A& Sci USA, 90:64446448 (1993) 00 1 4I "Single-chain F v" or "scFv" antibody fragments comprise the % and W dornains of antibody, wherein these domains are present in a sigle polypeptide chain, and optionally comprising a polypeptide linker between the VI; and V domains that enables the Fv to fBrm the desired structure for antigen binding (Bird et at, Science 242:423~426, t988, and Huston et al Proc, Natt Acd Sc USA 85:879-5883, 1988), An Fd fragment consists of the V> and Cul domains. 1001491 Additional antibody fragments include a domain antibody (dAb) fragment (Ward eta., NArWe 341544-546, 1989) which consists of a V domain. [0015501 "Linear antibodies' comprise a pair of tandei Fd segments (Vo -Cal -V 4 CH 1) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospeedic (Zapata et a, Protei Eng, 8:1057-62 (1995)), 04 1 5 1 A "mninibody" consisting of set fused to CH$ via a peptide linker (hingeless. or via an lgG hinge has been described in Giafsen, et al Protein Eg, Des, Sei, 2004 Apr; 1 7(4):315-23, 1 00 152 j The term "maxibody" refers to bivalent seFvs covalently attached to the Fc reIon of an inAnunoglobulin, see, for example, Fredericks et al, Protein Enginering Design & Selection, 17:95-106 (2004) and Powers et at, Journal f hmmunologicai Methods, 2511231 5 (2001). 1001531 Functional heavy-chain antibodies devoid of light chains are naturally oc-curng in certain species of aniak, such as nurse sharks, wobbegong sharks and c~ameiidlae such as canils, dromedarion, alpacas and lamas, The antigen-binding site is reduced to a single domain, the VH It domain, in these animals, These antibodies form antigen-binding regions in only heavy chain variable region i., these tunictional antibodies are homodimers of heavy chains Only having the structure Itt-t (referred to as 'heavy-chain antibodies" or "H !Abs"). Canelized VmQ reportedly recombines with lgG2 and 44- IgG3 constant regIins that contain hinge 4 CR2 4 arnd CR3 domains and lack a CR1l domain, Classical Vonly fragments are difficult to produce in soluble fom. but improvement in solubilhy and specific binding can be obtained when framework residues are altered to be more VIlike, (See, e~g., Reichman, e al, I immJnol. M ds. 1999 231:25-38,) C em have been fou to bid to antigen with high affinity (Desmyter et al] Hi w Chtemn. 276:26285-9, 200E) d possess high stability in solution (E wert et al Biochemisy 41:3628,36, 2002). Methods for Mgnrating antibodies having camelized heavy chains are described in, for example, in U.S. Patent Application Publication Nos, 2005/0136049 and 2005/0037421. Alternative scaffolds can be made from human variable like domaims that more closely match the shark VNAR scaffold and may provide a framework for a long penetrating loop strcture, 0(0 154] Because the vanable doomnain of the heavy-chain antibodies is the smallest fully functional antigenbinding fragment with a molecular mass of only 15 kDa, this entity is referred tuas a nanobody (Cortex- Retamozo et. al, Cacr Rewearch 64:2353-57, 2004), A nanobody library may he generated from an inmumzed dromedary as described in Conrath e al. ( Anrtnimcrb A ems Cheoncher 45: 2807-1.2, 2001 ) [4015 $Sjntrabodies are single chain antibodies which demonstrate intracellular expression andi can manuiulate iracellular protein function (Biocca, et al, HEMBO J~ 9'1.01 108, 1990; Colby et at, Proc Nad A cd Sct U SA 101:17616-21, 2004), intrabodies, which comprise cell sig~nau sequences which retain the antibody conitruct in intr acellular regions, may be produced as described in Mhashilkar et a! (EMBO 1 14:1542-51 1995) and Wheeler et al. (WASB j 17:1 733-. 2003). TransHodies are cellpermeabie antibodies in which a protein transduction domains (PTD) is fused with single chai variable fragment (se~v) antibodies H eng et al, (Md /paThese s-, 64:1105-8 2005). [00 1561 Purther contemplated are antibodies that are SMIPs or binding domain immiunoglobtalin fusion proteins specific for target protein, These constructs are single-chain polypeptides comprising antigen binding domains used to inmunoglobulin domains necessary to carr out antibody effetor functions. See eg;, W003/041600, U.S. Patent Application Publication No. 2003/0133939 and US Pant Application Publication No, Mudtialakit antibodies 100 1571 In some embodiments, it may be desirable to generate multivalert or even a multispecific (e g. bispecific. trispecific, etc.) monoclonal antibody Such antibody may have 45 binding specificties for at least two different epitopes of the target atgen, or alternatively it may bind to two different molecules, e.g. to the target antigen and to a cell surface protein or receptor For example, a bispecific antibody may include an arm that binds to the target and another arm that binds to a triggering molecule on a leukocyte such as a T-ceii receptor molecule (e.g. CD2 or CD1), or W; receptors (br igG (FcyRL) such as FeyRI (CD64) FeyRli (CD32) and FyRIt (CD16) so as to focus celdar dense mechanisms to the target expressing cell. As another example bispecific antibodies nay be used to localize cytotoxic agents to cells which express target antigen. These antibodies possess a targetbinding arm and an arm which binds the cytotoxic agent (eg sapoin, anti-interferon-60, vica alkaloid, ricin A chain, methotrexate or radioactive isotope happen), Multiapecific antibodies can be prepared as fAl length antibodies or antibody fragments. 0.158 ) A additionally, the antihepeidin antibodies disclosed herein can also be constructed t told ino multivalent forms, which may improve binding affinity, speciicity and/or increased half-ife in blood. Multivalent forms of anti-hepeidin antibodies can be prepared by techniques known in the art, 0 159 j Bispecific or multispecific antibodies include cross-linked or "heteroconjugate antibodies, For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin Heteroconjugate antbodies may be made using any convenient crosslinking methods. Suitable cross-linking agents are well known m the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques. Another method is designed to make tetramers by adding a streptavidin-coding sequence at the Cterminus of the scfv. Streptavidin is composed of four subunits, so when the scFv streptavidin is flded, tour subumits associate to form a tetramer (K;priyanov et al. um Antibodies Hvbridomas 6(3), 93-101 (1995), the disclosure of which is incorporated herein by reference in its entirety), {I0 1601 According to another approach for making bispecific anti bodies, the nterface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. One interface comprises at least a part of the C3 domain of an antibody constant domain, in this method, one or more small amino acid sde chains from the interface of the first antibody molecule are replaced with larger side echams (e.g, tyrosine or tryptophan). Compensatory "avities" of identical or similar size to the large side chain(s) are created on the intertace of the second antibody molecule by replacing large amino acid side chains with smaller ones (eg., alanine or -46threonmne), TPhi s provides a mechanism for increasing the yield of the. heterodimrer over other unwanted end-products such as homodimners. See W O 96/2701 1 published Sep. 6., 1996. 00161 ' Techniques for generating bispeciftie or multispeciflie antibodies from antiboody fragments have also been described in the literature, For example, hispecific or trispecitfic antibodies can be prepared using chemical linkage, Brnnan ct al, Science 229:81 (1985) describe a procedure wherein intact antibodies are proteol ytically cleaved to generate F(ab b fragmnents. These fragments are' reduced in. the presence of the dithiol coimplexing agent sodium arsenite to stabilize vicinal dithiols and prevent interolecular di sulfide fornati on, The Fab' fragments generated are then converted to tionitrohenzoate (TN B3) derivatives One of the FabUTN B derivatives is then reconverted to the Fabithi by reduction with mercaptoethyiamine and is mixed with an eqioaraonto heohr atN deriativ to form the bispecific aibdTe bispecific antibodies produced can be used as agents for the selective immrobilization of enzymes, Better et at, Science 240: 104 P1043 (1988) dtisclose secretion of functional antibody fragements frm bacteria (see, e.g. Better et al,. Skerra et at. Science 240: 1038-.1041 (1988)), F or example, Fab*'SH fragmnents can be directly recovered from Ei coli and chemrically coupled to form bispecific antibodies (Carter et al Bio/technology 19: 163-167 (1992'); Shataby et al , 1. Exp Med, 175:217~225 (1992)) 10162] Shalaby est al,t 54xp Med, 175:217225 (1992) describe the production of a fully humanized bispecific antibody F(ab')2amolecule, Each FablY fragment was separately secreted 'frm E5 cob and subjected to directed chemical coupling in'vitro to form the bispecfic antibody. The bispecific antibody thus fbrm ed was able to bind to cells overexpressmng the HIER2 receptor and normal human' cells as weastigger he ye ag gchvofhmnN cyvtotoxic lymphocytes against hunmam breast tumor targets, 00163) Various techniques for making~ and isolating bispecific or nmltispecinec antibody f'ragmnents directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leuceme zippers, e~g. QCN4. (See generally Kostelny et a l) hnmmnol 148(5):15471 553 ('19921.) The leucrrne zipper peptides from the Fos and. Jun proteins were linked to the Fab portions of' two di frerent antibodies by gene fusion. The antibody honmodimners were reduced at the hinge region to form mxonomners and then re-.oxidized to fin-n the antibody heterodirners, This method can also be utilized for the production of antibody' homodimners, [00164| Diabodies, described above, are one example oif a bispecinec antibody, See, fir example, lolliriger e~t atL Proc. Nat, A cad. Sec., USA, 90:6444-6448 (1993). Bivalent diabodies can be stabilized by disulfide linkage, .47'- UU I 651 Stable nmnospecific or bispecific Fv tetramers can also be generated by noncovalent association in (seFv 2 h configuration or as bis-tetrabodies Alternatively, two different se~ys can be joined in tandem to form a bis-soFv. 00 166) Another strategy for making bispecific antibody fragments by the use of single chan Fv (sAv) dimers has also been reported. See Gruber el at, j bnmunoi 152. 5368 (1994), One approach has been to link two srFv antibodies wish liners or disulfide bonds (Mallender and Voss, . Biof Chem , 269;199-2061994, WO 94/13806, and U Patent No. 5,98,q830, the disclosures of which are incorporated herein by referrence in their entirties). [0016 71 Alternatively, he specific anybody any be a linear antibody" produced as descrbed in Zapaa t A Proen En. 8(10):1057-1062 (1995) Brefy, these antibodies comprise a pai ofytandem Ed segma ent (V -C l-V -CH i) which forNM a pair of amien bmding regions. Linear anibodies can be bispecific or mronospecif. [00 1681 Antibodies with more than two valencies are also contemplated, For example:, trispecific antibodies can be prepared. (Tutt et al, J 1mmauna 147:60 (1991)). 1001691 A cheatingg recormbnant antibody" is a bispecific antibody that recognizes adjacent and non-overiapping epitopes of the target antigen, and is flexible enough to bind to both epitopes siunltanously (Nen et a, dM61.," 4 246.367-73, 1995), 1001701 Production ofbispecific Fab-scFv ("bibody") and trispecific Fab-(scFv)(2) bodydy) are described in Schoonjans et at (i ano/ 165:7050-57, 2000) and Wilems et at. (i Chromawgr B Analyt Technol .iomed Life Sat 786:161-76, 2003). For bibodies or tribodies. a scFv molecule is fused to one or both of the VICL (L) and V-CI-H (Fd) chains. e.g. to produce a tribody two scFvs are fused to -tern of Fab whie in a body one scFv is fsed to Cam of Fab 100 1711 In yet another method. dimers timers, and tesramers are produced after a free cysteine is introduced in the parental protein A peptide-based cross lnker witn varable numbers (two to thur) of maleimide groups was used to cross link the protein interest to the tree cysteines (Cochran en a,. immunity 12(3) 241-50 (2000), the disclosure of which is iorporated ein ints entire SPECIC IMNDIN2G AGENTS 001 721 Other bepcidin-specific binding agents can be prepared, for example, based on CDRs from an anibody or by screening libraries of diverse peptides or organic chemical compounds for peptides or compounds that exhibit the desired binding properties for human hepeidin, Hepcidin specific binding agent include peptides containing am ino acid sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, S6%, 87% 88%, 89%. 90%. 91%, 92%, 93%4 94%, 95%, 96%, 97% 98%. 99% or more identical to one or more CDRs of murne antibody Ab43 (SEQ D NOs: 16-21 ).; maurine antibody 2 7 (SEQ ID NOs: 28-33); murne antibody 2,41 (SEQ 1D NCs: 40-45), rat antibody R9 (SEQ ID NOs: 5257) or human antibody 1C9 (SEQ D NOs: I 11-1 16), human antibody 333 (SEQ DNs: 121-126), hunan antibody 4E1 (S EQ ID NO: 131-136), human atibody 7A3 (SEQ D NOs: 141-46)., human antibody t9D2 (SEQ MD NOs: 151-156, human antibody 12B9 (SEQ ID NOs: 161-166) human antiody 5E I (SEQ ID NOs 71-176), human antibody 18311 (SEQ ID)Nt s; 334 239), human antibody 18 E8 (SEQ ID NOs: 31, , humnaribody 1938 (SEQ ID NOs: 343-349), human antibody 19C1 (SEQ ID NO: 324-329 human antibody 9D12(SEQ ) D NO: 294-299), human antibody 1916 (SEQ ID Nis; 304-309) human antibody 2012 (SEQ HI s 353359, human antbody2 (SEQ ID NAs: 363-369), human antibody 22H0 (SEQ If D)Os: 373-379), human antibody 23A 11 (SEQ 1D NOs: 383-389), human antibody 23FI 1 (SEQ ID Os: 181-186) human antibody 24E4 (SEQ ID NOs: 393-399) human antibody 26FR I (SEQ ID NOs: 19196) or human antibody S" (SEQ ID NOs: 203-205 ard 131-133) or human antibody 1S2 (SEQ ID NOs: 214-216 and 144-146) or human antibody 183 (SEQ ID NOs: 225-227 and 164-166) or human antibody 154 (SEQ ID NnOs:236-38 ad 174-1S76) or human antibody 15 (SEQ ID NO; 247-249 and 184-186, 100173j Hepeidin-specifc binding agents also include pepubodies, The term "peptihody" refers to a molecule comprising an antibody Fc domain attached to at least one peptid. T production of peptibodies is generally desrbed in PCti publication WC 00/2472, published May 4, 2000. Any of these peptides ma' be linked in tandem (ie, sequentially, with or without liners, Peptides containing a cysteinyl residue may be cross linked with another ti peptid either or both of which Nmy be linked to a vehicle, Any peptide having more than one Cys residue may om1 an intapeptide disulfide bond, as web Any of these peptides may be denvatized, for example, the carboxyl terminus may be capped with an amino group, cysteires may be capped. or armio acid residues may substituted by moieties other than amino acid residues (see, e.g, Bhatnagar et al, I Med. Chem, 39: 3814-9 (1996), and Cuthbertson al.. I Med, Chem, 40: 2876-82 (1997) which are incorporated by reference herem in their entirety), The peptide sequences may be optimized, analogous to affinity maturation for antibodies, or otherwise altered by alanine scanning or random or directed mutagenesis followed by screening to identify the best binders, Lowman, Ann Rev Biophys. Biomat Struct, 26; 401-24 (1997), Various moleules can be inserted into the specific binding agent structure, e., within the peptde portion itself . 49 or between the peptde and vehicle portions of the specific binding agents, while retaining the desired activity of specific bin g agent, One can readily insert, for example, molecules such as an Fe domain or frgment thereof polyethylene glyc-o or other related molecules such as dextan, a fatty acid, a lipid, a cholesterol group, a small carbohydrate, a peptides a detectable moiety as described herem (including fluorescent agents. radiolabels such as radioisotopes) an oligosaccharide, oligonucleotide, a polIyucleotide, interference (or other) RNA. enzymes, hormones, or the like. Other mcdecules suitable for insertion in this fashion will be appreciated by those skilled in the art, and are encompassed within the scope ofhe invention. This includes insertion of for example, a desired molecule in between two consective amino acids optonatll joined by a suitable linker. jOl 74| The development of hepcidin peptibodies is also contemplated. The interaction of a protein ligand with its receptor often takes place at a relatively large interface, However as demonstrated for human growth hormone and its receptor, only a few key residues at the interface contribute to most of the binding energy. Clackson et at, Science 267: 383-6 (1995) The bulk of the protein igand merely displays the binding epitopes in the right topology or serves tuctions unrelated to binding. Thus, molecules of only "peptide" length (generally 2 to 40 amino acids) can bind to the receptor protein of a given large protein ligand. Such peptides may ii n the bioactivity of the large protein ligand ("peptide agonists") or, through competitive bid ,inhibit theb protein ligand ("peptide antagonists"), 00 175 Phage display technology has emerged as a powerful method in identifying such peptide agonists and antagonists. See, for exainple, Scott et t Science, 249: 386 (1990) Devlin et al, Science 249: 404 (1990); US Patent No. 5,223409, issued June 29, 1993; U.S. Patet No. 5,733,731, issued March 31. 1998; U S. Patent No 5,498,530. issued March 12, 1996; U S. Patent N>o 5,432,018, issued July 11,1995; US, Patent No, 5338665, issued August 16, 1994: U.S. Patent No. 5,922,545, issued July 13, 1999 WO 96/ 4 0 987, published December 19, 1996; and WO 98/15833, published April 16, 1998 (each of whicl is incorporated by reference in its entirety) In peptide phage d libraries, random pephde sequences can be displayed by fusion with coat proteins of filamentous phage The displayed pepti des can be affinity-eluted against an antibody-immobilized extracelular domain of a receptor, if desired. The retained phage may be enriched by successive rounds of affinity purification and repropagation. The best binding peptides may be sequenced to identify key residues within one or more structurally related families of peptides, See, eg.. (wiria et a, Science 276: 1696-9 (1 997), in which. two distinct families were identified. The peptude seqmC way also suggest whi residues may he safl repla-cedbn br - 50)mutagenesis at the DNA level, Mutagenesis libraries may be created and screened to further optimize the sequence of the be't binders, owman, Ann. Rev Biophys Biomol Siruct, 26: 401-24 (1997). 100 1761 Structural analysis of proteinprotein iteraction may also be used to suggest peptides tat mimic the binding av of a potei ligands, in such an analys the cryta) structure may suggest the identity and relative orientation of critical rescues large protein ligand, from which a peptide may he designed, See, eg. Takasaki et at, Nature Biotech 5: i,266-0 (1997), These analytical methods may also be used to investigate the interaction between a receptor proein and peptides selected by phage display, which may suggest further mod, ficti of the peptides to increase binding affinity. 0 0 177] Other methods compete with phage display m peptide research, A peptide library can be fused to the catboxyl terminus of the lac repressor and expressed in E col Another E co/-based method allows display on the ces outer membrane by fusion with a p i yn oatd lioprotin (PAL), Herkinafter, these and lt methods are collectively referred to as "E coli display." In another method, translation of random RNA is halted prior tr e a library of piypeptides with their associated RN A still attached. Hereinafter this and related methods are collectively referred to as "ribosoen display." Other methods employ chemical linkage of peptides to RNA. See, for ex ample, Roberts and Szastak, roc. ?2l. Acad Sci. USA, 94: 1 2297303 (1997), Hereinater, this and related methods are collectively referred to as "RNA-peptide screening." Chemically derived peptde libraries have been developed m which peptides are immobilzed on stable, nombiological materials, such as polyethylene rods or solven-permeable resins. Another chemically derived peptide library uses photolithography to scan peptides immobilized on glass slides, Heremafter, these and related rnethods are collectively referrred to as "cheRicabpeptide screening * Chemica peptide screening may be advantageous in that it. allows use of D-amino acids and other unnatural analogues, as well as nonpeptide elements. Both biological and chemical methods are reviewed in Wells and Lownan, Curr, -pin-. Botchnol, 3 2(1992). 100 178I Conceptually, one may discover peptide mumetics of any protein using phage display and the other methods mentioned above. These methods have been used for epitope mapping. for identification of critical amino acids in proteiriprotein interactions, and as leads for the discovery of new therapeutic agents, See, e, Cortese et al Curr, Opin, Riotech, 7: 616-21 (1996), Peptide libraries are now being used most often in immunological studies, sucn as epitope mapping. See Kreeger, The Scitmist 10(13):1920(1996), -51 0(1 179) Sources for compounds that rmay be screened for ability to bind to or modulate i e, increase or decrease) the activity of the hepcidin polypeptides described herein include ) inorganm and organic chemical libraries, (2) natural product libraries, and (3) combinatorial hbraries comprised of either random or mimetic peptides, oligonucleotides or orgamc. molecules. [00180I Chemical libraries may be readily synthesized or purchased from a number of commercial sources, and may include structural analogs of known compounds or compounds that are identified as "hits" or leads' via natural product screening. IOU 181} The sources of natural product libraries are microorganisms (including bacteria and fungi) anmais, plants or other vegetation, or marine organisms, and libraries of mixtures for screening may be created by: (1) fermentation and extraction of broths from soil, plant or marine rnicroorganiss or (2) e>:traction of the organsms themselves. Natural product libraries include polyketides, on-ribosornal peptides, ani (non-naturally occurring) variants thereof For a review, see Science 22.63-68 (1998), P00 182) Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds and can be readiRy prepared by traditional automated synthesis methods, PCR, clonig or proprietary synthetic methods. Of particular merest are peptide and oligonucleotide combinatorial libraries. Still other libraries of interest include peptide, protein, peptdomimetic, mltiparallel sythetic collection, recombinatorial, and polypeptide libraries For a review of cOMbinatorial chernstrv and libraries created therefarm, see Myers, Curr, (in, Bwioteo. 8:701-707 (1997). For reviews and examples ofpeptidonimetic libraries, see .AlObeidi et al, Mo Biotechno, 9(3)205-23 (1998); ruby et Al, CIh e. (lom. Bia, 1(1):1 14-19 (1997); Dorner et atSBoorg Md. Chem. 4(5):709-13 (1996) (aikylated dipeprides). 100831 Hepeidin-specti binding agents also include scaffolding proteins, as described by Hays et at Trends In Biotechnology 23(1OY514-522 (200 5 Y herein incorporated by reference in its entirety and Aimer protein technology as described in U.S, Publication Nos, 2006-0286603 and 2006-0223114, hoth herein incorporated by reference in their entireties, SCRENING METHO DS FOR ANTIBODIES OR SPECIFIC BINDING AGENTS 1001841 Methods of identifying antibodies or specific binding agents which bind hepeidin and/or which crossblock exemplary antibodies described herein, and/or which inhibit hepcidin activity are also provided 52 go i 185 Anatibodies or specific binding agents may be screened for binding affinity by methods known in the art For example, el-shift assays, Westem blots, radiolabeled comnpetitiont assay, co-fractionadion by chronmatography, co-precipitation, cross linking. EL~iSA, and the like may be used, which are described in, tor example, Current Protocols in Molecular Biology (1 999) John. Wiley & Sons, N Y, which is incorporated herein by reference in its entirety. 900186 To initially screen fur antibodies or specific binding agents which hind to the desired epitope on the target antigen, a routine cross-blocking assay such as that described in Antibodies, A. Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (18) cnb prormed, R outine competitive binding assays may also be used, in which the unknown antibody is characterized by its ability to ihibit binding of target to a target -specific antibody described herein, Intact antigen, fragments thereof such as the extracellular domain, or linear epitopes can be used, Epitope mapping is described in Chamnpe et al.. J, Biat CThem. 270: 1388-1394 (1995), Competitive binding assays may also be used to determine the off-rate of an antibody-antigen interaction, For example, one example ot a cornpetiti e binding assay is a radi oimmnunoassa~y comprising the incubation of labeled antigen (eg,3 or 0I), or fragment or variant thereof, with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen, The binding off-rates can be determined frm the data by scatchard plot analysis. 10O 1 87) In one variation of an in vitro bmdng ssay, method is provided comprising (a) contacting an immobilized hepcidin with a candidate antibody or specific bmtding agent and (b) detecting binding of the candidate antibody or specific binding agent to the hepcidin, in an alternative emibodinment, the candidate antibody or specific binding agent is immobilized and binding of hepcidini is detected. immobilization is accomplished using any of the methods well known in the art, including covalent bonding to a support, a bead, or a chromnatographic resmn, as well as non-covalent, high affinity interaction such as antibody binding, or use of streptavidi n/biotin binding wherein the immobilized compound includes a biotin moity, Detection of binding can be accomplished (i) using a radioactive label on the compound that is not immiobilized, (ii) using a fluorescent label on the non-irrmnohilized compound, (iii) using an antibody iimunospecific for the non-immobilized compound, (iv) using a label on the non-immobilized compound that excites a fluorescent support to which the immobilized compound is attached, as well as other techniques wel known and routinely practiced in the art.
(011 In sonme emibodimnents, antibodies or specific binding agents that inhibit or neutralize human hepcidin activity maiy be identified by contacting hepeidini with the antibody (or specific binding agent), comparinig hepcidin activity in the presence and absence of the test antibody (or specific binding agen t), and determining whether the presence of the antibody (or specific binding agent) decreases activity of the bepcidin, The biological activity of a particular antibody, or specific binding agent, or combination of antibodies or specific binding agents, may be evaluated in viva' using a suitable animal model, including any of those described herein. j001 89) In some em-bodimnents, high throughput screening (i-ITS) assays to identify anibodies that interact with or inhibit biological activity (ic. inhibit pbosphoryiation. dimeration, ligand induced-.receptor activation, or intracellular signaling, etc.) of target antigen are also contemplated, H TS assays permit screening of large numbers of compounds in an efficient manner, Cell-based H TS systems are contemplated to investigate the interaction between target antigen and its bmdimg partners. HITS as says are designed to identify "hits" or 'lead compounds" having the desired property, from which modifications can lie designed to irnprove the desired property. f101 90 in another embodiment, high throughput screening for antibody fragments or CDRs with 1, 2. 3 or more modifications to amino acids within the CDRs having suitable binding affiity to a target antigen polypeptide is employed. PRODUCTION Of AN BODY VARIANTS AND DERIVATIVES 100 1911 The anti-hepcidin antibodies disclosed herein can readily be modified by techniques well-known to one of ordinary skill in the art. Potential mutations include mnsertion, deletion or substitution of one or more residues, in some em~bodimnent, nsertions or deletions are in the range of about I to 5 amino acids, in thie range of about I to 3 amino acids. or in the range ofuabout I or Zamnino acids, 00 192 1 Deletion variants are polypeptides wherein at least one amino acid residue of any amino acid sequence is removed. Deletions can. be effteted at one or both termimi of the protinor ithremoval oftone or more residues within (i.e.. internal to) the polypeptide. Methods for preparation of deletion variants are routine in the art. See, e.g., Sambrook et at. (1989) Molecular Cloning: A Laboratory Guide, Vols I1-3, Cold Spring H-arbor Press, the disclosure of which is incorporated herein by reference in its entirety. (00 1 931 Ammno acid sequence insertions include amino- arnd/or car boxyl-erminual fusions ranging in length from one residue to polypeptides containing hundreds or more :At.4 residues, as well as intemao sequence insertions of one or more amino acids As with any of the different variant types described herein, insernional variants can be designed such that the resulitmg polypeptide reans the same biological properties or exhibits a new physical, chemical and/or biologica property not associated with the parental polypeptide from which it was derived. Methods fbr preparation of insertion variants arc also routine and weU known in the art (Sambrook et a" .'supra), 00 1941 1 Fusion proteins comprising a polypeptide comprising an anti-hepcidin antibody descied herein, and a heterologous polypeptide, are a specific p section variant contemplated herein. Monlimiting examples of heterologous pol ypeptides which can be fised to poypeptides of interest inycude proteins with long circulating half-hI, such as, but not hmited to, rriunoglobuin constant regions (e.g, Fe region); marker sequences that permit identification of the polypeptide of imerest; sequences that facihtate purification of the polypeptide of interest; and sequences that promote formation of mtimeric proteins. 100 195 Methods of making antibody fusion proteins are well known in the art See, e.g, U.S. Patent No, 6306393, the disclosure of which is incorporated heremn by reference in its entirety, in certain embodiments, fusion proteins are produced which may include a flexible linker, which connects the chimeric scFv antibody to the heterologous protein moety Appropriate linker sequences are those that do not affect the ability of the resulting fusion protein to be recoagnized and bind the epitope specifically bound by the V domain of the protein (see, e g., W() 98/25965, the disclosure of which is incorporated herein by reference in its entirety) 1001961 Substitution variants are those in which at least one residue in the polypeptide amino acid sequence is removed and a different residue is inserted in its place., Modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their efc on mairaing (a) the structure of the pb in the area of the substitution, for example, as a sheet or hehial conformation, (b) the charge or hydrophobicity of the molecule a the target site, or (Q the bulk o ide c i embodiments, substuution variants are designed. ie., one or more specir (as opposed to random) amino acid residues are substituted with a specific amino acid residue, Typical changes of thes types include conservative substitutions and/or substitution of one residue for another based on similar properties of the native and substitutmg residues. (00 197 Conservative substitutions are shown in Tabe 1 The most conservative substitton is found under the heading of "preferred substiturons" If such substitutions result in no chane in logical activity, hen more substantial changes may be intoduced aind the products screened Original Exemphry Prefred Residue Substuiins Ala (A) vat; leu; de Arg (R) lys; gin; asn lyv Asn (N) gin; his; aspls;gin e Asp (D) glu; asn gi Cys (C) ser; ala ser Gin (Q) asn; giu n Giu (E) asp; gin asp Gly (G) ala is (') amn; gin; lys; arg le () leu: val; met; ala; phc ; nodeeucrn Leu (U) nordeucine; ile; val; ile mnet; a) a; phe Lys (K) ar gin; asn arg he val i alay Pro () a Ser(S) tr Ihr (T se se 'flp y) tyr FA. Tyt rp; phe; hr scr hec aQa; nodunme 00 198 Amno acid residues which share common dechin properties ac oten grouped as fo lows. (I) hydrophobic: norleucine, met, ala, vkai leu, ilec: (2) neutral hydrophilic: cys, ser, thr:; (3) acidic; asp, gin; (d) basic aan, gin. h lys, arg, Sresidues that infuence chain orienta tirnely, pro; and (6) aomatc:o rp, tr phe Antibody Variants 00 1 99| In certain instances, antibody variants are prepared with the intent to modify those ammno acid residues which are directly involved in. epitope binding. In otter embodiments, modification of residues which are not directly involved in epitope binding or residues not involved in epitope binding in any way, is desirable. for purposes discussed herem. MIutagenesis within any of the CDR regions and/or framework regions is contemplated. l1 02 001 In order to determine which antibody amino acid residues are important for epitope recognition and binding, lanine scannig mutagenesis can be performed to produce sbstitution variants, See, for example, Cunningham et at, Science, 244:1081 1085 (1989) the disclosure of which is incorporated herein by reference m its entiety.n this method. individual amino acid residues are replaced one-at-a-time with an alanine residue and the resulting nt-eidnantibody is screen orisablt tobid~ itpecific Opitope relative antiiepedin.w. i as AttYtobn to the unmodifed antibody, Modified antibodies with reduced binding capacity are sequenced to determine which residue was changed, indicating its significance in binding or biological prop erltes, 100201) Substitution variants of antibodies can be prepared by affinity maturation wherein random aino acid changes are introduced into the parent antibody sequence See, for example, Ouwehand et at, Vox Sang 74 (Suppi 2):223-232, 199$; Rader et at, Proc. NAod Acad, Sci USA 95:8910-8915, 1998; .DaWAcqua et aLC cur. Opi Struct Bwl. 8444, 1998, the disclosures of whieb. are incorporated herein by reference in their entireties, Affinity maturation involves preparing and screening the ant-hepcidin antibodies, or variants thereof and selectigfron the resulting variants those that have modified biological properties. such as increased binding affinity relative to the parent i-hepcidin antibody. A convemnt way fvtor generating substitutional -vaiants is affinity maturation using phage display brefly, several hypervariable region sites are mutated to generate all possible amino substitutions at each site. The variants thus generated are expressed in. a monovalent fashion on the surface of tilamentous phage particles as sion to the gene I product of M 13 packaged with each particle. The phage-displayed variants are then screened for their biological activity (e g, binding affinity) See e.g> WO 92/01047, WO 93/ 12366, WO 95/1388 and WO 93/l 9172.
( 00202) Current antibody affinity maturation methods belong to two mutageniesis categories: stochastic and nonstochastic Error prone PCR, mutor r bacterial strains (Low et aL J. Mo/ Biot 26,396,19), and saturation mnutagenesis (Nishimniya et atr £, Biot. Chem. 273:128i320, 2000; Chowdhury, P. S. Methodr MotSlot 178, 269-83 2002) are typical examples of stochastic u tagenesis nc S U 02 8466- 71, 2005). Nonstochast techniques often use alanine-scanning or site directed mutagenesis to generate limited collections of specific muteins. Some methods are described in. further detad below [00203 Affirity mammron vi pannng methods-Affinity maturation of recombinant antibodies is commonly performed through several rounds of panning of candidate antibodies in the presence of decreasing amounts of antigen. Decreasing the amount of amigen per round selects the antibodies with the highest affinity to the antigen thereby yielding antibodies of high sffinity from a large pool of starting materiaL. Affinity maturation via panming is well known in the art and is described, for example, in Huls et at (Cancer bnmunol Immunother. 50:163-71, 2001) Methotds f atfnity maturation using phage display technologies are described elsewhere herein and known in the art (see eg, Daugherty et al. * Proc at/ Acod sci U S A 97:2029-34 2000), 100204 1 Look-khrough aagenles-Look-through mutagenesis (LTN) (R ajpal et at, Proc Nat Acad Sei US A. 102:8466-7 2005) provides a method for rapidly mapping the antibody-binding site For LTUM nine amino acids, representative of the major sidechain by the" 20) natu .rald ;amino, acids, r cerneistries provided by th.0ntrlann acd, are selected to dissect the functional side cnai contributions to bindimg at every position in all six CDRs of an antibody, LTM generates a positional series of single mutations within a CDR where each wild type" residue is systematically ubstituteAd by one of anne selected ammo acids. Muted CURs are ombned to generate co inatoria single-chain variable fragment (cFv) libraries of increasing complexity and size without becoming prohibitive to the quantitative display of all mutens. After positive selection, clones with improved binding are sequenced, and beneficial mutations are mapped. 00205 jEror-prone PCR-Error-prone PCR involves the randomization of nucleic acids between different selection rounds, The randomization occurs at a low rate by the intrinsic error rate of the polymerase used but can be enhanced by error-prone PCR (Zaccolo et al. . . Mo Bio. 285:775-783, 1999) using a polymerase having a high intrinsic error rate during transcription (Hlawkins et al, II Moi fBiol 226:889-96, 1992). After the mrutation egdes.e cones witiproved affinity ior the antigen are selected usig routine methods in the art. 1002061 Techniques utilizing gene shuffling and directed evolution may also be used to preopre and screen antihepcidin antibodies, or variants thereof for desired activF For exanple Jernutus et A, Proc NatI Acad Sci U 3 A, 98(1):75-80 (2001) showed that tailored ?n vitr selection strategies based on ribosorn display were combined with in vuro diversification by DNA shuffing to evolve either the o-rate or thermodynamics stability of scFvs; Fermer et at, Tumour Bioi, 2004 Jan-pr;2S(b2):743 reported that use of phage display in combination with DNA shuffling raised affinity by almost three orders of magnitude. Dougherty et al, Proc Nati Acad Sci U S A, 2000 Feb 29; 97 5 ):02-23 reported that (i) functional clones occur at an unexpectedly high frequency in hypermutated libraries, (ii) gain-of-function mutants are well represented in such libraries, and (iii) the majority of the scFv mutations leading to higher affinity correspond to residues distant from the binding site. 00 2071 Alternatively, or in addition, it may be beneficial to analvze a crystal structure of the antigenantibody complex to identify contact points between the antibody and antigen, orto use computer software to nmode suh contact points. Such contact residues and neighboring residues are candidates tor substatution according to the techniques elaborated herein. Once such varants are generated, ihey are subjected to screening as described herein an tiboies with superior properties Un one or more relevant assays may be selected for further development. Antibody h modded abohydrat [00208 Antibody variants can also be produced that have a modified glycosylation patten relative to the parent antibody, for example, adding or deleting one or more of the carbohydrade moieties bound. to the specific binding agent or antibody, and/or adding or deleting one or more glycosyation sites in the sp binding agent or antibody. 002091 Glycosylation of po lypep tides inciudng antibodies is typically either N-linked or ( inked, N1inked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-sere and asparagine-X threome, where X is any amina acid except police, are the recognition sequences fur enzymatic attachment of the carbohydrate moiety to the asparagine side chain. The presence of either of these tripeptide sequences in a poiypeptide creates a potential glycosylation site, Thus, N-linked glycosylation sites may be added to a specific binding agent or antibody by aterin the amino acid sequence such that it contains one or more of these tripeptide sequences. -linked glycosylation refers to the attachment of one of the sugars N aceylgalactosamine, galaciose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5hydroxylysine may also be used, C-linked glycosyiation sites may he added. to a specific binding agent or antibody by inserting or subsituting one or more sermne or threonine residues to the sequence of the original specif ic bindngu aendt or antibody, Atered Effctor Funtion 1002101 Cysteine residue(s) may be removed or introducedin the F! region of an antiboy or Fe-containinig polypeptide, thereby eliminating or increasing mterchain disulfide bond formation in this region, A homodimer speWcific binding agem or antibody thus generated may have improved internalization capability and/or inreased complement mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) See Caron et a, J Exp. Ae, 176:1 1914195 (1992) and Shopes, Bi inunal 148: 2918-2922 (1992). Hondimeric specific binding agents or antibodies may also be prepared using heterobhimctionai cross-linkers as described in Wolff et al. Cancer Research, 53:2560-2565 (1993), Aternatively, a specific binding agent or antiody can be engineered which has dual Fe regions and may thereby have enhanced complement lysis and ADCC capabilities See Stevenson et al Anti-Cancer Drug: Design 3:2i9-230 (1989). low 2 1] 1t has been shown that sequences within the CDR can cause an antibody to hind to MFIC Class it and trigger an unwanted helper T-cell response A conservative sbsAtion can allow the specific binding agent or antibody to retan btding activity ye reduce its abibty to trigger an unwanted ,-el response, it is also contemplated that one or more of the N-terminal 20 amno acids of the heavy or lght chain are removed, 1002121 in some embodinmnts, production of antibody molecules are contemplated with altered carbohydrate structure resulting in altered effctor activity, including antibody molecules with absent or reduced fucosylation that exhibit improved ADCC activity, A variey of ways are known in the art to accomplish this. For example, ADCC effector activity is mediated by bindmg of the antibody molecule to the FeyRIll receptor, which has been shown to be dependent on the carbohydrate structure of the N-linked glycosylation at the Aan 297 of the CH2 domain. Non-teosylated antibodies ind this receptor with increased affinity and trigger FcyRliLmediated e'ffetor functions more cfiiciently than native, fucosylated antibodies. For example, recombinant production of non-fheesylated an'tdy in CH-O cYlIs 60 in which the alph I ,6-fucosyi transferase enzyme has been knocked out results in antibody with 0fold increased ADCC activity (Yamaneohnuki et aL, Biotechnol Bioeng. 2004 Sep 5;87(5t):64-22), Similar effects canb beomplished through decreasing the activity of this or other enzymes in the fucosylation pathway, eg, through siRNA or antisense RNA treatment, engineering cell lines to knockout the enzyme(s), or culturing with selective glycosylation inhibitors (Rothnan et al, Mol hmmunol 1989 Dec;26(12) 13-23), Some host cell strains. eg. Leel3 or rat hybridoma YB2/0 cell line naturally produce antibodies with lower fucosylation levels. Shields At al, J Biol Chent 2002 Jul 26;27(30):26733-40; Shinkawa et alj I Biol Chem, 2003 Jan 3 1 278(5)346673, An increase i the level of bisected carbohydrate.g. through recombinantly producing antibody in cells that overexpress OnTill enzyme, has also been determined to increase ADCC activity. Umana et at, Nat Biotechnol 1999 Feb; 1(2:1 680. it has been predicted that the absence of only one of the two fuoose residues may be suficMent to increase ADCC activity. (Ferrara et at, J Bio ChM 205 Dec 5), Other oventModificadons [0021 3 Covalent modifications of a polypeptide, or antibody are also included within the scope of this invention, They may be made by chemical synthesis or by enzymatic or chemical cleavage of the polypeptide or antibody, if applicable. Other types of covalent nodliations can be introduced by reacting targeted amino acid residues with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-temnual residues |02141 Cysteinyl residues most commonly are reacted with a-haloacetates (and corresponding amines) such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are dervatized by reaction with bromotrd uoroacetone, alpha.bromo-0(5 imidozoypropionic acid, chloroacetyl phosphate N-.alk ylnalens des, 3-nitro,2-pridyl disulfide, methyl 2-pynidyl d isuifide, p-. chIoromnercunbenzoate, 2-chloromercuri-4-nitrophenol, or chlorm7nitrobenzo-2-oxal3 1002151 Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain, in some embodiments, para-bromophenacyi bromide also is useful; and the reaction is performed in 0l M sodim acdylte at pH 6.0 ~6 1002161 Lysiny and amino-ermnal residues are reacted with succinic or other carboxyh acid anhydides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivanizing .lphaamnino conainmg residues include imidoesters such as methyl picolinimidate. pyridoxal phosphate pyrndoxal, cloroborohydride, trimtrobenzenesulfonic acid, O-methylisourea, 2,4 pentanedione, and transamase-catalyzed reaction with glyoxylate. 1002171 Arginyl residues are modified hy reaction with one or several conventional reagents, among them phenyglyoxal, 23-butanedione, 1-cyclohexanedione, and ninhydrin Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKi of the guanidine fuctiona' group. Furthermore, these reagens may react with the groups of lysine as well as the arginine epsilon-amino group 1002181] The specific modification of trosyl residues may be made, with particular interest i introducing spectral labels into tyrosyl residues by reaction with aromatic di aaonium compounds or tetrani tromnethane, Most commonly, N -acetyhnmidizolie and tetranitromnethane arc used to form 0-acetyl tyrosyi species and 3nitro derivatives, respectively. Tyrosyl residues are odumated using Ql or 'Ito prepare labeled proteins for use in radiommunoassay, 00 2 191 Carboxyl side groups (asparty- or glutamyl) are selectively modified by reaction with carbodiimides (R Ndbd.C~dbd.N-R, where R and R are different alkyl groups, such as I-cyclohexyb-3- (2-morpho linyh-4-ethyl) carbodiimide or 1-ethyl-3K4-azomna-4+4 dimethylpentyl) carbodimide. Furthermore, aspartyl and glutamyl residues are converted to asparag iny and glutaminyl residues by reaction with ammonium ions. 100220 Giutamninyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. These residues are deamidated under neutral or basic conditions. The deamidated form of these residues falls within the scope of this invention, 1002211 Other modifications include hydroxylation oproine and lysine, phosphorylation of hydroxyl groups of serl or threonyl residues, methylation of the .alpha, amno groups of lysine, arginine, and histidine side chams (T, E Cretghton, Proteins; Structure and Molecular Properties, WiH, Freeman &. Co, San Francisco, pp. 79-86 (1983)) acetylatton of the N-ermninal amine, and amidation of any 0-terminal carboxyl group. 10022.21 Another type of covalent modification involves chemically or enzymatically coupling glycosides to the specific binding agent or antibody. 'These procedures are advantageous in that they do not require production of the polypeptide or antibody in a host -62 cell that has glycosylation capabilities for N-~ or C-linked glycosyiation, Depending on the coupingrmode used, the sugar(s) may be attached to (a) argiine and histide, (b) free carboxyl groups, (c) free sulthydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine threonine, or hydroxyproine, (e) aromatic residues such as those of tenaylaJanitne, yrosine, or tryptophan. or (f) tbe aide group ot glutamine. These methods are described in WOST5330 published I Sep. 1987, and in Aplin and Wristor. CR Cit Rey Bochem, pp. 259-306 (1981), 1002231 Removal of any carbohyrate moieties present on the polypeptide or antibody nmiay be accomplished chemically or enzymnatically. Chemical deglycosylation requires exposure of the specific binding agent or antibody to the compound trifluoronethanesulfonic acid, or an equivalent compound, This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosaine or N-cetylgalactosamine) while leaving the specify binding agent or antibody intact. Chemical deglcoslation is described by H akimuddm,~ et al.., Arch, Biochem, Biophysw 2.59: .52 (1987 ) and by Edge et al. AneL Bicsh e, I i8: 131 (1981). Enzymatic cleavage of carohydate moieties on a speci ic bindng agent or antibody can be achieved by the use of a varty of endo- and exo glycosidases as described by Thotakura et AlW Meh. EMyro, 138: 350 (1987). 90224 Another type of covalent modification of an anti-hepcidin an y body described herein comprises iing the polypptde, speci nd ig agent or antibody to one of a variety' of nonproteinaceous po.lymers, eg. polyethylene glycol, polypropylene glycol, polyoxt*hylated pol yola, polyoxyethylated sorbitol, polyo>tyethyated$ glucose, pol yoxyvethyat'ed glycerol, polyoxyalkylenes, or polysacchar ide polymers such as dextran, Such mnethods are known in the art, see, e~g. U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,7 ,7 .,791,i192. 4,179,337, 4,766106, 4,1~ 9337, 4,49,285 4,609546 or EP> 315 456. IAGNOST 'C METHODS9 FOR HEPCIDINIRELATE' lD DISORDERS ANDP MONITORING OF THERAPY WITH AN~T1-EPCIDEN ANTIBODIAES [ 00225) in another aspect, a. method is provided of detecting human bepcidin in a sample, comprising contacting a sample from a human wit any ot'the afrorenmentioned antibodies under condations that allow binding cf te anitody to human hepcidin, and detecting the bound antibody, in on~e embodiment, a firs atibody to hepcidin is immlobihzed on a so lid support, as a capture reagent, and a second antibody to hepcidin is used as a detetion reagens, in a related aspect the amount of hepeidin in the sample is quantitated by 639 measurng the amount of the bound antibody, The detection methods can be used in a variety of diagnostic. prognostic and monitoring methods, including methods of dianosing a hepcidin-eated disorder, methods of differentiating an inflammatory disease from, a non~ inflannatory disease and methods of monitoring therapy with an antiInhepidin antibody. in such methods, a level of hepcidin above a certain threshold is correated with the presence of hepcid irrelated disorder, such as hepcidin-related anemia, while a level below said threshold indicates that the patient is unlikely to have hepcidin-reiated disorder. Similarly, a level of hepcidin above a certain threshold is correlated with the presence of an inflarmnatory disease, while a level below said threshold indicates that the patient is unlikely to have an inflanmmatory disease In some embodiments, such methods will diagnose patients having iron defciency anenia, aneua of mflammation or mixed anemia, For monitoring of therapy aimed at suppressing hepcidin levels, a level of hepcidin below a certain threshold indicates that the dose of hepcidin antibody is therapeutically effective, and a level above said threshold indicates that the dose of hepcidin antibody is not therapeutically effective 100226] Also provided are methods for diagnosing bepcidnreiated disorders. soon as hepcidin-related anemia, or other diseases of hepcidin excess or hepcidin deficiency, and for monitoring the effectiveness of therapy for such a disease, including therapy with an anti hepcidin antibody described herenm To determme the presence or absence of hepcidi-reiated anemia, a biological sample from a patient is contacted with one or more of the ant-hopcidin antibodies disclosed herein under conditions and fOr a thme sufficient to allow imunoomplexes to term. immunocomplexes fOrmed between an anthepridin antibody and hepcidin m the biological sample are then detected. The amount of hepcidin in the sample is quantitated by measuring the amount of the inmunocomplex formed between the antibody and hepridin. Within certain methods, a biological sanple is isolated from a patient and is incubated with. one or more of the anti -hepidin antibodies disclosed herein, and the level of the antibodyhepoidin comnpex above a certain threshold is correlated with the presence of hepchdin-related anemia, and a level below said threshold indicates that the patient is unlikely to have hepcidirreated anemia. For example, a level within the normal range indicates the patient is unlikely to have hepeidin-related anemia. Normal range of serum hepidin is generally less than 10 ng/ml when determined by certain assays, ie., mass spectrometry techniques described in co-owned U.S, Patent Application No. 1 /880,313 and international Publication No. WO 2008/011158, the disclosures of which are incorporated herein by reference in teir entirety, but vill ary depending on the assay and depending on the subset of population tested, 64- )10227| Also provided are methods for differentiating an inflammatory disease from a non-inammatory disease. To determine the presence or absence of an inflammatory disease, a biological sample from a patient is contacted with one or more of the anti-hepcidin antibodies disclosed herein under conditions and for a time sufficient to allow immunocomplexes to for. Various inrunoassays known in the art can be used, including but are not imited to; competitive and noncompetitive assay systems using techniques such as radtoirnunoassays, ELISA (enzyme linked im munosorber assay) "sandwieif immaunoassays, imnoradionmetric as'says, gel diffusion precipitation reactions, immunodiffhsion assays, in situ immunoassays (using eolloidal gold, enzyme or radioisotope labels for example), Wester blots, precipitation reactions, agglutination assays (e.g, get agglutination assays, benaggutination assaysk cornplement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc, In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. in a further embodiment, the secondary antibody is labeled, Many means are known in the art for detecting binding in an .imunoassay and are within the scope of the present invention. Antibodies: A Laboratory Manual ( 9988) by Harlow & Lane or more recent editions; hmmimoassays: A Practical Approach, Oxford University Press, Gosling, I P. (ed,) (2001) or more recent editions; and/or Current Protocols in Molecular Biology (AusObel et at), which is regularly updated, Examples of such assays usually involve the antibody attached to a surface or matrix, patient serum added and time allowed for a complex to form suitable washing procedures to remove unbound complex followed by either the addition of a second antibody to allow detection of the complex (a sandwich ELI SA) or a detectable version of hepcidin to detect free hepcidin binding sites on the antibody surface (a competition E LISA), The level of hepcidi, as detected by the foregoing methods, above a certain threshld is correlated with th~e presence of' an inflammatory disease, and. a level below said threshold indicates that the patient is unlikely to have art inflanmmatory disease, A patient is unlikely to have an inflammatory disease when the hepeidin level is within the nomtal range. A patient is likely to have an inflammatorydisease when the hepeidin level exceeds the normal range, for example 20 ng/mi, in particular, when the leve is between 2.0 and 1000 Og/mI Exemplary hepcidin-related inflammatory diseases include anemia of cancer, anemia of chronic disease, anemia of inflammation, chemotherapy induWced anemia, chronic kidney disease (stage 1, 11, 1l, IV or V) end stage renal disease, chronic renal failure congestive heart failure cancer, rheumatoid arthritis, systemic lupus -65- Croh's dAs S perincion or Other hacterial HlY, and other viral illnesses, atiosclerosis, atherosclerosis, cirrhosis of the liver, pancreatitis. sepsis, vasculiis, iron-deficiency, hypochromic microcytOc anemia and conditions with bepeidn excess. 100228 Within other methods, a biological sample obtained from a patient is tested for the level of hepcidin. The biological sample is incubated with one or more of the ant hepcidin antibodies disclosed herein under conditions and for a tume sufficient to allow immnunocompexes tonrm. immunocompiexes formed between the hepcidin and anibodies in the biological sample that specifically bind to the hepcidin are then detected. A biological sanpa or use within such methods may be any sample obtained frn a patient that is expected to contain hepeidin, Suiable biological samples include blood, sera, plasma, urine and bone narrow. Suitable antibodies include antibodies from hnman cells, rodent, rabbit, goat, came or any other species. 002291 The biological sample is incubated with antibodies in a reaction inxture under conditions and fR a time sufficient to permit imiunocomplexes to forn between hepcidin and antibodies that are immunospecific for hepcidin For example, a biological sample and one or more anti-epcidin antibodies may be incubated at 4*C for 24-48 hours. 100230 Following the incubation, the reaction mixture is tested for the presence of imuno-compl exes, Detection of inmmunocomnplexes formed between an anti-hepcidin antibody and hepcidin present in the biological sample may be accomplished by a variety of known techniques, such as radioimmnunoassays (RiA) and enzyme linked immunosorbent assays (ELISA), Suitable assays are wel known in the art and are amply described in the scientific and patent literature (Harlow and Lane, 1988) Assays that may be used include, but are not limited to, the double monoclonal antibody sandwich immnoasay technique (U .S. Pat. No. 4,376,110); monoclonal-polyclonal antibody sandwich assays (Wide L, "Solid Phase Antigen-Antibody Systems" Radioimmunuossay Methods: European workshop September /5 /7 / 970 Edinburgh, Kirkham and Ihnter, eds. (Churchill Livingston, Edenburgh, (1971)) pp. 405-412; the "Vestern bloc method (U.S Pat, No. 4,45291); imnunoprecipitation of labeled igand thrownn et al J Biad (het., 4980-493m IP98); enzyme-inked immiunosorbenm assays, immunocytochemical techniques, including the use of fluorochromies (Brooks et al, Cll Exp, jtmmunn, 39: 477, 1980) and neutraliztion of activity (Howen Pope et al. Science, 226:701703,1984), Other inmunoassays include, but are not limited to those descnbed in US. Pat, Nos. 3U50,752; 3,901,654;3,9350,74; 3,984533; 399634 4.034,074; and 4,098,876. -66 [00231 For detection purposes, an anihepcidin antiody may either be labeled or unlabeled. Unlabeled antibodies rnay be used in agglutination assays or in combination with labeled detection reagents that hind to the immunocomplexes (e.g, anti-imnoglobulin, protein (, Protein A or a lectin and secondary antibodies, or antigen-binding tragments thereof, capable of binding to the antibodies that specifically bind to the hepcidin. If the anti hepcidin antibody is labeled, the reporter group may be any suitable reporter group known in itopes fluorescent groups (e.g. fluorescei or rhodamine), luninescen groups, enzymes, biotin and dye particles, Labels that are themselves directly detectable include fluorescent or luminescent dyes, metals or metal cheiates, electrochemnical labels, radionuclides (e.g, 32P, 14C 1251 3W, or 131), magnetic labels or beads (e., DYNABEADS) paranagnetic labels, or coloinetric labels (e.g., collodal goldteol ored glass or plastic beads). Such detectable labels nay be directly conjugated to the anti-hepcidn antibody or detection reagent or may be associated with a bead or particle that is attached to the antihhepcidia antibody or detection reagent, Labels that are detectable through binding of a labeled specific binding partner include biotin, digoxigenin, maltose, oligohistidine, 2,4 dinitrobenzene, phenylarsenate, ssDNA, or dsDN A). Indirect labels that can be indtrectv detected by their production of a detectable reaction product include various enzymes well known in the art, such as alkaline phosphatas, horseradish peroxidase, p-galactosidase, xanthine oxidase, glucose oxidase or other saccharide oxidases, or luciferases which cleave appropriate substrate to frmn a colored or fluorescent reaction product, 1002321 Within certain assays, an unlabeled ant-hepcidin antibody is immobiized on a solid support. fbr use as a "capture agent" (or reagent) that captures the hepeidin within a biological sample, The solid support may be any material known to those of ordinary skill in the art to which the antibody may be attached For example the solid support may be a test well in a rnicrotier plate or a nitrocellulose or other suitable membrane, Alernatively, dhe support may be a tube, bead, particle or disc, such as glass, fiberglass, latex or a plastic material such as polyethylene, polypropylene, polystyrene or polyvinylchloride or a porous matrix Other natenals micde agarose, dextran, polyacrylanide, nylon, Sephadex, cellulose or polysaccharides, The support may also be a magnetic particle or a fiber optic sensor> such as those disclosed, for example, in US. Pat, No, S,359,681. The immobilized ant-hepcidi antibody may he a polyclonal antibody, or one or more monoclonal antibodies such as those described herein, or a combmation of polyclonal and one or more monoclonal antibodies, The artibody may be irrobilizd on the solid support using a variety of techniques known to those of skil in the art, which are amply descrbed in the patent and scientific literature. In - 67 the cortemt4 the present invention, the ter, mobli refers to both noncowtlent assocedion such as adsorption. and covaent attachment (which may be a diect linkage between the artgen and fnional groupsonthe support r may be a linkage by may of a crosAinking agent), Immobilization by adsorption to a well in amcrOtitet plate or to a membrane is contemplated. In such case may e achieved by contacting the ant hepeidin antibody in a suitable buffer, wh the solid support for a suitable amount of time. The contact tme varies with temperatum, but is typically between about I hour and about day. In general cortactig a well of a plastic nuicrotiter plate (including pcdystyrene or pyiylchloride) with an amount of peptide ranging from about 10 ng to about ,ug, about 100 ng to about i pg, is sufcient to immobilize an adequate amount of peptide. 00 233| Following immobilization, the remaining protein binding sites on the support are typical blocked. Any suitable blocking agent known to those of ordmary skill in the art, icludmg bovine serum albumin, TweenT 20"M (Sigma Chemical C t. Louis. Ma), heat inactivated normal go t erum (NGS), or 'LOTTO (buffered solution of nonfat dry milk which also contains a preservative, salts, and an antitoaing agent) can be used. Thre supot is then incubated with a biological sample suspected of containing bepeidn. The sample can be applied neat or, more often, it can b diluted, usually in a buffered solution which contains a small amount (0,%5 0% by weight) ot protein, such a BSA, NGS, or BL OTTO. in general an appropriate contact time (ie, incubation time) is a period of time that is sufficient to detect the presence of antibody oar n aten finding ment that is immunospecific fo the hepcidin within a sample containing hepcidin. In some embodiments, the contact ime is sufcient to achieve a level of binding that is at teast about 95% of that achieved at equilibrium between bound and und und anybody or antibody fragment, Those of ordinary skill in the art will recognize th'at he time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of' time, At room temperature, an incubation time of about 30 minutes is general sufficient 1002341 Unbound sample may then be removed by washing the solid support with an appropriate buffer,such as P containing 0,1% TweenM 20 A detection reagent that binds to the hepeidin in the immunocomplexes (formed by binding of the capture agent and the hepeidin from the sample) may then be added, Suc.h detection reagent may be a polyclonal antibody, or one or more monoclonal antibodies such as those described herein, or a combination of polyclonal and one or more monoclonal antibodies such as those described herein or Fab fiaction of any antibody The detectun reagent ay be directly abed, comprises atleatirst detectable label or porter" molecule AMemntivelythe detectio reagent may be an labeled anti-bepeidin antibody, This unlabeled anti-hepcidin (primary) antibody n then detected by the binding of a labeled secondary antibody or reagent to the piayantibody. For examl, fthe primary antibody is a murine immutnogobulin the primary nniy FrIarnple. if o secondary st body may be a labeled annmurie immunoglobulin antibody, Smiiarly, it the primary antibody is a rabbit immunoglobulin, the secondary antibody may be a labeled anti rabbit inmmnunoglobulin antibody. 100235] The detection reagent is incubated with the tmmunocompex fr an amount of tine sufficient to detect the bound antibody or antigen binding fragment thereof An appropriate amount of ine may generally be determined by assa h level of binding that occurs over a period of time. Unbound label or detection reagent is then removed and bound label or detection reagent is detected using a suitable assay or analytical instrument The method employed for detecting the reporter group depends upon the nature ofthe reporter group. For radioactive labels, sctinlation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent or chemiummnescent moieties and various chronogens, fluorescent labels and such like. 13iotn nay he detected using avidin, coupled to a different reporter group (coIonly a radioactive or fluorescent group or an enzyme), Enzyme reporter groups (including horseradish perox idase, pgalactosidase. alkahne phosphate and glucose oxidase) may general be detected by the addition of substrate (generally for a speciti period of time)b fllow edby specroscopic or other analysis of the reaction products. Regardless of the specifiemethod employed, a level of bound detection reagent that is at least two fold greater than background (i.e, the level observed for a biological sample obtained from an individual with a normal level of hepeidin) indicates the presence of a disorder associated with expression of hepcidin 1002361 in alterative enbodrncnts. the sample and detection reagent may be contacted simult aneously with the capture agent, rather than sequentill added, In yet another alternative, the samle and detection reagent may be prc-incubated together, then added to the capture agent. Other variations are ready apparent to one of ordinary skll m the art 1002371 in another embodiment. the amount of hepcidin present in a sample is determined by a competitive binding assay. Competitive binding assays rely on the ability of a labeled standard (e.g. a hepidin polypeptide, or an immunologically reactive portion thereof) to compete with the test sample analyze (a hepeidin polypeptide) for binding with a limited amount of an ani-hepcidin anybody. Following separation of free and bound hepeidin, the hepein is quantitated by relating ratio of hound/unbound hepcidin to known standards, The amount of a hepcidin polypeptide in the test sample is inversely proportional -69 to the amount ofstandard that becomes bound to the antibodies. To facilitate determining the amount of standard that becomes bormd, the antibodies typically are inmobilized on a solid support so that the standard and analyte that are bound to the antibodies may conveniently he separated from the standard and analyze which remain unbound. Thus, in such eribodinents, also contemplated is contacting a biological sample with labeled mature hepctdin (or a beled framntv thereof that retains the antigenicity of hepcidin) and an antibody that binds to mature hepeidin, and detecting the arnount of arnibody-labeled hepcidisn complex formed, 1002381 Preparation of conugates to solid supports or detectable abels often comprise the use of chemical cross-inkers, Cross-linking reagents contain at least two reactive groups, and are divided generally into honofunctional cross-inkers (containing identical reactive groups) and heterounctional cross-nkers (containmg nowidentical reactive groups). Homnobifunctional cross-linkers that couple through anmines, sulihydryls or react non speefically are avail able from many commercial sources Maleimides, alkyl and arvi halides aipha-haloacyls and pyridyl disuifides are thiol reactive groups, Maleimdes, alky1 and aryl hades, and alphai-haloacyls react with sulthydryls to formn thiol ether bonds, whereas pyrndyl disulfides react with suifhydryls to produce mixed dtsuifides, The pynidyl dtstdfide product is cleavable. imidoesters are also very useful for proteinprotein cross-inks. 00239| Heterobifunctional cross-linkers possess two or more different reactive groups that allow for sequential conjugations with specific groups of proteins, minimizing undesirable polymerization or self-conjugation. Heterobitbnctional reagents are also used when modification of amines is problematic, Amines may sometimes be found at the aetve sites of macromoleeuies, and the modification of these may lead to the loss of activity, Other moieties such as sulfhvdryls, carboxylk, phenols and carbohydrates may be more appropriate targets. A two-step strategy allows fr the coupling of a protein that can tolerate the modification of its amines to a protein with other accessible groups. A variety of heterabi functional cross-linkers, each combining different attributes for successful conjugation, are commercially available. Cross-linkers that are amine-reactive at one end and sulThydryl -reactive at the other end are quite common, if using heterobifurnctional reagents. the most labile group is typically reacted first to ensure effective cross-inking and avoid unwanted polymerization. 1002401 As described in copendng US Patent Application No. 12/022,51l the disclosure of which is incorporated by reference herein in its entirety, it is the level of mature hepcidin aino acids 60-84 of SEQ ID NO: 8) rather than the level of prohepeidin (amno acids 2584 of SEQ iI NO: 8) which is diagnostic for certain disease states such as anemia of -70inflammation and anemia of cancer Thus, in one preferred embodiment, antibodyfies) that bind to matur, property toded, hepeidn (SEQ iD NO: 9) are used as both capture agent and detection reagent Antibodies that bind to the naturaly occurring N-terminaly truncated versions (eg. lacking up to two or up to five of the N-tenninal amino acids of mature hepcidin) may also be used. Various combinations of capture agent and detection reagent are contemplated, For example, the capture agent may be a monoclonal antibody that bids to a first epitope of mature hepeidin and the detection reagent may be a different monoclonal antibody that binds to a second epitope of mature hepcidin. in some embodiments, antibodies specific for different epitopes of hepidin are used, in order to minimize competition or intertorence between the capture agent and detection reagent. Aternati vely, the capture agent may be a polycional antibody that binds to mature hepcidin and the detection reagent may be a monoclonal antibody. As yet another alternative, the capture' agent may be a monoconal antibody that binds to mature hepcdin and the detection reagent may be a polyclonal antibody. in any of the preceding embodiments, either the capture agent or the detection reagent may be a combination of a polyclonal and a monoclonal antibody, |002411 in sone embodiments a mature-hepcidin-pecnic monoclonal anmibody is used as either the capture agent or detecion reagent or both A mature-hepcidinspecific antibody does not bind prohepeidin at all, or binds to probepeidin with such low affinity that the antibody can differentiate mature hepcidin from prohepcidin. For example, such a nmonoonal antibody may bind to the N-termmus of mature hepeidin, or it may bind an epitope of mature hepcidin that is not detectable in prohepcidin (eg due to masking by the prodomain). j00 2421 in embodiments utihzmg a monoclonal antibody that binds to an epitope present in both mature hepcidin and prohepcidin, an optional further refinement is contemplated, The amount of mature hepcidin alone is determined by subtracting the amount of probepcidin present in the sample from the amount of total hepeidin (prohepoidin plus mature hepeidin) present in the same sample. The amount of prohepcidin can be determined by using prohepcid mn-specifi polyclonal and/or moonaional antibodies in techniques like those: described above, A prohepcidin-specific antibody does not bind mature hepcidin at all or binds to mature hepcidn with such low affinity that the antibody can differentiate probepcidim from mature hepeidin. For example, such antibodies may bind to a linear or conformati onal epiope present. uniquely in the prodomnain of hepcidin (amino acids 25-59 of SEQ ID NO: 8). In such embodiments, the amount of total hepeidin and prohepeidin may be determined sequentially or simultaneously. Because prohepridin is rapidly degraded im serum w71 " to hepcadin, in ome fr inhibitors are added to the biological sample in order to prevent or reduce degradation of prohepcidin. [0024 31 In some embodiments utilizing a monoclonal antibody that binds to the 25 armno acid mature hepcidin, the monoclonal antibody does not bind the degradation products (i e. hepcidim22 and hepcidin-20), 100244i in one embodiment of a simultaneous assay for detecting total epdin and prohepcidin, the capture agent is an. antibody that binds to an epitope present in both mature hepcidin and prvhepcidin, and two detection reagents are apphed simultaneously, The first detection reagent is a labeled antibody that binds to an epitope present in both mature bepeidim and prohepcidin and the second dteteeton reagent is a differently labeled prohepcidin-speciic antibody, For example, the first detection reagent is labeled with a fluorescent dye detectable at a frst wavelength while the second detection reagent is labeled with a fluorescent dye deteciable at a second wavlength. Thus, in such an example, the capture agent vill bind total hepcidin (mature hepeidin plus prohepeidin) in the sample, the first detection reagent will detect the amount of total hepcidin, and the second detecton reagent will detect the amount of prohepcidin. Subtracting the amount of prohepidin from amount of the total hepciin will yield the amount of mature hepcidin, in other alternative embodiments, two different capture agents may be used; a first capture agent that binds to an epitope present in both mature hepcidm and prohepidm, and a second capture agent that is a prohepcidit-specifie antibody, optionally with a detection reagent that binds an epitope present in both mature hepcidin and prohepci dm, 1002451 Other embodiments for carrying out simultaneous assays are well known in the art includNg the multiplex system described e.g. in Khan et a', Cln. Vaccine Immunal, 13(1) 45-52 (Jan.2006) involving differentially coded sets of fluorecent microbeads, Other embodiments for performing multiple simultaneous assays on a single surfce include surfaces having a plurality of discrete, addressable locations for the detection of a pluraliy of different analytes, Such fonmats include protein microarrays, or "protein chips (see, eg, Ng and 1lag, 1. Cell Mol. Med, 6: 329-340 (2002)) and capillary devices (see, eg,, U.S. Pat, No, 6,{19,944), In these embodiment, each discrete surface location has a different antibody that imnobiiizes a different analyte for detection at each location. Surfaces can ahernatively have one or more discrete particles (e.g, microparticles or nanoparticies) immbilized at discrete locations of a surface, of which each set of particles contains a different capture agent for a different analyte.
100246 Cornpiementary antibody pairs (antibodies that bind to different eptopes on hepcidn such that the pairs are suitable for use in sandwich assays) were diffeul to identify, Use of complementary pairs that minimize competition or interference can increase sensitivity of the assay by 20-fold to 50-oid. In some embodiments, the immunoassays described herein are capable of measuring hepcidin levels ranging from 0 01 ng/mL to 10 pg/rmLn 100247} Antibody pairs suitable for use in sandwich immunoassays include the following: (i) when one antibody of the pair is an antibody binds to the same epitope as antibody is IS i, or competes wath antibody S1 forl binding to mature human hepcidin of SEQ 1D NO: 9 by at least about 75%, 80%, 85%, 90% or more, a suitable second antibody may be: (a) an antibody that bnds to the same epitope as antibody is 23F1 1 or competes with antibody 23F i for binding to mature human hepcidin of SEQ 1D NO: 9 by at least about 75%. 80%. 85%. 90% or more: or (b) an antibody that bnds to the Same epitope as antibody is 15E i. or conmetes with antibody 15E1 for bindig to mature human hepcidin of SEQ ID NO: 9 by at least about 75%, 80%, 85%, 90% or more; or (c an antibody that binds to the same epitope as antibody is 12B9, or competes with antibody 129 for binding to mature human hepcidm of SEQ [D NO: 9 by at least about 75%, 80%, 85%, 90% or more; (') when one antibody of the pair is an antibody that binds to the same epitope as antibody 12139 or competes with antibody 12B9 Pf binding to mature hman hepeidin of SEQ ID NO: 9 by 'at lstout 75%, 80%. 85%, 90% or more. a suitable second antibody may be: (a) an anybody that binds to the same epoope as antibody 8DS, or competes wih antibody 18D for binding to mature human hepcidin of SEQ iD, NO: 9 by at least about 75%, 80%, 85% 90t or more, orb) an antibody that binds to the same epitope as antibody 1901 or competes with antibody 19C1 for binding to mature human bepeidin of SEQ 1D NO: 9 by at least about 75%, 80%, 85%, 90% or more, or (c) antibody that binds to the same epitope as antibody 19D12, or competes with antibody 19112 for biding to mature human hepcidir of SQ (D NOS 9 by at least about 75%, 8%, 85%, 90% or more, or (d) an antibody that binds to the same epitope as antibody 1916, or competes with antibody k,16 for binding to matu human hepcidin of SEQ 11) NO, 9 by at least about 75%, 80%, 85%, 90%Y or more; or (c) anybody that binds to the same epitope as antibody 1St or competes with antibody 1S I for binding to mature human hepoidin of SEQ ID NO: 9 by at least about 75% 80%, 85%, 90% or more; or (3) when one antibody o the pair is an antibody that binds to the same epitope as anibody 23F11, or competes with anibody 23H 1 for binding to mature human hepcidin of SEQ ID NO: 9 by at least about 75%, 80%, 85%, 90% or more, a suitable second antibody may be: (a) an antibody that binds to the same epitope as antibody 1808, or competes with antibody 18DS for binding to mature 73 human hepcidini of S EQ ID NO: 9 by at least about 5%, 80%, 85%, 90% or more, or (b) an antibody that binds to the same epitope as anybody 19C, or competes with antibody 19C1 for binding to nature human hepeidin of SEQ ID NO: 9 b at least about 75%, 80%, 85%, 90% or more, or (c) an antibody that binds to the sarne epitope as antibody I9D2, or competes with anmibody 19D2 for binding to mature human hepidin of SEQ ID NO: 9 by at least about 75%, 80%, 85%, 90% or more, or (d) an antibody that binds to the same epitope as antibady 1986, or competes with antibody 191H6 for binding to mature human hepcidin of SEQ ID NO: 9 by at least about 75%, 80%, 85%, 90% or more; or (e) an antibody that binds to the same epitope as antibody I11 or competes with antibody 4EI for binding to mature human hepcidm of S EQ D NO: 9 by at least about 75%, 80%, 85%, 90% or more; or (0 an amtbody that binds to the same epitope as antibody 383 or competes with antibody 383 for binding to mature human. hepcidin o f SEQ ID NO: 9 by at least about 75%, 80%, 85%. 90% or more; (4) when one antibody of the pair is an antibody binds to the sane epitope as an tibody V5E, or competes with amibody 15 El for binding to mature human bepcidin of SEQ "D 80%, 85%, 90% or more, a suitable second antibody may be: (a) an antibody that binds to the same epitope as antibody 1S]_ or competes with antibody IS I for binding to mature human hepcidin of SEQ iD NO: 9 by at least about 75% 80%, 85%, 90% or more, 1002481 in soni embodiments, methods for monitoring the ewftiveness of therapy wth an ant-hepcidin antibody include monitoring changes in the level of hepcidin in a sample, or n an animal such as a human patient, Methods in which hepeidin levels are montored may comprise (a) incubating a irst biological sample, obtained from a patient pror to a therapy with one or more of the ant hepcidin antibodies disclosed herein, wherein the incubation is performed under conditions and for a time sufficient to allow immunocomplexes to form; (b) detecting immunocomplexes formed between the hepcidin in the biological sample and antibodies or antigen bindmg fragments that specifically bind hepeidin; and optionally (c) repeating steps (a) and (b) using a second biological sample taken f the patient at later time, such as for example, following therapy with one or wore of the anti hepeidin antibodies disclosed herein; and (d) comparing the number of immunocomplexes detected in the first and second biological samples, [00249 Other momiormg methods include measuring (a.) the blood (egg serum or plasma) circulating level of complexes between hepcidin and the therapeutic agent, and optionaly (b) the amount of freehepcidin presentin circulati For example complexes between bepeidinand therapeutcanibody can be detected using an ant-humancano 74 A that binds to the therapeutic antibody part of the complex and an Lab fragment of a "pairing" anti-hepcidin antibody that binds to the hepcidin part of the complex. Alternatively, an ant idiotypic antibody can be used in place of the antihurnan Fe antibody. As another ahematve, an anti-hepcidin antibody contaimng a non-human Fc (e. a human Fe is replaced with mnurinec Fe) can be used in place of the Fab agentn, o0(02504 As another example, free hepcidin can be detected after removmg hepoidm therapoutic antibody cornplexe f-om the biological sample, usin either an anti-human Fe antibody or an anti-idiotypic amibody that has been imobilhzed on a solid support The amount of free hepcidin which remains unbound to the solid support is then measured. This level of ree hepadin may reflect the effectiveness of the therapeutic antibody in removing available circulating hepeidin, 1002511 A biological sample ior use within such methods may be any sample obtained from a patient that would be expected to contain hepidin. Exemplary biologicalsanples include blood, plasma, sera, urne and bone marrow, A first biogical sample may be obtained prior to initiation of therapy or part way through a therapy regime, The second biological sample should be obtained in a similar nner, but at a time following additional therapy. The second biologist sample may be obtained at the completion of or part way through, therapy, provided that at least a portion of therapy takes place between the isolation of the first and second biological samples, 100252{ Incubation and detection procedures for both samples may generally be period as described above. A decrease in the number of immunocomplexes in the second sample relative to the first sample indicates a decrease in hepcidn levels and reflects successful therapy Free serum hepcidin may also be analyzed in a similar manner, and a decrease in free serum hepcidin indicates successful therapy, 1002534 Hepeidin-related disorders, inflammatory diseases, and diseases or disorders of iron homeostasis for which the diagnostic or monitoring methods may be useful include but are not limited to african iron overload, alpha thalassemia, Alzheimer's disease, anemia, anerma of cancer. anemia of chronic disease, anemia of inflammation, arteriosclerosis or atherosclerosis (including coronary artery disease, cerebrovascular disease or peripheral occlusive arteral disease), ataxias, ataxias related to iron atransferrinemia cancer, ceruloplasmin deficiency chemotherapyvinduced aneria, chronic renal/kidney disease (stage , T l it IV or V , including end stage renal disease or chromi renal/kidney failure, cirrhosis of liver, classic hemachromatosi s collagen-induced arthritis (CIA), conditions with hepeidin excess (elevated hepcidin), congenital dyserythropoietic anemia, congestive heart failure, Crohn' s disease, diabetes, disorders of iron biodistribution, disorders of iron homeostasis, disorders of iron metabolism. terronorti disease, rerroportin mutation hemochromatosis, folate deficiency, Friedrieh's ataxia, funicular nelosis, gracile syndrome, i pyelornfecition or other bacterial infections, Hal lervordan Spatz disease, emochromatosis, hemochromatasis from mutations in transfer receptor 2, tis, hepatitts (Brock), hepatitis C, hepatoceillular carcinoma, hepeidin deficiency, hereditary hemochromatosis, hIV or other viral illnesses Huntmington's disease, hyperferntiema, hypochromic microcytic anenia, h rr i in resistance, iron deficiency anemia, - a. tvnnfrrmmia insub re'ni't~i1i' iron deficiency disorders. iron overload disorders, iron-deficiency conditions with hepcidin excess, joiIe hemochroaltosis (H E2), multiple sclerosis. mutation in transfrria receptor 2, H hemojuvelin, ferropori or other genes of to : in etabolism. neonatal hemochromatosisa, neurode enerati v diseases related to iron, osteopenia, 0steOoos0s pancreatitis,. Pantothenate kimase-aissociated neurodegeneration, P arkinson's disease, pellagra,~ pica, porphyria, porphyria cutanea tarda, pseuedoenocephalitis, pulmonary hemosiderosis, red blood c arthritis, sepsis, sideroblastic anemia, systemic lupus erythematnsus, thalassemia, thalassemia intermedia, transusional iron overload, tumors, vasculitis, vitamin U6 deficiency, vitamin Bl deficiency, andor Wilsor's disease. 100254 Methods of setting an appropriate threshold for diagnosis of the disease states described herein and prognostic monitoring as described herem are Wl known in the art. By way of example, levels of hepcidin in a fluid sample fom a sufficient representative number oftnormal subjects (e.g healthy population without the condition to be detected) are analyzed relative to the hepcidin level from a sufficient representative number of diseased subjects (e.g population conrmed to have the disease or condition) using the sane protocols. A threshold cautoT can be determined that differentiates most of the normal population from most of the diseased population. Alternatively, useful end point values for negative, uncertain and positive results can be determined from the data. For example, a normal rage (indicative of a negative result) can be determned, which includes hepcidin of most of the normal population bu. which exclude almost all of the diseased population. Correspondingly, a range indicative ot a positive result can be determined, which includes hepcidin of most of the diseased population but which exclude almost all of the normal population. Similarly, a threshold differentiating hepidin levels in a population suffering from aneia of inflammation from hepidin levels im a population sufering from iron deficiency anemia can be determined. Useful endpoint values may indicate that the patient is suffering rm anemia of inflammation iron deficiency anermia or mixed anemia. Appropriate endpoint values for the threshold may be determined to optirmze the desired specificity or sensitivity, and may also take account of overall medical and epidemnologicai factors. Factors to he considered include the clinical objective of the laboratory test and whether i is necessary to have a high positive predtive vahie, or a high negative predictive value, as well as prevalence of the disease in the test population. THERhAPEUTIC USES FOR ANThI-H4EPCID[N ANTIPODES 100 2551 Also provided is the use of anthepcidi antibodies described herein that specifically bind human hepcidn, to treat subjects in need thereoE In some embodiments, the subject may be at risk of or suffering from an elevated level of hepeidin, a hepcidin-related disorder, a disorder of iron honeostasis, or anema. {[02 $46 As used herein. "treatment" or "treat refers to both prophylactic treatment of a subject at risk of, or having a predisposition toward, a disease or disorder, and to therapeutic treatment of a subject suffering from a disease or disorder. j00 2571 Administration of a therapeutic agent in a prophylactic. method can occur prior to the mantestation of symptoms of an Indesired disease or disorder, such that the disease or disorder is prevented or, ahtematively, delayed in its progression, Thus, when used n conjunction with prophylactic methods, the term "therapeuticaly effective" means that, after treatment, a fewer number of subjects (on average) develop the undesired disease or disorder or progress in seventy of symptoms, 100258 When used in conjunction with therapeutic methods involving, administration of a therapeutic agent after the subject manifests symptoms of a disease or disorder. the term "therapeutically effective" means that, after treatment, one or more signs or symptoms of the disease or disorder is amieiorated or eliminated, 1002591 '"Mammal" for purposes of tae ersto any animal classified as a manual, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cals, cows, etc. In some embodiments, the mammal is human. j00260] As used herein, a "hepcidin-related disorder" refers to a condition caused by or associated with an abnoral level of hepidin (e.g,, hepeidin excess or hepcidin deficiency relative to the degree of anemia or iron stored) which disrupts iron homeostasis. A disruption in iron homeostasis can in turn result in secondary diseases such as anemia. Acute or chronic inflammatory conditions can result in upregulation of hepcidin expression, which cn result in decreased circulating iron levels, which can cause anemia or worsen existing anemia. Exemplary hepcidin-related inflammatory diseases include anemia of cancer, anemia of 2C ?7- *crwn of ct'hrtvn~ idn chrorncldseaseg anemia o ifammnation, *he nterapnduce armi ehronic -ine diseasestage PH HIV or> end stage remldsease chonic red aiureongest heart fanlureg cancer ieumatoid arthritis, ystemic luus erythemnatsus, Cmohn disease, L yalari ifection or other bacterial infections, hepatitis C,. wlVl, arnd other viral illnesses, arteriosclerosis, atherosclerosis, cirrhosis of the liver, pancreattis, sepsis, vascutlis, piro deil ciency, hypochromic microcytic anenia and conditons wit hepeidin excess, 10026 1 As used herein, the phrase "disease (or disorder) of iron homeostasis" refers to a condition in which a subject's iron' ,v r re modulation. It includes hepcidin-related disorders: conditions not associated with elevated levels of hepcidin that nevertheless would benefit from inhibition of hepcidin activity, such as a disruption in iron homeostasis not caused by hepcidin; diseases where aberrant iron absorption, recycling, metabolism or excton. causes a disruption in. normal iron blood levels or tissue distribution; diseases where ron dysregulation is a consequence of another disease or condition, such. as inflammacn cancer or or therapy; diseases or disorders resulting from abnormal irn blood levels or tissue distribution; anl diseases or disorders that can be treated by moduiaing iron levels or distribution, Nonhmiiting examples of such diseases or disorders of iron homeostasis, hepcid m<elated disorders and inflammatory conditions which can result in hepcidin excess include afican iron overload, alpha thalassemia, Aizheimer's disease, anemia, anemia of cancer, anemia of chronic disease, anemia of inalamnation, arterioselerosis or atherosclerosis (including coronary artery disease, cerebrovascular disease or peripheral occlusive arterial disease) ataxias, ataxias related to iron, atransferrinema, cancer, eruiopiasmin deficiency, chmnotheapy- induced anemia, chronic renal/kidney disease (stage I, I , IV or V) including end stage renal disease or chronic ren alkidney failure, cirrhosis of liver, classic hemochromatosxs, coliagen-induced arthritis (CIA), conditions with hepcidin excess (elevated hepcidin), con genital dyserythropoietic anemia, congestive heart failure, Crohn's disease, diabet, disorders of iron biodistribution, disorders of iron homeostasi, disorders of iron metabolism, ferroportin disease, ferroportin mutation hemochromdtosis folate deficiency, Friedrchs ataxia, flmicular myeensis, graile syndrome, H pyori infection or other bacterial infections, Halervordan Spatz disease, hemochromatosis, hemochromatosis resulting from mutations n transferrin receptor.2, hemaglobinopathies, hepatitis, hepatitis (rock), hepatitis C, hepatocelluliar carcinoma. hereditary hemochromratosis, HIV or other viral illnesses, luntington's disease, hyperferritinernia, hypoehromic microcytic anemia, hypoferremia, insulin resistance, iron deficiency anemia, iron. deficiency disorders, iron overload disorders, irondficiency conditions. with hepeidin excess, juvenile hemochromatosis (FE2),muiiple sclerosis, mutation in transterrin receptor 2, HFE, hemojuvelin, ferroportin or other genes of mtron metatbolism, neonatal hemochromatosis, neurodegenerative diseases related to iron, oteopeni a, osteoporosis pancreahitis, Pantothenate kinase-associ aved neurodegeneration, Parkinson's disease, pellagra, pica, porphyria, porphyria cutanea tarta, pseudoencephlitis, pulmonary hemosiderosis, red blood cel disorders, rheumatoid arthritis, sepsis, siderabiastic anemia, systemic upus eryheadtosus, thalassemia, thalassenia intemedia, transfusional iron overload, tumors, vascubitis vim 86 deficiency, vitamin 1 2 deficiency, and/or Wilso's disease 100262)Non-infilammatory conditions which are implicated ina disruption of iron regulaton u but are not limited to, vitamin B6 deficiency vitamin B 12 dcenxy folate deficiency, peil agra, fuxnicular myelosisa, pseudoenceph aliis, Parkinson's dIsease (Fasano ev a, J Neurochera 96:909 (2006) and Kaur t al, A ng es. 3:32 (2004)), Alzhlimer's disease, coronary heart d as, osteopend steoporosis (G nht &Jsteopors. Int 16:1809 (2005)), hemoglobinopathies and other disorders of red cel metabolism (Papanikolaou et al, Blod 105:4103 (2005)), and peripheral occlusive arterial 041263 Various other iron indices and their normal ranges Of concentrations are ied Tablc2 ---- o-- .... c.. --- hnIndex Normal Level (Range) 5 rnm ron 50-17 V pgdL ______________ Hemoglob in 1___________ 18 ag/dL _________ Hem atori ------ -37-54 ited blood cel count (RBC) 4. A62 x 10. cells/L (men) 4.25,5.4 10 cells/ (w Mea-n C~orpuscular Hemoglobin (MCH) 27-3 pg ____________ Mean C orpusecular- Hmctoglobin 3-6 Concntraion (MCCC) Mean Corpuscular-Vlume(MCV) 809 - Red Cell Distribution Width (DW) II 5-14,5% (electricl impedence method) _or 10.211 8% (laser light med) Reticulicycte count 815 x 10* cells/h ___________(0.8-2.tO % in meni; Q,8~4% in women) Total iron Binding Capacity (TIBC) 250:0 Qg/dL Transferrin Iron Satration Percentage I 5-0% (Tsat)--------------- _ _ - - E erritin _____________- - 2
-
1 2 0 gLi~- Foiate 3i6 ngsrmL (scrum)l and ____________________ I 30-628 ng/m L (red blood cell) ___
-----------
--- --- ---- 9 1002641 A patient's iron index level outside of the normal ranges lsted in Table 2 idicaes that the patient ray benefit from treatment with an ant-hepcdmi anti body descrbed hierein Since hepcidin plays a key role in iron homeostasis> hepcidin levels and activity will correlate to a disruption of iron. homeostass and/or iron indices. Elevated hepcidin lOvels correrlate with serum iron levels below the normal ranges indicated in Table 2, low hemoglobin, and hmatocri t, reduced or normal Tsar higph or normal ferritin values, and elevated inflanmatory status as measured by C-reactive protein (CRP) elevation r other markers of mftlanmmation,. 1002651 As used heremn, t phrase "therapeutically effective amount" of an ant hepcidm antibody described here refers to an amount that results in the desired therapeutic eft'ct (iae. that provides "therapeutic etficacy"). Exemplary therapeutic effects include increased circulating iron levels or increased iron availability, increased red blood cell count increased red blood cell mean cell volume, increased red blood cell hemoglobin content, increased hemoglobin (eg, increased by 20'5 g/dL, increased hematocrit, increased Tst, increased reticulocyte count; increased or normalized retiCulioyte mean cell volume, increased reicdulocyte hemoglobin content 0or reduced free hepeidin levels in serum or plasma, or normalization of any of the parameters described above. Returning such a parameter to its normal range is not required for therapeutic efficacy; for example, a measurable change (increase or reduction) in the direction of normal can be considered to be a desired therapeutic effet by a clinician> When applied to an individual active ingredient, administered alone, the term eters to that ingredient alone. When applied to a combination, the term renters to combined amounts o f the active ingredients that result in die therapeutic effect, whether administered in combination, serially or simutaneous For example, in aspects where the anti hepeidin antibody is administered in conjunction with an enrythropoiesis stimuli atr-, a therapeutically effective amount is meant to refer to the combined amount that increases or normalizes any of the parameters staed above, 1002661 ifn order to faciItate the diagnosis of patients, decision trees, such as that of fIG.j 4B, can b7e used to interpret the level of the hepcidin, and which is used to assist the se or interpreter in determining a course of treatment and the significance of the concentration reading Hepcidin values are predicted to be elevated in patients with inflammaion iron overload and f'erroportin dises anrd suppressed in patients with hemochromatosis, hemogobmopathies, and other red cell disorders, The decision tree: of FIG. i1 shows how 80measureent of hepcidin levels simplifies diagnosis aid/or assessment of a patient suspected ofhaving irn metabolism disorders, FiCnl4A shows the decision tree assessment without a mnenarment of hepoidin evels. 1002671 The compositions for and methods of treatment described herein may utilize one or more antiepcidin antibodies described here used singutarly or in combination with other therapeutic agents to achieve the desired effects, COMINION THERAPY 002 6l It may be fbrberadvantageous to nix two or more antibodiestogether(which bind to the same or different target antigens) or to co-administer an antibody described herein with a second therapeutic agent to provide still improved efficacy. Concurrent administration of iwo therapeutic agens does not require that the agents be adminstered at the same time or by the same route, as long as there is an overIap in the tnme period during which the agents are exerting their therapeutic effect, Simultaneous or sequential administration is contemplated, as is administration on different days or weeks, [002691 in some embodimeMs, the methods described herein include the administration of single antibodies, as well as combinations, or "cocktails', of different antibodies. Such antibody cocktails may have certain advantages inasmuch as they contain anibodies which exploit different eftector mechanisms, Such antibodies in combination may exhibit synergistic therapeutic effects. [90 2701 Combination therapy using an antihepcidin antibody described herein and an erythlropoi esis stimulator is specifically contemplated. in various embodiments, anti-hepcid in antibodies and erytihropoiesis simulators can be used to improve treatment of a patient with anemia. In particular, patns wo are hypo-responsiv t incud unresponsive or resistant to, erythropoiesis stimulator therapy, such as erythropoietin or analogs thereof (Epoeti n ala, Epoetin beta, darbepoetin alfa) among others, will benefit from co-treatmen with an anti -hepidin antibody described herein, i one embodiment, combination therapy includes treatment with at least one antibody that binds to human hepeido and at least one erythropoiesi s stimulator, 1002711 Combination therapy using an anti-hepcidin antibody and an iron chelator to redistribute iron stores in the body is also contemphaed, An iron chelator is an agem capable oftbindin iron and removing it frn a tissue or orn circulation. Examples include deferox amine (Desferal@) and deferasirox (Exjade@), and deferiprone (1,2-dimethyyh3 hydroxypyridin-4one). in some embodiments, anti-hepcidin antibodies and erythropoiesis .81i stimulators can be used to improve treatment of a patient an iron loading diso~rder secondary to transfusion-dependent iron overload, or have an iron maldisribution disorder such as Friedreich's ataxia. 002721 As used herein, "erythropoiesis stimulator' means a chemical compound that directly or mdirecy causes act vation of the erythropoietin receptor. for example, by binding to and causing dimerization of the receptor or by stnmulatng endogenous erythropietin expression. Erythropoiesis stinalators 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 arid activate c rpoetin receptor; or small organc chemical compounds, optionally less than about 1000 Daltons in moleculrar weight, that bind to and activate erythropoietin receptor, Erythropoisis stinalators include, but are not limited to, epoetin alfa, epoetin beta, epoetin delta, epoetin omega, epoetin iota, epoerin zeta. and aalogs thereof pegylated erythropoietin carbamylated erythropoi etin, mimetic peptides (including EMP1I/hemsatide), mimetic antibodies and MF inhibitors (see US. Pant Application Publication No. 2005/0020487, the di scosure of which is incorporated by reerence in its entirety). Exemplary rythropoiesis stimulators include erythropoietn, darbepoetin. erythropoietin agonist varints, and pepties or antibodies that bind and activate erythropoietin receptor (and include opundreported in US. Patent A pplication Publicaton Nos. 2003/0215444 and 2006/0040858, the disclosures of each of which is incorporated herein by reference in its entirety) as well as erythropoietin molecules or variants or analogs thereof as disclosed in the folowing patents or patent applications, which arc each herein incorporated by reference in its entirety; U.S. Pat, Not 4,703,008; 5,441 86 5,547,933; 5,618,698; 5,621,080; 5,756,349; 5,767,078; 5,773,569; 5,955,422; 5,830,85 ; 5,856,298; 5,986,047; 6,310,078; 6391,633; 6,583,272; 6,586,398; 6,900292; 6750,369; 7,030,226; 784,245; 7,217689; PCT publication nos WO 91/05867; WO 95/05465 WE) 99/66054; WE 00/24893; WO 01 /81405; WO 00/61637; WEO 01/36489; WE) 02/014356; WO 02/19963; WEO 02/20034; WO 02/49673; WO 02/085940; WO 03/029291; WO2003/55526; WE 2003/084477; WE0 2003/094858; W) 20 400-'I", WE) 2004/002424; W) 2004/009627; W)O 2004/024761; WO0 2004/033651 ; WE) 2004/035603, WO 2004/043382; WO 2004/101600; WO 2004/101606; W) 2004/101611; WE 2004/106373; WO 2004/018667; WE) 2005/001025; WE) 20 05/001136; WO 2005/021579; WEO 2005/025606; WE) 2005/032460: W) 2005/051327; WO 2005/063808; WO 2005/63809; WO 2005/070451; WE) 2005/081687; WO 2003/08471 1; W 2005/103076; Wo 2005/100403; WE) 2005/092369; WO 2006/50959; WO 2006/02646; W) 2006/29094; and U.S, Patent Application Publication Nos. US 2002/0155998; US 2003/0077753; US 2003/0082749; US 2003/0143202; US 2004/0009902; US 2004/07 1694; US 2004/0091961; US 2004/0143857; US 2004/0157293; US 2004/0175379;US 2004/0175824; US 2004/02.29318; US 2004/0248815; US 2004/0266690; US 200510019914; US 2005/0026834; US 2005/0096461; US 2005/0107297; US 2005/0107591 US 2005/0124045; US 2005/0124564; US 2005/0137329; US 2005/0142642; US 2005/01 4329 U.S 2005/0153879; US 2005/0158822; U 2005 015832 US 2005/0170457; US 2005/0181359; US 2005/0181482; US 2005/092211; US 2.005/0202538; US 2005/0227289; US 2005/0244409; US 2J006/00806; US: 2006111 279. 1002731 Erythropoietin includes, but is not limited to, a polypeptide compising the aminO acid sequence as set fortn in SEQ H) NO: 72. Amino acids h '5 ofSEQ ID NO. 72 constitute the mare protein of any molecules designated as an epoetin, e go epoetin alfa, epoetin beta, epoerin delta, epoetin omega, epoetn iita. epoetin gamma, epoetm zeta, and the like. Additionally, an epoetin also includes any of the aforementioned epoetin which are chemically modified, e.g, with one or more water-soluble polymers such as, eg., polyethylene glycol includingg PEG-EPO-beta). Also contemplated are analogs of erythropoietin, with 65%. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96% 97%, 98%, or 99% identity to SEQ ID NO: 72 still retaining erythropoietic activity, 1002741 Exemplary sequences. manufacture, purification and use of recombinant human erythropoietin are described in a number of patent publications, including but not limited to Lin U.S. Patent 4,703008 and Lai et al.U. S, Patent 4,667,016 each of which is incorporate~dherein by reerence in its entirety. Darhepoetin is a hypergycosylated erythropotetin analog having five changes in the amino acid sequence of rHuEPO which provide tor two addi tional carbonydrate chains. More specifically, darbepoeti n a]lfa contains two additional N-inked carbohydrate chains at amino acid residues 30 and 88 ofSEQ ID NO; 73. Exemplary sequences, manufacture, purification and use of darbepoetin and other erythropoietir analogs are described in a number of patent publications, including Strickland et alW, WO 91/05867, Elliot e at., WO 95/05465, Egrie et at. WO 00/24893, and Egie et at, NYO 01/81405, each of which is incorporated herein by reference in its emirety, Denvatives of naturally occuring or analog polypepuides mclude those which have been chemically modified, for example, o attach water soluble polymers. (e.g, pegy t ated) radonuclides, or other diagnostic or targeting or therapeutic moieties.
002 7. The term "rythropoiiactivity nicans activity to stimute eythropoies as demonstrated in an in vivo a x the e-h y Seee.g., Cotes and BEnghai Nra ure 9i:1065 (96, ADMINISTRATION AND P REPARATION OF PHARMACEP-UTICAL FORMULATiONS 1002761 In another aspect, phamaceulical compotions are provided comprismg a therapeutically atfctive amount of any of the antibodies described herein and a phannaceutically acceptable sterile carrier, diluent or excipient, Also provided is the use of such antibodies in preparation of a medicament for treatment of a human wit an elevated level of hepeidin, a hepcidinrelated disorder, a disorder of iron homeostasis or an anemia, It is understood that co-administration methods mnvolvings administration of antibodies with a second therapeutic aent, as descibed herein, encompass not oney he use of the antibody in preparation of a medicament for co-adtministrmon with the second therapeutic agent, but also the use of the second therapeutic agent in preparation of a medicament for co-administration wth the antibody. (1002771 in some embodiments, the ati-hepcido antibodies or specific binding agents used in the practice of a. method described herein may be formulated into phamaceutical compositions comprising a carter suitable for dhe desired delivery method. Suitable carrers include any material which, when combined with an anti~bepcidin antibody or specific binding agent, retains the high-affinity binding of bpIcidin and is nonreactive with the subjects inroune systems Examples include but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate bufered saline solutions, bacteriostatic water, and the like. A variety of aqueous carriers may be used, e..g., water, buffered water, 0.4% saline, 0.3% glycine and the like, and may include other proteins for enhanced stability; such as album, lipoprotein, globuhn, etc, subjected to mild chemical modi fictions or the like. 10027g 1 Exemplary antibody concentration; in the formulation may range frn about 0.1 mg/mi to about 180 mg/mi or from about 0.1 mg/mL to about 50 mghnt, or from about .5 g/mL to about 25 m/min, or alternatively from about 2 mg/mL to about !0 mg/mL An aqueous formulation of the antibody may be prepared in a pH-buffered solution, for example, at pH ranging from about 4.5 to about 6.5, or from about 4,8 to about 5.5. or alteratively about 5,0. Examples of buffers that are suitable for a pH within this range include acetate (eg. sodium acetate), sutmate (such as sodium succinate) gluconate, histidine, citrate and other orgame acid buffers. The buffer concentration can be from about 1 mM to about 200 184-~ mM, or from about 10 mM to about 60 mM, depending, for example, on the buffer and the desird i soonicity of the formulation. 1002791 A onicity agent, which may also stabilize the antibody, may be included in the fbnnuiation. Exemplary toniciy agents include polyols, such as nanonitol, sucrose or trehalose. in some embodiments, the aqueous formation is tsotonic, although hypertolic or hypotonic solutions Maly be suitable. Exemplary concentrations of the polyoI in the fndruation may range from about 1% to about 15% wv. |00 280| A surfactant may also be added to the antibody formulation to reduce aggregation of the formulated antibody and/or minimize the formation of particulates in the tormuation and/or reduce adsorption. Exemplary surfactamts include noronic surfactants such as polysorbates (e.g, polysorbate 20, or polysorbate 80) or poloxamers (eg. poloxamer 188) Exemplary concenmraions of surfactant may range from about 0.001% to about 0.5%, or frim about 0 005% to about 0.2%, or alternatively from about 0.004% to about 0.01% w/v. [00 2811 in one embodiment, the formulation contains the aboveldentified agents (i.e. antibody, buffer, polyol and surfactant) and is essentially free of one or more preservatives, such as benzyl alcohol phenol, m-cresoi, chlorobutanol and benzethonium CL. in another embodiment, a preservative may be included in the formulation, e.g, at concentrations ranging &rom about 0,1% to about 2%, or alternatively from about 0,5% to about 1%. One or more other pharmaceuticaly acceptable carricts, excipients or stabilizers such as those described in Remingon's Pharmaceutical Sciences 16th edition, Osot A. Ed, (1980) may be mcl uded in the formulation provided that they do rnot adverselyv affect the desired characteristics of the formulation. Acceptable careers, excipiems or stabdizers are nontoxic to recipients at the dosages and concentrations employed and include; additional suffering agents; co-solvents; antioxidants including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e g. Zn-protein complexes); biodegradable polymers such as polyesters; and/or satforming counterons such as sodium, 100282{ Therapeutic fornulations of the anti-hepcidin antibody are prepared fo' storage by mixing the gody hvn the desired eree of purity with optional physiologically acceptable carriers, excipien ts or stable izers (Rtem ington's Pharrmaceutical Sciences 16th edito, Osol A, Ed. (1980)), in the form of lyophilized formulations or aqueous solutions, Acceptable carriers, excipients, or stabilizers are nontoxIc to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl armmron ium chloride; hexamiethoni unm chloride; benzalkonium chiornde, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as metyl or popy paraben; catechol; resorcinol; eyclohexanol 3-pentanol; and m-cresol; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin. gelatin, or irrnunogiobuhna; hydrophibc polymers such as polyvmylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine. arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates mcluding glucose, mannose, maltose, or dextrins: cheating agents such as EMDTA; sugars such as sucrose, mnannto , trehalose or sorbitol: sat forming counter-ions such as sodium; metal complexes (e.g.. Zn-protein complexes); and/or non-ionic surfactanas such as TW EENM PLURONICSTN or polyethylene glycol (PEO) 1002831 In one embodiment, a suitable tormulation contains an isotonic buffer such as a phosphate, acetate, or TRIS buffer in combination with a Zonicity agent such as a pol voly SorbitoL sucrose or sodium chloride which tonicifies and stabilizes. One example of such a tonieyv agent is 5% Sorbitol or sucrose, in addition, the formulation could optionally include a surfactant such as to prevent aggregation and for stabilization at 0,01 to 0.02% wi/vol. The pH of the fonulation may range from 4,5-6,5 or 4.5 - 5.5. Other exemplary descriptions of phannaceutical formulations for aMibodies may be found in US 2003/0113316 and US patent No.each ioror' 5ated herem by reference in its emirety. j00 2841 The formulation herein may also contain more Than one active compound as necessary for the particular indication being treated, those with complementary activities that do not adversely affect each other, For example, it ma be desirable to further provide an immunosuppressive agent Such molecules are suably present in combination n amounts that are elective for the purpose intended, 1002851 The active ingredients may also be entrapped in microcapsuie prepared, for example, by coacervation techniques or by interfacial polymerization. for example, hydroxymethyicellulose or gelatin -microc apsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, mnicroemulsions, nano-particles and nanocapsul es) or in m acroemul sions. Such techniques are disclosed in Reminton's Pharmaceutical Sciences 16th edition, Osol, A, Ed, (19%0) 100286 Suspensons and crystal forms of antibodies are also contemplated Methods to make suspensions and crystal forms are known to one of skill in the art [002871 The formulations to be used for in vivo adrnmtration must be sterile. In some embodiments, the compositions described herem may be sterilized by conventional, well known sterilization techniques. For example, sterilization is readily accomplished by filtration -86 through sterile filtration membranes, The resulting soutions may be packaged for use or fitered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sternle solution prior to adnistraton. 0T 288 The process of freeze-drying is often employed to stabilize polypeptides for storCe.nriu anyl A, m long-term s pn the polypeptide is relatively unstable in liquid A lyophilizaion cycle is usually composed of three steps: freezing, primary drying, and secondary drying; Williams and Poh, Journa/ of PareMneral Scence and Tcbhnlogy, Volurne 38, Number 2, pages 48-59 (1984), in the freezing step, the solution is cooled unti it is adequately &ozen. Bulk water in the solution forms ice at tis stage. Th ice sublimes in the primary drymg stage, which is conducted by reducing chamber pressure below the vapor pressure of the ice using a vacuum, Finally, sorbed or bound water is removed at the secondary drying stage under reduced chamber pressure and an elevated shelf temperature. The process produces a material known as a lyophized cake, Thereafter the cake can be reconstituted prior to use, (00 2891 The standard reconstitution practice tor lyophized material is to add back a volume of pure water (typically equivalent to the volume removed dunng lyophihzation) although dilute solutions of antibacterial agents are sometimes used in the production of pharmaceuticals for parenteral administration; Chen, Drug Develo pment and /dsra Pharmacy, Volume 18, Numbers 11 and 12, pages 13 11-1354 (1992) 1002901 Excipients have been noted in som cases to act as stabilizers for freezeAried products; Carpenter et al, DeAvelopmnts in Biological Srandardihation, Volume 74, pages 225-239(i991), For example, known excipients include polyols (including mannitol, sorbitol and glycerol); sugars (including glucose and sucrose); and amino acids (including alanine, glycine and ghatamic acid>, 0oo291 In addition, polyols and sugars are also often used to protect polypeptides from freezing and drying-nduced damage and to enhance stability during storage in the dried state hn general, sugars, in particular disaccharides, are effective in both the freeze-drying process and during storage, Other glasses of molecules, including mono- and disaccharides and polymers such as PVP, have also been reported as stabilizers of lyophilized products. 1002921 For injection, tihe pharmaceutical formulation and/or medicament may be a powder suitable for reconstituton with an appropriate solution as described above, Examples of these include, but are not limited to, freeze dried, rotary dried or spray dried powders, amorphous powders, granules, precipitates, or particulates. For injection, the formulations -387may optuoal ivconain stabiierspH modifiers sufctants bionailabi tt modifies and combinationsof these. i00 293] Sustained-release preparations may be prepared. Suitable examples of sustainemd-release preparations include senipenneable matrices of solid hydrophobic polymer containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsule, Examples of sustamed-release matrices include polyester hydrogels (for example, poly(2-hydroxyethyi-methacrylatet or poiy~vinyi alcohol)), pol ylactides (US, Patent No, 373,919), copolymers of Lglutamic acid andy ethylLglutamate, non-degradable ethylene-vinyl acetate, degradable lactic acidgjycoic acid copolymers such as the Lupron Depot'M (injectable microspheres composed of lactic acidgiycolic acid copolymer and Icuprolde acetate), and poly-D+)3-hydroxybutyric acid. While polymers such as ethylene vinyl acetate and iactic acid-giycolic acid enable release of molecules tbr over 100 days, certain hydrogels release proteins tr shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 3PC, resulting m a loss of biological activity and possible changes m imunogenicay. Rational strategies can be devised {or stabilization depending on the mechanism involved. For exampleif the aggregation mechanism is discovered to be intennolecular S--S bond formation through thiodisulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture uontt, Using appropriate additives, arid developing spcii ymerm compositions. 1002941 In sone embodiments, the formulations described herein may be designed to be shor-acting. fas-releasing, long-acting, or sstainedreleasing as described herein. Thus, the pharmaceutical formulations may also be formulated for controlled release or for slow release. 1002951 Therapeutically effective amounts of a comosition will vary and depend on the severity of the disease and the weight and general state of the subject being treated, but generally range from about 1,0 pg/kg to about 1.00 mg/kg body weight, or about 10 pg/kg to about 30 mg/kg, or about 0, I mg/kg to about 10 mg/kg or about 1 mg/kg to about 10 mg/kg per application. Administration can be daily, on alternating days, weekly, twice a month, monthly or more or less frequency, as necessary depending on the response to the disorder or condition and the subject's tolerance of the therapy. Mamtenance dosages over a longer period of time such as 4, 5, 6, 7, 8, 10 or 12 weeks or longer may be needed until a desired supp esuion of disorder symptoms occurs, and dosages may be adjusted as necessary, The progress of this therapy is easily monitored by conventional techniques and assays. j00296) Specific dosages may be adjusted depending on conditions of disease, the age, body weight, general health conditions, sex, and diet of the subject, dose intervals, administration routes, xcrcton rate, and combinations of drugs. Any of the above dosage forns containing effective amounts rewl within the bounds of routine experimentation and therefore, well within the scope of the instant ivention, [00297 The anti-hepcidin antibody or specific binding agent is administered by any suitable means) either systemnicaly or locally, including via parenteraL subcutaneous, intraperitoneal, intrapulmonary, and intranasal and, if desired for local treatment, intralesional admiaion, Parenteral routes include intravenous, intraartenal, intraperitoneal, epidural intratecal administration. in addition, the specific binding agent or is suitably administered by pose infusion, particularly with declining doses of the specific binding agent or anibody. "n sonc embodiments, the dosing is given by injections, eg, intravenous or subcutaneous injections, depending in par on whether the administration is brief or chronic, Other administration methods are contemplated, including topical, particularly tranudernal, transmucosal, rectal, oral or local administranon eg through a catheter placed close to the desired site. In some embodiments, the spec ie bndig agent or antibody deserted herein is administered intravenously mn a physiological solut on at a dose ranging between 0,01 mg/kg to 100 mg/kg at a frequency ranging from dany to weekly to monthly (e.g. every day, every other day, every third day, or 2, 3, 4, :, or 6 times per week} or a dose ranging from 0.1 to 45 mg/kg, 0.1 to 1 5 mg/kg or 0.1 to 10 mg/kg at a frequency of2 or 3 times per week, or up to 45mg/kg once a month DIAGNOSTIC AND THERAPEUTIC KITS 0098 In another related aspect, kits for treating a disorder associated with elevated hepcidin levels, or a hepcidin-related disorder, or a disorder of iron homeostasis or a mammal with anemia, are also provided,. in one embodiment, the kit includes (a) an anti-hepcidin antibody, and (b) an erythropoiesis stimulator, and optionally, iron, in another embodiment, the kit includes an anti-hepcidin antibody and a label attached to or packaged with the conainer, the label describing use of the anti-hepidi antibody with an erythrapoiesis stimulator, i vet another embodiment, the kit includes an erythropoiesis stimulator and a label attached to or packaged ith the container, the label describing use of the erythropoiesis stimulator with an anti-hepidin antibody. Also provided is the use of an anti-hepcidin -89.
antibody in preparation of a medicament for administration with an erythropoiesis stimul actor, as well as use of an erythropoicsis stimulator in preparation of a medicameIt tor administration with an anti hepcid in antibody. in any of these kits or uses, the anti-hepcidin antibody and the erythropoiesis stimulator can be in separate vials or can be combined together in a single pharmaceutical composition. In yet another embodiment, an antihepCidin antibody or erythropoiesis stimulator, or both, can be combined with iron in a single pharmaceutical composition or can be in separate vials, 100 2991 As a matter of convenincc, an antibody disclosed herein can be provided in a kit, i. , a packaged combination of reagents in predetermined amounts with instructions tor peromiing the diagnostic assay. Where the antibody is labeled with an enzyme, the kit will include substrates and cofactors required by the enzyme (e.g, a substrate precursor wh4i provides the detectalne chromophore or fuorophore in addition. other additives may be ind ided such as stabilizers, buffers (emg, a blok bffer or sis buffer) and the ike. The relative amounts of the various reagents may be varied widely to provide fbr concentrations in solution. of the reagents which substantially optim ze the sensitiv ity of the assay, Particularly. the reagents may be provided as dry powders ua sualy lyophilized icluding excipients which on dissolution will provide a reagent solution having the appropriate concentration. (00300| Also provided are diagnostic reagents and kits comnprisng one or more such reagents for use in a variety of diagnostic assays, including for example, immunoassays such as E LSA (sandwich-type or competitive formiat). In some embodiments, such kits may include at least a foir peptide (optionally a properly fided mature hepeidin standard as described herein) or a first antibody or antigen binding fragment described herein, a imnctionil fragment tieeof, or a cocktail thereof. and means tOr signal generation, The kit components may be preoattached to a solid support, or may he applied to the surface of a sohd support when the kit is used, in one embodiment, the signal generating means may come pre-associated with an antibody described herein or may require combination with one or more components, eg, buffers, antibody-enzyme conjugates, enzyme substrates, or the like, prior to use. Kits may also include additional reagents, e.g, blocking reagenuts fr reducing nonspecific binding to the solid phase surface, washing reagents, enzyme substrates, and the like. The solid phase sur ace may be in the form of a tube, a bead, a microtiter plate, a microsphere, or other materal suitable for immobilizing proteins, peptides, or poypeptidehs, In some embodiments, an enzym that catalyzes the formation of a chemiluminescent or chromogenic product or the reduction of a chemiluminescent or chromogenic substrate is a component of the signal generating means. Such enzymes are well known in the art. Kits 01 may comprise any of the capture agents and detection reagents described herein. Optionally the kit may also comprise instructions for carrying out the methods described herein. 100301[ Also provided is a kit comprising an anti-hepcidin antibody deserbed herein and an erythropoiesis stimulator packaged in a comainer, such as a vial or bottle, and further comprising a label attached to or packaged with the container, the label describing the conents of the container and providing indications and/or instructions regarding use of the contents ofthe container to treat one or more disease states as described herein. {00302| In one aspect, the kit is for treating a disorder associated with elevated hepcidiwn levels and comprises an anti-hepcidin antibody and an erythropoiesissixmulator, The kit may optionally further include iron for oral or parenteral, e~g. intravenous, administration, In another aspect, the kit comprises an anti-hepcidin anybody and a label attached to or packaged with the container describing use of the anti-hepcidin antibody with an erythropoiesis simulator. In yet another aspect, the kit conIprises an erythropoiesis stimulator and a label attached to or packaged with the container describig use of the erythropoiesis stimulator with an anti-hepcidin antibody. in certain embodirments,an a nthepeidin antibody and an erythropoiesis stimulator, and optionaly the iron, are in separate vials or are combined together in the same pharmaceutical composition. in yet another aspect, an antihepcidin antibody described herein is combined with iron in a single pharmaceutical composition In yet another embodiment, the erythropoiesis stimulator is combined with iron in a single pharmaceutical composition. 01 03 As discussed above in the combination therapy section, concurrent administration of two therapeutic agents does not require that the agents be administered at the same time or by the same rote. as long as there is an. overlap in the time period during which the agents are exerin g their therapeutic effect Simultaneous or sequential administration is contemplated, as is administration on diffrem days or weeks. 100304z The therapeutic and diagnostic kits disclosed herein may also be prepared that compise at least one of the antibody peptide, antigen binding fragment, or polynucientide disclosed herein and instructions for using the composition as a diagnostic reagent or therapeutic agent, Containers for use in such kits may typically comprise at least one vial, test tube, lask, bottle, syringe or other suitable container, into which one or more of the diagnostic and/or therapeutic compositions) may be placed, and suitably aliqucOted, Where a second therapeutic agent is also provided, the kit may also contain a second distinct container into which this second diagnostic and/or therapeutic comnposition may be placed, Altematively, a plurality of compounds may be prepared in a single pharmaceutical composition, and may be packaged in a single contaier means, such as a vial flask, syringe, bottle, or other suitable sigle continer. The kits ofthe present invemion will also typically include means for containing the via(s) in close confinement for commercial sale, such as, eg, injection or blow-mAded plastic containers into which the desired vial(s) are retained. Where a radiolabel chroogenic, fluorigenic,o other type of detectable label or detecting means is included within the kit, the labeling agent may be provided either in the sane container as the diagnostic or therapeutic composinon itself, or may alternatively be placed in a second distinct container means into second composition my be placed and suitably aliquoted, Alternatively, the detection reagent and the label may be prepared in a single container means, and in most cases, the kit will also typically include a means for combining the vials) in close confinement for commercial sale and/or convenient packaging and delivery. [00305I A device or apparatus for carrying out the diagnostic or monitoring methods described hereim is also provided, Such an apparatus may include a chamber or tube into which sample can be nu, a fluid handling system pinl including valves or pumps to direct low of the sample through the device, emotionally filters to separate plasma or serum from blood, mixing chambers for the addition of capture agents or detection reagents, and optonally a detection device fR detecting the amount of detectable label bound to the captme agent immunocomple The flow of sample may be passive (e.g, by capillary, hydrostatic, or other forces that do not require further manipulation of the device once sample is applied) or acive (eg, by application of force generated via mechanical pumps, electroosmotic pumps, centre ugai force, or increased air pressure), or by a cormbintation of active and passive forces, 1003061 in related embodiments, also provided is a processor, a computer readable memory, and a routine stored on the computer readable memory and adapted to be meecuted on the processor to perform any of the methods described herein, and/or to generate as output the detected level of hepidin and. a threshold or range of' threshold levels considered "normal', such that levels outside the "normal" range correlate with one or more of the conditions as desenbed herein. in somi embodiments, computer readable media containing programs or rotnst perform similar funtions are also provided, Extamples of suitable com~p utin5g systems, environm ents, and/or con figurations meclude personal computers, server computers, hand-held or laptop devices, multiprocessor systems, miicroprocessor-.based systems, set top boxes, programmable consumer electronics, network P~s, mnicomputers, maifame computers, distributed computing environments that include any of the above sysems or devices, or any other systems known in the art "'"' ud NUN,&IIHERAPEUTIC USES FOR ANTI-HEPCIDIN ANTIBODIES 1003071 The antibodies disclosed heren may be used as affinity purification agents for target antigen or in diagrostic assays for target antigen, ceg detecting its expression in speci ei cs tissues, or serum, The antibodies may also be used for in vivo diagnostic assays. Generally, for these purposes the antibody is labeled with a radionuclide (such as In . OTc. C ', . l ip or 35S) so that the site can be locahzed usmng runmunoscintiography. 1003081 'The antibodies disclosed herein may be employed in any known assay method, such as conpettive binding assays, direct and indirec sandwich assays, such as ELISAs, and immunoprecipitation assays, Zoa, Monoclonal Antibodies: A Manual of Techniques, ppj 47 158 (CRC Press, Inc. 1987) The antibodies may also be used for immunohistocheimstry, to label cell samples using methods known in the art. EXAMPLES EXA MP Fi PREPARATION OrANT-HUMAN ECIEN MONOCLONAL ANT [BODIE S 1003091 Monocional antibodies can be prepared by various procedures gender described in copending LES. Patent Application No. 142- 2 5 incorporated by reference herein in. its entirety. For example, Xenomouse gGd and igG4 d mice were i z with KLiH-conjugated human hepcidin (SEQ ID NO: 9) using standard methods. 23040 1g42 supernatants and I 1,520 IgG4 supematants were screened at a singe concentration against biotinylated human hepeidin anchored to a plate. From this screen 617 (gG2 and 1013 14 supemnatants were tested for binding to both human and mouse biotinylated bepcidin using an antibody capture ELISA in which the amount of amibody captured was limited to niniize the effect of concentration differences between supematants, Top-ranking samples (70 gG2 and 1 10 Ig4) were further characterized in a bridging EfLSA which measures solution-phase hepeidin-antibody binding over a range of antibody concentrations. This assay provided a relative affinity ranking of antibody binding 1003101 Superams &om each of the 1gG2 and 1gG4 panels were d a as follows, 1C9 (SEQ ID NOs: 107-1 16) 3W3 (SEQ ID NOs: 117-126, 4E 1 (SEQ ID NOs: 127 36 7A3 (SEQ ID NOs: 137-146, 9D1)2 (SEQ ID NOs: 147-i56 129 (SEQ ID NOs: 157 166), 15E1 (SEQ ID NOs: 167-176, 18DS (SEQ 11) NOs 310-319) 19C1 (SEQ ID NOs: 320329L 19D12 (SEQ ID NOs: 290-299), 191 (SEQ ID N Os- 300-309)23F1 1 (SEQ ID NUs 1?7-186) 26F1 (SEQ ID NOs: 187-196) 18311 (SEQ ID NOs: 331-339) I9E8 (SEQ ID NOs: 341,3149) 20E12 (SEQ 1D Ns: 351-359), 22E12 (SEQ ID Ns: 361-369), 22J 10 (SEQ 1D Nis: 371-379), 23A1 1 (SEQ ID Ns; 381-389) and 24E4 (SEQ iD N Os: 391-399) 0031 11 Generally, the binding affinities of these antibodies to human bepridin were determined by lAcore, whic were then confirmed by KinExA if the Ko as estimated by BlAcore was below 100 pM. The binding affnity of amibody M18 1, however, was determined by KinExA without the BlAcore assay, The K 0 for the lead antibodies were in the range of between I pM and more than 400 pM, |0031 2 Relative species cross-reactivity and binding to Hepc20 (SEQ ID NO: 96) was determined by competition ELSA. I &8 1 was observed to be cross-reactive with cynoniolgus monkey hepcidin and nosig cantly cross5reactive with mouse hepcidin, Antibody 18 11 competes with antibody 23Fl I for biding to human hepcidint EXAMPLE 2- GENERATION AND ELECTION O HUMAN ANBODIES WITH CERTAItNPHARMACOKENET C PROPERTY ES 1003131 2522 hepcidin-specific antibodies were screened for differential binding profiles to human hepcidin at pH 74 and pH 6.0 by ELISA. 50 pt of Neutravidin (Pierce) at 8 gnL in 1xPBS was coated upon a Nuc Maxisorp 334-well plate, and incubated at 37* for 1 hr. After blocking the wells with 0-1%BSA/PBS/0.05%Tween20 for I hour at room temperature, plates were washed si times with PBS5/0,05%Tween20, 25 gLk ofnmono biotinylated hepeidin at 50 ng/mL in 0.1 %BSAPBS/0,05%Tween2 was added to the 384 well plate, and incubated at room temperature for 1 hour, The plates were next washed six times with PB S/0,05%Tween20. Starting hepcidin antibody concentrations were norrnalzed to I gmL for pH 5.5 and 6' conditions and to100 rg/Lnk for pH 7,4 conditions. The hepcidin antibodies were serially diluted 3-fold in PBS/i%NFDM pH 7,4 and 4-fild in PBS/ I%NDM pH 6,0 and 5.5. The dilutions and titrations were performed in polypropylene .96-well dilution plates, and then were transferred in duplicate to a Netravidin-coated 384 well plate, The biotinylated hepcidin and antibodies were incubated for 2 hours at room temperature, T he plate was next washed six times with PBS/0.05%Tween20, 25 jiL of goat anmidruhgG-horseradish peroxidase at a 1.7000 dilution in 0.1 %BSA/PBS/0,05%Tween20 was next added to each well of the assay plate. The plate was finally washed six times in PBS/0.05%Tween20, Enhanced K-Blue 34 %355 -Ttramethylbenzidine (TMR) Substrate (Neogen) was added and the reaction stopped using 1 M H1PO4 after i0 minutes of incubation at room temperature, The absorption was measured at 450 nm on a plate reader, Binding data -94 were analyzed by non-hnear recession analysis (sigmoidal dose-response, vanable slope) to generate EC values using GraphPad Prism software, From this screen 243 antibodies demonstrated a > 2-fOld di fbrence in binding at pH 74 versus pH 6,0, The top 32 we supenmants were rescreened for a third time over a range of antibody dilutions at pHI 7,4 and pH 6,0. Antibodies 18B 1, 1918, 20E2, 22Cl 1, 2212, 22H 10. 23A 11, 24E4 and 25H6 were selected thR subcloning. 1003141 The binding affinities of these antibodies to human hepcidin were determined by KinExA and the offates were determined by BiAcore. At a 1:230 dilution, all of the antiodie teted demonstrated an abou 10-foid reduction in affinity for hepcidin at pH 6 coMpared to phi 74. EXAMPLE 3 -ENGINEER NG OF ANTIBODY WITh DIFFERENTIAL PH BINDING 100315 Introduction of one or more histidine residues in the light and/or heavy variable region of an antibody can provide antibodies that eAhibit diffbrential phi binding to its antigen Histidine is the amino acid most sensitive to pH shts from 7,4 to 6.0 as the jmidazole side chain of histidine has a pKa just over 6, varyng higher or lower depending on the environment of the amino acid, This technique can be applied to any anti-hepcidin antibodies, including those described herein. [00316& A crystal structure modei of the Fv portion of the anthepcidin antibody ISE I was prepared. Using this structure model, all 62 CDR residues of antibody SE1 using the Kahat. definition, were selected for mutation, along with framework residues that were at least 10% exposed and within 4 5 A of a CDR residue, resulting in an additional 31 residues for nmtationA Adiinlpositions were selected for mutation by visual inspection of the structure modd for amino acids in proximity to the CDRs or selected framework residues, The encoding DNA was mutated to provide histidine mutations at single or multiple positions withi the amino acid sequence. Mutations which produced some pH differential binding effect as amgle mutations can be combined as double, triple or more multiple mutations. The histidine mutatons displayed collectively below were engmeered at any one or more amino acids mi which the "'Mutants" sequence identifies a change to a histidine in the following 150 i M th t uutTQPn rs HH L } PSVSVS P OQTA T 1 C S O ISIHH$Y V qQR Pi$QS. 2 V V H HW , 22222 light Nua332 1. ga 2 iii LigAt SRO{EFS iTT 1 &'A iEOYQ~MST~W F ' -95.i Li ght MUMS zg iTVLCQP ThU~~~~M CiAR A_'at ~LYtQ QV06 SCGGGVCPGR SLRLsCSG 'EIt WV PG~iM Asi b-eavv mtn HA ER AEyANiAsiFtTsMRKSKNTLYQaNSbA. AYA 1SE aHTavyaMttanrn 'teM eAS0tYCARBF I5MU heavy Mtmants hmami uuIa cTvss E:<pression of Mutant Constructs 00317i MtItions were introduced into wild-ype constrcts in vector pT5 (heavy and ight chains on separat vectors) using a Quickchange 11 ku (Stratagene #200523) and were transiemlyv transfected into 293-E cells (NRCC), Y2H Q6H Q6H Y2311 A2 H C33H Q49H -s-- - .. .... .... .. -- -- -- -- -- -- -- -- -- -- -- -- ---- ---- -- .... .. _ .. .. ----- ---- ---- - I - - - - - - - - -- 4,-- " --- --- - --------------- ----- -------- ....... K)5211 ___ Ki2H_________96 __ 11(2. .1 .......... -5 4 ------------ -- ---- --- -- -- .----------.- -.------------ ----- S6SH_ _ _ _ ......... -~1- ----------- .. ... ... ... .. -- -- -- ----- --- -- - --- Q ---------------------------- ... .. .. .. .. ..... . ---- ------ ----- --- S9 6, 1 _____ .. . .... .... -------------- .. G .6 ------- -11- -- ------- -N9----SH --------- - v -- 7 --- ------- ------------------------- -- ---- -- C----i- N-- utitionl .............. .. ..... 5.. .... .-. ..-- -- ---- -- -- --------------- -------------------------- ......... - ------------- --- ---- -- -- ----- $1- --- 3 -- - - ---------------------- ------- 6 -I -_ _ _ _ ---------- ------- ---- ----- .. .... ... .. ...-- -- -- -- -- \.. . ...... .. .... ..... -- -- --- -- -- ------ ------ -- U- -- ----- - --- -- S ---- - --- - ----- --------- -- -t --- -- -- --- .... ... ... .. --- - -----.----- 1)7 V64H ________* K6SH 66H1 ~D73H N74H Nt) A 104H At 107H ___ KinExsA Solution Equilibriu Bi nding Analysis for Ant boi 13E, 15$El Vriants and 188311 to Binding to Hurnan Hepc. I)3 18j SA-Sepharose beads were pre-coated. with biotinylated bun bepidin (SEQ 11D NO: 9) and blocked with BISA according tonmanufacturer's instructions, Antibodies and hepcidin were diluted in PBS8/0,1% BISA! 0.05% NaN; buffer. Fixed concentrations of aunotides 15E1, 15E1 W52H, 1$E1 A99.H, 1521 N52 .5E. A-07- and- -8-3- were incubated with various concentrations of human hepcidin at room temperature for 8 hours before being run through the human hepcidin-coated beads. The amnount of the bead-bound antibody was quantified bytfluorescently (Cy5)-Iabeled goat anti-mrinedgG (H+L-h) antibody (Jackson Emmuno Research, West Grove, PA). The binding signal is proportional to the concentration of tree antibody at equilibrium, Dissociation equilibrium constant (K(o) was obtained from nonlinear regressio n of the competition curves using a dual-curve one~site homogeneous binding model (KinEx ATM Pro software), The results are set forth below in Table 3. -- ------ ---- 9 8 --- ---------- Tabke3 k~ bd ______KL ______ Ko range 3311 u2,23,4 nM ___ 15E1 (wild type) 37pM ___ 13-75 pM 15E1 AI07H 31___ pM | 35 %pM 15E'1A99H >10 nl~M _____ N/A _ 1IEI N57H 3n____ M |____ 1L6-4, onMi______ T(ET___ISL7__ I.-6,5nM 003 191 Differen tia pH binding of the antibodies bised atbove in Tablde 3 was then determined by ELISA. 50 pL. of Neutravidin (Pierce) at 8 pg/mL in I xPBS5 was coated upon a Nune Maxisorp 384-well plate, and incubated at 37"C for 1 hr. A fter blocking. the wells with o01%B5AiPS/t005%Tween20 for hour at room temperature, pltswere washed six times with PB1S/0.-05%Tween20.- 25 pL. of mono-biotinyiated hepeidin at 50 ng/mL in 0. %BiSAfPBiSI0,0S%Tween20 was added to the 3834-well plate, and incubated at room temperature tbr 1 hour, The' plates were next washed sux times with PBS/0,05%Tween20, Startingt bepcidmn antibody concentrations were normalized to I ptgkmL for pH 5.5 and 6 0 conditions and to 100 ng/mL~ for pH 7 4 conditions. The hepcidin antibodies were serially dihuted 3-fbid in PBS/l%NFDM pH- 7,4 and 4-fold in PBS/I %NFDM pH 6.0 and 5.5. The dilutions and titrations were pertormoed in polypropylene 961-well dilution plates, and then were transferred in duplicate to a Neutravidin-coated 384-well plate, The biotinylated hepcidin and antibodies were incubated for 2 hours at room temperature, The plate was next washed six times with PB3S/.05%Tween20, 25 p L of g oat anti-hulgG-horseradish peroxidase at a 1:7000 dilution in 0.1%BSA!PBS/0.05%Tween20 was next added to each well of the assay plate The plate was finally washed six times mn PBS/.05%Tween20, Enhanced K Blue- 33 ------- Tetranmethybenzidine (TMB) Substrate (Neogen) was added ad - the reaction stopped using I M H 3
P
4 after 10 minutes of incubation at room temperature. The absorption was measured at 450 ore on a plate reader. Binding data were analyzed by non-linear regression analysis (sigmoia dose--response. variable slope) to generate ECve values using~ GiraphPad Prnsm* software, Single mutations of wild type 15E31 that produced at least 1.5 foid increase in EC(50 as the pH4 was lowered to 6.0 included L.27H (light chain), A89H (light chain), W52 H (heavy chamn), N57H (heavy chain), A99H- (heavy chain), and A 107H (heavy chain). Double combinations of these nmutants were made, Multiple mutants of wild type I 5EI with at least a 5.5 fold increase in EC(50 as the pH1 was lowered to 6.0 included A 1079 (heavy chain)/A89H (light chain), A1 07H1 (heavy cham)/KL27H (light chain), Al079 (heavy 99 cain)!N57H Iheavy Chai), and A 0' H heavy chain)A99H (heavy chn) Representafve resuhs are set forth in Tab bebow Tabe 4 5 nlepH H 7.4 pH 6 pH 5,5 188F1 1 2.7 ______244. NC --- -v'~ --- ---- ks- --- .. ............ Si II 22733 2_____ 3 1 5E1! 89 54 10,6 2 15 N57H __ 8 4 3,2 1MB A99H1 4____ I, 10272, 154 A7H 2 3,6 3,7 1 -- -- 1 H 7- ----- - .75.6 1SE1 A99H A-7H- 30- i 43' "--__2_'1_5 16.1 ISI XIHIA89H 60 34.7 NC IEA O 10 .L27H .3. 0, 193 316 003 01 Resuls indicaed that antibody 8B demonstrated a 2og lower apparent bindng affinity and that 5E 53 A571 l07 demonstraed a og lower apparent ding aftin> for hepeidin at pH 6.0 compared to p-I 7A EXAMPLE 14 - OFF4RATH BINDING ANALYSIS FOR H iUMAN ANTIBODY 1813 1 00321 fHrate anayss of dissociation at di feret ps was also performed, A slow offli-rate is to increouased binding onterar a off-ateis xpetedto redctncrase bidin interact over a longer period of time, while a faster off-rate is expected to predict decreased binding interaction, For example, a faster off rate at lower pH is expected to predict greater release of antigen at lower pi. Solution euil ibrium bwnding analysis was performed using BiAcore to study the off-rates of antibodies I S1, 1 3, 2 7, 18311, 23F 11 and 26F 11 with recombinant human hepcidin (SEQ ID NO: 9), Preparationl cni -Acore Chip Surfeces (103221 immobilization ofrecombinant human hepcidin (.huHep') to a BtAcore sensor chip surface was performed according to manufacturer's instructions at a flow rate 10 L/in of running buffer (DPBS' Dulbecco's Phosphate Buffer Salinel X, no CaC or ACL. with 0.005% Bis'ore surfactant P-20 ). The carboxylated matrix of the sensor chip was first activated with a 60 p injection of a mixture containig 0.2 M! EDC (NethylN-. (dimethylamine-propylcarbodiinide in water, from BAcore) and 0,05M NHS (N hydroxysuccinimide in water, from Biacore). 55 L of recominbant human hepcidin (I pg/mil 100in 10 M aN-a ietate pH4- as injected to immobi ze onto the sensor chip The excess eactve groups of the sensor chip were deactivated with an injection of60 gL of ethanolamine (IJ)M from Bracore). 3lAcre Any s i 1003231 Aner rhulHepe was immobilized on the CMS chip with low density 50 nM of antibodies I S 1 S3, 2.7, 188111, 23F.1 1 and 26F11 were injected over and bound the ruHepe surface at pH 7, Dissociation buffers with pH 7A, p16 ad pH 5,5 wer rn7 over the bound surface. The dissociation curves were obtained. Results indicated that antibody 18B11 demonstrated a significant difference in off rate at pH 7,4 (>1x0) compared to p-1 5 S. The other antibodies tested did not demonstrate a significant difference in off rate at p- 7,4, 6,0 or 5. See Figure 1. EXAMPLE 5 -YN VITRO HE.PCIN ACIIV FT UN AN IRON~R ESPONS VE B-LACTAMASE ASSAY CAN BE NEUTRALUZED BY ANTI4I-EPCIDIN AN'TIBODES 1003241 Hepcidin causes feroportin to be internalized and removed from the cell surface, thus inhibiting release of iron and raising intracellular iron concentraions, The effect of anti-human hepCidin antibodies on this hepcidin-mediated iron sequestration was evaluated nvito, A 293 c line containing a doxycyclin-inducible ferroporin (Epn) expression construct as well as a beta-lactamase (BLA) expression construct containing one copy of the 51 iron response element. (IRE) hmn fenitin havmg the following nucleotide sequence: tcggcccgecctgccaccaga'te ggecetagcccteccogagegccetgcctcgagggcggcgC accataaaga agegocctagecacgiy etgattgegicggtteetgtcaa tgtttggacggaacagatceegggga ctctctuccagcctecgacegccetcgattectetc gcttgeaacctcc gggaccatcttctcggecatctcctgettctgggarctgccagiacegttttgtggttageteettettgecaace (SEQ lID NO; 103) that regulates nRNA translation was constructed. These .293/FprVBLA cells, taken from a 70~ 80% confluent culture, were plated at 2,8A10 cells/nL in DMFM (Invitrogen Cat# 11965) 5% FBS (lnvitrogen Cat# 10099-141) PSQ (Invitrogen Cat 103=7-016), 90 ghl/weh(, cells/well) in BioCoat Poly-) yine coated plates (Becton-Dickinson Cat 35-6640) and incibated at 37C with 5%1 CO At the end of the same day, a solution of assay medium (DMEM 5%FBS PSQ) wit 100 ug/mL doxycycine was made, 10 gL/well of it added to the plate, and the plate incubated overmght or for at least 20 hours. The next day, media was A0 removed rrtm the wells and replaced with premnade ixes of DMEM 5% FBS PSQ. 2. pghnL ferric citrate. 0 ng/nL synthetic hurnan hepcidin and serial dilutions of the antibodies (24E4, 23F11 k 188&&1, 27, 2.41, and Ab43), all prepared o a 96-well polypropylene deep-well block plate immediately before addition to the assay plate. Mixtures were added at 100 UTwel and incubated ovemight at 37C, %C0 2 in a cell culture incubator. Plates were then removed from the incubator and equilibrated to room temperature for 10 minutes before adding 20 pLUwell of the prepared invtrogen GeneBhuer CCF4 A/M development reagent (Invitrogen Kt# K 108S) and incubating for 90 minutes in the dark. development reagent was also added to 16 wens of a control assay plate without cells containing (10 L assay mediurn (DMEM 5%FBS PSQ and incubated for the same time. Blue & Green fluorescence signals were then read on an Envision Multilabel Reader (Perkin-Elmer Inc.) by exciting at 409nm and reading emissions at 447nm (b9ue) and 520nm (green). The results are depicted in Figures 2 and 3. It was determined that mAb 43, 2.7. 241, 81 7 , 23F 1, 24E4 decreased ntracellular concentration of iron at an ECi of 1.3801 ^ x 1 i .7 x 10", 636 x lA2,0 > l0t 2.3 x i0 and 5.0 xt respectively. EXA 6 -A IT I4EPCIDIN ANTI BODIES NE S lRAnS UZE HUMAN H EPGID N 1003251 Activity of ant-human hepcdin antibodies was evaluated m vivo in mice that were administered human hepcidin in an amount sufficient to generate a hypoferremic response. On day 0. female C57BL/6 mice were inJected subcutaneously with a urine monoclonal antibody (Ab2 7) directed against human hepcidir. Control mice received marine 1gG1 as an isotypic control At dcay 3, the mice received a single intrapeitoneal injection of 25 g recombinant an Hepcidin ( Serum iron leve w analyzed two hours later. Control animals treated with saline had normal serum iron levels, while animals treated with hepcidin and an isotype control antibody showed hypoferremia, Results are set forth in Figure 4. Both 1mg and 0.5mg of mAb2:7 provided statistically significant protection from the hypoferremic response, Although a reduction in hypoferremin was observed at the 0,25 mng dose of Ab 2.7, the lower doses (0.2 and 0, mg) were defined as non-neutraizing doses. Statistics represent ANOVA with a Dunnett's post-hoc test comparing all groups against the saline control, 102 EXAM PLE 7-- AN ,ODY NEUTRALIZATION OF AADELNERED IEt DIN RESTORES NORMAL EARY IED BLOOD CELL CARACERISTICS [003261 AAV-mdiated human hepcido expression in mice produces a microcytc, hypochromic anemia consistent with iron deprivation. The activity of andti-human hepcidin antibodies was evaluated in vivo in these Mice overex pressing human hepcidin. Male C57F/6 mice were injected with A AV (L x10" particles/mouse, LV:) containing expression cassettes fbr either human hepeidin or beta-alactosidase (fgai) as a negative control The mice were left frS two weeks to allow constitutive production of huHepc before being treated with 1mg/mouse of Ab 27 or isotype control (mulg 1) at varous dosing frequencies (IX, 2?X and 4X per week) as shown in Figure 5A Blood was drawn on the fifth day for serum iron levels and d nation of early red blood cell (reticulocyte) characteristics (reticulocyte count, reticulocyte hemoglobin content (CHr), and reticulocyte mean cell volume (Reti. MCV) 100327] Results are set forth in Figures SB5E, Serur iron levels werestored to normal in mice receding 4X dosing of Ab2,7 but not isotype control. All mice receiving Ab2 showed increased reticulocyte production, The reticulocyte hemoglobin content (CHr) was normal in mice given the 4 and 2x dosing of Ab 2,7, but hypochromicity is still seen in groups with lx dosing, or the isotype control group, Treatment with Ab2. at the 4X and 2X dose restored normal volume to reticulocytes (Retic. MCV) but microcytosis was still present in the IX and isotype control groups. Statistical comparisons to -gal injected animals with isotype control treatment were determined to look for restoration of normal red cell characteristics (ANOVA with Dunnett's post-hoc test), 100328 I in another experiment, the activity of ant-human hepcidin antibodies 131, ISI 1 and 24E4 was evaluated in wvo in mice overexpressing human hepcidin. C5781/6 tmce (4 weeks of age) were obtained from Charles River Laboratories, On Veek 0, mice (t= per group) were injected via the tail vein with AAV containing human hepcidini (hepe) or green fluorescence protein ( FP) as an expression control. Mice were maintained for 2 weeks after viral introduction to a1ow for protein expression before treatment with antibody, Mice were treated with either I mg or 01 mg of each antibody t 1, 188311 and 24E4 (subcutaneous injection, 0.2rinimouse in PBS) on Days 14 and 16 following viral introduction. Blood was collected on Day 18, and response to antibody administration was measured as a change in teulcA yte .OW ,epo'~ cae) g ean ADVIA 2120} retiuloytecharacteristics (reticulocyte cellular hemoglobin content) usingar DVA22 Hematology Analyzer (Bayer Corporation, Tarrytown, NY), Total serur bepeidin levels (free and bound) were measured by 3LISA to determine the degree of complex formation All results were expressed as the mean standard error of the mean, ANOVA and a Dunnen's post test uning Gahad Prism sonware v4.0 (San Diego, CA) assessed statistical significance of differences (* denotes p<0,05, and ** denotes p<Q0 compared to AA V hiHept isotype control group). 1003291 After 18 days, the reticulocytes in the AAVdbHepc + isotype treated control mice had reduced hemoglobin content (CHr), rendering them hypochromic, Animals treated with anti-hepidin antibodies 1S!, 188311 or 24114 at either 1mg or 0.5mg/mouse had normal CHr values as compared to AAV-GFP control mice, indicating that these antibodies are effiacious in this model in restoring normal earn red cell characteristics. See Figures 6A and 6B, 1003301 Results indicated that mice treated with the 1 mg dose of antibody 188I had a 1 0-fid reduction in total serum bepcidin compared to animals treated with antibody 1$1 or antibody 24E14 (Figure 7A), Similar results were obtained at the .Smg/mouse dose (Figure 713). The markedly reduced amount of total hepeidin seen with antibody 18811, is consistent with hepidin clearance through endosomes, EXAMPLE' - VIRAL HEPCIDIN OVEREXPRESSION RESULTS IN HY PO-RESPON SI VENES S1 TO ER YTHROPOfTIN 10033 The following Example investigated the role of thepcidin and anti-hepcidin an tibodies in erythropoi etmn hypo-responsive mice, 10093321 Titration of AAV-mediated human hepcidin expression in oice causes an increase in serum hepcidin levels and dose-dependent hypobrreimiaS as shown in Figure 8, Doses of AAV-humnan hepcidin were selected that gave an erythropoietin resistant phenotype and expressed levels of hepcidin in a similar range to that detected in cancer patient samples in previous studies (as described in co-pending co-owned U,, Patent Apphiation No. 11/880,313 and international! P i o No. WO 2008/011158. the disclosures of which are incorporated herein by reference in their entirety). Male C57BL/6 mice were injected with AAV expressing human hepeidin or GFP as an expression control (n=4 per group) The mice were injected through the tail vein (human hepcidin, irom I x010 to A 102 particles/mouse; GFP 3x 10 particles/mouse). Protein expression was allowed to develop tor two weeks prior to harvest, At two weeks, seram was collected from the mice and iron and hepcidin levels were determined, Results are reported in Fig.re 8 1003331 In order to evaluate heocidin's effet on erythropoietin resistance, male C5783/6 mice were injected with AAV (3x W particles/mouse, hepatic portal vein delivery) 104 containg expression cassettes fr either human hepcidin or GFP as a negative control (1=5 per group), The mice were lef for three weeks to aow constitutive production of human hepeidin, and then bled to determine baseine hemoglobin (Hb) levels. The mice were treated with darbepoetin alfa (100 Qg/kg/mouse) or saline as a negative control at four weeks, At five weeks, hemoglobin levels were again measured. Results are shown in Figure 9 Mice ove expressing human hepcidin are resistant to high doses of darbepoetin alfW. Resistance to darbepoetm alfa demonstrates that elevated hepeidin levels are sufficient to cause hypo rponsiveness to erythtopoetifl EXAMPLE 9 COMBINAION THERAPY V H it iIJECID N AN BOD0 AN) AN FdYTIROPOIESIS SIIMULAT n 3?O N AVRAL HBPbIN OVER EXt~ WSSION MODEL ff0334) Treating mice that possessed an erythropoetin resistant phenotype with an aniheUcidin antibody restored responsiveness to treatment with darbepoetin alfa. Male C57..6 mice were infected with AAV ($xI0 p articles/mouse L.) containing genes coding fbr either human hepcidin or GFP as an expression control (n=5 per group), After allowinAg two weeks to estabhsh constitutive protein expression, mice were bled to determine baseline hemoglobin (Nb) levels, then treated with Ab 2.7 (1 mg/mouse) or isotype control at various dose frequencs, On the dy after the first dose, they were treated with darbepotin alta (100pg/kg, subcutaneous), A schematic of the dosmg schedule appears in Figure 1GA. 100335 Neutralization of hepcidin restores responsiveness to darbepoetr a&fI. Monday- Wednesday-r iday dosing of the antibody led to a partial response to darbepoetn alfa treatment as measured by an increase in hb levels; a cohort with the same antibody dosing without. darbepoetin alfa treatment showed no rise in Ib levels. (See Figure 10B) A maximal response to darbepoetin alfa was achieved in mice receiving daily (Monday through Friday) dosing of Ab 2 7 (See Figare 10) Two and three doses of anibody in combination with darbepoetin aifa treatment ed to a partial response, as measured by Hb levels (See Figure 10D) Antibody dose and proximity of antibody dose to darbepoetin alfa treatment affected overall ib response to anti-epcidin antibody treatment, as shown in Figure KOE (results varying from the control where p <0,01 by ANDOVA with Dunneit's post-hoe test are noted with double asterisks). Thus antibody-mediated neutralizaton of hepcidin was shown to be an effective treatment for anemia caused by elevated hepeidin levels, lOS EXAMPLE 10 CO BNAIONTURAPY WITH AN ANT IIEPiOIN ANTIMY N D ERY ROPOIESIS STIMULATOR IN A MOUSE MODEL OF NFLANIATORY ANEMIA 1003361 Combination therapy with an antilbepeidin antibody and an erythropoiesis stimulator was also evaluated in a urine inflammatory anemia model as follows 100337i Mice were generated such that murnine epcidin I was knocked out and replaced with human hepeidin. Female mice, both homozvgous for human hepcidin expression and widtype lttermate controls, were injected with Brucefa aborfus 02x10 particles/mouse, LP.) on day 0 and then bied on day 6 to assess hemoglobi levels, The mice were then treated with ether Antibody 27 or an isotype control ab (Img/mouse/day) o days 6 through 9. Darbeoetm alfLa was administered (100pgfkg/mouse) on day 7, and 1Hlb levels evaluated on day 13 A schematic of the protocol is shown in Figure 11 A, 1003381 Wild-ype control mice which still possessed the mouse hepcidin I gene did not respond to darbepoetin alfa either with or without Ab 2.7. (See Figure 1i B) Human [nock -m mice treated with Antibody 2.7 exhibited a restored responsiveness to darbepoetin aO treatment, as shown by the maintenance of stable hemoglobin levels. (See Figure I 2C), 1003391 These results demonstrate that ami-hepcidin antibodies can be used to neutralize hepcidin under conditions of hepcidin excess and restore responsieness to erythropoietic agents in nepcidin-mnediated antmia~s such as the anenia of intRammrration, EXAMPLE 11 MEASUREMENT OF HiEPCIDiN LEVEL LN PATIENTS 1003401 The level of hepcidin in human patients was measured by spectrometry techniques as previously described in co-pending co-owned U.S. Patent Application No. I 1/880213 and international Publication No, WO 2008/011158, the disclosures of each of these applications are incorporated herei by reference in their entirety The method is repoduced bdow. 1003411 Samples from patients suffering from anemia of cancer (obtained from ProteoGenex) or volunteers (control) were collected. l00 p L of each sample, serum blanks and calibration standards consistig of seven non-zero concentrations in duplicates (10, 25 50, 100, 250, 500, 1000 ng/ML) were extracted by SPE using an Oasis HLB mElution 96wel plate (Waters, Milford, MA), Washing sol vent was 30% methanol/water with a pH of about 10 adjusted with amoniumi hydroxide. Elution solvent was 90% methanol/water solution with a pH- of about 5 adjusted with acetic acid, The SPE plate was activated wah $00 pL -106methanol and conditioned with 500 g L water, then 100 p L serum sample and 200 L intemal standard were loaded onto the elution p1ate washed with 350 p LV water and 350 pL washmg solvent Elution was done using 1 00 pL elution solvent and diluted with 100 PL water. The suing 200 p L eluate was analyzed by LCMS/MS. 1003421 20 p1 of each extracted sample was injected onto a Polaris C18A 5 m HAL.C column (2. x M50 m, ovarian . The L flow rate was set to 300 p1/mi. The HPLC mobile phase A was 5:95 methanol/water, and miobile phase B was 95:5$ methanol/water, both containing 0 1% formic acid. The gradient conditions were set as follows: 0-0, 1 mm, isocrati 2% /98% A; 2% B to 95% B at 0145 rain; 95%B at 145-49 min; 95% B to 2.%B at 4.95.0 in; 5,0-6,0 min, isocratic 2% B. 00343 A Sciex AP14000 triple quadruple mass spectrometer from Applied Biosvstems (Foster City, CA) with Turbo ESI source was used for hepcidin detection in MRM mode with ion transition of ma 930.60 to maz 10 i5 Quantification was achieved by compariOg the ratio of the 1C peak areas of the hepeidin and the internal standard to the ratios obtained from a series of standards where the amounts of hepeidin. and internal standard were known 1003441 This experiment allowed fow the detemunation of the serum levels of hepeidin in a control population presumed to contain a large number of healthy individuals as well as the serum level of hepcidin from patients anemia of cancers (AoC), The results are shown in Figure 12. 10034,S Each patients sample was then analyzed or other iron index concentrations to determine whether a patient had inflammation or iron deficiency anemia (Figure 13). The parameters were measured as tcilows: serum iron, UBC, ferritin. and CRP were measured on an Olympus AU400 clinical laboratory analyzer using standard procedures; sTfR was measured using a standard F LI SA method (R&D systems) 1003461 As described in copending U.S. Patent Application No. 12/022,515, inco rpo ratedby preference herein in its entirety, prohepcidin levels measured using the DRO prohepcidin EUSA kit, however, do not correlate with the mature hepcidin levels of the patients, nor do prohepcidi levels correlate with the inflammatory status of patients Hepeidmn, but not prohepcidin, shows a relationship with C'P in anemia of cancer patients and can therefore be used as a marker of inflamaion. 100347 1 Distinguishing the anemia of flammation (Al) from iron deficiency anemia (IDA) and mixed anemia (components of both Al and IDA) is comphcatd since most of the commonly used lab parameters are influenced by acute phase responses. A ratio utilizing 147soluble vransferrin receptor (sTfR) and ferritin (Ft) values has been described in the literatxre as a means to provide a more accurate diagnosis, See Punnonen et at, Blood. 89:05-7 1997. Anemia orfinflammation is characterized by a low sTfR/log Ft quotient (values less than one), while a high ratio is indicative of IDA, Hence, the sTIR/log Ft ratio may serve as an accurate predictor of the three conditions when combined with an inflammatory marker to ahd diagnosis of mixed anemia from absolute mDA. I 00348) Hepeidin levels are strongly related to sTtR/log Ft levels in AoC patients (r"' 0,6407; P<Q.000)O, thus aiding patient diagnosis, 00349) Using a decision tree combining CRP as a marker of inflammation and sTfPJiogFt, anemia of cancer patients could be sub-divided into those with A L with mixed anemia, with IDA and with an anemia or unknown origin, designated *othet (Figure 4A), Patients with elevated hepcidin levels were all observed to have either Al or a mixed anemia, (Ftgure 15). Patients with low or absent hepcidin levels were observed to have either IDA or anemia of unknown origin, Hepeidin levels, as meassured by the antibody-based jmmuinoassay methods described in copending U lS. Patent Application No, 12/022,515, incorporated by reference herein in its entirety, or the mass spectrometry-based method quantitation method descmbed in co-pending co-owned US, Patent Application No, 1/8380,313 and international Publication No, WO 2008/01 1158, the disclosures of which are incorporated herein by reference in their entirety. and discussed in detail above, can be used to diagnose in flamiator anemia. EXAMPLE 12 -MONOCLON AL ANTIBODIES IN A SANDWICH IMMUNOA SSAY FOR HEPCIDIN 100350 The following Example describes a sandwich immunoassay to determine hepcidin levels in a sample. 101)351 1 Using Biacore analysts, a surface coated with antibody 1S1 was tested for the concurrent binding ofh hepcidin and another antibody (Figure 16). Immobilization of anti Hepc antibody I SI to the seso chip surface was performed according to maniufacturer's instructions uising a continuous flow of 0.005% P-20/P BS8 buffer, Brie-fly, carboxyl wgrps on the sensor chip surfaces were activated by injecting 60 pLE of a mixture containing 0.2 M N ethyl -N -(dimethylainopropyl~carbodiimnide (EDC) and 0.05 N J-hydroxyvsuccinimi de (NHIS), This was followed by inj ectin~g 1S1 dilut-ed in I 0mM acetate, pH 4,0 at concenitraions between 20 p g/mL Excess reactive groups on the surfaces were deactivated by injecting 60 ygL of I M ethanolaminie Final immobilized levels were 5J000-6,000 resonance units (RU)1 108 for the Ab I S1 surface. A blank, mock -coupled reference surface was also prepared on the sensor chip. 2CnM coli-derived human hepcidi was injected over and bound to the 1 S antbdy surface, Then 5nM antibody 2, 23F11, 26F01, and 11 were injected over the hepcidin /11 surface. A fter the antibody injecton, the surfaces were r a byinrectimg 30 pL 10mM HCI pH 2.0. 100352| There was a high selectivity of binding in the form of complexes. The muine antibody 2.7., which was used in the competitive assay above, was not able to form a sandwich pad with 18 1, and 26F11 showed m e lower a y to bind to hepcidi concurrently with S! than did 23F1 1. 1003531 When 1S1 and 23Fi l were assembled into a sandwich ELISA format, the sensitivity of the imnunoassay for detecting hepcidin levels was improved by 50fo1d. As shown in Figure 17, the assay proved capable of measuring levels of hepcidin in normal sera after a 50-fold prewdilution step. The axis represents the hepcidin levels pre-dilution EXAMPLE 13 -COMPETITVE BIN DING ASSAY 1003541 The flowing Example describes a competitive binding assay to determine hepcidin levels, in one protocol, unlabeled hepeidin present in serum competes with biotinylated hepcidin tor binding to an anti-hepcidin antibody (e~g.. Antibody 2,7). 1003551 Hepcidin levels were determined using hepetdtn standards of varying concentrations (from I .400 ng/ml) spiked into buffer(5% BSA ibiock) rabbit serum, or pooled human serum, Hepcidin was added to equal volumes of 40ng/mL o Ab2.7 and incubated tor 120 minutes. 25 pu/well of mixed solution was added to Black half area plates coated with 1-2pg.mL CxM capture antibody. 25 L/well of btnated hepcidin was added at 0.25nM. The plate was covered with plate film sealer and icubated at room temperature (25"C) on a plate shaker at around 200RPM for around 60 minutes. The plate was washed and then 50 pL/well of Polv horseradish peroxidase amplification reagent at 1:2000 was added, The plate was allowed to sit for 30 minutes and was then washed with a plate washer using PBS or KPL butffr 6 times. The plate was patted dry and a urninescent substrate (Femto or Pico) was quickly added, The plate was read with luminometer (ex: mnax 340) for 1 second using Feto or Picn Substrate. Results indicated that hepeidin was measurable at a concentration range of 1- 100 ng/mi in both the the rabbit serum and buffer. (Figure 18). 1003561 Pooled human serum appeared to have an existing hepcidin level of greater than 20 ng/n. It was determined that the levels of hepcidin varied substantially in human sera, over the range of 130 ng/mifo various randomly selected sera (Figure 19) 1090 100357 1 Using hepidin standards i rabbit serum determined above, 24 random sera from normal human subjects was tested. The hepcidin levels varied fror undetecta 50 ng/ml See Figure 20. These vales were at variance with the results from the levels of hepcidin measured through the mass spectrometry-ased quantitation method described in co pending co-owned U.S. Patent Application No. 11/880313 and Intemational Publication No, WO 2008/01 158 the disclosures of which are incorporated herem by reference in their entirety, which generally gave much lower values. EXAMPLE 14 AMACOKINETICTUDY OF ANTIBODYFOLLOW 4 NO SINGLE DOS O' 0 ANIBODYH EPC lOIN COMP hEX 1003581 2757 BL/6 mice were pr-dosed with either the control antibody or antibodies iS? or 1Bu i on Day 0 as a single intraperitoneal injection at a dose of I mg/mouse to ensure that the antibody concentation was above the antibody K On Day 1, the mice were dosed with an antibody-hepcidi n complex ( e either iS 1 iepcidin complex or 18B 1-hepcidin eompelex. Urine samples for determinaton of hepcidin concentrations were collected prior to hepcidin administration and at I hour 24 and 96 hours antibody-hepeidin complex administration. The resuhs are set forh in Iable 5 below, Table 5 1S__ __ _ 1S431_ _ _ ---- __ _ _ _ ----.......... .....------ - ... _ _ _ _ .. ..... --- ------ ___(hours Hidi ocntainHepii Cnetrto Not detectable 20ng/m L 24 Not detectable _______INot detcctabhle 96 Not detectable Not detectable {003591 Serum samples for determination of semm antibody and serum hepeidin concentrations were collected at 5 minutes, I hour, 24 hours, 96 hu 16 hours, 264 hours and 336 hours after adaimstration of the antibody-hepcidin complex Serum antibody and hepeidin concentrations were calculated by ELISA and the results are set forth vin Figures 21 and 22, respectively. Results indicated that the concentration of serm hepcidin at the 5 minute timepoint in mice that received the 1831!-hepcidim complex was lower compared to the IS ihepcidin complex. interestingly, hepidin was not detectable after 24 hours in mice that received the 18 R1I -hepcidin complex, while mice treated with the IS1-hedin complex still had detectable levels of serum hepcidin at 168 hours, -110- EXAMPL E $ - PH AR MACOK1NETIC STUDY OF ANTILBODiES FOLLOWING SlNGLEt DOSE OF FREE NEPOIDIN TO MICE 100360! 257 BL/6 mice were pre-dozed wih either the control antibody or antibodies IS! or ISBI I on Dayas a sin intraperitonea irection at a dose of 1 mgnouse. On Day 1, the mice were predosed with the antibodies as a single intravenous injection at a dose of i mg/mouse. On Day 4, human hepcidin (3.72pg/mouse) was administered to the me by intravenous injection. Urine samples fr deterination of heperdm coctrations were collected prior to hepcidin administration and at 1 hour, 6 hours and 24 hours post-hepcidin adinistrration, Results indicated that hepcidin was not detected in mice pre-dosed wih either 181 r 1381. e gre2 {00361] Serum samples for determination of antibody I or , B1 and hepcidin concentrations were collected at 5 mnuus, 1 hour, 24 hours, 96 hours, 168 hours, 264 hours and 336 hours after administration of the hepeidin, Serum antibody and hepcidm concentration were calculated by E LISA and the results are set torth in Figures 24 and 25, respectively. Results indicated that antibody 1811 cleared all detectable serm hepcidin by 24 hours, while hepcidin levels stabilized in mice treated with antibody 11 EXAMPLE 16 - DETECTION OF HEPCIDIN INTRACELLULAR ACCUMULATiON BY ANT BODIES COMPACTED WETH CELLS EXPRESS NG FCRN 1003621 FeRn is the salvage receptor involved in recycling antibodies by rescuing them from cndosomal degradation. Thi Example examined the effect of antibodies on relative levels of intracellular hepcidin compared to total hepeidin, providing an indication of the iernalization nd subsequent degradation oby cnels expressing FcRn Alexa-647 labeled IS! or i11 I antibodies were completed with excess of biotinylated-hepcidin by icubation for 10 irutes at roor temperature. Free hepcidin was removed using spi columns 293T/FcRn eells were incubated with the antibody-epcidin complexes for 6 hours at 37C, 5% CC0 in (0% FBS medium. At the end of the incubation cells were harvested in cold FAs buffer (PBS 2% FBS) Cells rom each group were either fixed only (detection of extracelluldar hepeidin) or fixed and perreabilized (detection of total epcidin) using R&D's CytoFix and CytoPern reagents. All samples were stained with SA-PE and read on FAGS Results indicated that antibody 18B i caused greater intracellular accumulation of hepcidin compared to antibody I Sh See Figure 26. Of the total hepcidin detected in association with cells contacted with 181, all of the hepcidin was extracellular, Of the total hepcidin detected in associaton wih cels contacted with O a 1 8 1, only about onethird of the hepeidin was extracellular ad the remainder was intracellular. 1003631 For the sake of completeness of disclosure, al patent documents and literature articles cited herein are expressly incorporated in this specification by reference in their entireties. j0034 The foregoing description arnd exarnples have been; set forth merely to illustrate the invention and are not intended to be limiting. S moe modifications of the described embnodimnents mocorporatinrg the spirit and substance of the in vention may occur to persons sailed in the art, the invention should be construed broad>y to include all variations within the scope of the appended claims and equivalents thereof WOUNS A'd "r O11>

Claims (12)

  1. 2. An isolated antibody that binds to human hepcidin of SEQ ID NO: 9 with an affinity K 0 of ess than about I 0YM that exhibis at least one of the properties selected from the group consisting Of: (a) reduces the level of totahuman heidin in serum by at least 90% in a C57J6 mouse about 24 hours after the administration to said mouse of (i) a 1 mg doses of said antibody and (ii) a precomplexed single dose of 32 tg of human hepcidin with a I mg dose of said antibody (b) reduces the level of total human hepcidin in serum in a mouse by at least 90% about 24 hours after said mouse is adminstered a single dose of 3, pg of hum an hepcidin whdern said hepcidin is administered three days after said mouse is pre-dosed with said antibody; (C) results in a greater than50% reduction in overallaccumulation of total serum hepcidin in mice treated with said antibody compared to antibody I S1; and (d) results in at least about a 2--old higher intraceliulr accumulation of hepcidin in FecRn transfected HEK293 cells incubated with said antibody compared to antibody 15s)
  2. 3. The antibody of daim I that exhibits at least one of the properties selected from the group consisting of: (a) reduces the level of total human hepcidin in serum by at least about 90% in a C57BU6 mouse about 24 hours after the administration to said mouse of(i) a ig doses of said antibody and (ii) a pre-omnplexcd single dose of 3,7 pg of human hepcidin with a I mg dose of caid antibody, (b) reduces thelvel of total human hpeidin in Serm in a mouse by at least about 90% about 24 hours after said mouse is administered a single dose of 3, g of human hepoidin, wherein said hepcidin is administered three days after said mouse is predosed with said antibody; (c) results in a greater than about 50% reduction in overall accumulation of total serun Iepcidin in mice treated with said antibody compared to antibody ISl; and (d) results in at least about a 2-fbld higher intracelular accumulation of hepeidin in FeRn transfected HEK293 cells incubated with said antibody compared to antibody 181
  3. 4. An isolated antibody according to any of claims 1-3 that binds to human hepoidin of SEQ !I NO: 9 with an affinity K 0 of less than about I 0M, wherein said antibody increases circulating iron level or Tsat in a mouse overexpressing human hepcidin for at least I day after a single dose of antibody,
  4. 5. The isolated antibody according to any of claims 1-3, wherein said antibody decreases iron in ferroportin-expressing cells stimulated with 50 ng/mL hepeidin at an ECs 5 of about 20 nM or less.
  5. 6. An isolated antibody that binds to human hepeidin of SEQ ID NO: 9, with an affinity K 0 of at least 01 M0 wherein said antibody is obtained by: (a) replacing an amino acid in the heavy orlight chain of said antibody with a hi stidine; (b) screening the antibody obtained in (a) for differential pH binding; (c) replacing another amino acid in the heavy or light chain of said antibody with a histidine; and (d) screening said antibody for having at least one of the properties selected from the group consisting of: (i) at least about 50- fold higher K 0 at about pH 5.5 compared to its Kr for said hepcidin at about pH '7., and (ii) an off rate of about 6x104 sa or higher at about pH 5. The isolated antibody according to any of claims 1-6, wherein said antibody increases the level in a subject of one of at least hemodobin or heaintocrit, or both.
  6. 8. The isolated antibody according to any of claims 1-6, wherein said antibody increases In a subject one of at least the red blood cell count, the red blood cell hemoglobin content or the red blood cell iean cell volume of red blood cell count, or any combinations thereof
  7. 114- 9 The isolatedantibody according to any of claims 1-6, wherein said antibody increases in a subject one of at least the reticulocyte count, the reticulocyte hemoglobin content or the reticulocyte mean cell volume of reticulocyte count, or any combinations thereof 10, The isolated antibodyaccording to any of claims 1-6, wherein said antibody inhibits the iron-regulating activity of hepeidin, 1 L An isolated antibody according to any of claims 1-6 comprising an amino acid sequence at least 90%.identical to SEQ ID NO: 170 or to SEQ ID NO: 168, said polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NIs: 3 1.71-76, and any sequences comprising at least one amino acid change to any of SEQ ID NOs: 171~176 12. An isolated antibody according to any of claims 1-6, comprising SEQ ID NOs: 171 -173. 13. An isolated antibody according to any of claims I-6 or 12. compnsingSEQ ID NOs: 174-176. 14. An isolated antibody according to any of claims 1-6, comprising an amino acid sequence at least 90% ientical to SEQ ID NO: 333 or to SEQ ID NO: 331 said polypeptide comprising at least one anino acid sequence selected from the group consisting of SEQ ID Ns: 334-349. arid any sequences comprising at least one amino acid change to any of SEQ ID NOs: 334-349, 15. An isolated antibody according to any of claims 1-6, comprising SEQ ID NOs:
  8. 334-346, 1 An isolated antibody according to any of claims 1 -6 or 15, comprising SEQ ID NOs: 347-349, 17 An isolated antibody of according to any of claims 1-6, comprising an amino acid sequence at least 90% identical to SEQ ID NO: 343 or to S EQ ID NO: 341, said polypeptide comprising at least one amino acid sequence sdected from the group consisting of SEQ ID NOs: 344-349, and any sequences comprising at least one amino acid change to any of SEQ ID NOs; 344-349, 18, An. isolated antibody according to any of claims 1-6 comprising SEQ ID NOs:
  9. 344-346 19. An isolated antibody according to any of claims 1-6 or 1$, comprising SEQ ID NOs: 347-149, 20, An isolated antibody according to any of claims 1-6, comprising an amino acd sequence at least 90% identical to SEQ 1D NO: 33 or to SEQ ID NO: 351, said polypeptide -115- composing at least one amino acid sequence seeded from the group consisting of SEQ I) NOs: 354-359, and any sequences comprising at least one amino acid change to any of SEQ ID NOs: 354-359 21, An isolated antibody according to any of claims 1 -6, composing SEQ ID NOs:
  10. 354-356 22, An. isolated antibody according to any of claims 1-6 or 21, comprising SEQ iD NOs: 357-359, 23. An isolated antibody according to any of claims 1-6 com.prising an amino acid sequence at least 90% identical to SEQ ID NO: 363 or to SEQ ID NO: 361 said polypeptide comprising at least one amino acid sequence selected from the group consistng of SEQ ID NOs: 364-369, and any sequences comprising at least one amino acid change to any of SEQ I) NOs: 364369, 24. An isolated antibody according to any of claims 1-6 comprising SEQ ID NOs; 364366, 25. An isolated antibody according to any of claims 1-6 or 24, comprising SEQ ID NOs: 367069 26. An isolated antibody according to any of claims 1-6, comprising an amino acid sequence at least 90% identical to S EQ ID NO: 373 or to SEQ ID NO: 371, said polypeptide comprising at last one amino acid sequence selected from the gaoup consisting of SEQ ID Ns: 374379, and any sequences comprising at least one amino acid change to any of SEQ ID NOs: 374279. 27, An isolated antibody according to any of daims 1-6, comprising SEQ ID NOs: 28 An isolated antibody according to any of claims 1-6 or 27, comprising SEQ ID NOs: 377379. 29. An isolated antibody according to any of claims 1-6,.comprising an amino acid sequence at least 90% identical to SEQID NO 383 or to SEQ ID NO: 381, said polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 384-389, and any sequences comprising at least one amino acid change to any of SEQ ID NOs: 384-389, 30 An isolated antibody according to any of claims 1-6, comprnsing SEQ ID) N()s:
  11. 384-386, 31 An isolated antibody according to any of claims 1-6 or 30, comprising SEQ ID NOs: 387-389, 32. An isolated antibody according to any of claims 1-6, comprising an amino acid sequence at least 90% identical to SEQ ID NO: 393 or to SEQ ID NO: 391, said polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 394-399, and any sequences comprising at least one amino acid change to any of SEQ ID NOs: 394-399, 33, An isolated antibody according to any of claims 1-6, comprising SEQ ID NOs:
  12. 394-396, 34. An isolated antibody according to any of claims 1-6 or 33, comprising SEQ ID NOs: 387-389 35, An isolated antibody according to any of claims 1-6, wherein said anybody comprises an amino acid sequence of SEQ ID NO: 170 wherein at least one of the amino acids at positions 52, 57, 99 and 107 of said amino acid sequence are replaced with. a istidine 36, An isolated antibody according to any of aims 1 -6 or 20 wherein said antibody comprises an amino acid sequence of SEQ ID NO 168 wherein at least one of the amino acids at positions 2? and 89 of said amino acid sequence are replaced with a histidine. 3 The isolated antibody according to claim 35 or 36 wherein an amino acid at position 107 of*SEQ ID NO: 170 is replaced with a histidine. 38. The isolated antibody according to claim 37, wherein the amino acids at positions 57 and 107 of SEQ ID NO: 170 are both replaced with a histidine. 39. The isolated antibody according to claim 36, wherein the amino acid at position 107 of SEQ ID NO: 170 and the amino acid at position 27 of SEQ ID NO: 168 are both replaced with a histidine 40: The isolated antibody according to claim 37, wherein the amino acid at position 107 of SEQ ID NO: 170 and the amino acid at position 89 of SEQ ID NO: 168 are both replaced with a histidine, 41. The isolated antibody according to claim 37, wherein the anmino acids at positions 99 and 107 of SEQ ID NO: 170 are both replaced with a histidine, 42, The isolated antibody according to any one of claims 1-41, wherein the antibody is a monoclonal antibody 43. The isolated anybody according to daim 42 wherein said antibody is a chimeric, humanized, or human antibody, -117- 44 The isolated anybody according to claim 42, wherein said antibody is a human antibody. 45. The isolated antibody according to claim 42, wherein the antibody is of an IgG isotype, 46, The isolated antibody according to claim 42, wherein the antibody is of angG I Ig6 2 , 1g03 or IgG4 isotype 47. An isolated nucleic acid molecule comprising a ncleotide sequence that encodes the antibody according to any one of caims 146. 48, An expression vector comprising the nucleic acid molecule according to claim 47 operably linked to a regulatory control sequence. 49 A host cell comprising the vector according to claim 48 or a nucleic acid molecule according to claim 47 50, A method of using the host cell according to claim 49 to produce an antibody, comprising culturing the host cell according to claim 49 under suitable conditions such that the nucleic acid is expressed to produce the antibody. 51 The method according to claim 50, further comprising recovering the anybody from the host cell culture. 52 A composition comprising the antibody according to any one of claims 1-46 and pharmaceutically acceptable carrier diluent or recipient. 53. A method of treating a disorder of iron homeostasis in a subject in need thereof comprising administering to said subject the antibody according to any one of claims 1-46. 54 -1, The method according to claim 53, wherein the disorder of iron hom ostasis is selected from the group consisting of: anemia, sepsis, anemia of inflammation, anemia of cancer, chemotherapy induced anemia, chronic inflammatory anemia, congestive heart failure end stage renal disorder chronic kidney disease (stage 1, 11, 11, IV or V), iron deficiency anemia, a disorder of iron homeostasis ferroportin diseasehemochromatosis diabetes, nflammationheumatoid arthritis, arterioscierosis, tumors, vasculitis, systemic lupus erythematosus, hemogiobinopaihies and red blood cell disorders 55. A method of treating a human with an elevated levelof hepeidin comprising administerng tJhe composition according to claim 52, 56. A method of treating a human with anemia composing administering the composition according to claim 52, 57. The method according to claim 56, wherein the human suffering from anenia, sepsis, anemia of inflammation, anemia of cancer, chronic inflammatory anemia, congestive -118- heart failure, end stage renal disorder chronic kidney disease (s 1a , g , IV or V), iron deficiency anemia, a disorder of iron homeostasis; ferroportin disease hemochromatosis diabetes, inflanmation, rheumatoid arthritis, arteriosclerosis, tumors, vasculitis, systemic 1upus eryhernatosus, hemoglobinopathies, red blood cell disorders 58. The method according to any one of claims $5-56, Further comprising administering to said human an erytlhropoiesis stimator selected from the group consisting of erythropoletin, erythropoietin variants and antdbodies that bind erythropoi etin receptor, 59. The method according to claim 58, wherein the erythropoesis stimulator is human erythropoietin of SEQ ID NO: 72, 60 The method according to claim 58, wherein the erythropoiesis stimulator is darbepoetin alfa of SEQ ID NO: 73. 61. The method according to claim 58, wherein the method further comprises adinii sting' iron to said patient.
AU2013227987A 2008-05-01 2013-09-10 Anti-hepcidin antibodies and methods of use Abandoned AU2013227987A1 (en)

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