Composition and method for diagnosing and treating iron-related disorder

Abstract

To provide a composition and method for treating an iron-related disease which are very specific for a subject who does not react with epoetin and related analog thereof, shows excellent tolerability, and can be useful as a useful therapy.SOLUTION: Provided are an isolated antibody that has a specific amino acid sequence, and binds to Repulsive Guidance Molecule c (RGMc), or an antibody fragment thereof. Provided is a method for treating an iron metabolism disease including a step of administering a therapeutically or preventively effective amount of the antibody to a subject who needs it.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 61 / 570,715, filed December 14, 2011, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application has been submitted in ASCII format via EFS-Web and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The ASCII copy, created on February 13, 2013, is named 029996-1767-WOO1_SL.txt and is 105,782 bytes in size.

[0003] The present invention relates to antibodies and methods of using the antibodies for treating and diagnosing iron-related disorders. [Background technology]

[0004] Iron homeostasis is essential for the normal functioning of the body. Since iron is central to hemoglobin production, insufficient levels of iron result in iron deficiency anemia. Iron overload can also disrupt iron balance by inappropriately increasing iron absorption from the intestine. This increase often results in iron deposition in the liver, pancreas, heart, pituitary gland and other organs, causing tissue damage and impaired normal function of these organs.

[0005] A variety of iron-related disorders are due, at least in part, to iron misregulation and can be difficult to diagnose and treat. Such disorders include liver disease, hypogonadism, diabetes, cirrhosis, cardiomyopathy, iron deficiency anemia, and anemia of chronic disease ("ACD"), which are characterized by unequal distribution of iron associated with infection, malignancy, and / or chronic inflammation. Current treatments often fail to adequately diagnose and treat iron disorders, as symptoms associated with iron-related disorders are often unclear and tend not to have immediate effects. These difficulties can cause delays in administering appropriate treatment. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there is a need for reliable methods of diagnosis and treatment for iron-related disorders. Current treatment options for iron-related disorders, including anemia of chronic disease, include administration of erythropoietic agents such as epoetin alpha, epoetin beta and darbepoetin. Additional treatments include oral or parental iron therapy and / or blood transfusion. However, iron therapy has limited efficacy and is not usually recommended for ACD subjects. In addition, blood transfusions have ongoing problems with multiple organ dysfunction and high mortality in critical care patients. Therefore, there is a need for novel methods of treating iron-related diseases that can serve as a highly specific, well-tolerated and useful treatment for subjects who do not respond to epoetin and its related analogues in a satisfactory manner. [Means for solving the problem]

[0007] Summary of the Invention In one aspect, the present invention relates to an isolated antibody or antibody fragment thereof that binds to Repulsive Guidance Molecule c ("RGMc"). The antibody comprises (a) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:3, (b) a variable light domain region comprising the amino acid sequence of SEQ ID NO:4, (c) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:5, (d) a variable light domain region comprising the amino acid sequence of SEQ ID NO:6, (e) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:7, (f) a variable light domain region comprising the amino acid sequence of SEQ ID NO:8, (g) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:9, (h) a variable light domain region comprising the amino acid sequence of SEQ ID NO:10, (i) a variable heavy chain comprising a complementarity determining region (CDR)1 comprising the amino acid sequence of SEQ ID NO:11, a CDR2 comprising the amino acid sequence of SEQ ID NO:12, and a CDR3 comprising the amino acid sequence of SEQ ID NO:13, (j) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:14, a CDR2 comprising the amino acid sequence of SEQ ID NO:15, and a CDR3 comprising the amino acid sequence of SEQ ID NO:16, (k) a CDR1 comprising the amino acid sequence of SEQ ID NO:17, a CDR2 comprising the amino acid sequence of SEQ ID NO:18, 2 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 19; (l) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (m) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25; (n) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28; (o) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 31; (p) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR2 comprising the amino acid sequence of SEQ ID NO: 33, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 34; (q) a CDR1 comprising the amino acid sequence of SEQ ID NO: 11;(r) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 19, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a CDR2 comprising the amino acid sequence of SEQ ID NO: 21 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (s) a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR2 comprising the amino acid sequence of SEQ ID NO: 24 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25; (t) a variable heavy chain comprising a CDR2 comprising the amino acid sequence of SEQ ID NO:24 and a CDR3 comprising the amino acid sequence of SEQ ID NO:25, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:26, a CDR2 comprising the amino acid sequence of SEQ ID NO:27 and a CDR3 comprising the amino acid sequence of SEQ ID NO:28, (t) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:29, a CDR2 comprising the amino acid sequence of SEQ ID NO:30 and a CDR3 comprising the amino acid sequence of SEQ ID NO:31, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:32, a CDR2 comprising the amino acid sequence of SEQ ID NO:33 and a CDR3 comprising the amino acid sequence of SEQ ID NO:34. The antibody may be an immunoglobulin molecule, a disulfide linked Fv, an affinity matured antibody, a scFv, a chimeric antibody, a single domain antibody, a CDR grafted antibody, a diabody, a humanized antibody, a human antibody, a multispecific antibody, a Fab, a bispecific antibody, a DVD, a Fab', a bispecific antibody, a F(ab')2 and an Fv. 3. The isolated antibody or antibody fragment of claim 2, wherein the antibody or antibody fragment is a monoclonal antibody, a humanized antibody or a human antibody. The antibody or antibody fragment comprises a heavy chain immunoglobulin constant domain selected from the group consisting of a human IgM constant domain, a human IgG4 constant domain, a human IgG1 constant domain, a human IgE constant domain, a human IgG2 constant domain, a human IgG3 constant domain and a human IgA constant domain.

[0008] The isolated antibody or antibody fragment may comprise a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:3, a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:5, a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:7 or a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:9. The antibody may comprise a variable light domain region comprising the amino acid sequence of SEQ ID NO:4, a variable light domain region comprising the amino acid sequence of SEQ ID NO:6, a variable light domain region comprising the amino acid sequence of SEQ ID NO:8 or a variable light domain region comprising the amino acid sequence of SEQ ID NO:10. The antibody may comprise a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:3 and a variable light domain region comprising the amino acid sequence of SEQ ID NO:4.

[0009] The antibody may comprise a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:5 and a variable light domain region comprising the amino acid sequence of SEQ ID NO:6. The antibody may comprise a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:7 and a variable light domain region comprising the amino acid sequence of SEQ ID NO:8. The antibody may comprise a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:9 and a variable light domain region comprising the amino acid sequence of SEQ ID NO:10. The antibody may comprise a variable heavy chain comprising a complementarity determining region (CDR)1 comprising the amino acid sequence of SEQ ID NO:11, a CDR2 comprising the amino acid sequence of SEQ ID NO:12 and a CDR3 comprising the amino acid sequence of SEQ ID NO:13.

[0010] The antibody may comprise a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 16. The antibody may comprise a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 19.

[0011] The antibody may comprise a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:20, a CDR2 comprising the amino acid sequence of SEQ ID NO:21 and a CDR3 comprising the amino acid sequence of SEQ ID NO:22.

[0012] The antibody may comprise a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:23, a CDR2 comprising the amino acid sequence of SEQ ID NO:24, and a CDR3 comprising the amino acid sequence of SEQ ID NO:25. The antibody may comprise a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:26, a CDR2 comprising the amino acid sequence of SEQ ID NO:27, and a CDR3 comprising the amino acid sequence of SEQ ID NO:28. The antibody may comprise a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:29, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31. The antibody may comprise a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:32, a CDR2 comprising the amino acid sequence of SEQ ID NO:33, and a CDR3 comprising the amino acid sequence of SEQ ID NO:34. The antibody may comprise a variable heavy chain comprising a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 16.

[0013] The antibody may comprise a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 19, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22.

[0014] The antibody may comprise a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:23, a CDR2 comprising the amino acid sequence of SEQ ID NO:24, and a CDR3 comprising the amino acid sequence of SEQ ID NO:25, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:26, a CDR2 comprising the amino acid sequence of SEQ ID NO:27, and a CDR3 comprising the amino acid sequence of SEQ ID NO:28.

[0015] The antibody may comprise a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:29, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:32, a CDR2 comprising the amino acid sequence of SEQ ID NO:33, and a CDR3 comprising the amino acid sequence of SEQ ID NO:34. The antibody or antibody fragment may further comprise an agent selected from the group consisting of an immunoadhesion molecule, an imaging agent, and a therapeutic agent. The imaging agent may be a radioactive label, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, or biotin. The radioactive label may be 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, or 153Sm.

[0016] In another aspect, the present invention also relates to an isolated nucleic acid encoding any one of the antibodies or fragments thereof described herein. The present disclosure also relates to a pharmaceutical composition comprising the antibodies, antibody fragments, mixtures or derivatives thereof described herein.

[0017] In another aspect, the present invention also relates to a method of treating an iron metabolism disorder. The method includes administering to a subject in need thereof a therapeutically or prophylactically effective amount of an antibody, wherein the antibody comprises: (a) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:3; (b) a variable light domain region comprising the amino acid sequence of SEQ ID NO:4; (c) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:5; (d) a variable light domain region comprising the amino acid sequence of SEQ ID NO:6; (e) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:7; (f) a variable light domain region comprising the amino acid sequence of SEQ ID NO:8; (g) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:9; (h) a variable light domain region comprising the amino acid sequence of SEQ ID NO:10; (i) a variable heavy chain comprising a complementarity determining region (CDR)1 comprising the amino acid sequence of SEQ ID NO:11, a CDR2 comprising the amino acid sequence of SEQ ID NO:12, and a CDR3 comprising the amino acid sequence of SEQ ID NO:13; (j) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:14, a CDR2 comprising the amino acid sequence of SEQ ID NO:15, and a CDR3 comprising the amino acid sequence of SEQ ID NO:16; (k) a CDR1 comprising the amino acid sequence of SEQ ID NO:17; (l) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a CDR2 comprising the amino acid sequence of SEQ ID NO: 21 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (m) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR2 comprising the amino acid sequence of SEQ ID NO: 24 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25; (n) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 27 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 29; (o) a variable light chain comprising a CDR1 comprising an amino acid sequence of SEQ ID NO:29, a CDR2 comprising an amino acid sequence of SEQ ID NO:30, and a CDR3 comprising an amino acid sequence of SEQ ID NO:31; (p) a variable light chain comprising a CDR1 comprising an amino acid sequence of SEQ ID NO:32, a CDR2 comprising an amino acid sequence of SEQ ID NO:33, and a CDR3 comprising an amino acid sequence of SEQ ID NO:34; (q) a complementarity determining region (CDR)1 comprising an amino acid sequence of SEQ ID NO:11;(r) a variable heavy chain comprising a CDR2 comprising the amino acid sequence of SEQ ID NO: 12 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a CDR2 comprising the amino acid sequence of SEQ ID NO: 15 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 16; (r) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 19, and a CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a CDR2 comprising the amino acid sequence of SEQ ID NO: 21 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22; and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR2 comprising the amino acid sequence of SEQ ID NO: 24 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 27 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28, and (t) a CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a CDR2 comprising the amino acid sequence of SEQ ID NO: 30 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 31. (u) a variable heavy domain region comprising an amino acid sequence of SEQ ID NO: 43; (v) a variable light domain region comprising an amino acid sequence of SEQ ID NO: 44; (w) a variable heavy domain region comprising an amino acid sequence of SEQ ID NO: 51; (x) a variable light domain region comprising an amino acid sequence of SEQ ID NO: 52; (y) a variable heavy domain region comprising an amino acid sequence of SEQ ID NO: 53; (z) a variable light domain region comprising an amino acid sequence of SEQ ID NO: 54; (aa) a variable light domain region comprising the amino acid sequence of SEQ ID NO:57; (bb) a variable light domain region comprising the amino acid sequence of SEQ ID NO:58; (cc) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:69; (dd) a variable light domain region comprising the amino acid sequence of SEQ ID NO:70; (ee) a variable heavy chain comprising a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO:95, a CDR2 comprising the amino acid sequence of SEQ ID NO:96 and a CDR3 comprising the amino acid sequence of SEQ ID NO:97; (ff) a CDR1 comprising the amino acid sequence of SEQ ID NO:98;(gg) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 119, a CDR2 comprising the amino acid sequence of SEQ ID NO: 120 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 121; (hh) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 122, a CDR2 comprising the amino acid sequence of SEQ ID NO: 123 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 124; (ii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 125, a CDR2 comprising the amino acid sequence of SEQ ID NO: 126 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 127; (jj) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 128, a CDR2 comprising the amino acid sequence of SEQ ID NO: 129 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 130; (kk) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 137, a CDR2 comprising the amino acid sequence of SEQ ID NO: 138 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 139; (ll) a CDR1 comprising the amino acid sequence of SEQ ID NO: 140, a CDR2 comprising the amino acid sequence of SEQ ID NO: 141 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 142; (mm) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 173, a CDR2 comprising the amino acid sequence of SEQ ID NO: 174 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 175; (nn) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR2 comprising the amino acid sequence of SEQ ID NO: 177 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 178; (oo) a CDR1 comprising the amino acid sequence of SEQ ID NO: 95, a CDR2 comprising the amino acid sequence of SEQ ID NO: 96 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 97 a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 98, a CDR2 comprising the amino acid sequence of SEQ ID NO: 99 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 100; (pp) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 119, a CDR2 comprising the amino acid sequence of SEQ ID NO: 120 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 121 and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 122, a CDR2 comprising the amino acid sequence of SEQ ID NO: 123 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 124;(qq) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 125, a CDR2 comprising the amino acid sequence of SEQ ID NO: 126, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 127, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 128, a CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 130; (rr) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 137, a CDR2 comprising the amino acid sequence of SEQ ID NO: 138, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 139, and a CDR1 comprising the amino acid sequence of SEQ ID NO: 140; (ss) a variable light chain comprising a CDR2 comprising the amino acid sequence of SEQ ID NO: 141 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 142, (ss) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 173, a CDR2 comprising the amino acid sequence of SEQ ID NO: 174 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 175, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR2 comprising the amino acid sequence of SEQ ID NO: 177 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 178, and the iron metabolism disorder in the subject is treated therapeutically or prophylactically. For example, the iron metabolism disorder treated in this manner may be selected from the group consisting of anemia of chronic disease (ACD), iron-refractory iron deficiency anemia, anemia of chronic kidney disease, resistance to erythropoietin and β-thalassemia.

[0018] In another aspect, the present invention also relates to a method for determining whether a subject has an iron-related disorder, the method comprising: a. measuring the level of membrane-bound or soluble RGMc in a sample from a subject; and b. comparing the level of RGMc in the sample with the level of RGMc in a normal control or calibrator, wherein an altered level of RGMc indicates that the subject has an iron-related disorder; and c. Diagnosing the subject with an iron-related disorder The altered level of RGMc compared to the subject may indicate that the subject has an iron-related disorder. In the above method, a decreased level of membrane-bound RGMc compared to the RGMc level of a normal control indicates that the subject has an iron-related disorder associated with iron overload. In the above method, a decreased level of membrane-bound RGMc compared to the RGMc level of a normal control indicates that the subject has an iron-related disorder associated with iron overload. In the above method, an increased level of membrane-bound RGMc compared to the RGMc level of a normal control indicates that the subject has an iron-related disorder associated with iron deficiency. In the above method, a decreased level of soluble RGMc compared to the RGMc level of a normal control indicates that the subject has an iron-related disorder associated with iron deficiency. In the above method, an increased level of soluble RGMc compared to the RGMc level of a normal control indicates that the subject has an iron-related disorder associated with iron overload. In the above method, the subject may have been previously diagnosed or has been diagnosed with a disorder selected from the group consisting of cancer, acute infection, chronic infection, autoimmune disease, liver disease and chronic kidney disease. In the above method, the sample may be selected from the group consisting of a blood sample and a serum sample.In the above method, step a) is an immunoassay, such as an enzyme-linked immunosorbent assay (ELISA).

[0019] In particular, the ELISA may be a sandwich ELISA.In the above methods, the level of membrane-bound or soluble RGMc in a sample may be determined using any of the isolated antibodies described above.

[0020] In another aspect, the present invention also relates to a method for determining the presence, amount or concentration of RGMc or a fragment thereof in a test sample. The method includes the steps of assaying a test sample for RGMc (or a fragment thereof) by an immunoassay utilizing at least one antibody and at least one detectable label, and comparing a signal generated by the detectable label as a direct or indirect indication of the presence, amount or concentration of RGMc in the test sample with a signal generated by the detectable label as a direct or indirect indication of the presence, amount or concentration of RGMc in a control or calibrator, wherein one of the at least one antibody is an isolated antibody that specifically binds to RGMc or a fragment thereof, and the antibody comprises: (a) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:3; (b) a variable light domain region comprising the amino acid sequence of SEQ ID NO:4; (c) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:5; (d) a variable light domain region comprising the amino acid sequence of SEQ ID NO:6; (e) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:7; (f) a variable light domain region comprising the amino acid sequence of SEQ ID NO:8; (g) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:9; (h) a variable light domain region comprising the amino acid sequence of SEQ ID NO:10. (i) a variable heavy chain comprising a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 13; (j) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 16; (k) a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 19. (l) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (m) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25; (n) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28.(o) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:29, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; (p) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:32, a CDR2 comprising the amino acid sequence of SEQ ID NO:33, and a CDR3 comprising the amino acid sequence of SEQ ID NO:34; (q) a variable heavy chain comprising a complementarity determining region (CDR)1 comprising the amino acid sequence of SEQ ID NO:11, a CDR2 comprising the amino acid sequence of SEQ ID NO:12, and a CDR3 comprising the amino acid sequence of SEQ ID NO:13, and a CDR3 comprising the amino acid sequence of SEQ ID NO:14; (r) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 19 and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a CDR2 comprising the amino acid sequence of SEQ ID NO: 21 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (s) a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR2 comprising the amino acid sequence of SEQ ID NO: 24 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25; a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28; (t) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 31, and a CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR2 comprising the amino acid sequence of SEQ ID NO: 33, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 34; (u) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO: 43; (v) a variable light domain region comprising the amino acid sequence of SEQ ID NO: 44; (w) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO: 51; (x) a variable light domain region comprising the amino acid sequence of SEQ ID NO: 52; (y) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO: 53; (z) a variable light domain region comprising the amino acid sequence of SEQ ID NO: 54; (aa) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO: 57; (bb) a variable light domain region comprising the amino acid sequence of SEQ ID NO: 58.(cc) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:69; (dd) a variable light domain region comprising the amino acid sequence of SEQ ID NO:70; (ee) a variable heavy chain comprising a complementarity determining region (CDR)1 comprising the amino acid sequence of SEQ ID NO:95, a CDR2 comprising the amino acid sequence of SEQ ID NO:96, and a CDR3 comprising the amino acid sequence of SEQ ID NO:97; (ff) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:98, a CDR2 comprising the amino acid sequence of SEQ ID NO:99, and a CDR3 comprising the amino acid sequence of SEQ ID NO:100; (gg) an amino acid sequence of SEQ ID NO:119. (hh) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 122, a CDR2 comprising the amino acid sequence of SEQ ID NO: 123 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 124; (ii) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 125, a CDR2 comprising the amino acid sequence of SEQ ID NO: 126 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 127; (jj) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 128, a CDR2 comprising the amino acid sequence of SEQ ID NO: 129 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 130; (kk) a variable heavy chain comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 137, a CDR2 comprising an amino acid sequence of SEQ ID NO: 138 and a CDR3 comprising an amino acid sequence of SEQ ID NO: 139; (ll) a variable light chain comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 140, a CDR2 comprising an amino acid sequence of SEQ ID NO: 141 and a CDR3 comprising an amino acid sequence of SEQ ID NO: 142; (mm) a CDR1 comprising an amino acid sequence of SEQ ID NO: 173; a variable heavy chain comprising a CDR2 comprising the amino acid sequence of SEQ ID NO: 174 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 175; (nn) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR2 comprising the amino acid sequence of SEQ ID NO: 177 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 178; (oo) a variable heavy chain comprising a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 95, a CDR2 comprising the amino acid sequence of SEQ ID NO: 96 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 97, and a CDR1 comprising the amino acid sequence of SEQ ID NO: 98;(pp) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 119, a CDR2 comprising the amino acid sequence of SEQ ID NO: 120 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 121, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 122, a CDR2 comprising the amino acid sequence of SEQ ID NO: 123 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 124; (qq) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 125, a CDR2 comprising the amino acid sequence of SEQ ID NO: 126 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 127, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 128, a CDR2 comprising the amino acid sequence of SEQ ID NO: 129 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 130; (rr) a sequence A variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 137, a CDR2 comprising the amino acid sequence of SEQ ID NO: 138 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 139, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 140, a CDR2 comprising the amino acid sequence of SEQ ID NO: 141 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 142; (ss) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 173, a CDR2 comprising the amino acid sequence of SEQ ID NO: 174 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 175, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR2 comprising the amino acid sequence of SEQ ID NO: 177 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 178, thereby determining the presence, amount or concentration of RGMc or a fragment thereof in a test sample.

[0021] In the above method, the presence, amount or concentration of RGMc or a fragment thereof in the test sample is used to determine or assess whether a subject is at risk or at risk of developing an iron-related disorder. In the above method, the RGMc is membrane-bound RGMc or soluble RGMc. In the above method, a decreased level of membrane-bound RGMc compared to the RGMc level of a normal control indicates that the subject has an iron-related disorder associated with iron overload. In the above method, an increased level of membrane-bound RGMc compared to the RGMc level of a normal control indicates that the subject has an iron-related disorder associated with iron deficiency. In the above method, a decreased level of soluble RGMc compared to the RGMc level of a normal subject indicates that the subject has an iron-related disorder associated with iron deficiency. In the above method, an increased level of soluble RGMc compared to the RGMc level of a normal control indicates that the subject has an iron-related disorder associated with iron overload. In the above method, the iron-related disorder is selected from the group consisting of cancer, acute infection, chronic infection, autoimmune disease, liver disease and chronic kidney disease. Furthermore, the above method comprises the steps of: a. contacting the test sample with at least one capture antibody that binds to an epitope on RGMc (or a fragment thereof) to form a capture antibody / RGMc (or a fragment thereof) complex; b. contacting the capture antibody / RGMc (or fragment thereof) complex with at least one detection antibody that contains a detectable label and that binds to an epitope on the RGMc (or fragment thereof) that is not bound by the capture antibody, to form a capture antibody / RGMc (or fragment thereof) / detection antibody complex; and c. Determining the presence, amount or concentration of RGMc (or a fragment thereof) in the test sample based on a signal generated by the detectable label in the capture antibody / RGMc (or a fragment thereof) / detection antibody complex formed in (b). whereby the presence, amount or concentration of RGMc (or a fragment thereof) in the test sample is determined.

[0022] Alternatively, the method may include the steps of: a. contacting the test sample with at least one capture antibody that binds to an epitope on RGMc (or fragment thereof) to form a capture antibody / RGMc (or fragment thereof) complex, and simultaneously, or sequentially in any order, contacting the test sample with a detectably labeled RGMc (or fragment thereof) that can compete with any RGMc (or fragment thereof) in the test sample for binding to the at least one capture antibody, such that any RGMc (or fragment thereof) present in the test sample and the detectably labeled RGMc compete with each other to form a capture antibody / RGMc (or fragment thereof) complex and a capture antibody / detectably labeled RGMc (or fragment thereof) complex, respectively; and b. may further comprise determining the presence, amount or concentration of RGMc in the test sample based on a signal generated by the detectable label in the capture antibody / detectably labeled RGMc (or fragment thereof) complex formed in (b), where the signal generated by the detectable label in the capture antibody / detectably labeled RGMc (or fragment thereof) complex is inversely proportional to the amount or concentration of RGMc in the test sample, thereby determining the presence, amount or concentration of RGMc in the test sample. The above method may further comprise assaying the test sample for hepcidin.

[0023] In another aspect, the present invention also relates to a method for determining whether a subject has an iron-related disorder, the method comprising: a. measuring the level of membrane-bound or soluble RGMc in a first sample from a subject; b. measuring the level of hepcidin in a second sample from the subject; c. comparing the level of RGMc in the first sample to the level of RGMc in a normal control or calibrator; and d. comparing the level of hepcidin in the second sample to the level of hepcidin in a normal control or calibrator, wherein altered levels of RGMc and hepcidin, respectively, indicate that the subject has an iron-related disorder; and e. Diagnosing the subject as having an iron-related disorder Includes.

[0024] In the above method, a decreased level of membrane-bound RGMc compared to the level of membrane-bound RGMc in a normal subject indicates that the subject has an iron-related disorder associated with iron overload. In the above method, an increased level of membrane-bound RGMc compared to the level of membrane-bound RGMc in a normal control indicates that the subject has an iron-related disorder associated with iron deficiency. In the above method, a decreased level of soluble RGMc compared to the level of soluble RGMc in a normal control indicates that the subject has an iron-related disorder associated with iron deficiency. In the above method, an increased level of soluble RGMc compared to the level of soluble RGMc in a normal control indicates that the subject has an iron-related disorder associated with iron overload. In the above method, a decreased level of hepcidin compared to the level of hepcidin in a normal control indicates that the subject has an iron-related disorder associated with iron overload. In the above method, an increased level of hepcidin compared to the level of hepcidin in a normal control indicates that the subject has an iron-related disorder associated with iron deficiency.

[0025] In the above method, the subject is diagnosed with a disorder selected from the group consisting of cancer, acute infection, chronic infection, autoimmune disease, liver disease and chronic kidney disease.In the above method, the level of membrane-bound RGMc or soluble RGMc and the level of hepcidin in each of first and second samples are continuously determined.In the above method, the level of membrane-bound RGMc or soluble RGMc and the level of hepcidin in each of first and second samples are continuously determined.

[0026] In the above method, the sample is selected from the group consisting of a blood sample and a serum sample. In the above method, step a) is an enzyme-linked immunosorbent assay (ELISA). For example, the ELISA is a sandwich ELISA. In the above method, the level of membrane-bound or soluble RGMc in the sample is determined using any of the above isolated antibodies.

[0027] Any assays for RGMc (such as membrane-bound RGMc, soluble RGMc, fragments of membrane-bound RGMc, fragments of soluble RGMc, variants of RGMc (membrane-bound RGMc or soluble RGMc) or any combination thereof) and hepcidin may be performed simultaneously or sequentially in any order using the same types of methods or different methods and using the same or different test samples obtained from the same source, such as the same patient. Alternatively, the method may also include using data obtained from assays of test samples obtained from the same source, such as the same patient, but assayed for hepcidin at different time points, or obtained and assayed for hepcidin at different time points.

[0028] In another aspect, the present invention also relates to a kit for assaying a test sample for RGMc (or a fragment thereof). The kit may comprise at least one component for assaying a test sample for RGMc (or a fragment thereof) and instructions for assaying a test sample for RGMc (or a fragment thereof), wherein the at least one component comprises at least one composition comprising an isolated antibody that specifically binds to RGMc (or a fragment thereof), wherein the antibody comprises (a) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:3, (b) a variable light domain region comprising the amino acid sequence of SEQ ID NO:4, (c) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:5, (d) a variable light domain region comprising the amino acid sequence of SEQ ID NO:6, (e) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:7, (f) a variable light domain region comprising the amino acid sequence of SEQ ID NO:8, (g) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:9, (h) a variable light domain region comprising the amino acid sequence of SEQ ID NO:10, (i) a variable heavy chain comprising a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO:11, a CDR2 comprising the amino acid sequence of SEQ ID NO:12, and a CDR3 comprising the amino acid sequence of SEQ ID NO:13, (j) a variable light chain comprising the amino acid sequence of SEQ ID NO:14, (k) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:15, (l) a variable light chain comprising the amino acid sequence of SEQ ID NO:16, (l) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:17, (l) a variable light chain comprising the amino acid sequence of SEQ ID NO:18, (l) a variable light chain comprising the amino acid sequence of SEQ ID NO:19, (m) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:20, (m) a variable light chain comprising the amino acid sequence of SEQ ID NO:21, (n) a variable light chain comprising the amino acid sequence of SEQ ID NO:22, (n) a variable heavy chain comprising (k) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 19; (l) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a CDR2 comprising the amino acid sequence of SEQ ID NO: 21 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (m) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR2 comprising the amino acid sequence of SEQ ID NO: 24 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25; (n) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 27 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28; (o) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a CDR2 comprising the amino acid sequence of SEQ ID NO: 30 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 31; (p) a CDR1 comprising the amino acid sequence of SEQ ID NO: 32;(q) a variable heavy chain comprising a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 16; (r) a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18, a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a CDR2 comprising the amino acid sequence of SEQ ID NO: 21 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22, (s) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR2 comprising the amino acid sequence of SEQ ID NO: 24 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25, and a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 27 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28; (t) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 31, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR2 comprising the amino acid sequence of SEQ ID NO: 33, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 34; (u) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO: 43; (v) a variable light domain region comprising the amino acid sequence of SEQ ID NO: 44; (w) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO: 51. main region, (x) a variable light domain region comprising the amino acid sequence of SEQ ID NO: 52, (y) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO: 53, (z) a variable light domain region comprising the amino acid sequence of SEQ ID NO: 54, (aa) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO: 57, (bb) a variable light domain region comprising the amino acid sequence of SEQ ID NO: 58, (cc) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO: 69, (dd) a variable light domain region comprising the amino acid sequence of SEQ ID NO: 70, (ee) a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 95,(ff) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 98, a CDR2 comprising the amino acid sequence of SEQ ID NO: 99 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 100; (gg) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 119, a CDR2 comprising the amino acid sequence of SEQ ID NO: 120 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 121; (hh) a CDR1 comprising the amino acid sequence of SEQ ID NO: 122, a CDR2 comprising the amino acid sequence of SEQ ID NO: 123 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 124; (ii) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 125, a CDR2 comprising the amino acid sequence of SEQ ID NO: 126 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 127; (jj) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 128, a CDR2 comprising the amino acid sequence of SEQ ID NO: 129 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 130; (kk) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 137, a CDR2 comprising the amino acid sequence of SEQ ID NO: 138 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 139; (ll) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 140, a CDR2 comprising the amino acid sequence of SEQ ID NO: 141 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 142; (mm) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 173, a CDR2 comprising the amino acid sequence of SEQ ID NO: 174 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 175; (nn) a CDR1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR2 comprising the amino acid sequence of SEQ ID NO: 177 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 178; (oo) a variable heavy chain comprising a CDR2 comprising the amino acid sequence of SEQ ID NO: 96 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 97, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 98, a CDR2 comprising the amino acid sequence of SEQ ID NO: 99 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 100; (pp) a CDR1 comprising the amino acid sequence of SEQ ID NO: 119,(qq) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 125, a CDR2 comprising the amino acid sequence of SEQ ID NO: 126 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 127, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 128, a CDR2 comprising the amino acid sequence of SEQ ID NO: 129 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 130; (rr) a CDR1 comprising the amino acid sequence of SEQ ID NO: 137, a CDR2 comprising the amino acid sequence of SEQ ID NO: 138 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 139; The antibody comprises a domain or region selected from the group consisting of a variable heavy chain comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 140, a CDR2 comprising an amino acid sequence of SEQ ID NO: 141 and a CDR3 comprising an amino acid sequence of SEQ ID NO: 142, a variable light chain comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 173, a CDR2 comprising an amino acid sequence of SEQ ID NO: 174 and a CDR3 comprising an amino acid sequence of SEQ ID NO: 175, and a variable light chain comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 176, a CDR2 comprising an amino acid sequence of SEQ ID NO: 177 and a CDR3 comprising an amino acid sequence of SEQ ID NO: 178, and the antibody is optionally detectably labeled. In the above kit, the RGMc or a fragment thereof assayed in the test sample is used to determine or evaluate whether a subject is at risk or at risk of developing an iron-related disorder. Furthermore, the RGMc assayed is membrane-bound RGMc or soluble RGMc. The kit may further include at least one component for assaying a test sample for hepcidin and instructions for assaying the test sample for hepcidin. [Brief description of the drawings]

[0029] [Figure 1] FIG. 1 shows a simplified diagram of signaling pathways related to iron homeostasis. [Diagram 2]FIG. 2 shows another simplified diagram of signaling pathways related to iron homeostasis. [Diagram 3] 3 is a histogram showing that humanized 5F9.23 (h5F9.23) increases blood iron levels in rats. FIG. 3 shows data from rats treated with 0, 20, 60 or 200 mg / kg intravenous injection once a week. Blood iron levels were significantly increased at all h5F9.23 doses used (significance: **p<0.01; ***P<0.001). This data shows that h5F9.23 increases blood iron levels in rats. [Figure 4] 1 is a histogram showing that h5F9.23 increases saturable transferrin 1 levels (%) in rats. Saturable transferrin 1 levels were measured in rats treated with 0, 20, 60 or 200 mg / kg h5F9.23 by IV injection once a week. Saturable transferrin 1 levels were significantly increased at all h5F9.23 doses (significance: ***p<0.001). [Diagram 5] 1 is a histogram showing that h5F9.23 increases saturable transferrin 2 levels (%) in rats. Saturable transferrin 2 levels were measured in rats treated with 0, 20, 60 or 200 mg / kg h5F9.23 by IV injection once a week. Saturable transferrin 2 levels were significantly increased at all h5F9.23 doses (significance: **p<0.01; ***p<0.001). [Figure 6] 1 is a histogram showing that h5F9.23 reduces unsaturated iron-binding capacity (UIBC) in rats. Unsaturated iron-binding capacity (UIBC) was also measured in rats treated with 0, 20, 60 or 200 mg / kg h5F9.23 by IV injection once a week. UIBC levels were significantly reduced at all h5F9.23 doses (significance: ***p<0.001). [Figure 7] A fixed rat liver sample (control) stained with Prussian blue is shown (100x magnification), with the arrow pointing to the periportal region of the liver lobe. [Figure 8]A fixed rat liver sample (treated with 200 mg / kg / week h5F9.23) stained with Prussian blue is shown (100x magnification). Arrows point to periportal regions of liver lobes. Black granules represent iron. [Figure 9] A fixed rat spleen sample (control) stained with Prussian blue is shown (40x magnification). Iron-loaded macrophages are shown in the red pulp between lymphoid follicles (F). [Figure 10] Fixed rat spleen samples (treated with 200 mg / kg / week h5F9.23) stained with Prussian blue are shown (40x magnification). Macrophages in the red pulp between lymphoid follicles (F) released iron into the serum. [Figure 11] Figure 1 shows an RGMc-mediated BMP reporter assay in 293HEK cells to evaluate selected h5F9 affinity matured Abs for blocking RGMc function. (A) Schematic of the RGMc-mediated BMP reporter assay. (B) Rat hybridoma mAbs 5F9, h5F9.23 and the affinity matured Abs dose-dependently inhibited RGMc-mediated luc activity. IC50 values ​​are shown next to the legend. The Y-axis represents luciferase activity as relative luminescence units (RLU). [Figure 12] 1 is a histogram showing the results of Example 5, in which female cynomolgus monkeys were treated with different doses of humanized antibody 5F9.23 (h5F9.23). Monkeys were injected subcutaneously (sc) at 60 mg / kg or intravenously (iv) at 20, 60, 200 mg / kg once a week for 4 weeks. Primate serum was collected 0.5, 4, and 24 hours after antibody administration (4th dose) on day 22. Hepcidin was measured using mass spectrometry. ***p<0.001: significant vs. pre-test, *p<0.05: significant vs. pre-test, *p<0.05: significant vs. control. [Figure 13A]13A is a histogram showing the results of Example 6, in which female Sprague Dawley rats were treated with different doses of antibody h5F923.AM8. Rats were injected intravenously with 2.5 mg / kg, 5 mg / kg, 10 mg / kg, or 20 mg / kg once a week for four weeks. At the end of the fourth week, serum was collected to measure: (1) free blood iron levels (FIG. 13A). ***Significance vs. phosphate buffered saline (PBS) (light grey), ***p<0.01: significance vs. monoclonal antibody human IgG (black). The control was a human IgG antibody directed against IL-18 obtained from Abbott Laboratories, Worcester, Mass. The control did not cross-react with rat IL-18 protein. [Figure 13B] 13 is a histogram showing the results of Example 6, in which female Sprague Dawley rats were treated with different doses of antibody h5F923.AM8. Rats were injected intravenously with 2.5 mg / kg, 5 mg / kg, 10 mg / kg, or 20 mg / kg once a week for four weeks. At the end of the fourth week, serum was collected and the following was measured: (2) Unsaturated iron binding capacity (FIG. 13B). ***Significance vs. phosphate buffered saline (PBS) (light grey), ***p<0.01: significance vs. monoclonal antibody human IgG (black). The control was a human IgG antibody directed against IL-18 obtained from Abbott Laboratories, Worcester, Mass. The control did not cross-react with rat IL-18 protein. [Figure 14A]14A is a histogram showing the results of Example 7, in which female Sprague Dawley rats were treated with different doses of antibody h5F923.AM8. Rats were injected intravenously with 0.02 mg / kg, 0.2 mg / kg, 2.0 mg / kg, or 20 mg / kg once a week for four weeks. At the end of the fourth week, serum was collected and the following were measured: (1) Free blood iron levels (FIG. 14A). ***p<0.001, significant vs. vehicle control; **p<0.01, significant vs. vehicle control; *p<0.05, significant vs. vehicle control. Vehicle control contained 30 mM histidine, 8% w / v sucrose, pH 6.0 plus 0.02% Tween 80 in water. [Figure 14B] 14 is a histogram showing the results of Example 7, in which female Sprague Dawley rats were treated with different doses of antibody h5F923.AM8. Rats were injected intravenously with 0.02 mg / kg, 0.2 mg / kg, 2.0 mg / kg, or 20 mg / kg once a week for four weeks. At the end of the fourth week, serum was collected and the following was measured: (2) Unsaturated iron binding capacity (FIG. 14B). ***p<0.001, significant vs. vehicle control; **p<0.01, significant vs. vehicle control; *p<0.05, significant vs. vehicle control. Vehicle control contained 30 mM histidine, 8% w / v sucrose, pH 6.0 plus 0.02% Tween 80 in water. [Figure 15] 1 is a histogram showing the results of the first set of experiments described in Example 8, demonstrating that h5F923.AM8 and 1A-2989 improved anemia in ACD rats at day 30 by increasing hemoglobin levels. Also shown in the figure, dorsomorphin was inactive. [Figure 16A] 16A-16C are histograms showing the results of the second series of experiments described in Example 8. In particular, FIG. 16A shows that the control antibody hIgG does not significantly alter the low hemoglobin levels of anemic rats on days 41, 47 and 51. *p<0.05: significance relative to D0 hemoglobin. [Figure 16B]16A-B are histograms showing the results of the second series of experiments described in Example 8. FIGURE 16B shows that a humanized monoclonal antibody that was selective for RGM A does not significantly alter the low hemoglobin levels of anemic rats at days 41, 47 and 51. *p<0.05, **p<0.01, significance relative to D0 hemoglobin levels. [Figure 16C] Figure 16C is a histogram showing the results of the second series of experiments described in Example 8. Figure 16C shows that antibody h5F9.AM8 significantly increases low hemoglobin levels (D24) in anemic rats on days 41, 47 and 51. ***p<0.001, significance relative to hemoglobin levels on day 0 (D0). D41: *p<0.05, significance relative to D24, D47 / 55: *p<0.05, significance relative to D24. [Figure 16D] 16D is a histogram showing the results of the second series of experiments described in Example 8. FIG 16D shows that antibody h5F9.23 increases low hemoglobin levels (day 24 (D24)) in anemic rats on days 41, 47 and 51. *p<0.05; **p<0.001, significance relative to D0 hemoglobin levels on day 41: *p<0.05, significance relative to days 24, 47 and 51: *p<0.05, significance relative to day. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] RGMc is a glycosylphosphatidylinositol ("GPI")-anchored membrane protein expressed in muscle, retina and periportal hepatocytes. RGMc works with hepcidin through signaling proteins to maintain iron homeostasis in the body. See, for example, Severyn et al., Biochem.J., 422:393-403 (2009) and Pietrangelo, J.Hepatology, 54:173-181 (2011). Cell membrane RGMc binds to neogenin and promotes signaling through bone morphogenetic proteins (BMPs), which induce intracellular signaling through downstream effectors to promote hepcidin gene expression. Again, see, for example, Pietrangelo, J.Hepatology, 54:173-181 (2011). Soluble RGMc is released by cleavage of membrane-bound RGMc by the serine protease, matriptase-2 (TMPRSS6). Release of soluble RGMc is induced by decreasing the extracellular concentration of iron and, conversely, is inhibited by increasing the extracellular concentration of iron. Again, see, e.g., Severyn et al., Biochem. J., 422:393-403 (2009) and FIG. 1. The soluble form of RGMc sequesters BMP6 from membrane-bound RGMc, thereby preventing induction of hepcidin expression. See FIG. 2.

[0031] When BMP binds to BMP receptors I and II, a membrane-bound complex is formed with neogenin, BMP6 and RGMc. Together with intracellular proteins called Smads (Smads1, 5 and 8), this complex transduces intracellular signals, thereby initiating a signaling pathway that governs hepcidin expression and ultimately systemic iron metabolism. Again, see, e.g., Pietrangelo, J. Hepatology, 54:173-181 (2011) and FIG. 1. Hepcidin binds to ferroportin, the exclusive mammalian iron transporter. When hepcidin binds to ferroportin, it blocks the iron transport pathway, as ferroportin is internalized by macrophages and by duodenal cells where ferroportin is degraded. See, e.g., Hentz et al., Cell, 142:24-38 (2010) and Cheng et al., Clin. Exp. Med., 11:33-42 (2011).

[0032] Both macrophages and duodenal cells express ferroportin, and at high hepcidin levels, hepcidin-induced degradation of ferroportin blocks the only available iron transport pathway. As a result, both macrophages and duodenal cells accumulate large amounts of intracellular iron. See FIG. 1. Because these cells can no longer release iron into the blood, anemia of chronic disease ("ACD") is a common outcome. Again, see, e.g., Cheng et al., Clin. Exp. Med., 11:33-42 (2011).

[0033] RGMc-specific antibodies block the normal expression of hepcidin, which directly regulates iron concentration in plasma and distribution of iron to various tissues. The antibodies can prevent binding between BMP and RGMc. The antibodies can prevent binding between BMP and the N-terminus of RGMc. As a result of this action, the expression of hepcidin is reduced or inhibited. As hepcidin levels decrease, ferroportin-dependent transport of iron increases because hepcidin is no longer available to bind to ferroportin and induce its internalization and degradation. See Figure 2.

[0034] The present inventors have made a surprising discovery that antibodies that bind to repulsive guidance molecule c ("RGMc") can be used to regulate iron metabolism.Antibodies that block the normal expression of hepcidin are provided in the present invention, which directly regulate the iron concentration in plasma and the distribution of iron to various tissues.Excessive levels of hepcidin cause iron-restricted anemia.For example, a marked increase in hepcidin levels has been reported in patients with ACD and patients with acute inflammation (AI).Slightly increased hepcidin levels have been observed in patients with ACD and iron deficiency anemia (ACD-IDA).Patients with only iron deficiency anemia (IDA) showed a tendency to low serum hepcidin levels. For example, serum hepcidin levels have been shown to be 177,58 μg / l (+ / - 119,84) in healthy controls, 434,83 ​​μg / l (+ / - 217) in ACD patients, 410,08 μg / l (+ / - 299,96) in AI patients, 238,32 μg / l (+ / - 93,85) in ACD-IDA patients and a slightly reduced serum hepcidin level of 110,79 μg / l (+ / - 19,22) in IDA patients. In contrast, hemochromatosis is characterized by low serum hepcidin levels. In addition, β-thalaassaemia is a disease in which hepcidin levels can be low.

[0035] The antibodies disclosed herein are useful for treating disorders of iron metabolism. Additionally, the antibodies disclosed herein are useful in diagnostic assays for determining whether a subject has an iron-related disorder.

[0036] 1.Definition The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise.

[0037] a.About As used herein, "about" can refer to approximately a + / - 10% variation from the stated value, which is understood to always be included in any given value provided herein, whether or not a specific reference is made.

[0038] B. Affinity matured antibodies "Affinity matured antibody" is used herein to refer to an antibody with one or more changes in one or more CDRs to increase the affinity of the antibody for a target antigen (i.e., K D , k d or k a ) resulting in improved affinity maturation. Exemplary affinity matured antibodies have nanomolar or even picomolar affinities for the target antigen. Various means for producing affinity matured antibodies are known in the art, including screening combinatorial antibody libraries that have been prepared using biodisplay. For example, Marks et al., BioTechnology, 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described in Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169:147-155 (1995); Yelton et al., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol., 154(7):3310-3319 (1995); and Hawkins et al., J. Mol. Biol., 226:889-896 (1992). Selective mutagenesis positions with activity enhancing amino acid residues and selective mutations at contact or hypermutation positions are described in U.S. Patent No. 6,914,128 B1.

[0039] C. Antibodies and antibodies As used herein, "antibody" and "antibodies" refer to a monoclonal antibody, a multispecific antibody, a human antibody, a humanized antibody (fully or partially humanized), an animal antibody (e.g., a mammal or non-human primate (e.g., monkey, chimpanzee, etc.), including, but not limited to, an avian (e.g., duck or goose), shark, whale, and non-primate (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, etc.), a recombinant antibody, a chimeric antibody, a single chain Fvs ("scFv"), a single chain antibody, a single domain antibody, a Fab fragment, a F(ab') fragment, a F(ab') 2 It refers to fragments, disulfide-linked Fvs ("sdFv") and anti-idiotypic ("anti-Id") antibodies, dual domain antibodies, dual variable domain (DVD) or triple variable domain (TVD) antibodies (dual variable domain immunoglobulins and methods for making them are described in Wu, C. et al., Nature Biotechnology, 25(11):1290-1297 (2007) and PCT International Application WO2001 / 058956, the contents of each of which are incorporated herein by reference), as well as functionally active epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an analyte binding site. Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. For simplicity, antibodies to an analyte are frequently referred to herein as being either "anti-analyte antibodies" or simply "analyte antibodies" (e.g., anti-RGMc antibodies or RGMc antibodies).

[0040] d. Antibody fragment As used herein, "antibody fragment" refers to a portion of an intact antibody that contains the antigen binding site or variable region. This portion does not contain the constant heavy chain domains of the Fc region of the intact antibody (i.e., CH2, CH3 or CH4, depending on the antibody isotype). Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab') fragments, and the like. 2 These include fragments, Fd fragments, Fv fragments, diabodies, single chain Fv (scFv) molecules, single chain polypeptides containing only a light chain variable domain, single chain polypeptides containing the three CDRs of a light chain variable domain, single chain polypeptides containing only a heavy chain variable region, and single chain polypeptides containing the three CDRs of a heavy chain variable region.

[0041] e. binding constant "Binding constant" is defined herein. As used herein, "association rate constant", "k on " or "k a " refers to the binding rate of an antibody to its target antigen or the following formula: Antibody (Ab) + Antigen (Ag) → Ab-Ag This refers to a value indicating the rate of complex formation between an antibody and an antigen as indicated by:

[0042] As used interchangeably herein, "dissociation rate constant," "k off " or "k d " refers to the dissociation rate of an antibody from its target antigen or the following formula: Antibody (Ab) + Antigen (Ag)←Ab-Ag This refers to a value that indicates the separation of the Ab-Ag complex into free antibody and antigen over time as indicated by:

[0043] Methods for determining the association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques provides high sensitivity and ability to test samples in physiological buffer at equilibrium. Other experimental approaches and instruments such as BIAcore® (Biomolecular Interaction Analysis) assays may also be used (e.g., instruments available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). In addition, KinExA® (Kinetic Exclusion Assay) assays available from Sapidyne Instruments (Boise, Idaho) may also be used.

[0044] As used interchangeably herein, the term "equilibrium dissociation constant" or "K D The term "dissociation rate (k off ) to the association rate (k on The association rate, dissociation rate and equilibrium dissociation constant are used to express the binding affinity of an antibody to an antigen.

[0045] f. binding protein "Binding protein" is used herein to refer to a monomeric or multimeric protein that binds to and forms a complex with a binding partner, such as, for example, a polypeptide, an antigen, a chemical compound or other molecule, or any type of substrate. A binding protein specifically binds to a binding partner. Binding proteins include antibodies and antigen-binding fragments thereof, as well as various other forms and derivatives thereof known in the art and described herein below, and other molecules that contain one or more antigen-binding domains that bind to an antigen molecule or a specific site (epitope) on an antigen molecule. Thus, binding proteins include, but are not limited to, antibodies, tetrameric immunoglobulins, IgG molecules, IgG 1Molecules include monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, affinity matured antibodies and fragments of any such antibodies which retain the ability to bind antigen.

[0046] g. Bispecific antibodies "Bispecific antibodies" are used herein to refer to full-length antibodies generated by quadroma technology (see Milstein et al., Nature, 305(5934):537-540 (1983)), by chemical conjugation of two different monoclonal antibodies (Staerz et al., Nature, 314(6012):628-631 (1985)), or by "knob-into-hole" or similar approaches that introduce mutations in the Fc region resulting in multiple different immunoglobulin species, only one of which is a functional bispecific antibody (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90(14):6444-6448 (1993)). A bispecific antibody binds one antigen (or epitope) in one of its two binding arms (one HC / LC pair) and a different antigen (or epitope) in its second arm (a different HC / LC pair). By this definition, a bispecific antibody has two distinct antigen-binding arms (in both specificity and CDR sequences) and is monovalent for each antigen to which it binds.

[0047] h.CDR "CDR" is used herein to refer to the "complementarity determining region" in an antibody variable sequence. There are three CDRs in each of the heavy and light chain variable regions, which are designated as "CDR1", "CDR2" and "CDR3" for each variable region. As used herein, the term "CDR set" refers to a group of three CDRs occurring in a single variable region that binds to an antigen. The exact boundaries of these CDRs are defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. These CDRs are sometimes referred to as "Kabat CDRs." Chothia and coworkers (Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); and Chothia et al., Nature, 342:877-883 (1989)) have used the Kabat CDRs to It has been found that certain sub-portions within the CDRs adopt nearly identical peptide backbone conformations, despite the large diversity at the level of amino acid sequence. These sub-portions are designated "L1", "L2" and "L3" or "H1", "H2" and "H3", where "L" and "H" designate the light chain region and the heavy chain region, respectively. These regions are sometimes referred to as "Chothia CDRs", which have overlapping boundaries with Kabat CDRs. Other boundaries that define CDRs that overlap with Kabat CDRs are described by Padlan, FASEB J., 9:133-139 (1995) and MacCallum, J.Mol.Biol., 262(5):732-745 (1996).Still other CDR boundary definitions may not strictly follow one of the systems herein, but still overlap with the Kabat CDRs, but they may be shortened or lengthened in light of predictions or experimental findings that certain residues or groups of residues or even entire CDRs do not significantly affect antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use CDRs defined by Kabat or Cothia.

[0048] i. component or components "Component," "component," or "at least one component" generally refers to capture antibodies, detection or conjugate calibrators, controls, sensitivity panels, containers, buffers, diluents, salts, enzymes, cofactors for enzymes, detection reagents, pretreatment reagents / solutions, substrates (e.g., in solution), stop solutions, and the like that may be included in a kit for assaying a test sample, e.g., a patient urine, serum, or plasma sample, according to the methods described herein and other methods known in the art. Some components may be in solution or lyophilized for reconstitution for use in the assay.

[0049] j. consensus or consensus sequence As used herein, "consensus" or "consensus sequence" refers to a synthetic nucleic acid sequence or corresponding polypeptide sequence that is constructed based on the analysis of the alignment of multiple subtypes of a particular antigen. This sequence can be used to induce broad immunity against multiple subtypes or sertypes of a particular antigen. Synthetic antigens, such as fusion proteins, can be engineered into consensus sequences (or consensus antigens).

[0050] K. Control As used herein, "control" refers to a composition known not to contain the analyte of interest, e.g., RGMc (such as membrane-bound RGMc, soluble RGMc, a fragment of membrane-bound RGMc, a fragment of soluble RGMc, a variant of RGMc (membrane-bound RGMc or soluble RGMc), or any combination thereof) ("negative") or a composition known to contain the analyte of interest, e.g., RGMc (such as membrane-bound RGMc, soluble RGMc, a fragment of membrane-bound RGMc, a fragment of soluble RGMc, a variant of RGMc (membrane-bound RGMc or soluble RGMc), or any combination thereof) ("positive control"). A positive control may comprise a known concentration of RGMc. "Control", "positive control" and "calibrator" may be used interchangeably herein and refer to a composition comprising a known concentration of RGMc. A "positive control" may be used to establish assay performance characteristics and is a useful indicator of the integrity of a reagent (e.g., an analyte). A "normal control" can refer to a sample or subject that is not susceptible to an iron-related disease or disorder.

[0051] l. Derivatives As used herein, a "derivative" of an antibody may refer to an antibody that has one or more modifications to its amino acid sequence compared to the pure or parent antibody, and exhibits a modified domain structure. The derivative may still adopt the typical domain arrangement found in natural antibodies and amino acid sequences, and may bind to the target (antigen) with specificity. Typical examples of antibody derivatives are antibodies bound to other polypeptides, rearranged antibody domains or antibody fragments. The derivative may also comprise at least one further compound, such as a protein domain, said protein domains being linked by covalent or non-covalent bonds. Linking may be based on gene fusion according to methods known in the art. The additional domains present in the fusion protein comprising the antibody utilized according to the present invention may preferably be linked by a flexible linker, advantageously a peptide linker, comprising a plurality of hydrophilic peptide-bound amino acids of sufficient length to span the distance between the C-terminus of the further protein domain and the N-terminus of the antibody or vice versa. The antibody may be linked to an effector molecule having a suitable configuration for selective binding with biological activity or, for example, a solid support, a biologically active substance (e.g., a cytokine or growth hormone), a chemical agent, a peptide, a protein or a drug.

[0052] m.Bispecific antibodies "Bispecific antibodies" are used herein to refer to full-length antibodies that can bind to two different antigens (or epitopes) in each of their two binding arms (a pair of HC / LC) (see PCT Publication WO02 / 02773). Thus, a bispecific binding protein has two identical antigen-binding arms with identical specificity and identical CDR sequences, and is bivalent for each antigen to which it binds.

[0053] Dual variable domain "Dual variable domain" is used herein to refer to two or more antigen binding sites in a binding protein, which may be a bivalent (two antigen binding sites), tetravalent (four antigen binding sites) or multivalent binding protein. A DVD may be monospecific, i.e., capable of binding one antigen (or one specific epitope), or multispecific, i.e., capable of binding two or more antigens (i.e., two or more epitopes of the same target antigen molecule or two or more epitopes of different target antigens). A preferred DVD binding protein comprises two heavy chain DVD polypeptides and two light chain DVD polypeptides and is referred to as a "DVD immunoglobulin" or "DVD-Ig". Such DVD-Ig binding proteins are thus tetrameric, reminiscent of IgG molecules, but providing more antigen binding sites than IgG molecules. Thus, each half of a tetrameric DVD-Ig molecule is reminiscent of half of an IgG molecule and contains a heavy chain DVD polypeptide and a light chain DVD polypeptide, but unlike the pair of heavy and light chains of an IgG molecule that provides a single antigen-binding domain, the pair of heavy and light chains of a DVD-Ig provides two or more antigen-binding sites.

[0054] Each antigen binding site of a DVD-Ig binding protein may be derived from a donor ("parent") monoclonal antibody and therefore contains a heavy chain variable domain (VH) and a light chain variable domain (VL) that contain all six CDRs involved in antigen binding per antigen binding site. Thus, a DVD-Ig binding protein that binds to two different epitopes (i.e., two different epitopes on two different antigen molecules or two different epitopes on the same antigen molecule) contains an antigen binding site derived from a first parent monoclonal antibody and an antigen binding site of a second parent monoclonal antibody.

[0055] Descriptions of the design, expression and characterization of DVD-Ig binding molecules are provided in PCT Publication No. WO2007 / 024715, U.S. Patent No. 7,612,181, and Wu et al., Nature Biotech., 25:1290-1297 (2007). A preferred example of such a DVD-Ig molecule comprises a heavy chain comprising the structural formula VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first heavy chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker (but is not CH1), X2 is an Fc region, and n is 0 or 1, but preferably is 1; and a light chain comprising the structural formula VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first light chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker (but is not CH1), X2 does not comprise an Fc region, and n is 0 or 1, but preferably is 1. Such a DVD-Ig may comprise two such heavy chains and two such light chains, where each chain comprises variable domains linked in tandem, with no intervening constant region between the variable regions, and the heavy and light chains may associate to form a functional antigen binding site in tandem, and one pair of heavy and light chains may associate with another pair of heavy and light chains to form a tetrameric binding protein having four functional antigen binding sites. In another example, a DVD-Ig molecule may comprise heavy and light chains each comprising three variable domains (VD1, VD2, VD3) linked in tandem, with no intervening constant region between the variable regions, and the heavy and light chains may associate with another pair of heavy and light chains to form a tetrameric binding protein having six antigen binding sites.

[0056] In a preferred embodiment, the DVD-Ig binding protein of the present invention not only binds to the same target molecule bound by the parent monoclonal antibody, but also has one or more desirable properties of the parent monoclonal antibody or antibodies. Preferably, such additional properties are antibody parameters of the parent monoclonal antibody or antibodies. Antibody parameters that can be attributed to the DVD-Ig binding protein derived from the parent monoclonal antibody or antibodies include, but are not limited to, antigen specificity, antigen affinity, potency, biological function, epitope recognition, protein stability, protein solubility, production efficiency, immunogenicity, pharmacokinetics, bioavailability, tissue cross-reactivity, and ortholog antigen binding.

[0057] A DVD-Ig binding protein binds to at least one epitope of RGMc. Non-limiting examples of DVD-Ig binding proteins include DVD-Ig binding proteins that bind to one or more epitopes of RGMc, DVD-Ig binding proteins that bind to an epitope of human RGMc and an epitope of RGMc of another species (e.g., mouse), and DVD-Ig binding proteins that bind to an epitope of human RGMc and an epitope of another target molecule (e.g., VEGFR2 or VEGFR1).

[0058] o. epitope or epitopes "Epitope" or "epitopes" or "epitope of interest" refers to a site on any molecule that can be recognized and bind to a complementary site on its specific binding partner. The molecule and specific binding partner are part of a specific binding pair. For example, an epitope may be on a polypeptide, a protein, a hapten, a carbohydrate antigen (such as, but not limited to, a glycolipid, a glycoprotein, or a lipopolysaccharide), or a polysaccharide. The specific binding partner may be, but is not limited to, an antibody.

[0059] p. Framework or framework array As used herein, "framework" (FR) or "framework sequence" can refer to the remaining sequence of the variable region minus the CDRs. The exact definition of the CDR sequence can be determined by different systems (see, for example, above), so the meaning of the framework sequence is subject to different interpretations. The six CDRs (CDR-L1, -L2 and -L3 of the light chain and CDR-H1, -H2 and -H3 of the heavy chain) also divide the framework region in the light and heavy chain into four subregions (FR1, FR2, FR3 and FR4) in each chain, with CDR1 located between FR1 and FR2, CDR2 located between FR2 and FR3, and CDR3 located between FR3 and FR4. Without specifying a particular subregion as FR1, FR2, FR3 or FR4, the framework region as otherwise referred to represents the combined FRs in the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR refers to one of the four subregions, and FRs refers to two or more of the four subregions that make up a framework region.

[0060] Human heavy and light chain FR sequences are known in the art and can be used as heavy and light chain "acceptor" framework sequences (or simply "acceptor" sequences) to humanize non-human antibodies using techniques known in the art. In one embodiment, the human heavy and light chain acceptor sequences are selected from framework sequences described in public databases such as V-base (hypertext transfer protocol: / / vbase.mrc-cpe.cam.ac.uk / ) or the International ImMunoGeneTics® (IMGT®) information system (hypertext transfer protocol: / / imgt.cines.fr / texts / IMGTrepertoire / LocusGenes / ).

[0061] q.Functional antigen binding site As used herein, a "functional antigen-binding site" may refer to a site in a binding protein (e.g., an antibody) that can bind a target antigen. The antigen-binding affinity of an antigen-binding site may not be as strong as that of the parent binding protein, e.g., the parent antibody from which the antigen-binding site is derived, but the ability to bind to an antigen must be measurable using any one of a variety of methods known to evaluate protein, e.g., antibody binding to an antigen. Furthermore, the antigen-binding affinity of each of the antigen-binding sites of a multivalent protein, e.g., a multivalent antibody, herein does not need to be quantitatively the same.

[0062] r. Humanized antibody "Humanized antibody" is used herein to refer to an antibody that comprises heavy and light chain variable region sequences derived from a non-human species (e.g., mouse), but at least a portion of the VH and / or VL sequences have been altered to be sufficiently "human-like", i.e., sufficiently similar to human germline variable sequences. A "humanized antibody" is an antibody or variant, derivative, analog, or fragment thereof that immunospecifically binds to an antigen of interest and comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementarity determining region (CDR) having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" with respect to a CDR refers to a CDR that has an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises at least one, typically two variable domains (Fab, Fab', F(ab') 2, FabC, Fv), with all or substantially all of the CDR regions corresponding to the CDR regions of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions being those of a human immunoglobulin consensus sequence. In one embodiment, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. In some embodiments, the humanized antibody contains at least the variable domains of the light and heavy chains. The antibody may also include the CH1, hinge, CH2, CH3 and CH4 regions of the heavy chain. In some embodiments, only the humanized antibody contains a humanized light chain. In some embodiments, only the humanized antibody contains a humanized heavy chain. In certain embodiments, only the humanized antibody contains a humanized variable domain of the light chain and / or the humanized heavy chain.

[0063] The humanized antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. The humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions using techniques well known in the art.

[0064] The framework regions and CDRs of a humanized antibody do not have to correspond exactly to the parental sequences, e.g., donor antibody CDRs or consensus frameworks may be mutagenized by substitution, insertion and / or deletion of at least one amino acid residue, so that the CDR or framework residues at this site do not correspond to either the donor antibody or the consensus framework. In preferred embodiments, such mutations are not, however, extensive. Usually, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues correspond to the humanized antibody residues of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region in a consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently in a family of related immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, 1987)). Thus, a "consensus immunoglobulin sequence" may contain a "consensus framework region" and / or a "consensus CDR". In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.

[0065] s. Identical or identical As used herein with respect to two or more polypeptide or polynucleotide sequences, "identical" or "identity" can mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions where identical residues are present in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying this result by 100 to obtain the percentage of sequence identity. If the two sequences have different lengths or the alignment produces one or more alternating ends and the specified region of comparison contains only a single sequence, the residues of the single sequence are included in the denominator, not the numerator, of the calculation.

[0066] t. Isolated polynucleotides As used herein, an "isolated polynucleotide" can refer to a polynucleotide by its origin (e.g., of genomic, cDNA or synthetic origin, or a combination thereof), where an isolated polynucleotide is not associated with all or a portion of a polynucleotide with which the "isolated polynucleotide" is found in nature, is operably linked to a polynucleotide with which it is not naturally linked, or is not naturally occurring as part of a larger sequence.

[0067] u. Labels and detectable labels As used herein, "label" and "detectable label" refer to a moiety that is attached to an antibody or analyte to render the reaction between the antibody and the analyte detectable, and the antibody or analyte so labeled is referred to as "detectably labeled." The label can generate a signal that is detectable by visual or instrumental means. Various labels include signal generating substances, such as chromogens, fluorescent compounds, chemiluminescent compounds, radioactive compounds, and the like. Representative examples of labels include moieties that produce light, such as acridinium compounds, and moieties that produce fluorescence, such as fluorescein. Other labels are described herein. In this regard, the moiety itself may not be detectable, but may become detectable upon reaction with yet another moiety. The use of the term "detectably labeled" is intended to encompass such labels.

[0068] Any suitable detectable label known in the art may be used. For example, the detectable label may be a radioactive label (e.g. 3 H, 125 I, 35 S, 14 C. 32 P and 33P), enzyme labels (e.g., horseradish peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase, etc.), chemiluminescent labels (e.g., acridinium esters, thioesters or sulfonamides; luminol, isoluminol, phenanthridinium esters, etc.), fluorescent labels (e.g., fluorescein (e.g., 5-fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, etc.)), rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (e.g., zinc sulfide-capped cadmium selenide), temperature labels, or immuno-polymerase chain reaction labels. Label introduction, labeling procedures and detection of labels can be found in Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd Edition, Springer Verlag, NY (1997) and Haugland, Handbook of Fluorescent Probes and Research Chemicals (1996), a combined handbook and catalog published by Molecular Probes, Inc., Eugene, Oregon. Fluorescent labels may be used in FPIA (see, for example, U.S. Patent Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093 and 5,352,803, which are incorporated herein by reference in their entirety).Acridinium compounds can be used as detectable labels in homogeneous chemiluminescent assays (see, e.g., Adamczyk et al., Bioorg. Med. Chem. Lett. 16:1324-1328 (2006); Adamczyk et al., Bioorg. Med. Chem. Lett. 4:2313-2317 (2004); Adamczyk et al., Bioorg. Med. Chem. Lett. 14:3917-3921 (2004); and Adamczyk et al., Org. Lett. 5:3779-3782 (2003)).

[0069] In one embodiment, the acridinium compound is acridinium-9-carboxamide. Methods for preparing acridinium 9-carboxamide are described in Mattingly, J. Biolumin. Chemilumin. 6:107-114 (1991); Adamczyk et al., J. Org. Chem. 63:5636-5639 (1998); Adamczyk et al., Tetrahedron 55:10899-10914 (1999); Adamczyk et al., Org. Lett. 1:779-781 (1999); Adamczyk et al., Bioconjugate Chem. 11:714-724 (2000); Mattingly et al., In Luminescence Biotechnology: Instruments and Applications; Dyke, KV Ed.; CRC Press: Boca Raton, pp. 77--105 (2002); Adamczyk et al., Org. Lett. 5:3779-3782 (2003); and U.S. Pat. Nos. 5,468,646, 5,543,524, and 5,783,699, each of which is incorporated by reference in its entirety for the teachings therein.

[0070] Another example of an acridinium compound is acridinium-9-carboxylate aryl ester. An example of the acridinium-9-carboxylate aryl ester of formula II is 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (available from Cayman Chemical, Ann Arbor, MI). Methods for preparing acridinium-9-carboxylate aryl ester are described in McCapra et al., Photochem. Photobiol. 4:1111-21 (1965); Razavi et al., Luminescence 15:245-249 (2000); Razavi et al., Luminescence 15:239-244 (2000); and U.S. Patent No. 5,241,070, each of which is incorporated herein by reference in its entirety for its teachings. Such acridinium-9-carboxylate aryl esters are effective chemiluminescent indicators for hydrogen peroxide generated in the oxidation of analyte by at least one oxidase in terms of signal intensity and / or signal speed. The time of chemiluminescence for acridinium-9-carboxylate aryl esters is rapid, i.e., completed in less than one second, whereas acridinium-9-carboxamide chemiluminescence extends for more than two seconds. However, acridinium-9-carboxylate aryl esters lose their chemiluminescent properties in the presence of protein. Therefore, their use requires the absence of protein during signal generation and detection. Methods for separating or removing proteins in samples are well known to those skilled in the art and include, but are not limited to, ultrafiltration, extraction, precipitation, dialysis, chromatography and / or digestion (see, for example, Wells, High Throughput Bioanalytical Sample Preparation. Methods and Automation Strategies, Elsevier (2003)). The amount of protein removed or separated from the test sample may be about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95%.Further details regarding acridinium-9-carboxylate aryl esters and their uses are described in U.S. Patent Application Publication No. 11 / 697,835, filed April 9, 2007. The acridinium-9-carboxylate aryl esters can be dissolved in any suitable solvent, such as degassed anhydrous N,N-dimethylformamide (DMF) or aqueous sodium cholate.

[0071] v. Linking sequences and linking peptide sequences A "linking sequence" or "linking peptide sequence" refers to a natural or artificial polypeptide sequence that is connected to one or more polypeptide sequences (e.g., full length, fragments, etc.) of interest. The term "connected" refers to the attachment of the linking sequence to the polypeptide sequence of interest. Such polypeptide sequences are preferably linked by one or more peptide bonds. The linking sequence may have a length of about 4 to about 50 amino acids. Preferably, the linking sequence is about 6 to about 30 amino acids in length. A natural linking sequence may be modified by amino acid substitution, addition, or deletion to create an artificial linking sequence. Exemplary linking sequences include, but are not limited to, (i) histidine (His) tags, such as 6X His tag (SEQ ID NO: 197), having an amino acid sequence of HHHHHH (SEQ ID NO: 197), which are useful as linking sequences to facilitate the isolation and purification of polypeptides and antibodies of interest; (ii) enterokinase cleavage sites, such as His tags, which have been used in the isolation and purification of proteins and antibodies of interest. Often, enterokinase cleavage sites are used together with His tags in the isolation and purification of proteins and antibodies of interest. A variety of enterokinase cleavage sites are known in the art. Examples of enterokinase cleavage sites include, but are not limited to, the amino acid sequence of DDDDK (SEQ ID NO: 198) and its derivatives (such as ADDDDK (SEQ ID NO: 199)); (iii) various sequences may be used to link or connect the light and / or heavy chain variable regions of the single chain variable region fragment. Other examples of linking sequences can be found in Bird et al., Science 242:423-426 (1988); Huston et al., PNAS USA 85:5879-5883 (1988); and McCafferty et al., Nature 348:552-554 (1990). Linking sequences may also be modified for additional functions, such as attachment of drugs or attachment to solid supports. In the context of the present disclosure, monoclonal antibodies may contain linking sequences, such as, for example, His tags, enterokinase cleavage sites, or both.

[0072] W multivalent binding protein "Multivalent binding protein" is used herein to refer to a binding protein that contains two or more antigen binding sites (also referred to herein as "antigen binding domains"). Multivalent binding proteins are preferably engineered to have three or more antigen binding sites and are generally not naturally occurring antibodies. The term "multispecific binding protein" refers to a binding protein that can bind to two or more related or unrelated targets, including a binding protein that can bind to two or more different epitopes of the same target molecule.

[0073] x.Prescribed cut-offs and prescribed levels "Predetermined cut-off" and "predetermined level" generally refer to assay cut-off values ​​that are used to evaluate diagnostic / prognostic / therapeutic outcomes by comparing assay results to the predetermined cut-off / level, and the predetermined cut-off / level always relates or correlates with various clinical parameters (e.g., disease severity, progression / non-progression / improvement, etc.). The present disclosure provides exemplary predetermined levels. However, it is well known that cut-off values ​​may vary depending on the nature of the immunoassay (e.g., the antibody utilized, etc.). Furthermore, it is well within the skill of the art to adapt the disclosure herein to other immunoassays to obtain immunoassay-specific cut-off values ​​for these other immunoassays based on the present disclosure. The exact values ​​of the predetermined cut-off / level may vary between assays, but the relationships described herein should be generally applicable.

[0074] y. Pretreatment reagent As used in the diagnostic assays described herein, a "pretreatment reagent", e.g., a lysis, precipitation and / or solubilization reagent, is a reagent that lyses any cells and / or solubilizes any analyte present in the test sample. Pretreatment is not necessary for all samples further described herein. In particular, solubilization of the analyte (i.e., RGMc (membrane-bound RGMc, soluble RGMc, fragments of membrane-bound RGMc, fragments of soluble RGMc, variants of RGMc (membrane-bound RGMc or soluble RGMc) or any combination thereof, etc.)) is accompanied by release of the analyte from any endogenous binding proteins present in the sample. Pretreatment reagents may be homogeneous (no separation step required) or heterogeneous (requiring a separation step). Heterogeneous pretreatment reagents are used to remove any precipitated analyte binding proteins from the test sample before proceeding to the next step of the assay. The pretreatment reagent may optionally include (a) one or more solvents and salts, (b) one or more solvents, salts and detergents, (c) detergents, (d) detergents and salts, or (e) any reagent or combination of reagents suitable for cell lysis and / or solubilization of the sample.

[0075] z. Quality control reagents "Quality control reagents" in the context of the immunoassays and kits described herein include, but are not limited to, calibrators, controls, and sensitivity panels. "Calibrators" or "standards" (e.g., one or more, such as a plurality) are typically used to establish a calibration (standard) curve for interpolating the concentration of an analyte, such as an antibody or analyte. Alternatively, a single calibrator near a pre-defined positive / negative cutoff may be used. Multiple calibrators (i.e., more than one calibrator or varying amounts of an antibiotic) may be used together to comprise a "sensitivity panel."

[0076] aa. Recombinant antibodies and multiple recombinant antibodies "Recombinant antibody" and "recombinant antibodies" refer to antibodies prepared by one or more steps, including cloning a nucleic acid sequence encoding all or part of one or more monoclonal antibodies into a suitable expression vector by recombinant techniques and subsequently expressing the antibody in a suitable host cell. The terms include, but are not limited to, (i) recombinantly produced monoclonal antibodies, chimeric antibodies, humanized antibodies (fully or partially humanized), multispecific or multivalent structures formed from antibody fragments, bivalent antibodies, heteroconjugate Abs, DVD-Ig® and other antibodies described herein (dual variable domain immunoglobulins and methods for preparing them are described in Wu, C. et al., Nature Biotechnology, 25:1290-1297 (2007)). As used herein, the term "bivalent antibody" refers to an antibody that includes a first arm having specificity for one antigenic site and a second arm having specificity for a different antigenic site. That is, a bivalent antibody has dual specificity.

[0077] bb. Samples, Test Samples and Patient Samples "Sample," "test sample," and "patient sample" may be used interchangeably herein. When obtained from a patient, a sample, such as a urine, serum, plasma, amniotic fluid, cerebrospinal fluid, placental cell or tissue, endothelial cell, leukocyte, or monocyte sample, may be used directly or may be pretreated by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., to modify the characteristics of the sample in some manner described herein or otherwise known in the art.

[0078] cc. A series of calibration compositions A "series of calibration compositions" refers to a plurality of compositions containing known concentrations of Cys-RGMcC, each of which differs from the other compositions in a sequence by the concentration of Cys-CRGMc.

[0079] dd.solid phase "Solid phase" refers to any material that is insoluble or can be made insoluble by a subsequent reaction. The solid phase may be selected for its inherent ability to attract and immobilize the capture agent. Alternatively, the solid phase may have a linking agent attached thereto that has the ability to attract and immobilize the capture agent. The linking agent may, for example, comprise a charged substance that is oppositely charged to the capture agent itself or to a charged substance conjugated to the capture agent. In general, the linking agent may be any binding partner (preferably specific) that is immobilized (attached) to the solid phase and has the ability to immobilize the capture agent via a binding reaction. The linking agent allows the indirect binding of the capture agent to the solid phase material before or during the performance of the assay. The solid phase may, for example, be plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon, including, for example, test tubes, microtiter wells, sheets, beads, microparticles, chips and other structures known to those skilled in the art.

[0080] ee.Specific binding As used herein, "specific binding" or "specifically binds" can refer to the interaction of an antibody, protein, or peptide with a second chemical species, which interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) in the chemical species; for example, antibodies generally recognize and bind to specific protein structures other than proteins. If an antibody is specific for epitope "A," then in a reaction containing labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0081] ff. Specific binding partners A "specific binding partner" is a member of a specific binding pair. A specific binding pair comprises two different molecules that specifically bind to each other through chemical or physical means. Thus, in addition to the antigen and antibody specific binding pair of a typical immunoassay, other specific binding pairs may include biotin and avidin (or streptavidin), carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, multiple enzymes and enzyme inhibitors, and the like. Additionally, specific binding pairs may include members that are analogs of the original specific binding member, e.g., analyte analogs. Immunoreactive specific binding members include antigens, antigen fragments, and antibodies, including monoclonal and polyclonal antibodies, whether isolated or recombinantly produced, as well as complexes and fragments thereof.

[0082] gg. Stringent conditions "Stringent conditions" is used herein to refer to hybridization with filter-bound DNA in 6x sodium chloride / sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2x SSC / 0.1% SDS at about 50-65° C. The term "highly stringent conditions" refers to hybridization with filter-bound nucleic acid in 6x SSC at about 45° C., followed by one or more washes in 0.1x SSC / 0.2% SDS at about 68° C., or under other stringent hybridization conditions. See, e.g., Ausubel, FM et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, pages 6.3.1-6.3.6 and 2.10.3.

[0083] hh. to treat, to be treating or to cure "Treating", "treating" or "treatment" are used interchangeably herein to indicate reversing, alleviating or inhibiting the progression of the disease to which such term applies or one or more symptoms of such disease. Depending on the condition of the subject, the term also refers to preventing the disease, including preventing the onset of the disease or preventing symptoms associated with the disease. Treatment may be performed in either an acute or chronic manner. The term also refers to reducing the severity of the disease or symptoms associated with such disease prior to the onset of the disease. Such prevention or reduction of the severity of the disease prior to onset refers to administration of the antibody or pharmaceutical composition of the present invention to a subject who is not at the time of administration suffering from the disease. "Preventing" also refers to preventing the recurrence of the disease or one or more symptoms associated with such disease. "Treatment" and "therapeutically" refer to the action of treatment, as "treating" is defined above.

[0084] ii. Tracer As used herein, "tracer" refers to an analyte or analyte fragment conjugated to a label, such as Cys-CRGMc conjugated to a fluorescein moiety, where the analyte conjugated to the label can effectively compete with the analyte for sites on an antibody specific for the analyte.

[0085] jj.Variant "Variant" is used herein to refer to a peptide or polypeptide that differs from an amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. Representative examples of "biological activity" include the ability to be bound by a specific antibody or the ability to stimulate an immune response. Variant is also used herein to refer to a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., replacement of an amino acid with an amino acid of different similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are recognized in the art as typically involving minor changes. These minor changes can be identified in part by considering the hydropathic index of the amino acid as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on this hydrophobicity and charge considerations. It is known in the art that amino acids of similar hydropathic indexes may be substituted and still retain protein function. In one embodiment, amino acids with hydropathic indexes of ±2 are substituted. Hydrophilicity of amino acids may also be used to identify substitutions that result in proteins that retain biological function. Consideration of the hydrophilicity of amino acids in relation to a peptide allows calculation of the greatest local average hydrophilicity of the peptide, a useful criterion that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101 is incorporated herein by reference in its entirety. Substitution of amino acids with similar hydrophilicity values ​​may result in peptides that retain biological activity, such as immunogenicity, as understood in the art. Substitution may be performed with amino acids with hydrophilicity values ​​within ±2 of each other. Both the hydropathic index and hydrophilicity value of an amino acid are influenced by the particular side chain of that amino acid. Consistent with this observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of amino acids, particularly the side chains of these amino acids as expressed by hydrophobicity, hydrophilicity, charge, size and other properties."Variant" may also be used to refer to an antigenically reactive fragment of an anti-RGMc antibody that differs in amino acid sequence from the corresponding fragment of an anti-RGMc antibody, but is still antigenically reactive and can compete with the corresponding fragment of an anti-RGMc antibody for binding to RGMc. "Variant" may also be used to refer to a polypeptide or fragment thereof that has been processed differently, such as by proteolysis, phosphorylation or other post-translational modification, but still retains this antigenic reactivity.

[0086] kk.vector A "vector" is used herein to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, into which additional DNA segments may be ligated within the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of inducing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As the plasmid is the most commonly used form of vector, "plasmid" and "vector" may be used interchangeably. However, other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions may be used. In this regard, RNA types of vectors, including RNA viral vectors, may also find use in the context of the present disclosure.

[0087] With respect to the recitation of numerical ranges herein, each intervening number is specifically contemplated to the same degree of precision. For example, with respect to the range 6-9, the numbers 7 and 8 are specifically contemplated in addition to 6 and 9, and with respect to the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are specifically contemplated.

[0088] 2. Anti-RGMc antibody Provided herein are antibodies that bind to RGMc and block normal expression of hepcidin, which directly regulates iron concentrations in plasma and distribution of iron to various tissues.

[0089] a.RGMc As previously described herein, RGMc is a glycosylphosphatidylinositol ("GPI")-anchored membrane protein expressed in muscle, retina, and periportal hepatocytes. RGMc acts with hepcidin through signaling proteins to maintain iron homeostasis in the body. Hepcidin is a small peptide (20-25 amino acids) that regulates whole-body iron metabolism by binding to ferroportin, the exclusive mammalian iron transporter. Because these cells can no longer release iron into the blood, anemia of chronic disease ("ACD") is a common outcome. Hepcidin-induced degradation of ferroportin can occur at levels of greater than 300 mg / l hepcidin, greater than 325 mg / l hepcidin, greater than 350 mg / l hepcidin, greater than 375 mg / l hepcidin, greater than 400 mg / l hepcidin, greater than 425 mg / l hepcidin, greater than 450 mg / l hepcidin or greater than 475 mg / l hepcidin.

[0090] Human RGMc, a 426 amino acid protein with a predicted 31 amino acid N-terminal signal peptide and 45 amino acid C-terminal GPI attachment signal, was first proposed to be involved in whole-body iron metabolism when mutations in the human gene were associated with the severe iron overload disorder, juvenile hemochromatosis. Hepcidin expression is controlled by membrane-bound RGMc expressed on the surface of hepatocytes and by soluble RGMc, which is present in human blood at a concentration of approximately 1 μg / ml. Soluble RGMc arises from cleavage of membrane-bound RGMc by matriptase-2 (TMPRSS6). The soluble form of RGMc binds and sequesters BMP-6, thus preventing induction of hepcidin expression. The membrane form of RGMc has the opposite effect, which is to increase hepcidin expression.

[0091] The human RGMc may have the following amino acid sequence:

[0092] [ka] The human RGMc may be a fragment or variant of SEQ ID NO:1.

[0093] A fragment of RGMc may be between about 5 and about 425 amino acids, between about 10 and about 400 amino acids, between about 50 and about 350 amino acids, between about 100 and about 300 amino acids, between about 150 and about 250 amino acids, between about 200 and about 300 amino acids or between about 75 and about 150 amino acids in length. The fragment may comprise a contiguous number of amino acids from RGMc.

[0094] The fragment of RGMc may have the amino acid sequence:

[0095] [ka] The RGMc fragment may be a variant of SEQ ID NO:2.

[0096] b.RGMc-recognizing antibody The antibody is an antibody that binds to human RGMc (such as membrane-bound RGMc, soluble RGMc, a fragment of membrane-bound RGMc, a fragment of soluble RGMc, a variant of RGMc (membrane-bound RGMc or soluble RGMc) or any combination thereof). The antibody may be a fragment of an anti-RGMc antibody or a variant or derivative thereof. The antibody may be a polyclonal or monoclonal antibody. The antibody may be a chimeric antibody, a single chain antibody, a humanized antibody, a fully human antibody or an antibody fragment, such as a Fab fragment or a mixture thereof. The antibody may be an immunoglobulin molecule, a disulfide-linked Fv, an affinity matured antibody, a scFv, a chimeric antibody, a single domain antibody, a CDR-grafted antibody, a diabody, a humanized antibody, a fully human antibody, a multispecific antibody, a Fab, a bispecific antibody, a DVD, a Fab', a bispecific antibody, a F(ab') 2 or Fv. The antibody fragment or derivative may be F(ab') 2 , Fv or scFv fragments. Antibody derivatives may be produced by peptidomimetics. Furthermore, techniques described for producing single chain antibodies may be adapted to produce single chain antibodies according to methods known in the art. Transgenic animals may also be used to express humanized or fully human antibodies.

[0097] The antibody can recognise and specifically bind to an epitope present in the RGMc polypeptide or variant described above (e.g. an epitope contained in SEQ ID NO: 1 or a variant of SEQ ID NO: 1). The epitope may also be an epitope contained in SEQ ID NO: 2 or a variant of SEQ ID NO: 2.

[0098] The antibody is preferably distinguishable from known anti-RGMc antibodies by possessing a different biological function from anti-RGMc antibodies known in the art.For example, in addition to recognizing and binding to membrane-bound RGMc, the antibody preferably has a further biological activity, such as the ability to increase or decrease hepcidin expression.In addition or alternatively, the antibody has the ability to block RGMc-neogenin interaction and / or RGMc-BMP-6 (bone morphogenetic protein 6) interaction.

[0099] (1) Antibody binding characteristics The antibody can immunospecifically bind to RGMc (membrane-bound RGMc, soluble RGMc, or a combination thereof), a fragment thereof, or a variant thereof, and has a specific binding affinity of at least about 1.0×10 -3 s -1 , at least about 1.0 × 10 -4 s -1 , at least about 1.0 × 10 -5 s -1 , at least about 1.0 × 10 -6 s -1 k off (or k d ) or about 1.0 × 10 -3 s -1 to about 1.0×10 -6 s -1 , about 1.0×10 -3 s -1 to about 1.0×10 -5 s -1 Or about 1.0 x 10 -3 s -1 to about 1.0×10 -4 s -1 k in the range off (or k d ). The fragment may have SEQ ID NO:2.

[0100] The antibody can immunospecifically bind to RGMc (membrane-bound RGMc, soluble RGMc, or a combination thereof), a fragment thereof, or a variant thereof, and has a specific binding capacity of at least about 2.4×10 4 M -1 s -1 , at least about 2.5 × 10 4 M-1 s -1 , at least about 3.3 × 10 4 M -1 s -1 , at least about 5.0 × 10 4 M -1 s -1 , at least about 1.25 × 10 7 M -1 s -1 , at least about 1.35 × 10 7 M -1 s -1 , at least about 1.0 × 10 8 M -1 s -1 , at least about 1.0 × 10 9 M -1 s -1 k on (or k a ) or about 5.0 × 10 4 M -1 s -1 to about 1.0×10 8 M -1 s -1 , about 3.3×10 4 M -1 s -1 to about 1.0×10 9 M -1 s -1 , about 2.5×10 4 M -1 s -1 From about 1.25 x 10 7 M -1 s -1 , about 2.4×10 4 M -1 s -1 From about 1.35 × 10 7 M -1 s -1 is in the range f k on (or k a ). The fragment may have SEQ ID NO:2.

[0101] (2) Antibody structure (a) Variable heavy and light chain regions and heavy and light chain CDRs The antibody can immunospecifically bind to RGMc, a fragment thereof or a variant thereof and comprises a variable heavy chain and / or a variable light chain as shown in Table 1. The antibody can immunospecifically bind to RGMc, a fragment thereof or a variant thereof and comprises one or more of the heavy or light chain CDR sequences as also shown in Table 1 and / or Table 2.

[0102] [Table 1] TIFF2025028985000004.tif236166TIFF2025028985000005.tif243165TIFF2025028985000006.tif142166

[0103] [Table 2] TIFF2025028985000008.tif239162TIFF2025028985000009.tif240163TIFF202 5028985000010.tif240161TIFF2025028985000011.tif240161TIFF20250289850 00012.tif240160TIFF2025028985000013.tif237162TIFF2025028985000014.t if237161TIFF2025028985000015.tif237161TIFF2025028985000016.tif237161

[0104] The above humanized antibodies in Table 2 and methods for making them are described in US Patent Application Publication No. 2010 / 0028340, the contents of which are incorporated herein by reference.

[0105] As shown in Table 2, the CDR-H1 domains of the variable regions (VH and VL) are all identical in sequence. In addition, the CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 domains are all identical in sequence (i.e., the CDR-H2 domain of the VH h5F9.1 region is identical to the CDR-H2 domain of the VH h5F9.2 region, the CDR-H2 domain of the VH h5F9.2 region is identical to the CDR-H2 domain of the VH h5F9.3 region, etc.; the CDR-H3 domain of the VH h5F9.1 region is identical to the CDR-H3 domain of the VH h5F9.2 region, the CDR-H3 domain of the VH h5F9.2 region is identical to the CDR-H3 domain of the VH h5F9.3 region, etc.).

[0106] The isolated antibody that specifically binds to RGMc (or a fragment thereof) of the present disclosure comprises (a) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:3, (b) a variable light domain region comprising the amino acid sequence of SEQ ID NO:4, (c) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:5, (d) a variable light domain region comprising the amino acid sequence of SEQ ID NO:6, (e) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:7, (f) a variable light domain region comprising the amino acid sequence of SEQ ID NO:8, (g) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:9, (h) a variable light domain region comprising the amino acid sequence of SEQ ID NO:10, (i) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:11, (ii) a variable light domain region comprising the amino acid sequence of SEQ ID NO:12, (iii) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:13, (iv) a variable light domain region comprising the amino acid sequence of SEQ ID NO:14, (v) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:15, (vi) a variable light domain region comprising the amino acid sequence of SEQ ID NO:16, (vii) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:17, (viii) a variable light domain region comprising the amino acid sequence of SEQ ID NO:18, (viii) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:19, (viii) a variable light domain region comprising the amino acid sequence of SEQ ID NO:20, (viii) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:21, (viii) a variable light domain region comprising the amino acid sequence of SEQ ID NO:22, (viii) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:23, (viii) a variable light domain region comprising the amino acid sequence of SEQ ID NO:24, (viii) (i) a variable heavy chain comprising a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 13; (j) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 16; (k) a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 19. (l) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (m) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25; (n) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28; (o) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 31; (p) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR2 comprising the amino acid sequence of SEQ ID NO: 33 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 34; (q) a variable heavy chain comprising a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR2 comprising the amino acid sequence of SEQ ID NO: 12 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR1 comprising the amino acid sequence of SEQ ID NO: 14;(r) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 19, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a CDR2 comprising the amino acid sequence of SEQ ID NO: 21 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (s) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR2 comprising the amino acid sequence of SEQ ID NO: 24 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25. (t) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28; (t) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 31, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR2 comprising the amino acid sequence of SEQ ID NO: 33, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 34.

[0107] The antibody, fragment, variant or derivative thereof may contain one or more amino acid sequences having about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55% or about 50% identity to one or more of SEQ ID NOs: 3-34. The antibody or variant or derivative thereof may be encoded by one or more nucleic acid sequences having about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55% or about 50% identity to one or more nucleic acid sequences encoding one or more of SEQ ID NOs: 3-34. Polypeptide identity and homology may be determined, for example, by the algorithm described in the report: Wilbur, WJ and Lipman, DJ Proc. Natl. Acad. Sci. USA 80, 726-730 (1983). The antibodies, fragments, variants or derivatives thereof described herein may be encoded by nucleic acids that hybridize under stringent conditions to one or more complements of a nucleic acid sequence encoding one or more of SEQ ID NOs: 3-34. The antibodies, fragments, variants or derivatives thereof described herein may be encoded by nucleic acids that hybridize under highly stringent conditions to one or more complements of a nucleic acid sequence encoding one or more of SEQ ID NOs: 3-34.

[0108] c. Antibody preparation / production Antibodies may be prepared by any of a variety of techniques. In general, to allow for the production of antibodies, which may be recombinant, antibodies may be produced by conventional techniques or by cell culture techniques, including the generation of monoclonal antibodies by transfection of antibody genes, heavy and / or light chains into suitable bacterial or mammalian cell hosts. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although it is possible to express the antibodies of the present invention in either prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells is preferred, and expression of the antibodies in mammalian host cells is most preferred, since such eukaryotic cells (especially mammalian cells) are more likely than prokaryotic cells to assemble and secrete properly folded and immunologically active antibodies.

[0109] Exemplary mammalian host cells for expressing a recombinant antibody of the invention include Chinese hamster ovary (CHO cells) used with the DHFR selectable marker, NS0 myeloma cells, COS cells and SP2 cells, e.g., as described in Kaufman and Sharp, J. Mol. Biol., 159:601-621 (1982), including dhfr-CHO cells as described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980). When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a sufficient time to allow for expression of the antibody within the host cell or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibody may be recovered from the culture medium using standard protein purification methods.

[0110] The host cells may also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. Modifications to the above procedures are understood to be within the scope of the present invention. For example, it may be suitable to transfect the host cells with DNA encoding functional fragments of either the light and / or heavy chains of the antibody of the present invention. Recombinant DNA techniques may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that are not necessarily required to bind the antigen of interest. Molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the present invention. Furthermore, bivalent antibodies may be produced in which one heavy chain and one light chain is an antibody of the present invention (i.e., binds human RGMc) and the other heavy and light chains are specific for an antigen other than human RGMc by crosslinking the antibody of the present invention to a second antibody by standard chemical crosslinking methods.

[0111] In a preferred system for recombinant expression of the antibody of the invention or antigen-binding portion thereof, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operably linked to a CMV enhancer / AdMLP promoter regulatory element to drive high level transcription of the genes. The recombinant expression vector also carries a DHFR gene, allowing for selection of CHO cells transfected with the vector using methotrexate selection / amplification. The selected transformant host cells are cultured to allow expression of the antibody heavy and light chains, and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select the transformants, culture the host cells, and recover the antibody from the culture medium. Still further, the invention provides a method of synthesizing a recombinant antibody of the invention by culturing the host cells of the invention in an appropriate culture medium until the recombinant antibody of the invention is synthesized. The method may further comprise isolating the recombinant antibody from the culture medium.

[0112] Methods for preparing monoclonal antibodies include the preparation of immortal cell lines capable of producing antibodies with the desired specificity. Such cell lines may be produced from spleen cells obtained from immunized animals. Animals may be immunized with RGMc or fragments and / or variants thereof. For example, either SEQ ID NO:1, SEQ ID NO:2, a fragment of SEQ ID NO:1 or SEQ ID NO:2, or a variant of SEQ ID NO:1 or SEQ ID NO:2 may be used to immunize animals. Animals may be immunized with RGMa or fragments and / or variants thereof. For example, either SEQ ID NO:35, SEQ ID NO:36, a fragment of SEQ ID NO:35 or SEQ ID NO:36, or a variant of SEQ ID NO:35 or SEQ ID NO:36 may be used to immunize animals. RGMa may have the following amino acid sequence:

[0113] [ka] RGMa may be a fragment or variant of SEQ ID NO:35.

[0114] The fragment may be between 5 and 425 amino acids, between 10 and 400 amino acids, between 50 and 350 amino acids, between 100 and 300 amino acids, between 150 and 250 amino acids, between 200 and 300 amino acids or between 75 and 150 amino acids in length. The fragment may comprise a contiguous number of amino acids from SEQ ID NO:35.

[0115] A fragment of RGMa can have the amino acid sequence:

[0116] [ka] The RGMa fragment may be a fragment of SEQ ID NO: 36. The RGMa fragment may be a variant of SEQ ID NO:36.

[0117] The peptide used to immunize the animal may include amino acids encoding a human Fc, e.g., a fragment crystallizable region or tail region of a human antibody. The spleen cells may then be immortalized, e.g., by fusion with a myeloma cell fusion partner. A variety of fusion techniques may be utilized. For example, spleen cells and myeloma cells may be mixed with a non-ionic detergent for a few minutes and then plated at low density on a selection medium that supports the growth of hybrid cells, but not myeloma cells. One such technique uses hypoxanthine, aminopterin, thymidine (HAT) selection. After a sufficient time, usually about 1 to 2 weeks, hybrid colonies are observed. Single colonies are selected and their culture supernatants are tested for binding affinity to the polypeptide. Hybridomas with high reactivity and specificity may be used.

[0118] Monoclonal antibodies may be isolated from the supernatant in which the hybridoma colonies are growing. In addition, various techniques may be utilized to improve the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. The monoclonal antibodies may then be collected from the ascites fluid or blood. Contaminants may be removed from the antibody by conventional techniques, such as chromatography, gel filtration, precipitation and extraction. Affinity chromatography is one example of a method that may be used in the process of purifying the antibody.

[0119] The proteolytic enzyme papain selectively cleaves IgG molecules to produce several fragments, two of which (F(ab) fragments) each contain a covalently linked heterodimer with an intact antigen-binding site. The enzyme pepsin cleaves IgG molecules to produce F(ab') fragments, which contain both antigen-binding sites. 2 Several fragments containing the fragments can be given.

[0120] Fv fragments can be generated by selective proteolytic cleavage of IgM and rarely IgG or IgA immunoglobulin molecules. Fv fragments can be derived using recombinant techniques. Fv fragments contain a non-covalent VH:VL heterodimer that contains an antigen-binding site that retains much of the antigen recognition and binding ability of the native antibody molecule.

[0121] An antibody, antibody fragment or derivative may comprise a set of heavy and light chain complementarity determining regions ("CDRs"), each interposed between a set of heavy and light chain frameworks ("FRs") that provide support for the CDRs and define the spatial relationship of the CDRs to each other. A set of CDRs may contain three hypervariable regions of the heavy or light chain V region. Starting from the N-terminus of the heavy or light chain, these regions are designated as "CDR1", "CDR2" and "CDR3", respectively. Thus, an antigen-binding site may comprise six CDRs, including a set of CDRs from each of the heavy and light chain V regions. A polypeptide comprising a single CDR (e.g., CDR1, CDR2 or CDR3) may be referred to as a "molecular recognition unit". Crystallographic analysis of antigen-antibody complexes has demonstrated that the amino acid residues of the CDRs make extensive contacts with the bound antigen, with the most extensive antigen contact being with the heavy chain CDR3. Thus, the molecular recognition unit may be primarily responsible for the specificity of the antigen-binding site. In general, the CDR residues are directly and most substantially involved in influencing the antigen-binding site.

[0122] Humanized antibodies can be designed to minimize undesired immune responses against rodent anti-human antibodies, limiting the duration and effectiveness of therapeutic applications of these moieties in human recipients. A humanized antibody may have one or more amino acid residues introduced into it from a non-human source. These non-human residues are often referred to as "import" residues, typically taken from the variable domain. Humanization can be performed by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Such "humanized" antibodies are thus chimeric antibodies in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. See, e.g., U.S. Pat. No. 4,816,567, the contents of which are incorporated herein by reference. A humanized antibody may be a human antibody in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Humanization or engineering of the antibodies of the present invention may be carried out using any known method, such as, but not limited to, those described in U.S. Pat. Nos. 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539 and 4,816,567.

[0123] Humanized antibodies may retain high affinity for RGMc and other beneficial biological properties. Humanized antibodies may be prepared by a process of analysis of parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are publicly available. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the possible role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues may be selected and combined from the recipient and import sequences to achieve the desired antibody characteristic, such as increased affinity for RGMc. In general, the hypervariable region residues may be directly and most substantially involved in influencing antigen binding.

[0124] As an alternative to humanization, human antibodies (also referred to herein as "fully human antibodies") may be generated. For example, it is possible to produce transgenic animals (e.g., mice that, upon immunization, are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production). For example, the expression of the antibody heavy chain joining region (J) in chimeric and germline mutant mice can be used to generate human antibodies. II Homozygous deletion of the .DELTA..DELTA.. gene results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array in such germline mutant mice results in the production of human antibodies upon antigen challenge. Humanized or fully human antibodies can be prepared according to the methods described in U.S. Patent Nos. 5,770,429, 5,833,985, 5,837,243, 5,922,845, 6,017,517, 6,096,311, 6,111,166, 6,270,765, 6,303,755, 6,365,116, 6,410,690, 6,682,928 and 6,984,720, the contents of each of which are incorporated herein by reference.

[0125] Other suitable methods of producing or isolating antibodies of the required specificity may also be used, including methods of selecting recombinant antibodies from peptide or protein libraries (e.g., but not limited to, display libraries, such as, but not limited to, bacteriophage, ribosomal, oligonucleotide, RNA, cDNA, etc.), available from a variety of commercial vendors, such as Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martinsreid / Planegg, Del.), Biovation (Aberdeen, Scotland, UK), Biolnvent (Lund, Sweden), using methods known in the art. U.S. Pat. No. 4,704,692; See US Pat. Nos. 5,723,323, 5,763,192, 5,814,476, 5,817,483, 5,824,514, and 5,976,862. An alternative method relies on immunization of transgenic animals capable of producing a repertoire of human antibodies (e.g., SCID mice, Nguyen et al. (1977) Microbiol. Immunol. 41:901-907 (1997); Sandhu et al. (1996) Crit. Rev. Biotechnol. 16:95-118; Eren et al. (1998) Immunol. 93:154-161), as known in the art and / or described herein.Such techniques include, but are not limited to, ribosome display (Hanes et al. (1997) Proc. Natl. Acad. Sci. USA, 94:4937-4942; Hanes et al. (1998) Proc. Natl. Acad. Sci. USA, 95:14130-14135); single cell antibody production techniques (e.g., selected lymphocyte antibody method ("SLAM") (U.S. Patent No. 5,627,052; Wen et al. (1987) J. Immunol. 17:887-892; Babcook et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848); gel microdroplets and flow cytometry (Powell et al. (1990) Biotechnol. 8:333-337; One Cell Systems, (Cambridge, Mass); Gray et al. (1995) J. Imm. Meth. 182:155-163; Kenny et al. (1995) Bio / Technol. 13:787-790); B cell selection (Steenbakkers et al. (1994) Molec. Biol. Reports 19:125-134 (1994).

[0126] Affinity matured antibodies can be produced by any one of many procedures known in the art. See, for example, Marks et al., BioTechnology, 10:779-783 (1992), which describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues has been described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169:147-155 (1995); Yelton et al., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol., 154(7):3310-3319 (1995); Hawkins et al., Mol. Biol., 226:889-896 (1992). Selective mutagenesis positions with activity enhancing amino acid residues and selective mutation at contact or hypermutation positions are described in US Pat. No. 6,914,128 B1.

[0127] The antibody variants of the invention may also be prepared using delivery of a polynucleotide encoding an antibody of the invention to a suitable host to provide a transgenic animal or mammal, such as a goat, cow, horse, sheep, etc., that produces such an antibody in its milk. These methods are known in the art and are described, for example, in U.S. Patent Nos. 5,827,690, 5,849,992, 4,873,316, 5,849,992, 5,994,616, 5,565,362, and 5,304,489.

[0128] Antibody variants may also be prepared by delivering the polynucleotides of the present invention to provide transgenic plants and cultured plant cells (such as, but not limited to, tobacco, corn, and duckweed) that produce such antibodies, specific parts, or variants in plant parts or cells cultured therefrom. For example, Cramer et al. (1999) Curr. Top. Microbol. Immunol. 240:95-118 and references cited therein describe the production of transgenic tobacco leaves that express large amounts of recombinant proteins, for example, using inducible promoters. Transgenic corn has been used to express mammalian proteins at commercial production levels with biological activities equivalent to those produced in other recombinant systems or purified from natural sources. See, for example, Hood et al., Adv. Exp. Med. Biol. (1999) 464:127-147 and references cited therein. Antibody variants have also been produced in large quantities from transgenic plant seeds containing antibody fragments such as single chain antibodies (scFv's), including tobacco seeds and potato tubers. See, for example, Conrad et al. (1998) Plant Mol. Biol. 38:101-109 and references cited therein. Thus, the antibodies of the present invention may also be produced using transgenic plants according to known methods.

[0129] Antibody derivatives may be produced, for example, by adding exogenous sequences to modify immunogenicity or to decrease, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life or any other suitable characteristic. Generally, some or all of the non-human or human CDR sequences are maintained, but the non-human sequences of the variable and constant regions are replaced with human or other amino acids.

[0130] The small antibody fragment may be a diabody having two antigen binding sites, the fragments being bound to the same polypeptide chain (V H V L ) in the light chain variable domain (V L ) connected to a heavy chain variable domain (V H ). See, e.g., EP 404,097; WO 93 / 11161; and Hollinger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, generating two antigen-binding sites. See also U.S. Pat. No. 6,632,926 by Chen et al., which is incorporated herein by reference in its entirety, and discloses antibody variants having one or more amino acids inserted into the hypervariable region of the parent antibody and a binding affinity for a target antigen that is at least about two-fold stronger than the binding affinity of the parent antibody for the antigen.

[0131] The antibody may be a linear antibody. Procedures for generating linear antibodies are known in the art and are described in Zapata et al. (1995) Protein Eng. 8(10):1057-1062. Briefly, these antibodies comprise a pair of tandem Fd segments (V H -C H1 -V H -C H1 ). Linear antibodies may be bispecific or monospecific antibodies.

[0132] Antibodies may be recovered and purified from recombinant cell cultures by known methods, including, but not limited to, Protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") may also be used for purification.

[0133] It may be useful to detectably or therapeutically label antibodies. Methods for conjugating antibodies to these agents are known in the art. For illustrative purposes only, antibodies may be labeled with detectable moieties such as radioactive atoms, chromophores, fluorophores, and the like. Such labeled antibodies may be used for diagnostic techniques either in vivo or in an isolated test sample. Antibodies may also be conjugated to pharmaceutical agents or toxins, such as, for example, chemotherapeutic agents. These may be linked to cytokines, ligands, or other antibodies. Agents suitable for binding to antibodies to achieve antitumor effects include cytokines such as interleukin 2 (IL-2) and tumor necrosis factor (TNF); photosensitizers for use in photodynamic therapy, including aluminum (III) phthalocyanine tetrasulfonate, hematoporphyrin, and phthalocyanine; iodine-131 ( 131 I), Yttrium-90( 90 Y), Bismuth-212( 212 Bi), Bismuth-213( 213 Bi), Technetium-99m ( 99m Tc), rhenium-186( 186 Re) and rhenium-188( 188antibiotics such as doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin, neocarzinostatin and carboplatin; bacterial, plant and other toxins such as diphtheria toxin, Pseudomonas aeruginosa exotoxin A, Staphylococcus aeruginosa enterotoxin A, abrin-A toxin, ricin A (deglycosylated ricin A and native ricin A), TGF-alpha toxin, cytotoxin from the Chinese cobra (naja naja atra) and gelonin (a plant toxin); restrictocin (a ribosome-inactivating protein produced by Aspergillus restrictus), saporin (a ribosome-inactivating protein produced by Saponaria officinalis), and cytotoxin (a cytotoxin produced by Aspergillus restrictus). officinalis) and RNase; tyrosine kinase inhibitors; ly207702 (a defluorinated purine nucleoside); liposomes containing anticystic agents (e.g., antisense oligonucleotides, plasmids encoding toxins, methotrexate, etc.); and other antibodies or antibody fragments such as F(ab).

[0134] Antibodies may be sequenced and replicated by recombinant or synthetic means. They may also be further sequenced down to the linear sequence of the nucleotides that encode them. Thus, the present invention provides these polynucleotides, alone or in combination with the above-mentioned carriers, vectors or host cells that encode the sequences of the antibodies of the present invention.

[0135] Antibody production through the use of hybridoma technology, selected lymphocyte antibody assay (SLAM), transgenic animals and recombinant antibody libraries are described in more detail below.

[0136] (1) Anti-RGMc monoclonal antibody produced using hybridoma technology As mentioned above, monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant and phage display techniques or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques known in the art, including those taught in, for example, Harlow et al., Antibodies: A Laboratory Manual, 2nd ed., (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988); Hammerling et al., In Monoclonal Antibodies and T-Cell Hybridomas, (Elsevier, NY, 1981). It should also be noted that the term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma techniques. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic or phage clone, and not the method by which it is produced.

[0137] In one embodiment, a method is provided for producing monoclonal antibodies and antibodies produced by a method comprising culturing hybridoma cells secreting the antibodies, preferably hybridomas are generated by fusing splenocytes isolated from an animal, e.g., rat or mouse, immunized with RGMc with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones secreting antibodies capable of binding to RGMc (or fragments or variants thereof). Briefly, rats can be immunized with RGMc antigens (see, e.g., Examples below). In a preferred embodiment, the RGMc antigens are administered with adjuvants to stimulate the immune response. Such adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide) or ISCOM (immunostimulating complex). Such adjuvants can protect the polypeptide from rapid dispersal by sequencing the polypeptide in local deposits or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. Preferably, if a polypeptide is being administered, the immunization schedule will involve two or more administrations of the polypeptide, spread out over several weeks, although a single administration of the polypeptide may also be used.

[0138] After immunization of an animal with an RGMc antigen, antibodies and / or antibody-producing cells can be obtained from the animal. Serum containing anti-RGMc antibodies can be obtained from the animal by bleeding or sacrificing the animal. The serum can be used as obtained from the animal, an immunoglobulin fraction can be obtained from the serum, or the anti-RGMc antibodies can be purified from the serum. The serum or immunoglobulins obtained in this manner are polyclonal and therefore have a heterogeneous sequence nature.

[0139] Once an immune response is detected, for example, antibody specificity against the antigen RGMc is detected in rat serum, the rat spleen is collected and splenocytes are isolated.The splenocytes are then fused with any suitable myeloma cells, for example cells from cell line SP20 available from American Type Culture Collection (ATCC, Manassas, Va., US), by well-known techniques.Hybridomas are selected and cloned by limiting dilution.The hybridoma clones are then assayed by methods known in the art for cells secreting antibodies that can bind to RGMc (or its fragments or variants).Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing rats with positive hybridoma clones.

[0140] In another embodiment, antibody-producing immortalized hybridomas may be prepared from immunized animals. After immunization, the animals are sacrificed and splenic B cells are fused with immortalized myeloma cells as is well known in the art. See, for example, Harlow and Lane, supra. In a preferred embodiment, the myeloma cells do not secrete immunoglobulin polypeptides (non-secretory cell lines). After fusion and antibiotic selection, the hybridomas are screened using RGMc or a fragment or variant thereof or cells expressing RGMc (or a fragment or variant thereof). In a preferred embodiment, initial screening is performed using an enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA), preferably an ELISA. An example of ELISA screening is provided in PCT Publication No. WO00 / 37504.

[0141] Anti-RGMc antibody producing hybridomas are selected, cloned and further screened for desired characteristics, including strong hybridoma growth, high antibody production and desired antibody characteristics as further described below. Hybridomas may be cultured and expanded in vivo in syngeneic animals, animals lacking an immune system, such as nude mice, or in cell culture in vitro. Methods for selecting, cloning and expanding hybridomas are well known to those skilled in the art.

[0142] In a preferred embodiment, the hybridoma is a rat hybridoma as described herein. In another embodiment, the hybridoma is produced in a non-human, non-rat species, such as mouse, sheep, pig, goat, cow or horse. In yet another preferred embodiment, the hybridoma is a human hybridoma, in which a human non-secretory myeloma is fused with a human cell expressing an anti-RGMc antibody.

[0143] Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, Fab and F(ab') 2 The fragments are purified using papain (to produce two identical Fab fragments) or pepsin (to produce F(ab') 2 These can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as F(ab') of IgG molecules. 2 The fragment retains two antigen-binding sites of the larger ("parent") IgG molecule, including both light chains (containing the variable light and constant light chain regions), the CH1 domain of the heavy chain, and the disulfide-forming hinge region of the parent IgG molecule. Thus, the F(ab') 2 The fragments are capable of cross-linking antigenic molecules such as the parent IgG molecule.

[0144] (2) Anti-RGMc monoclonal antibody using SLAM Recombinant antibodies can be produced from single isolated lymphocytes using procedures referenced in the art such as the selected lymphocyte antibody method (SLAM) as described in U.S. Patent No. 5,627,052; PCT Publication No. WO92 / 02551; and Babcook et al., Proc. Natl. Acad. Sci. USA, 93; 7843-7848 (1996). In this method, a single cell secreting an antibody of interest, for example a lymphocyte from any one of the immunized animals described in Section IA1 (above), is screened using an antigen-specific hemolytic plaque assay in which the antigen RGMc, a subunit of RGMc or a fragment thereof is bound to sheep red blood cells using a linker such as biotin and used to identify a single cell secreting an antibody having specificity for RGMc. After identifying the antibody-secreting cells of interest, heavy and light chain variable region cDNAs are recovered from the cells by reverse transcriptase-PCR (RT-PCR), and these variable regions can then be expressed in mammalian host cells, such as COS or CHO cells, in association with appropriate immunoglobulin constant regions (e.g., human constant regions). Host cells transfected with the amplified immunoglobulin sequences derived from in vivo selected lymphocytes can then be subjected to further analysis and in vitro selection, for example, by panning the transfected cells to isolate cells expressing antibodies against RGMc. The amplified immunoglobulin sequences can be further manipulated in vitro, such as by in vitro affinity maturation methods. See, for example, PCT Publication No. WO97 / 29131 and PCT Publication No. WO00 / 56772.

[0145] (3) Anti-RGMc monoclonal antibody using transgenic animals Antibodies can also be produced by immunizing non-human animals that contain part or all of the human immunoglobulin locus with RGMc antigens.In one embodiment, the non-human animal is a XENOMOUSE® transgenic mouse, which is an engineered mouse strain that contains large fragments of the human immunoglobulin locus and is deficient in mouse antibody production.See, for example, Green et al., Nature Genetics, 7:13-21 (1994) and U.S. Patent Nos. 5,916,771; 5,939,598; 5,985,615; 5,998,209; 6,075,181; 6,091,001; 6,114,598; and 6,130,364. See also PCT Publication Nos. WO91 / 10741; WO94 / 02602; WO96 / 34096; WO96 / 33735; WO98 / 16654; WO98 / 24893; WO98 / 50433; WO99 / 45031; WO99 / 53049; WO00 / 09560; and WO00 / 37504. XENOMOUSE® transgenic mice produce an adult-like human repertoire of fully human antibodies and generate antigen-specific human monoclonal antibodies. XENOMOUSE® transgenic mice contain approximately 80% human antibody repertoires through the introduction of megabase pair sized germline conformation YAC fragments of human heavy chain loci and x light chain loci. See Mendez et al., Nature Genetics, 15:146-156 (1997); Green and Jakobovits, J. Exp. Med., 188:483-495 (1998), the disclosures of which are incorporated herein by reference.

[0146] (4) Anti-RGMc monoclonal antibody using a recombinant antibody library In vitro methods may also be used to generate antibodies, and antibody libraries screened to identify antibodies with the desired RGMc binding specificity. Such methods of screening recombinant antibody libraries are well known in the art and are described, for example, in U.S. Patent No. 5,223,409 (Ladner et al.); PCT Publication No. WO 92 / 18619 (Kang et al.); PCT Publication No. WO 91 / 17271 (Dower et al.); PCT Publication No. WO 92 / 20791 (Winter et al.); PCT Publication No. WO 92 / 15679 (Markland et al.); PCT Publication No. WO 93 / 01288 (Breitling et al.); PCT Publication No. WO 93 / 01288 (Breitling et al.); PCT Publication No. WO 93 / 01289 ... 92 / 01047 (McCafferty et al.); PCT Publication No. WO92 / 09690 (Garrard et al.); Fuchs et al., Bio / Technology, 9:1369-1372 (1991); Hay et al., Hum. Antibod. Hybridomas, 3:81-85 (1992); Huse et al., Science, 246:1275-1281 (1989); McCafferty et al., Nature, 348:552-554 (1990); Griffiths et al., EMBO J., 12:725-734 (1993); Hawkins et al., J. Mol. Biol., 226:889-896 (1992); Clackson et al., Nature, 352:624-628 (1991); Gram et al., Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992); Garrard et al., Bio / Technology, 9:1373-1377 (1991); Hoogenboom et al., Nucl. Acids. Res., 19:4133-4137 (1991); Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991); U.S. Patent Application Publication No. 2003 / 0186374; and PCT Publication No. WO 97 / 29131, the contents of each of which are incorporated herein by reference.

[0147] The recombinant antibody library may be derived from a subject immunized with RGMc or a portion of RGMc. Alternatively, the recombinant antibody library may be derived from a subject not immunized with RGMc, such as a human antibody library derived from a naive subject, i.e., a human subject not immunized with human RGMc. Antibodies are selected by screening the recombinant antibody library with peptides comprising human RGMs to select those antibodies that recognize RGMc. Methods for performing such screening and selection are well known in the art, as described in the references in the previous paragraph. Specific K off To select antibodies with specific binding affinity to RGMc, such as antibodies that dissociate from human RGMc with a desired K off may be used to select antibodies with specific IC 50 To select antibodies with specific neutralizing activity for hRGMc, such as an antibody having:

[0148] In one aspect, the isolated antibody or antigen-binding portion thereof binds to human RGMc. Preferably, the antibody is a neutralizing antibody. In various embodiments, the antibody is a recombinant antibody or a monoclonal antibody.

[0149] For example, antibodies may also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying the polynucleotide sequences encoding them. Such phage may be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing antigen-binding domains that bind to the antigen of interest may be antigen-selected or identified, for example, using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage containing fd and M13 binding domains expressed from phage with Fab, Fv or disulfide-stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to generate antibodies include those described in Brinkmann et al., J. Immunol. Methods, 182:41-50 (1995); Ames et al., J. Immunol. Methods, 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol., 24:952-958 (1994); Persic et al., Gene, 187:9-18 (1997); Burton et al., Advances in Immunology, 57:191-280 (1994); PCT Publication Nos. WO92 / 01047; WO90 / 02809; WO91 / 10737; WO92 / 01047; WO92 / 18619; WO93 / 11236; WO95 / 15982; WO95 / 20401; and U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,4 84; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743; and 5,969,108.

[0150] As described in the above references, after phage selection, the antibody coding region from the phage is isolated and used to generate whole antibodies, including human antibodies or any other desired antigen-binding fragment, which may be expressed in any desired host, including, for example, mammalian cells, insect cells, plant cells, yeast and bacteria, as described in detail below. For example, Fab, Fab' and F(ab') fragments. 2 Techniques for recombinantly producing fragments may also be utilized using methods known in the art, such as those disclosed in PCT Publication No. WO92 / 22324; Mullinax et al., BioTechniques, 12(6):864-869 (1992); Sawai et al., Am. J. Reprod. Immunol., 34:26-34 (1995); and Better et al., Science, 240:1041-1043 (1988). Examples of techniques that can be used to produce single chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203:46-88 (1991); Shu et al., Proc. Natl. Acad. Sci. USA, 90:7995-7999 (1993); and Skerra et al., Science, 240:1038-1041 (1988).

[0151] As an alternative to screening recombinant antibody libraries by phage display, other methods known in the art for screening large combinatorial libraries may be applied to identify antibodies. One type of alternative expression system is one in which recombinant antibody libraries are expressed as RNA-protein fusions, as described in PCT Publication No. WO98 / 31700 (Szostak and Roberts) and Roberts and Szostak, Proc. Natl. Acad. Sci. USA, 94:12297-12302 (1997). In this system, covalent fusions are made between mRNAs and the peptides or proteins they code for by in vitro translation of synthetic mRNAs that carry puromycin, a peptidyl acceptor antibiotic, at their 3' ends. Thus, specific mRNAs may be enriched from a complex mixture of mRNAs (e.g., combinatorial libraries) based on the properties of the encoded peptides or proteins, for example, antibodies or fragments thereof, such as the binding of the antibodies or portions thereof to bispecific antigens. Nucleic acid sequences encoding antibodies or portions thereof recovered from screening of such libraries may be expressed by recombinant means as described above (e.g., in mammalian host cells) and may be subjected to further affinity maturation, either by additional rounds of screening of mRNA-peptide fusions in which mutations have been introduced into the original selected sequence, or by other methods for in vitro affinity maturation of recombinant antibodies as described above, a preferred example of which is the PRO fusion display technique.

[0152] In another approach, antibodies can also be produced using yeast display methods known in the art. In yeast display methods, genetic methods are used to constrain antibody domains to yeast cell walls and display them on the surface of the yeast. In particular, such yeast can be utilized to display antigen-binding domains expressed from repertoire or combinatorial antibody libraries (e.g., human or mouse). Examples of yeast display methods that can be used to generate antibodies include those disclosed in U.S. Patent No. 6,699,658 (Wittrup et al.), which is incorporated herein by reference.

[0153] (5) Synthetic products Once sequenced, polypeptides such as monoclonal antibodies (or fragments thereof) that specifically bind to RGMc can be synthesized using methods known in the art, such as, for example, exclusive solid-phase synthesis, partial solid-phase synthesis, fragment condensation, and classical solution synthesis. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149 (1963). On solid phase, synthesis typically begins at the C-terminal end of the peptide using an alpha-amino protected resin. Suitable starting materials may be prepared, for example, by attaching the required alpha-amino acid to a chloromethylated resin, a hydroxymethyl resin, or a benzhydrylamine resin. One such chloromethylated resin is sold under the trade name BIO-BEADS SX-1 by Bio Rad Laboratories (Richmond, CA), and the preparation of hydroxymethyl resin is described in Bodonszky et al., Chem. Ind. (London) 38:1597 (1966). Benzhydrylamine (BHA) resin is described in Pietta and Marshall, Chem. Comm. 650 (1970) and is commercially available in the hydrochloride form from Beckman Instruments, Inc. (Palo Alto, Calif.) Automated peptide synthesis is commercially available, as are custom peptide production services.

[0154] Thus, polypeptides may be prepared, for example, by coupling an alpha-amino protected amino acid to a chloromethylated resin using cesium bicarbonate catalysis according to the method described in Gisin, Helv. Chim. Acta. 56:1467 (1973). After initial coupling, the alpha-amino protecting group is removed with a selection of reagents including trifluoroacetic acid (TFA) or hydrochloric acid (HCl) in an organic solvent at room temperature.

[0155] Suitable alpha-amino protecting groups include those known to be useful in the field of stepwise synthesis of peptides. Examples of alpha-amino protecting groups are acrylic type protecting groups (e.g., formyl, trifluoroacetyl and acetyl), aromatic urethane type protecting groups (e.g., benzyloxycarbonyl (Cbz) and substituted Cbz), aliphatic urethane protecting groups (e.g., t-butyloxycarbonyl (Boc), isopropyloxycarbonyl and cyclohexyloxycarbonyl) and alkyl type protecting groups (e.g., benzyl and triphenylmethyl). Boc and Fmoc are preferred protecting groups. The side chain protecting groups remain intact during coupling and do not separate during deprotection of the amino terminal protecting group or during coupling. The side chain protecting groups must be removable upon completion of the synthesis of the final peptide and under reaction conditions that do not alter the target peptide.

[0156] After removal of the alpha-amino protecting group, the remaining protected amino acids are coupled stepwise in the desired order, typically using an excess of each protected amino acid in solution with a suitable carboxyl group activator, such as dicyclohexylcarbodiimide (DCC) in a mixture of methylene chloride and dimethylformamide (DMF).

[0157] After the desired amino acid sequence is completed, the desired peptide is separated from the resin support by treatment with a reagent such as TFA or hydrogen fluoride (HF), which not only cleaves the peptide from the resin, but also cleaves any remaining side-chain protecting groups. If a chloromethylated resin is used, HF treatment results in the formation of the free peptide acid. If a benzhydrylamine resin is used, HF treatment directly results in the free peptide amide. Alternatively, if a chloromethylated resin is utilized, the side-chain protected peptide can be separated by treatment of the peptide resin with ammonia to obtain the desired side-chain protected amide or by treatment of the peptide resin with an alkylamine to obtain the side-chain protected alkylamide or dialkylamide. The side-chain protection can then be removed in the usual manner by treatment with hydrogen fluoride to obtain the free amide, alkylamide, or dialkylamide.

[0158] These and other solid phase peptide synthesis procedures are well known in the art. Such procedures are also described in Stewart and Young, Solid Phase Peptide Syntheses (2nd ed., Pierce Chemical Company, 1984).

[0159] 3. Pharmaceutical Compositions The antibody can be incorporated into a pharmaceutical composition suitable for administration to a subject (e.g., a patient, which may be human or non-human). Typically, the pharmaceutical composition comprises an antibody and a pharma- ceutical acceptable carrier. As used herein, "pharma- ceutical acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic absorption delaying agents, and the like that are physiologically compatible. Examples of pharma- ceutical acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it is preferred to include an isotonic agent in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, or potassium chloride. The pharma- ceutical acceptable carrier may further comprise minimal amounts of auxiliary substances, such as wetting emulsifiers, preservatives, or buffers, which enhance the life or effectiveness of the antibody.

[0160] In a further embodiment, the pharmaceutical composition comprises at least one additional therapeutic agent for treating, preventing, modulating or alleviating the disorders described herein. The additional therapeutic agent can be erythropoietin or other erythropoietin-stimulating agent (ESA). The additional therapeutic agent can be one or more other antibodies that activate the EPO receptor, such as bispecific or bivariable antibodies, and / or one or more other antibodies that bind to IL-6, BMP-2, BMP-4 and / or BMP-6. Other therapeutic agents, such as hepcidin-reducing compounds, can be used. Examples of hepcidin-reducing compounds include spiegelmer NOX-H94 and / or dorsomorphin.

[0161] Various delivery systems are known and can be used to administer one or more antibodies or a combination of one or more antibodies and a prophylactic or therapeutic agent useful for treating or ameliorating a disorder or one or more symptoms thereof, for example, encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or antibody fragment, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), constructs of nucleic acids as part of retroviruses or other vectors, etc. Methods of administering prophylactic or therapeutic agents include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural administration, intratumoral administration and mucosal administration (e.g., intranasal and oral routes). Additionally, pulmonary administration can be used, for example, by use of an inhalation device or nebulizer and formulation with an aerosolizing agent. See, for example, U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540 and 4,880,078; and PCT Publication Nos. WO92 / 19244, WO97 / 32572, WO97 / 44013, WO98 / 31346 and WO99 / 66903, each of which is incorporated herein by reference in its entirety. In one embodiment, the antibody, combination therapy or composition is administered using AlkermesAIR® pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.). In certain embodiments, the prophylactic or therapeutic agent is administered intramuscularly, intravenously, intratumorally, orally, intranasally, pulmonary or subcutaneously. The prophylactic or therapeutic agent may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucosal subcutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. Administration may be systemic or local.

[0162] In certain embodiments, it may be desirable to administer the antibody locally to the site in need of treatment; this may be accomplished, for example, but not limited to, by local infusion, by injection, or by means of an implant, which is a porous or non-porous material, including membranes and matrices, such as sialastic membranes, polymers, fibrous matrices (e.g., Tissuel®), or collagen matrices. In one embodiment, an effective amount of one or more antibodies is administered locally to the affected site of a subject to prevent, treat, manage, and / or alleviate a disorder or a symptom thereof. In another embodiment, an effective amount of one or more antibodies is administered locally to the affected site in combination with an effective amount of one or more therapeutic agents (e.g., one or more prophylactic or therapeutic agents) other than the antibodies of the present invention to prevent, treat, manage, and / or alleviate a disorder or one or more symptoms thereof.

[0163] In another embodiment, the antibody can be delivered in a controlled or sustained release system. In one embodiment, a pump can be used to achieve controlled or sustained release (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of therapeutic agents (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas (1983) J. Macromol. Sci. Rev. Macromol. Chem. 23:61; Levy et al. (1985) Science 228:190; During et al., (1989) Ann. Neurol. 25:351; Howard et al., (1989) J. Neurosurg. 71:105); U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,597; U.S. Patent No. 5,912,015; U.S. Patent No. 5,989,463; U.S. Patent No. 5,128,326; PCT Publication No. WO 99 / 15154; and PCT Publication No. WO 99 / 20253. Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters.In certain embodiments, the polymers used in sustained release formulations are inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In yet another embodiment, controlled or sustained release systems can be placed near prophylactic or therapeutic targets, requiring only a fraction of the systemic dosage (see, e.g., Goodson, Medical Applications of Controlled Release, supra, pp. 2:115-138 (1984)).

[0164] Controlled release systems are discussed in the review by Langer (1990, Science 249:1527-1533). Any technique known to those of ordinary skill in the art can be used to create sustained release formulations containing one or more antibodies. See, e.g., U.S. Pat. No. 4,526,938; PCT Publication No. WO 91 / 05548; PCT Publication No. WO 96 / 20698; Ning et al., 1996, "Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel," Radiotherapy & Oncology 39:179-189; Song et al., 1995, "Antibody Mediated Lung Targeting of Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek et al., 1997, "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application," Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854; and Lam et al., 1997, "Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery," Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which is incorporated herein by reference in its entirety.

[0165] In certain embodiments where the composition is a nucleic acid encoding an antibody, the nucleic acid can be constructed as part of a suitable nucleic acid expression vector and administered in vivo to promote expression of the antibody it encodes, for example, by using a retroviral vector (U.S. Pat. No. 4,980,286), or by direct injection, or by using particle bombardment (e.g., a gene gun; Biolistic, Dupont), such that the nucleic acid is intracellular, or by coating with lipids or cell surface receptors or transfection agents, or by administering the nucleic acid linked to a homeobox-like peptide known to enter the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868). Alternatively, the nucleic acid can be introduced intracellularly and incorporated into the host cell DNA for expression by homologous recombination.

[0166] The pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation), transdermal (e.g., topical), transmucosal and rectal administration. In certain embodiments, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic, e.g., lignocaine, to ease pain at the site of injection.

[0167] When the composition is to be administered topically, it can be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, liquid, emulsion, or other forms known to those skilled in the art.See, for example, Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th Edition, Mack Pub.Co., Easton, Pa. (1995).For non-sprayable topical dosage forms, a viscous to semi-solid or solid form that contains a carrier or one or more excipients compatible with topical application and has a dynamic viscosity coefficient greater than that of water is typically used.Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, etc., which are sterilized as desired or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) to affect various properties, such as osmotic pressure. Other suitable topical dosage forms include, for example, sprayable aerosol preparations, in which the active ingredient combined with solid or liquid inert carrier is packaged in a mixture with a pressurized volatile substance (e.g., gaseous propellant such as Freon) or in a squeeze bottle.If desired, moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms.Examples of such additional components are well known in the art.

[0168] When the method includes intranasal administration of the composition, the composition can be formulated in aerosol form, spray, mist, or in the form of nasal drops.In particular, the prophylactic or therapeutic agent for use according to the present invention can be conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer with a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas).In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount.Capsules and cartridges (e.g., composed of gelatin) for use in an inhaler or insufflator can be formulated, containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0169] When the method includes oral administration, the composition can be orally formulated in the form of tablet, capsule, cachet, gel cap, liquid, suspension, etc. Tablet or capsule can be prepared by conventional means with pharma- ceutical acceptable excipients such as binder (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); filler (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricant (e.g., magnesium stearate, talc or silica); disintegrant (e.g., potato starch or sodium starch glycolate); or wetting agent (e.g., sodium lauryl sulfate). Tablet can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, but are not limited to, solution, syrup or suspension, or can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharma- ceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl p-hydroxybenzoate or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow, controlled, or sustained release of the prophylactic or therapeutic agent.

[0170] Method can include pulmonary administration, for example, by using inhalation device or nebulizer, by using the composition formulated with aerosolizing agent.See, for example, U.S. Patent No. 6,019,968, U.S. Patent No. 5,985,320, U.S. Patent No. 5,985,309, U.S. Patent No. 5,934,272, U.S. Patent No. 5,874,064, U.S. Patent No. 5,855,913, U.S. Patent No. 5,290,540 and U.S. Patent No. 4,880,078; and PCT Publication No. WO92 / 19244, WO97 / 32572, WO97 / 44013, WO98 / 31346 and WO99 / 66903.Each of these is incorporated herein by reference in its entirety. In a specific embodiment, antibodies, combination therapies and / or compositions are administered using Alkermes AIR® pulmonary drug delivery technology (Alkermes, Inc. Cambridge, Mass.).

[0171] The method may include administering a composition formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Injectable preparations may be provided in unit dosage form (e.g., in ampoules or multi-dose containers) with added preservatives. The composition may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use. The method may further include administering a composition formulated as a depot preparation. Such long-acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the composition may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).

[0172] The methods include administration of compositions formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations such as those derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0173] In general, the components of the composition are supplied separately or mixed together in unit dosage form, for example as lyophilized dry powder or water-free concentrate in a sealed container, such as a syringe or sachet indicating the amount of active agent.When the mode of administration is injection, the composition can be dispensed using an injection bottle containing sterile water or saline of pharmaceutical grade.When the mode of administration is injection, a syringe of sterile water or saline for injection can be provided so that the components can be mixed before administration.

[0174] In particular, the antibody or pharmaceutical composition may be packaged in a sealed container, such as a syringe or sachet indicating the amount of antibody. In one embodiment, the antibody or pharmaceutical composition is supplied as a lyophilized dry sterile powder or water-free concentrate in a sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. In one embodiment, the antibody or pharmaceutical composition is supplied as a lyophilized dry sterile powder in a sealed container in a unit dosage of at least 5 mg, e.g., at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg. The lyophilized antibody or pharmaceutical composition should be stored in its original container at between 2° C. and 8° C., and the antibody or pharmaceutical composition should be administered within one week, e.g., within 5 days, within 72 hours, within 48 hours, within 24 hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours, or within 1 hour after reconstitution. In another embodiment, one or more of the antibodies or pharmaceutical compositions are provided in liquid form in a sealed container indicating the amount and concentration of the antibody. In a further embodiment, the liquid form of the administered composition is provided in a sealed container at at least 0.25 mg / ml, e.g., at least 0.5 mg / ml, at least 1 mg / ml, at least 2.5 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / kg, at least 25 mg / ml, at least 50 mg / ml, at least 75 mg / ml or at least 100 mg / ml. The liquid form should be stored in its original container at between 2°C and 8°C.

[0175] The antibody can be incorporated into a pharmaceutical composition suitable for parenteral administration. In one embodiment, the antibody is prepared as an injectable solution containing 0.1-250 mg / mL of antibody. The injectable solution can consist of liquid or lyophilized dosage forms in flint or amber containers, syringes or pre-filled syringes. The buffer can be L-histidine (1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0 (optimally pH 6.0). Other suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. Sodium chloride can be used to modify the toxicity of the solution at concentrations of 0-300 mM (optimally 150 mM for liquid dosage forms). For lyophilized dosage forms, a cryoprotectant can be included, primarily 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. For lyophilized dosage forms, bulking agents may be included, primarily mannitol at 1-10% (optimally 2-4%). ​​In both liquid and lyophilized dosage forms, stabilizers may be used, primarily L-methionine at 1-50 mM (optimally 5-10 mM). Other suitable bulking agents include glycine and arginine, and may be included as 0-0.05% polysorbate-80 (optimally 0.005-0.01%). Additional surfactants include, but are not limited to, polysorbate 20 and BRIJ surfactants. Pharmaceutical compositions containing antibodies prepared as injectable solutions for parenteral administration may further include agents useful as adjuvants, such as those used to increase the absorption or dispersion of antibodies. A particularly useful adjuvant is hyaluronidase, such as Hylenex® (recombinant human hyaluronidase). The addition of hyaluronidase to injectable solutions improves human bioavailability following parenteral administration, particularly subcutaneous administration.Larger injection site volumes (i.e., greater than 1 mL) are also possible with less pain and discomfort and minimal incidence of injection site reactions (see PCT Publication WO04 / 078140 and U.S. Patent Application No. 2006104968, which are incorporated herein by reference).

[0176] The compositions may be in various forms. These include liquid, semi-solid and solid dosage forms, such as, for example, liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The form selected depends on the intended mode of administration and therapeutic application. The compositions may be in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies. In one embodiment, the antibodies are administered by intravenous infusion or injection. In another embodiment, the antibodies are administered by intramuscular or subcutaneous injection.

[0177] Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., binding protein, e.g., antibody) in the required amount in a suitable solvent with one or a combination of ingredients listed above, followed by filtration sterilization as required. In general, dispersions are prepared by incorporating the active compound in a sterile vehicle containing a basic dispersion medium and the required other ingredients obtained from those listed above. In the case of freeze-dried sterile powders for the preparation of sterile injectable solutions, the method of preparation includes vacuum drying and powder drying, which gives a powder of the active ingredient plus any additional desired ingredients from the previously sterile-filtered solution. The proper fluidity of the solution can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.

[0178] Antibodies can be administered by various methods known in the art. In many therapeutic applications, the route / mode of administration may be subcutaneous injection, intravenous injection or infusion. As understood by those skilled in the art, the route and / or mode of administration varies depending on the desired results. In certain embodiments, the active compound may be prepared with a carrier that protects the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches and microencapsulated delivery systems. Biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. Many methods for preparing such formulations are patented or generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, edited by JR Robinson, Marcel Dekker, Inc., New York, 1978.

[0179] In certain embodiments, the antibody may be administered orally, for example, with an inert diluent or an assimilable edible carrier. The antibody (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the patient's diet. For oral therapeutic administration, the antibody may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. In order to administer the antibody by other than parenteral administration, it may be necessary to coat the antibody with, or co-administer the antibody with, a material to prevent its inactivation.

[0180] Supplementary active compounds can also be incorporated into the composition. In certain embodiments, the antibody is co-formulated and / or co-administered with one or more additional therapeutic agents that are useful for treating the disorders or diseases described herein. For example, an anti-RGM globulomer antibody can be co-formulated and / or co-administered with one or more additional antibodies that bind to other targets (e.g., antibodies that bind to other soluble antigens or antibodies that bind to cell surface molecules). Furthermore, one or more antibodies can be used in combination with two or more of the aforementioned therapeutic agents. Such combination therapy can advantageously use lower dosages of the therapeutic agents administered, thus avoiding possible toxicities or complications associated with various monotherapies.

[0181] In some embodiments, antibody is linked to a half-life extension vehicle known in the art.Such vehicles include, but are not limited to, Fc domain, polyethylene glycol and dextran.Such vehicles are described, for example, in US patent application Ser. No. 09 / 428,082 and PCT application publication WO99 / 25044, which are incorporated herein by reference for this purpose.

[0182] In certain embodiments, a nucleic acid sequence comprising a nucleotide sequence encoding an antibody is administered to treat, prevent, manage, or alleviate a disorder or one or more symptoms thereof by gene therapy. Gene therapy refers to a therapy performed by administration of an expressed or expressible nucleic acid to a subject. In this embodiment, the nucleic acid produces the encoded antibody that mediates a prophylactic or therapeutic effect.

[0183] Any method for gene therapy available in the art can be used in the present invention.For general reviews of gene therapy methods, see Goldspiel et al., Clinical Pharmacy 12:488-505, (1993); Wu and Wu, Biotherapy 3:87-95, (1991); Tolstoshev, Ann.Rev.Pharmacol.Toxicol.32:573-596, (1993); Mulligan, Science 260:926-932, (1993); and Morgan and Anderson, Ann.Rev.Biochem.62:191-217 (1993); May, 1993, TIBTECH 11(5):155-215. Methods generally known in the art of recombinant DNA technology that can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990). A detailed description of various methods of gene therapy is disclosed in US20050042664A1, which is incorporated herein by reference.

[0184] It should be further understood that the combination is a combination that is useful for their intended purpose. The above-mentioned agents are for illustrative purposes and are not intended to be limiting. The combination can include an antibody and at least one additional agent selected from the following list. The combination can also include more than one additional agent, for example, two or three additional agents, so long as the composition formed in the combination can perform its intended function.

[0185] The pharmaceutical composition may include a "therapeutically effective amount" or a "prophylactically effective amount" of the antibody. A "therapeutically effective amount" refers to an amount effective at a dosage and for a period of time necessary to achieve a desired therapeutic result. The therapeutically effective amount of an antibody can be determined by one skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the toxic or detrimental effects of the antibody, if any, are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective at a dosage and for a period of time necessary to achieve a desired prophylactic result. Typically, the prophylactically effective amount is less than the therapeutically effective amount, since prophylactic dosing is used in subjects before or at an earlier stage of disease.

[0186] Dosage regimen can be adjusted to give the optimum desired response (e.g., therapeutic or prophylactic response). For example, a single bolus can be administered, multiple divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. As used herein, dosage unit form refers to physically discrete units suitable as a unitary dosage for the mammalian subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect together with the required pharmaceutical carrier. The specifications for dosage unit form are dictated by and directly depend on (a) the unique characteristics of the active compound and the specific therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the technical field of compounding such active compound for the treatment of hypersensitivity in an individual.

[0187] A typical non-limiting range for a therapeutically or prophylactically effective amount of an antibody is a dose of 0.1 to 200 mg / kg, for example, 0.1 to 10 mg / kg. A therapeutically or prophylactically effective amount of an antibody may be 1 to 200 mg / kg, 10 to 200 mg / kg, 20 to 200 mg / kg, 50 to 200 mg / kg, 75 to 200 mg / kg, 100 to 200 mg / kg, 150 to 200 mg / kg, 50 to 100 mg / kg, 5 to 10 mg / kg, or 1 to 10 mg / kg. It should be noted that dosage values ​​may vary depending on the type and severity of the condition to be alleviated. Furthermore, the dose of the antibody may be determined by one skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual and the ability of the antibody to elicit a desired response in the individual. The dose is also an amount in which the therapeutically beneficial effects outweigh any toxic or detrimental effects of the antibody, if any. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual's needs and the professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges set forth herein are exemplary only and are not intended to be limiting to the scope or practice of the claimed compositions.

[0188] 4. Methods for Treating, Preventing, Regulating, or Attenuating Iron Metabolism In any subject, the subject may be evaluated for whether it has an iron metabolism-related disorder using routine techniques known in the art (for example, such evaluation may include one or more tests to determine hemoglobin level, red blood cell count, reticulocyte count, serum ferritin, serum iron, saturated serum transferrin, serum hepcidin, serum RGM, etc.). The subject may be evaluated for whether it has an iron-related disorder related to iron deficiency or iron overload, and this evaluation can therefore indicate an appropriate course of treatment, such as preventive therapy, maintenance therapy, or modulation therapy. For reference, hematologists can use the following reference numbers to indicate that a patient has normal levels of the corresponding parameters. See Table 2.

[0189] [Table 3]

[0190] Therefore, a method for treating, preventing, regulating or alleviating a disease of iron metabolism is provided herein. The antibody can be administered to a subject in need thereof. The antibody can be administered to a subject in a therapeutically effective amount, which can be easily determined by a person skilled in the art. The method for treating, preventing, regulating or alleviating a disease can be to increase or decrease the level of hepcidin protein in a cell or tissue, compared to the level of hepcidin in a normal control or calibrator. The method for treating, preventing, regulating or alleviating a disease can be to decrease the level of hepcidin protein in a cell or tissue, compared to the level of hepcidin in a normal control or calibrator.

[0191] A. Disorders of iron metabolism The disease or disorder of iron metabolism may be any disease or disorder in which iron homeostasis is seriously disturbed in a subject. This homeostasis depends on the proper regulation of adequate plasma iron level. Iron binds to transferrin, which is a vehicle for iron delivery to cells, and circulates in plasma. Plasma transferrin is normally saturated with iron by about 30%. Therefore, transferrin saturation must be maintained at an appropriate physiological level in response to various signals from pathways involved in iron consumption.

[0192] Hepcidin regulates whole body iron flux and regulates plasma iron levels by binding to ferroportin and inducing its degradation. As ferroportin is degraded, macrophages and duodenal enterocytes can no longer release iron into the blood, resulting in reduced iron transport to transferrin. Thus, genetic and acquired disorders that disrupt normal hepcidin production can cause iron deficiency (high hepcidin levels) or iron overload (hepcidin deficiency).

[0193] This disruption may result in iron deficiency, iron overload, or iron overload with anemia. This disruption may also result in anemia of chronic disease, where subjects with the disease show high levels of blood hepcidin. Subjects may suffer from fatigue, joint pain, bone or joint disease (osteoarthritis, osteoporosis), rheumatoid arthritis, inflammatory bowel disease, shortness of breath, arrhythmia, liver damage, diabetes, infertility, impotence, depression, mood or mental disorders, poor cognitive skills or neurodegenerative diseases, ACD, iron-refractory iron deficiency anemia, anemia of chronic kidney disease, resistance to erythropoiesis-stimulating agents, aplastic anemia, myelodysplastic syndromes, sideroblastic anemia, hypoplastic anemia, paroxysmal nocturnal hemoglobinuria, von Willebrand's disease, hemophilia, and the like. You may have or be at risk for a disease or disorder such as Hereditary Hemorrhagic Telangiectasia, Red Blood Cell Enzymopathies; Glucose-6-phosphate Dehydrogenase (G6PD) or Pyruvate Kinase Deficiency (PKD), Atransferrinemia or Hypotransferrinemia, Aceruloplasminemia or Hypoceruloplasminemia, CDAII (Congenital Dyserythroid Anemia) (also called HEMPAS (Hereditary Polymorphonuclear Erythroblastosis with Positive Acid Hemolysis)).

[0194] Inhibition of hepcidin in the liver is associated with increased expression of ferroportin in peritoneal macrophages and decreased intracellular iron and oxidative stress, and is associated with increased expression of the ABC transporters, macrophage cholesterol efflux proteins, ABCA1 and ABCG1. See Saeed et al., Arterioscler.Thromb.Vasc.Biol, 32 (February 2012), accepted November 5, 2011. Inhibition of hepcidin increases expression of ABCA1 and ABCG1, which can result in increased lipid efflux and decreased foam cell formation. Thus, the antibody can be administered to a subject in need thereof. The antibody can be administered to a subject in a therapeutically effective amount, where the antibody can limit the formation of foam cells and atherosclerosis, and / or can treat, prevent, modulate or reduce atherosclerosis. The antibody can decrease macrophage intracellular iron, which results in increased expression of ABC transporters and lipid efflux capacity.

[0195] The cyclic activity of hair follicles can be controlled by signal molecules that are normally expressed in the macroenvironment of skin.See, for example, US Patent Application No. 2011 / 0293526, the contents of which are incorporated herein by reference in their entirety.For example, BMP expression can be negatively correlated with hair growth.Antibody can be used to stimulate quiescent hair follicles to reactivate and grow again.Antibody can directly or indirectly disrupt the signal transduction of molecules that are normally expressed in the macroenvironment of skin.

[0196] (1) Iron deficiency Iron metabolism disorders may be those in which the body has too little iron.For example, a subject may be diagnosed as having iron deficiency if serum iron is found to be below 60μg / dl, below 55μg / dl, below 50μg / dl, below 45μg / dl or below 40μg / dl.A subject may be diagnosed as having iron deficiency if total iron binding capacity ("TIBC") is lower than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15% or 10%.A subject may be diagnosed as having iron deficiency if ferritin level is increased compared to subjects who are not iron deficient. A subject may be diagnosed as having iron deficiency if hemoglobin level is less than 15.5, 15, 14.5, 14, 13.5, 13, 12.5, 12, 11.5, 11, 10.5, 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5 or 6 g / dl. A transferrin saturation of less than 25%, less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8% or less than 7% may indicate iron deficiency. A subject may be diagnosed as having iron deficiency based on one or more of the factors described above.

[0197] Iron deficiency during critical periods of growth and development can result in premature birth, low birth weight, growth and developmental retardation, and retardation of normal infant activity and movement; iron deficiency can result in poor memory or cognitive ability (mental function) resulting in underperformance in school, work, the military, or recreation. Low IQ has been linked to iron deficiency occurring during critical periods of growth.

[0198] Iron deficiency anemia ("IDA") is a condition in which a subject has insufficient amounts of iron to meet the body's needs. IDA results from a reduced amount of red blood cells in the blood associated with a subject having too little iron. IDA can result from an iron-deficient diet or from blood loss. IDA is the most common form of anemia. Approximately 20% of women, 50% of pregnant women, and 3% of men are iron deficient.

[0199] Subjects with iron-refractory iron anemia ("IRIDA") suffer from microcytic anemia that is unresponsive to oral therapy and refractory, in part, to parenteral iron, due to inappropriately high hepcidin levels. IRIDA is caused by mutations in the matriptase-2 gene (TMPRSS6), which encodes a serine protease that negatively regulates hepcidin expression by cleaving membrane-bound RGMc.

[0200] (2) Iron overload Examples of disorders associated with iron overload include chronic fatigue, joint pain, abdominal pain, liver disease (cirrhosis, liver cancer), diabetes, irregular heart rhythm, heart attack, or heart failure, skin color changes (bronze, gray-gray-green), cessation of menstruation, loss of interest in sex, osteoarthritis, osteoporosis, hair loss, enlarged liver or spleen, impotence, infertility, hypogonadism, hypothyroidism, hypopituitarism, depression, adrenal insufficiency, early onset neurodegenerative disease, hyperglycemia, elevated liver enzymes, and elevated iron (serum iron, serum ferritin).For example, if serum iron is determined to be greater than 150 μg / dl, greater than 155 μg / dl, greater than 160 μg / dl, greater than 165 μg / dl, or greater than 170 μg / dl, a subject may be diagnosed with iron overload. A subject may be diagnosed with iron deficiency if the subject's total iron binding capacity ("TIBC") is greater than 50%, 55%, 60%, 65%, 70%, 75% or 80%. A subject may be diagnosed with iron deficiency if the subject has increased ferritin levels compared to subjects who are not iron deficient. A subject may be diagnosed with iron deficiency if the subject has a hemoglobin level greater than 18.5, 18, 17.5, 17, 16.5, 16, 15.5, 15, 14.5, 14, 13.5, 13, 12.5 or 12 g / dl. A transferrin saturation greater than 35%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65% or 70% may indicate iron overload. A subject can be diagnosed as having iron overload based on one or more of the factors described above.

[0201] Hemochromatosis (HH) is another disease caused by an excess amount of iron in the body (iron overload). Hereditary (genetic) hemochromatosis (HHC) is an inherited disorder of abnormal iron metabolism. Individuals with HHC absorb too much dietary iron. Once absorbed, the body has no efficient way to eliminate the iron overload. Over time, these excesses lead to a state of iron overload that is toxic to cells. Glands and organs loaded with excess iron, such as the liver, heart, pituitary gland, thyroid gland, pancreas, synovium (joints), and bone marrow, cannot function normally. Symptoms develop and the disease progresses.

[0202] There are several types of HHC. These include type I or classical (HHC); type II a, b or juvenile (JHC); type III or transferrin receptor mutations; and type IV or ferroportin mutations.

[0203] HHC is an autosomal recessive disease that can result in iron overload of the liver and other organs. Four genes are involved in hemochromatosis: HFE(C282Y), TfR2, hemojuvelin (HJV), and the HAMP gene (hepcidin). The recessive form of the disease is the result of inappropriately low hepcidin expression, whereby disease severity and age of onset can be correlated with the degree of hepcidin expression.

[0204] Dominant forms of hereditary hemochromatosis are caused by missense mutations in ferroportin, the cellular iron exporter and hepcidin receptor. For example, mutations that reduce the membrane localization or ability of ferroportin to export iron lead to macrophage iron overload or retention, resulting in normal to low plasma iron levels, and in some cases iron-restricted erythropoiesis.

[0205] Hemochromatosis-associated disorders with high plasma iron and hepatocellular iron accumulation may be caused by hepcidin-resistant ferroportin mutations, whereby hepcidin does not bind to ferroportin (C326S) or the internalization and degradation of ferroportin and subsequent hepcidin binding are impaired.

[0206] Hepcidin levels can also be inappropriately low in "iron-loaded anemia," whereby erythropoietic signals repress hepcidin transcription even when total body iron is high. β-thalassemia intermedia is one example of such an anemia, characterized by transfusion-independent iron overload and low to nonexistent hepcidin levels.

[0207] (3) Iron overload accompanied by anemia Iron overload with anemia (IOA), also called aceruloplasminemia, is a recessive disease and can be characterized by anemia, iron overload and neurodegeneration. The disorder is caused by mutations in the gene encoding the copper-containing ferroxidase ceruloplasmin. Patients suffering from aceruloplasminemia have low serum hepcidin levels and reduced ferroportin expression in the liver due to the absence of the stabilizing function of mutant ceruloplasmin on ferroportin.

[0208] Iron overload is considered the main cause of mortality and morbidity in anemias with poor erythropoiesis (e.g., β-thalassemia and congenital dyserythroplastic anemia). In these disorders, high levels of erythropoietin stimulate extensive but poor erythropoiesis. Severe iron overload, similar to juvenile hemochromatosis, can develop in subjects who rarely or never receive transfusions, indicating that dietary iron is excessively absorbed under these conditions. These non-transfused patients usually have low hepcidin levels despite high serum ferritin concentrations and high hepatic iron overload. It may be hypothesized that poor erythropoiesis results in mediators such as GDF-15 that may suppress hepatic hepcidin synthesis. See Ganz, T., Blood, 117:4425-33, 2011, Hepcidin and Iron Regulation: 10 Years Later.

[0209] IOA is often caused by the environment, whereby the subject has very high biological iron, which may be due to whole blood transfusion or blood cell injury that causes chronic hemolytic anemia (early turnover or destruction of red blood cells). This process may cause biological iron accumulation similar to that seen in hemochromatosis patients. Various environments (including high food consumption) may cause iron surplus to build up rapidly. Erythropoietin, hepcidin and / or growth differentiation factor-15 (GDF-15) levels may be used to distinguish subjects with IOA. In β-thalassemia, for example, subjects have dramatically elevated GDF-15 levels. A subject with β-thalassemia may have a GDF-15 level of more than 45,000pg / ml, more than 50,000pg / ml, more than 55,000pg / ml, more than 60,000pg / ml, more than 65,000pg / ml, more than 66,000pg / ml, or more than 70,000pg / ml. For example, a subject with β-thalassemia may have a mean level of GDF-15 at or near 66,000+ / -9,600pg / ml, compared to a level of 450+ / -50pg / ml or near in a healthy subject. There may be too few red blood cells in hemoglobin to sustain life, and whole blood transfusion may be required for the subject to survive. Examples of diseases associated with iron overload accompanied by anemia include sickle cell anemia, thalassemia, sideroblastic anemia and enzyme deficiencies.

[0210] b. Target The subject may be a mammal, which may be human or non-human. The subject may be an emergency patient undergoing chemotherapy, recovering from surgery, or may be at risk of or have an infection, cancer, an autoimmune disease or disorder, chronic organ disease and / or inflammation, and / or chronic rejection of an organ after solid organ transplantation. The infection may be acute or chronic. The infection may be viral, bacterial, parasitic, or fungal. The cancer may be any cancer, such as a hematological tumor or a solid tumor. The autoimmune disease may be any autoimmune disease, such as rheumatoid arthritis, systemic lupus erythematosus and connective tissue disease, vasculitis, sarcoidosis, and inflammatory bowel disease. The chronic organ disease may be a chronic kidney disease, in which the subject may or may not be undergoing dialysis. The viral infection may be a hepatitis B or C virus infection, or a human immunodeficiency virus infection. Any of the diseases and disorders may be the underlying cause of the ACD. The surgery may be intraoperative or postoperative. The surgery may be tumor surgery.

[0211] 5. Diagnostic Methods Provided herein is a method for determining whether a subject has an iron-related disorder. The level of membrane-bound RGMc or soluble RGMc can be measured in a sample from a subject and can be compared to the RGMc level in a control sample or a calibrator, such as a series of calibrators. The control sample can be from a normal tissue or body fluid (e.g., whole blood, serum, plasma, etc.). The altered level of RGMc compared to the control can indicate that the subject has an iron-related disorder. For example, a decreased level of membrane-bound RGMc compared to the level of membrane-bound RGMc in a normal control can indicate that the subject has an iron-related disorder associated with iron overload. Alternatively, an increased level of membrane-bound RGMc compared to the level of membrane-bound RGMc in a normal control can indicate that the subject has an iron-related disorder associated with iron deficiency. Furthermore, an increased level of soluble RGMc or a soluble fragment thereof can indicate that the subject has an iron-related disorder associated with iron overload. A decreased level of soluble RGMc or a soluble fragment thereof can indicate that the subject has an iron-related disorder associated with iron deficiency. Levels of RGMc (membrane-bound or soluble) can be measured using the antibodies described herein.

[0212] Also, the method for determining whether a subject has an iron-related disorder may include measuring the level of hepcidin in addition to measuring the level of membrane-bound or soluble RGMc in one or more samples obtained from the subject. In particular, in one embodiment, the level of membrane-bound or soluble RGMc in a sample from the subject may be measured and compared with the level of RGMc in a control sample or a calibrator, for example, a series of calibrators. The control sample for membrane-bound or soluble RGMc may be from normal tissue or body fluid (e.g., whole blood, serum, plasma, etc.). The method may also include measuring the level of hepcidin in the same sample and comparing the level of hepcidin in a control sample or a calibrator, for example, a series of calibrators. The control sample for hepcidin may be from normal tissue or body fluid (e.g., whole blood, serum, plasma, etc.). The altered level of RGMc compared with the level of RGMc in the control may indicate that the subject has an iron-related disorder. For example, a decreased level of membrane-bound RGMc compared to the level of membrane-bound RGMc in a normal control can indicate that the subject has an iron-related disorder associated with iron overload. Alternatively, an increased level of membrane-bound RGMc compared to the level of membrane-bound RGMc in a normal control can indicate that the subject has an iron-related disorder associated with iron deficiency. Furthermore, an increased level of soluble RGMc or a soluble fragment thereof can indicate that the subject has an iron-related disorder associated with iron overload. A decreased level of soluble RGMc or a soluble fragment thereof can indicate that the subject has an iron-related disorder associated with iron deficiency. The level of RGMc can be measured using the antibodies described herein. An altered level of hepcidin compared to the level of hepcidin in a normal control can indicate that the subject has an iron-related disorder. For example, a decreased level of hepcidin compared to the level of hepcidin in a normal control can indicate that the subject has an iron-related disorder associated with iron overload. Alternatively, an increased level of hepcidin, as compared to the level of hepcidin in a normal control, can indicate that the subject has an iron-related disorder associated with iron deficiency.The order of measuring the levels of membrane-bound RGMc or soluble RGMc and hepcidin is not important. They can be measured simultaneously or sequentially in any order. Moreover, the levels of membrane-bound RGMc or soluble RGMc and hepcidin can be measured in the same reaction vessel or in different reaction vessels. In another embodiment, the levels of membrane-bound RGMc or soluble RGMc and hepcidin do not have to be determined in the same sample from the subject. For example, the level of membrane-bound RGMc or soluble RGMc can be determined in a first sample obtained from the subject. The level of hepcidin can be determined in a second sample obtained from the subject. Alternatively, the level of hepcidin can be determined in a first sample obtained from the subject, and the level of membrane-bound RGMc or soluble RGMc can be determined in a second sample obtained from the subject. The first and second samples obtained from the patient can be obtained simultaneously or at different times. Levels of RGMc (membrane-bound or soluble) may be measured using the antibodies described herein.

[0213] The method may further comprise assaying the test sample for the presence, amount or concentration of hepcidin, where (i) the test sample assayed for hepcidin may be the same test sample assayed for RGMc, or (ii) the test sample assayed for hepcidin is a different test sample from the test sample assayed for RGMc, but the test sample source assayed for hepcidin and the test sample source assayed for RGMc are the same. The test sample, or multiple test samples, are assayed for RGMc and hepcidin simultaneously or sequentially in any order using the same type of method or different types of methods described herein and known in the art. Alternatively, this method can further comprise using the result of the assay of test sample for the presence, amount or concentration of hepcidin, where (i) the test sample that is assayed for hepcidin is the same test sample that is assayed for RGMc, or (ii) the test sample that is assayed for hepcidin is a different test sample from the test sample that is assayed for RGMc, but the source of the test sample that is assayed for hepcidin and the source of the test sample that is assayed for RGMc are the same.In this regard, the assay of the test sample for hepcidin, the result of which is used in the context of the above method, can be carried out at different time points, for example, several hours (for example, 12 hours), one day, two days, three days, one week, two weeks, three weeks or even one month, either before or after the time of assaying the test sample for RGMc, provided that the result is still considered to be representative and reliable.It may be preferable that the assay of the test sample for hepcidin allows the determination of the presence, amount or concentration of hepcidin-25. For example, a sample, such as a plasma sample, serum sample, or urine sample, can be assayed for hepcidin-25 using, for example, TOF-MS and an internal standard.

[0214] Hepcidin levels may be measured using hepcidin antibodies known in the art, such as those commercially available from ABCAM® (Cambridge, MA) and BACHEM® (Torrance, CA), e.g., hepcidin-25 antibodies. Hepcidin levels in a sample can be determined using a variety of formats (e.g., immunoassays) as described herein.

[0215] The level of hepcidin and the level of RGMc determined by any of the methods described herein may be compared to indicate the presence of iron-related disorder.For example, the ratio of membrane-bound RGMc and hepcidin in test sample that is different from the ratio of membrane-bound RGMc and hepcidin in normal control can indicate that the subject from which the test sample is derived has iron-related disorder.For example, the increased ratio of soluble RGMc and hepcidin in test sample compared to the ratio of soluble RGMc and hepcidin in normal control can indicate that the subject from which the test sample is derived has iron-related disorder, such as iron overload.The decreased ratio of membrane-bound RGMc and hepcidin in test sample compared to the ratio of soluble RGMc and hepcidin in normal control can indicate that the subject from which the test sample is derived has iron-related disorder, such as iron deficiency.

[0216] A sample The sample may be any tissue sample of the subject. The sample may contain a protein of interest.

[0217] Any cell type, tissue or body fluid can be utilized to obtain the sample. Such cell types, tissues and body fluids may include sections of tissue, such as biopsy and autopsy samples, frozen sections taken for histological purposes, blood (e.g., whole blood), plasma, serum, sputum, stool, tears, mucus, saliva, hair and skin. Cell types and tissues may also include lymph, ascetic fluid, gynecological fluid, urine, serum, plasma, peritoneal fluid, cerebrospinal fluid, fluids collected by vaginal rinse or fluids collected by vaginal lavage. Tissues or cell types may be provided by removing a sample of cells from an animal, but may also be achieved by using previously isolated cells (e.g., isolated by another human, at another time, and / or for another purpose). Archival tissues, such as tissues with treatment or prognosis history, may also be used. Protein purification may not be required.

[0218] Methods known in the art for collecting, handling and processing urine, blood, serum and plasma, and other bodily fluids are used in the practice of the present disclosure, for example, when using the antibodies provided herein as immunodiagnostic reagents and / or in RGMc immunoassay kits. The test sample may contain additional components in addition to the RGMc analyte of interest, such as antibodies, antigens, haptens, hormones, drugs, enzymes, receptors, proteins, peptides, polypeptides, oligonucleotides or polynucleotides. For example, the sample may be a whole blood sample obtained from a subject. It may be necessary or desirable for the test sample, particularly whole blood, to be treated prior to the immunoassays described herein, for example, with a pretreatment reagent. In cases where pretreatment is not required (e.g., most urine samples), pretreatment may optionally be performed for mere convenience (e.g., as part of a formulation on a commercial platform). The sample obtained from the subject or the sample after pretreatment may be used directly to modify the characteristics of the sample. Pretreatment may include extraction, concentration, inactivation of interfering components and / or addition of reagents.

[0219] The pretreatment reagent can be any reagent suitable for use with the assays, e.g., immunoassays and kits described herein. The pretreatment optionally includes (a) one or more solvents (e.g., methanol and ethylene glycol) and a salt, (b) one or more solvents, salts and detergents, (c) detergents, or (d) detergents and salts. Pretreatment reagents are known in the art and such pretreatments can be used, for example, as used in assays on the Abbott TDx, AxSYM® and ARCHITECT® analyzers (Abbott Laboratories, Abbott Park, Ill.), as described in the literature (see, e.g., Yatscoff et al., Abbott TDx Monoclonal Antibody Assay Evaluated for Measuring Cyclosporine in Whole Blood, Clin. Chem. 36:1969-1973 (1990) and Wallemacq et al., Evaluation of the New AxSYM Cyclosporine Assay: Comparison with TDx Monoclonal Whole Blood and EMIT Cyclosporine Assays, Clin. Chem. 45:432-435 (1999)) and / or are commercially available. Additionally, pretreatment can be performed as described in U.S. Patent No. 5,135,875 to Abbott, European Patent Publication No. 0471293, and U.S. Patent Application Publication No. 2008 / 0020401 (which are incorporated by reference in their entirety for their teachings regarding pretreatment). The pretreatment reagent can be a heterogeneous agent or a homogeneous agent.

[0220] When a heterogeneous pretreatment reagent is used, the pretreatment reagent precipitates analyte binding proteins present in the sample (e.g. proteins capable of binding to RGMc (membrane-bound or soluble RGMc) or fragments thereof). Such a pretreatment step involves removing any analyte binding proteins by separating the supernatant of the mixture formed by adding the pretreatment agent to the sample from the precipitated analyte binding proteins. In such an assay, the supernatant of the mixture free of any binding proteins is used in the assay and proceeds directly to the antibody capture step.

[0221] When using homogeneous pretreatment reagents, there is no such separation step. The entire mixture of test sample and pretreatment reagent is contacted with a labeled specific binding partner for RGMc (membrane-bound RGMc, soluble RGMc, a fragment of membrane-bound RGMc, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc) or any combination thereof), such as a labeled anti-RGMc monoclonal antibody (or an antigenically reactive fragment thereof). The pretreatment reagent used in such an assay is typically diluted in the pretreated test sample mixture either before or during capture by the first specific binding partner. Despite such dilution, a certain amount of pretreatment reagent (e.g., 5M methanol and / or 0.6M ethylene glycol) is still present (or remains) in the test sample mixture during capture.

[0222] b. RGMc detection The presence or amount of RGMc (membrane-bound RGMc, soluble RGMc, fragments of membrane-bound RGMc, fragments of soluble RGMc, variants of RGMc (membrane-bound or soluble RGMc) or any combination thereof) present in a biological sample can be readily determined using any suitable assay known in the art. Examples include, but are not limited to, immunoassays, such as sandwich immunoassays (e.g., monoclonal-polyclonal sandwich immunoassays, including radioisotope detection (radioimmunoassays (RIA)) and enzyme detection (enzyme immunoassays (EIA) or enzyme-linked immunosorbent assays (ELISA) (e.g., Quantikine ELISA assays, R&D These include competitive inhibition immunoassays (e.g., forward and reverse), fluorescence polarization immunoassays (FPIA), enzyme-amplified immunosorbent assays (EMIT), bioluminescence resonance energy transfer (BRET) and homogeneous chemiluminescence assays. In a SELDI-based immunoassay, a capture reagent that specifically binds to the RGMc of interest (or a fragment thereof) is attached to the surface of a mass spectrometry probe, such as a pre-activated ProteinChip array. RGMc (membrane-bound RGMc, soluble RGMc, fragments of membrane-bound RGMc, fragments of soluble RGMc, variants of RGMc (membrane-bound or soluble RGMc) or a combination of both may be used. or any combination thereof) is specifically captured on a biochip and the captured RGMc (membrane-bound RGMc, soluble RGMc, a fragment of membrane-bound RGMc, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc) or any combination thereof) is detected by mass spectrometry. Alternatively, RGMc (membrane-bound RGMc, soluble RGMc, a fragment of membrane-bound RGMc, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc) or any combination thereof) can be eluted from the capture reagent and detected by conventional MALDI (matrix-assisted laser desorption / ionization) or SELDI.Chemiluminescent microparticle immunoassays, particularly those using the ARCHITECT® automated analyzer (Abbott Laboratories, Abbott Park, Ill.), are one example of a preferred immunoassay. Other methods include mass spectrometry and immunohistochemistry (e.g., using sections from tissue biopsies) using, for example, antibodies (monoclonal, polyclonal, chimeric, humanized, human, etc.) against RGMc as described herein or fragments thereof. Anti-RGMc antibodies and fragments thereof can be produced as described above. Other methods of detection include those described, for example, in U.S. Patent Nos. 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and 5,480,792, each of which is incorporated by reference herein in its entirety.

[0223] (1) Immunoassay RGMc and / or peptides or fragments thereof (e.g., membrane-bound RGMc, soluble RGMc, a fragment of membrane-bound RGMc, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof) may be analyzed using an immunoassay. The presence or amount of RGMc (e.g., membrane-bound RGMc, soluble RGMc, a fragment of membrane-bound RGMc, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof) can be determined by detecting specific binding to RGMc using an antibody described herein. For example, an antibody or fragment thereof may specifically bind to a polypeptide comprising SEQ ID NO: 1, or a fragment thereof. An antibody or fragment thereof may specifically bind to a polypeptide comprising SEQ ID NO: 2, or a fragment thereof. If desired, one or more antibodies described herein may be used in combination with one or more commercially available monoclonal / polyclonal antibodies. Such antibodies are available from companies such as R&D Systems, Inc. (Minneapolis, MN) and Enzo Life Sciences International, Inc. (Plymouth Meeting, PA).

[0224] Any immunoassay can be used. The immunoassay can be, for example, an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), a competitive inhibition assay, such as a forward or reverse competitive inhibition assay, a fluorescence polarization assay, or a competitive binding assay. The ELISA can be a sandwich ELISA.

[0225] Heterogeneous formats may also be used. For example, a test sample is obtained from a subject, and a first mixture is prepared. The mixture comprises a test sample to be evaluated for RGMc (e.g., membrane-bound RGMc, soluble RGMc, a fragment of membrane-bound RGMc, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof) and a first specific binding partner, where the first specific binding partner and any RGMc contained in the test sample form a first specific binding partner-RGMc complex. Preferably, the first specific binding partner is an anti-RGMc antibody or a fragment thereof. The order in which the test sample and the first specific binding partner are added to form the mixture is not important. Preferably, the first specific binding partner is immobilized on a solid phase. The solid phase used in the immunoassays (for the first specific binding partner and optionally the second specific binding partner) may be any solid phase known in the art, including, but not limited to, magnetic particles, beads, test tubes, microtiter plates, cuvettes, membranes, scaffold molecules, films, filter paper, discs and chips.

[0226] After the mixture containing the first specific binding partner-RGMc complex is formed, any unbound RGMc is removed from the complex using any technique known in the art. For example, unbound RGMc can be removed by washing. However, desirably, the first specific binding partner is present in excess of any RGMc present in the test sample, so that all RGMc present in the test sample binds to the first specific binding partner.

[0227] After any unbound RGMc has been removed, a second specific binding partner is added to the mixture to form a first specific binding partner-RGMc-second specific binding partner complex. The second specific binding partner is preferably an anti-RGMc antibody that binds to an epitope on RGMc that is different from the epitope on RGMc bound by the first specific binding partner. Also, more preferably, the second specific binding partner is labeled with or contains a detectable label as described above.

[0228] The use of immobilized antibodies or fragments thereof can be incorporated into immunoassays. Antibodies may be immobilized on a variety of supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (e.g., microtiter wells), a piece of solid substrate material, and the like. An assay strip can be prepared by coating an antibody or a plurality of antibodies in an array on a solid support. The strip can then be dipped into a test biological sample and then rapidly processed through washing and detection steps to generate a measurable signal, such as a colored spot.

[0229] (a) Sandwich ELISA Sandwich ELISA measures the amount of antigen between two layers of antibodies (i.e., a capture antibody (i.e., at least one capture antibody) and a detection antibody (i.e., at least one detection antibody). The capture antibody and the detection antibody bind to different epitopes on the antigen, e.g., RGMc. Desirably, binding of the capture antibody to the epitope does not interfere with binding of the detection antibody to the epitope. Either monoclonal or polyclonal antibodies may be used as the capture and detection antibodies in sandwich ELISA.

[0230] Generally, at least two antibodies are used to separate and quantify RGMc (e.g., membrane-bound RGMc, soluble RGMc, a fragment of membrane-bound RGMc, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof) in a test sample. In particular, the at least two antibodies bind to specific epitopes of RGMc or RGMc fragments forming an immune complex called a "sandwich". One or more antibodies can be used to capture RGMc in a test sample (e.g., membrane-bound RGMc, soluble RGMc, a fragment of membrane-bound RGMc, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof) (these antibodies are often referred to as a "capture" antibody or antibodies), and one or more antibodies are used to bind a detectable (i.e., quantifiable) label to the sandwich (these antibodies are often referred to as a "detection" antibody or antibodies). In a sandwich assay, binding of an antibody to an epitope desirably is not reduced by binding of any other antibody in the assay for its respective epitope. In other words, the antibodies are selected so that one or more first antibodies contacted with a test sample suspected of containing RGMc (e.g., membrane-bound RGMc, soluble RGMc, fragments of membrane-bound RGMc, fragments of soluble RGMc, variants of RGMc (membrane-bound or soluble RGMc) or any combination thereof) do not bind to all or a portion of the epitope recognized by a second or subsequent antibody, thereby not interfering with the ability of one or more second detection antibodies to bind to RGMc (e.g., membrane-bound RGMc, soluble RGMc, fragments of membrane-bound RGMc, fragments of soluble RGMc, variants of RGMc (membrane-bound or soluble RGMc) or any combination thereof).

[0231] The antibody can be used as the first antibody in the immunoassay. Preferably, the antibody immunospecifically binds to an epitope comprising at least three contiguous amino acids of SEQ ID NO:2 and has a K D is 4.2 x 10 -11 M to 7.4 x 10-13 The immunoassay may include a second antibody that immunospecifically binds to an epitope comprising at least three contiguous amino acids of SEQ ID NO:2, where the three contiguous amino acids bound by the second antibody differ from the three contiguous amino acids bound by the first antibody.

[0232] In a preferred embodiment, a test sample suspected of containing RGMc (e.g., membrane-bound RGMc, soluble RGMc, a fragment of membrane-bound RGMc, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc) or any combination thereof) may be contacted simultaneously or sequentially with at least one capture antibody (or multiple antibodies) and at least one detection antibody. In a sandwich assay format, a test sample suspected of containing RGMc (membrane-bound RGMc, soluble RGMc, a fragment of membrane-bound RGMc, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc) or any combination thereof) is first contacted with at least one capture antibody that specifically binds to a particular epitope under conditions that allow for the formation of an antibody-RGMc complex. When more than one capture antibody is used, multiple capture antibody-RGMc complexes are formed. In a sandwich assay, antibodies, preferably at least one capture antibody, are used in molar excess over the maximum amount of RGMc or RGMc fragment expected in the test sample, for example, about 5 μg / mL to about 1 mg / mL antibody per mL of microparticle-containing buffer may be used.

[0233] Optionally, prior to contacting the test sample with the at least one first capture antibody, the at least one capture antibody can be bound to a solid support that facilitates the separation of the antibody-RGMc complex from the test sample. Any solid support known in the art that can be used includes, but is not limited to, a solid support made of a polymeric material in the form of a well, a tube, or beads. The antibody (or antibodies) can be bound to the solid support by adsorption, by covalent binding using a chemical coupling agent, or by other means known in the art, provided that such binding does not interfere with the ability of the antibody to bind to the RGMc or RGMc fragment. Furthermore, if necessary, the solid support can be derivatized to allow reaction with various functional groups on the antibody. Such derivatization requires the use of specific coupling agents, including, but not limited to, maleic anhydride, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0234] After contacting a test sample suspected of containing RGMc (e.g., membrane-bound RGMc, soluble RGMc, a fragment of membrane-bound RGMc, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc) or any combination thereof) with at least one capture antibody, the test sample is incubated to allow for the formation of a capture antibody (or capture antibodies)-RGMc complex. Incubation may be carried out at a pH of about 4.5 to about 10.0, at a temperature of about 2° C. to about 45° C., for a period of at least about 1 minute to about 18 hours, for a period of about 2 to 6 minutes, or for a period of about 3 to 4 minutes.

[0235] After the formation of the capture antibody-RGMc complex, the complex is contacted with at least one detection antibody (under conditions that allow the formation of a capture antibody-RGMc-detection antibody (detection antibodies) complex). If the capture antibody-RGMc complex is contacted with more than one detection antibody, a capture antibody-RGMc-detection antibody (detection antibodies) detection complex is formed. If at least one detection (and subsequent) antibody is contacted with the capture antibody-RGMc complex as well as the capture antibody, an incubation period under conditions similar to those described above is required for the formation of the capture antibody-RGMc-detection antibody (detection antibodies) complex. Preferably, at least one detection antibody comprises a detectable label. The detectable label may be attached to at least one detection antibody before, simultaneously with or after the formation of the capture antibody-RGMc-detection antibody (detection antibodies) complex. Any detectable label known in the art can be used as discussed herein and is known in the art.

[0236] Chemiluminescence assays can be performed according to the method described in Adamczyk et al., Anal. Chim. Acta 579(1):61-67 (2006). While any suitable assay format can be used, a microplate chemiluminometer (Mithras LB-940, Berthold Technologies USA, LLC, Oak Ridge, TN) can rapidly assay multiple samples in small volumes. The chemiluminometer can be equipped with multiple reagent injectors using 96-well black polystyrene microplates (Costar #3792). Each sample is added to a separate well, followed by simultaneous / sequential addition of other reagents as determined by the type of assay used. Desirably, the formation of pseudobases in neutral or basic solutions with acridinium aryl esters is avoided, for example, by acidification. The chemiluminescence reaction is then recorded for each well. In this regard, the time to record the chemiluminescence reaction depends, in part, on the delay between the addition of the reagents used and the particular acridinium used.

[0237] The order of adding test sample and specific binding partner to form a mixture for chemiluminescence assay is not important.When the first specific binding partner is detectably labeled with an acridinium compound, a detectably labeled first specific binding partner-RGMc complex is formed.Alternatively, when a second specific binding partner is used and the second specific binding partner is detectably labeled with an acridinium compound, a detectably labeled first specific binding partner-RGMc-second specific binding partner complex is formed.Any unbound specific binding partner, whether labeled or unlabeled, can be removed from the mixture using any technique known in the art, such as washing.

[0238] Hydrogen peroxide can be generated in situ in the mixture or provided or supplied to the mixture prior to, simultaneously with, or after the addition of the acridinium compound described above. Hydrogen peroxide can be generated in situ in a number of ways as will be apparent to those skilled in the art.

[0239] Alternatively, a source of hydrogen peroxide can simply be added to the mixture. For example, the source of hydrogen peroxide can be one or more buffers or other solutions known to contain hydrogen peroxide. In this regard, a solution of hydrogen peroxide can simply be added.

[0240] Adding at least one base solution to the sample simultaneously or sequentially generates a detectable signal, i.e., a chemiluminescent signal, indicative of the presence of RGMc or a fragment thereof. The base solution comprises at least one base and has a pH of 10 or more, preferably 12 or more. Examples of base solutions include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide, magnesium hydroxide, sodium carbonate, sodium bicarbonate, calcium hydroxide, calcium carbonate, and calcium bicarbonate. The amount of base solution added to the sample depends on the concentration of the base solution. Based on the concentration of the base solution used, one skilled in the art can easily determine the amount of base solution to add to the sample.

[0241] The chemiluminescent signal generated can be detected using routine techniques known to those skilled in the art. Based on the intensity of the signal generated, the amount of RGMc (e.g., membrane-bound RGMc, soluble RGMc, a fragment of membrane-bound RGMc, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof) in the sample can be quantified. In particular, the amount of RGMc in the sample is proportional to the intensity of the signal generated. The amount of RGMc present can be quantified by comparing the amount of light generated to a standard curve of RGMc or to a reference standard. A standard curve can be generated using serial dilutions or solutions of RGMc of known concentration by mass spectrometry, gravimetry, and other techniques known in the art.

[0242] In a chemiluminescent microparticle assay using the ARCHITECT® (or successor) analyzer, the pH of the conjugate diluent should be about 6.0 + / - 0.2, the buffer containing the microparticles should be maintained at room temperature (i.e., about 17 to about 27°C), the pH of the microparticle-containing buffer should be about 6.5 + / - 0.2, and the pH of the microparticle diluent should be about 7.8 + / - 0.2. Solids are preferably less than about 0.2%, e.g., less than about 0.15%, less than about 0.14%, less than about 0.13%, less than about 0.12%, or less than about 0.11%, e.g., about 0.10%.

[0243] (b) Forward competitive inhibition In a forward competitive inhibition format, an aliquot of known concentration of labeled RGMc (e.g., a membrane-bound RGMc peptide, a soluble RGMc peptide, a fragment of a membrane-bound RGMc peptide, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc) or any combination thereof) is used to compete with RGMc in the test sample for binding to an RGMc antibody (e.g., an antibody).

[0244] In a forward competitive assay, the immobilized antibody (e.g., antibody) can be contacted sequentially or simultaneously with the test sample and the labeled RGMc, RGMc fragment or RGMc variant. The RGMc peptide, RGMc fragment or RGMc variant can be labeled with any detectable label, including the detectable labels discussed above in connection with anti-RGMc antibodies. In this assay, the antibody can be immobilized on a solid support. Alternatively, the antibody can be bound to an antibody, such as an anti-species antibody, immobilized on a solid support, such as a microparticle.

[0245] The labeled RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, fragment of membrane-bound RGMc peptide, fragment of soluble RGMc, variant of RGMc (membrane-bound or soluble RGMc) or any combination thereof), test sample and antibody are incubated under conditions similar to those described above in connection with the sandwich assay format. Two different species of antibody-RGMc complexes may then be generated. In particular, one of the generated antibody-RGMc complexes contains a detectable label, while the other antibody-RGMc complex does not contain a detectable label. The antibody-RGMc complex can, but need not, be separated from the remainder of the test sample prior to quantification of the detectable label. The amount of detectable label in the antibody-RGMc complex is then quantified, regardless of whether the antibody-RGMc complex is separated from the remainder of the test sample. The concentration of RGMc (e.g., membrane-bound RGMc, soluble RGMc, fragments of soluble RGMc, variants of RGMc (membrane-bound or soluble RGMc), or any combination thereof) in the test sample can then be determined by comparing the quantitative value of the detectable label in the antibody-RGMc complex with the standard curve. The standard curve can be generated by mass spectrometry, gravimetry, and other techniques known in the art using serial dilutions of known concentrations of RGMc (e.g., membrane-bound RGMc, soluble RGMc, fragments of soluble RGMc, variants of RGMc (membrane-bound or soluble RGMc), or any combination thereof).

[0246] The antibody-RGMc complex can be separated from the test sample by binding the antibody to a solid support, such as those solid supports discussed above in connection with the sandwich assay format, and then removing the remainder of the test sample from contact with the solid support.

[0247] (c) Reverse competition assay In a reverse competition assay, immobilized RGMc (e.g., a membrane-bound RGMc peptide, a soluble RGMc peptide, a fragment of a membrane-bound RGMc peptide, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc) or any combination thereof) can be contacted sequentially or simultaneously with a test sample and at least one labeled antibody. Preferably, the antibody specifically binds to an epitope comprising at least three amino acids of SEQ ID NO:2, or to an epitope comprising amino acids 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 7-13, 7-13, 7-11, 7-10, 7-9, 8-13, 8-12, 8-11, 8-10, 9-13, 9-12, 9-11, 10-13, 10-12 or 11-13 of RGMc (SEQ ID NO:1).

[0248] RGMc (e.g., a membrane-bound RGMc peptide, a soluble RGMc peptide, a fragment of a membrane-bound RGMc peptide, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc) or any combination thereof) may be bound to a solid support, such as the solid supports discussed above in connection with the sandwich assay format. Preferably, the RGMc (membrane-bound or soluble) peptide fragment comprises amino acids 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 7-13, 7-13, 7-11, 7-10, 7-9, 8-13, 8-12, 8-11, 8-10, 9-13, 9-12, 9-11, 10-13, 10-12 or 11-13 of RGMc (SEQ ID NO:1).

[0249] The immobilized RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, fragment of membrane-bound RGMc peptide, fragment of soluble RGMc, variant of RGMc (membrane-bound or soluble RGMc) or any combination thereof), test sample and at least one labeled antibody are incubated under conditions similar to those described above in connection with the sandwich assay format. Two different species of RGMc-antibody complexes are then generated. Specifically, one of the generated RGMc-antibody complexes is fixed and contains a detectable label, while the other RGMc-antibody complex is not fixed and contains a detectable label. The non-fixed RGMc-antibody complex and the remaining portion of the test sample are removed from the presence of the fixed RGMc-antibody complex through techniques known in the art, such as washing. Once the non-fixed RGMc-antibody complexes are removed, the amount of detectable label in the fixed RGMc-antibody complex is quantified. The concentration of RGMc (e.g., membrane-bound RGMc, soluble RGMc, a fragment of membrane-bound RGMc, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof) in the test sample can then be determined by comparing the quantitative value of the detectable label in the RGMc-complex with the standard curve. The standard curve can be generated by mass spectrometry, gravimetry, and other techniques known in the art using serial dilutions of known concentrations of RGMc or RGMc fragments.

[0250] (d) Fluorescence polarization In a fluorescence polarization assay, an antibody or functionally active fragment thereof may first be contacted with an unlabeled test sample suspected of containing RGMc (e.g., a membrane-bound RGMc peptide, a soluble RGMc peptide, a fragment of a membrane-bound RGMc peptide, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof) to form an unlabeled RGMc-antibody complex. The unlabeled RGMc-antibody complex is then contacted with fluorescently labeled RGMc (e.g., a membrane-bound RGMc peptide, a soluble RGMc peptide, a fragment of a membrane-bound RGMc peptide, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc). The labeled RGMc (e.g., a membrane-bound RGMc peptide, a soluble RGMc peptide, a fragment of a membrane-bound RGMc peptide, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof) detects any target RGMc in the test sample for binding to the antibody or a functionally active fragment thereof. The amount of labeled RGMc-antibody complex formed is determined and the amount of RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, fragment of membrane-bound RGMc peptide, fragment of soluble RGMc, variant of RGMc (membrane-bound or soluble RGMc) or any combination thereof) in the test sample is determined using a standard curve.

[0251] The antibodies used in the fluorescence polarization assays specifically bind to an epitope comprising at least three amino acids of SEQ ID NO:2, or to an epitope comprising amino acids 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 7-13, 7-13, 7-11, 7-10, 7-9, 8-13, 8-12, 8-11, 8-10, 9-13, 9-12, 9-11, 10-13, 10-12 or 11-13 of RGMc (SEQ ID NO:1).

[0252] The antibody, labeled RGMc (e.g., a membrane-bound RGMc peptide, a soluble RGMc peptide, a fragment of a membrane-bound RGMc peptide, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc) or any combination thereof) and the test sample and at least one labeled antibody may be incubated under conditions similar to those described above in connection with the sandwich immunoassay.

[0253] Alternatively, the antibody or functionally active fragment thereof may be simultaneously contacted with fluorescently labeled RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, fragment of membrane-bound RGMc peptide, fragment of soluble RGMc, variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof) and an unlabeled test sample suspected of containing RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, fragment of membrane-bound RGMc peptide, fragment of soluble RGMc, variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof), forming both labeled and unlabeled RGMc-antibody complexes. The amount of labeled RGMc-antibody complex formed is determined, and the amount of RGMc in the test sample is determined using a standard curve. The antibody used in this immunoassay may specifically bind to an epitope having the amino acid sequence of SEQ ID NO: 1 or 2, or to an epitope having an amino acid sequence including amino acids 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 7-13, 7-13, 7-11, 7-10, 7-9, 8-13, 8-12, 8-11, 8-10, 9-13, 9-12, 9-11, 10-13, 10-12 or 11-13 of RGMc (SEQ ID NO: 1 or 2).

[0254] Alternatively, the antibody or functionally active fragment thereof is first contacted with fluorescently labeled RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, fragment of membrane-bound RGMc peptide, fragment of soluble RGMc, variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof) to form a labeled RGMc-antibody complex. The labeled RGMc-antibody complex is then contacted with an unlabeled test sample suspected of containing RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, fragment of membrane-bound RGMc peptide, fragment of soluble RGMc, variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof). Any unlabeled RGMc in the test sample competes with labeled RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, a fragment of a membrane-bound RGMc peptide, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof) for binding to the antibody or a functionally active fragment thereof. The amount of labeled RGMc-antibody complex formed is determined and the amount of RGMc in the test sample is determined using a standard curve. The antibodies used in this immunoassay specifically bind to an epitope comprising at least three amino acids from amino acids 5-13 of RGMc (SEQ ID NO:2) or to an epitope comprising amino acids 13-20, 13-19, 13-18, 13-17, 13-16, 14-20, 14-19, 14-18, 14-17, 14-16, 15-20, 15-19, 15-18, 16-20, 16-19, 17-24, 17-23, 17-22, 17-21, 17-20, 17-19, 18-24, 18-23, 18-22, 18-21, 18-20, 19-24, 19-23, 19-22 or 19-21 of RGMc (SEQ ID NO:1 or 2).

[0255] (e) Mass spectrometry Mass spectrometry (MS) may be used alone or in combination with other methods. Other methods include immunoassays and those described above for detecting specific polynucleotides. Mass spectrometry may be used to determine the presence and / or amount of one or more biomarkers. MS analysis may include matrix-assisted laser desorption / ionization (MALDI) time-of-flight (TOF) MS analysis, such as directed spot MALDI-TOF or liquid chromatography MALDI-TOF analysis. In some embodiments, MS analysis includes electrospray ionization (ESI) MS, such as liquid chromatography (LC) ESI-MS. Mass spectrometry may be accomplished using commercially available spectrometers. Methods utilizing MS analysis, including MALDI-TOF MS and ESI-MS, may be used to detect the presence and amount of biomarker peptides in biological samples. See, for example, U.S. Patent Nos. 6,925,389; 6,989,100; and 6,890,763 for guidance. Each of these is incorporated herein by reference.

[0256] C. Control It may be desirable to include a control sample or calibrator, for example a series of calibrators. The control sample may be analyzed simultaneously with the sample from the subject described above. The results obtained from the subject sample can be compared with the results obtained from the control sample. A standard curve may be provided with which the assay results for the biological sample are compared. Such a standard curve presents the levels as a function of assay units, i.e., as a function of fluorescent signal intensity if fluorescent labels are used. Using samples from multiple donors, a standard curve may be provided for control levels of RGMc in normal tissues as well as for "at risk" levels of RGMc in tissues from donors that may have one or more of the characteristics described above.

[0257] In view of the above, therefore, there is provided a method for determining the presence, amount or concentration of RGMc (e.g. membrane-bound RGMc peptides, soluble RGMc peptides, fragments of membrane-bound RGMc peptides, fragments of soluble RGMc, variants of RGMc (membrane-bound or soluble RGMc) or any combination thereof) in a test sample, which comprises assaying the test sample for RGMc (e.g. membrane-bound RGMc peptides, soluble RGMc peptides, fragments of membrane-bound RGMc peptides, fragments of soluble RGMc, variants of RGMc (membrane-bound or soluble RGMc) or any combination thereof) by immunoassay, for example using at least one antibody and at least one detectable label, and comparing a signal generated by the detectable label as a direct or indirect indication of the presence, amount or concentration of RGMc in the test sample with a signal generated in a calibrator as a direct or indirect indication of the presence, amount or concentration of RGMc. The calibrator is optionally, but preferably, part of a series of calibrators, where each of the calibrators differs from other calibrators in the series by the concentration of RGMc. One of the at least one antibody is an isolated antibody that specifically binds to RGMc (e.g., a membrane-bound RGMc peptide, a soluble RGMc peptide, a fragment of a membrane-bound RGMc peptide, a fragment of a soluble RGMc peptide, a variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof), where the antibody has (a) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:3, (b) a variable light domain region comprising the amino acid sequence of SEQ ID NO:4, (c) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:5, (d) a variable light domain region comprising the amino acid sequence of SEQ ID NO:6, (e) a variable light domain region comprising the amino acid sequence of SEQ ID NO:7, (f) a variable light domain region comprising the amino acid sequence of SEQ ID NO:8, (g) a variable light domain region comprising the amino acid sequence of SEQ ID NO:9, (h) a variable light domain region comprising the amino acid sequence of SEQ ID NO:10, (i) a variable light domain region comprising the amino acid sequence of SEQ ID NO:11, (j) a variable light domain region comprising the amino acid sequence of SEQ ID NO:12, (j) a variable light domain region comprising the amino acid sequence of SEQ ID NO:13, (j) a variable light domain region comprising the amino acid sequence of SEQ ID NO:14, (j) a variable light domain region comprising the amino acid sequence of SEQ ID NO:15, (j) a variable light domain region comprising the amino acid sequence of SEQ ID NO:16, (j) a variable light domain region comprising the amino acid sequence of SEQ ID NO:17, (j) a variable light domain region comprising the amino acid sequence of SEQ ID NO:18, (j) a variable light domain region comprising the amino acid sequence of SEQ ID NO:19, (j) (e) a variable light domain region comprising the amino acid sequence of SEQ ID NO: 7; (f) a variable light domain region comprising the amino acid sequence of SEQ ID NO: 8; (g) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO: 9; (h) a variable light domain region comprising the amino acid sequence of SEQ ID NO: 10; (i) a variable heavy chain comprising a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 13; (j) a CDR1 comprising the amino acid sequence of SEQ ID NO: 14;(k) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 19; (l) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (m) a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, and a CDR2 comprising the amino acid sequence of SEQ ID NO: 24. and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25; (n) a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28; (o) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 31; (p) a CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR2 comprising the amino acid sequence of SEQ ID NO: 33, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 34. (q) a variable heavy chain comprising a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR2 comprising the amino acid sequence of SEQ ID NO: 12 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a CDR2 comprising the amino acid sequence of SEQ ID NO: 15 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 16, (r) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 19, and (s) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR2 comprising the amino acid sequence of SEQ ID NO: 24 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 27 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28; (t) a CDR1 comprising the amino acid sequence of SEQ ID NO: 29,A variable heavy chain having a CDR2 comprising the amino acid sequence of SEQ ID NO: 30 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 31, and a variable light chain having a CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR2 comprising the amino acid sequence of SEQ ID NO: 33 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 34.

[0258] Optionally, the antibody has a domain or region selected from the sequences shown in Table 2. For example, the antibody may comprise: (a) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:37; (b) a variable light domain region comprising the amino acid sequence of SEQ ID NO:38; (c) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:39; (d) a variable light domain region comprising the amino acid sequence of SEQ ID NO:40; (e) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:41; (f) a variable light domain region comprising the amino acid sequence of SEQ ID NO:42; (g) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:43; (h) a variable light domain region comprising the amino acid sequence of SEQ ID NO:44; (i) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:45; (j) a variable light domain region comprising the amino acid sequence of SEQ ID NO:46; (k) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:47; (l) a variable light domain region comprising the amino acid sequence of SEQ ID NO:48; (m) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:49; (n) a variable light domain region comprising the amino acid sequence of SEQ ID NO:50; (o) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:51; (p) a variable light domain region comprising the amino acid sequence of SEQ ID NO:52. (q) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:53; (r) a variable light domain region comprising the amino acid sequence of SEQ ID NO:54; (s) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:55; (t) a variable light domain region comprising the amino acid sequence of SEQ ID NO:56; (u) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:57; (v) a variable light domain region comprising the amino acid sequence of SEQ ID NO:58; (w) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:59; (x) a variable light domain region comprising the amino acid sequence of SEQ ID NO:60; (y) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:61; (z) a variable light domain region comprising the amino acid sequence of SEQ ID NO:62; (aa) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:63; (bb) a variable light domain region comprising the amino acid sequence of SEQ ID NO:64; (cc) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:65; (dd) a variable light domain region comprising the amino acid sequence of SEQ ID NO:66; (ee) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:67; (ff) a variable light domain region comprising the amino acid sequence of SEQ ID NO:68.(gg) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:69, (hh) a variable light domain region comprising the amino acid sequence of SEQ ID NO:70, (ii) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:71, (jj) a variable light domain region comprising the amino acid sequence of SEQ ID NO:72, (kk) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:73, (ll) a variable light domain region comprising the amino acid sequence of SEQ ID NO:74, (mm) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO:75, (nn) a variable light domain region comprising the amino acid sequence of SEQ ID NO:76, (oo) a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO:77, a CDR comprising the amino acid sequence of SEQ ID NO:78, (pp) a variable heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 80, a CDR2 comprising the amino acid sequence of SEQ ID NO: 81, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 82; or (qq) a variable heavy chain comprising a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 77, a CDR2 comprising the amino acid sequence of SEQ ID NO: 78, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 79, and a variable light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 80, a CDR2 comprising the amino acid sequence of SEQ ID NO: 81, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 82.

[0259] The method includes (i) combining a test sample with RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, fragment of membrane-bound RGMc peptide, fragment of soluble RGMc, modified RGMc (membrane-bound or soluble RGMc), or any combination thereof) to form a capture antibody / RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, fragment of membrane-bound RGMc peptide, fragment of soluble RGMc, modified RGMc (membrane-bound or soluble RGMc) or any combination thereof) complex. (ii) contacting the RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, fragment of membrane-bound RGMc peptide, fragment of soluble RGMc, variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof) with at least one capture antibody that binds to an epitope on the RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, fragment of membrane-bound RGMc peptide, fragment of soluble RGMc, variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof) to form a capture antibody / RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, fragment of membrane-bound RGMc peptide, fragment of soluble RGMc, variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof) / detection antibody complex. (iii) contacting the capture antibody / RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, fragment of membrane-bound RGMc peptide, fragment of soluble RGMc, variant of RGMc (membrane-bound or soluble RGMc) or any combination thereof) complex with at least one detection antibody that comprises a detectable label and binds to an epitope on RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, fragment of membrane-bound RGMc peptide, fragment of soluble RGMc, variant of RGMc (membrane-bound or soluble RGMc) or any combination thereof) that is not bound by the capture antibody, and (iv) determining the amount of RGMc (or fragment thereof) in the test sample based on a signal generated by the detectable label in the capture antibody / RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, fragment of membrane-bound RGMc peptide, fragment of soluble RGMc, variant of RGMc (membrane-bound or soluble RGMc) or any combination thereof) / detection antibody complex formed in (ii).

[0260] Alternatively, the method may comprise the steps of: (i) contacting the test sample with at least one capture antibody that binds to an epitope on RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, fragment of membrane-bound RGMc peptide, fragment of soluble RGMc, variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof) simultaneously or sequentially to form a capture antibody / RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, fragment of membrane-bound RGMc peptide, fragment of soluble RGMc, variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof) complex. The method may include contacting the test sample, in any order, with a detectably labeled RGMc (e.g., a membrane-bound RGMc peptide, a soluble RGMc peptide, a fragment of a membrane-bound RGMc peptide, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof) that can compete with any RGMc in the test sample for binding to the at least one capture antibody (e.g., a membrane-bound RGMc peptide, a soluble RGMc peptide, a fragment of a membrane-bound RGMc peptide, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof). Any RGMc present in the test sample (e.g., membrane-bound RGMc peptides, soluble RGMc peptides, fragments of membrane-bound RGMc peptides, fragments of soluble RGMc, variants of RGMc (membrane-bound or soluble RGMc) or any combination thereof) and detectably labeled RGMc compete with each other to form a capture antibody / RGMc (e.g., membrane-bound RGMc peptides, soluble RGMc peptides, fragments of membrane-bound RGMc peptides, fragments of soluble RGMc, variants of RGMc (membrane-bound or soluble RGMc) or any combination thereof) complex and a capture antibody / detectably labeled RGMc (e.g., membrane-bound RGMc peptides, soluble RGMc peptides, fragments of membrane-bound RGMc peptides, fragments of soluble RGMc, variants of RGMc (membrane-bound or soluble RGMc) or any combination thereof) complex, respectively.The method further comprises (ii) determining the presence, amount or concentration of RGMc in the test sample based on a signal generated by the detectable label in the capture antibody / detectably labeled RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, fragment of membrane-bound RGMc peptide, fragment of soluble RGMc, variant of RGMc (membrane-bound or soluble RGMc) or any combination thereof) complex formed in (ii). The signal generated by the detectable label in the capture antibody / detectably labeled RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, fragment of membrane-bound RGMc peptide, fragment of soluble RGMc, variant of RGMc (membrane-bound or soluble RGMc) or any combination thereof) complex is inversely proportional to the amount or concentration of RGMc in the test sample.

[0261] In one embodiment, mouse anti-RGMc antibodies can be bound directly or indirectly to a solid support, for example, via a sheep (or other species) anti-mouse Ab. Any RGMc present in the sample is contacted with the solid support and bound by the mouse anti-RGMc Ab. Biotin-labeled goat anti-RGMc Ab also binds to RGMc. Streptavidin is linked to horseradish peroxidase (HRPO) and binds to the biotin on the goat anti-RGMc Ab. When contacted with o-phenylenediamine, HRPO converts o-phenylenediamine to 2,3-diaminophenazine, which is orange-brown in color and can be measured spectrophotometrically at 492 nm.

[0262] The method may further comprise a step of diagnosing, predicting or evaluating the effectiveness of the therapeutic / prophylactic treatment of the patient from whom the test sample was obtained. When the method further comprises a step of evaluating the effectiveness of the therapeutic / prophylactic treatment of the patient from whom the test sample was obtained, the method may further comprise a step of modifying the therapeutic / prophylactic treatment of the patient in need of improving the effectiveness. The method may be adapted for use in an automated or semi-automated system.

[0263] Generally, the pre-determined level can be used as a benchmark against which to assess the results obtained by assaying a test sample for RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, fragment of membrane-bound RGMc peptide, fragment of soluble RGMc, variant of RGMc (membrane-bound or soluble RGMc) or any combination thereof). Generally, in making such a comparison, the pre-determined level is obtained by performing a sufficient number of specific assays under suitable conditions such that the presence, amount or concentration of the analyte can be linked or associated with a particular stage or endpoint or a particular clinical sign of a disease, disorder or condition (e.g., an iron-related disorder such as those associated with iron overload or iron deficiency as discussed herein and / or known in the art). Typically, the pre-determined level is obtained using an assay of a reference subject (or population of subjects). The measured RGMc can include fragments thereof, degradation products thereof and / or enzymatic cleavage products thereof.

[0264] In particular, with respect to a given level used to monitor disease progression and / or treatment, the amount or concentration of RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, fragment of membrane-bound RGMc peptide, fragment of soluble RGMc, variant of RGMc (membrane-bound or soluble RGMc) or any combination thereof) may be "unaltered", "favorable" (or "favorably altered") or "unfavorably" (or "unfavorably altered"). "Elevated" or "increased" refers to an amount or concentration in a test sample that is higher than a typical or normal level or range (e.g., a predetermined level) or higher than another reference level or range (e.g., an initial or baseline sample). The term "decreased" or "reduced" refers to an amount or concentration in a test sample that is lower than a typical or normal level or range (e.g., a predetermined level) or lower than another reference level or range (e.g., an initial or baseline sample). The term "altered" refers to an amount or concentration in a sample that is changed (increased or decreased) above a typical or normal level or range (e.g., a predetermined level) or above another reference level or range (e.g., an initial or baseline sample).

[0265] A typical or normal level or range of RGMc is defined according to standard convention. A so-called altered level or change can be considered to have occurred when there is any net change (that cannot be explained by experimental error or sample variation) compared to a typical or normal level or range or reference level or range. Thus, the level measured in a particular sample is compared to the level or range of levels determined in a similar sample obtained from a so-called normal subject. In this context, a "normal subject" is an individual who does not have a detectable disease or disorder, and a "normal" (sometimes referred to as a "control") patient or population is, for example, one that does not exhibit a detectable disease or disorder, respectively. An "apparently normal subject" is one whose RGMc has not been evaluated or is currently being evaluated. An analyte level is said to be "elevated" when the analyte is normally undetectable (e.g., normal levels are 0 or within about the 25th to about the 75th percentile of the normal population) but is detected in a test sample as well as when the analyte is present in the test sample at a higher than normal level. Thus, among other things, the present disclosure provides methods of screening for subjects having or at risk of having an iron-related disorder, such as those associated with iron overload or iron deficiency, as discussed herein and / or known in the art.

[0266] Generally, the predetermined level can be used as a benchmark for which to evaluate the results obtained by assaying test sample for hepcidin.Generally, when making such a comparison, the predetermined level is obtained by carrying out a sufficient number of specific assays under suitable conditions, so that the presence, amount or concentration of analyte can be linked or associated with a specific stage or end point or specific clinical sign of disease, disorder or condition (e.g., iron-related disorder, such as those related to iron overload or iron deficiency, as discussed herein and / or known in the art).Typically, the predetermined level is obtained by using the assay of a reference subject (or a group of subjects).Measured hepcidin can include its fragments, its degradation products and / or its enzymatic cleavage products.

[0267] In particular, with respect to a given level used to monitor disease progression and / or treatment, the amount or concentration of hepcidin may be "unchanged," "favorable" (or "favorably altered"), or "unfavorably" (or "unfavorably altered"). "Elevated" or "increasing" refers to an amount or concentration in a test sample that is higher than a typical or normal level or range (e.g., a predetermined level) or higher than another reference level or range (e.g., an initial or baseline sample). The term "decreased" or "reduced" refers to an amount or concentration in a test sample that is lower than a typical or normal level or range (e.g., a predetermined level) or lower than another reference level or range (e.g., an initial or baseline sample). The term "altered" refers to an amount or concentration in a sample that is altered (increased or decreased) above a typical or normal level or range (e.g., a predetermined level) or above another reference level or range (e.g., an initial or baseline sample).

[0268] A typical or normal level or range of hepcidin is defined according to standard convention. A so-called altered level or change can be considered to have occurred when there is any net change (that cannot be explained by experimental error or sample variation) compared to a typical or normal level or range or reference level or range. Thus, the level measured in a particular sample is compared to the level or range of levels determined in a similar sample obtained from a so-called normal subject. In this context, a "normal subject" is an individual who does not have a detectable disease or disorder, and a "normal" (sometimes referred to as a "control") patient or population is, for example, one that does not exhibit a detectable disease or disorder, respectively. An "apparently normal subject" is one in which hepcidin has not been evaluated or is currently being evaluated. An analyte level is said to be "elevated" when the analyte is normally undetectable (e.g., normal levels are 0 or within about 25 to about 75 percentiles of the normal population) but is detected in a test sample as well as when the analyte is present in a test sample at a higher than normal level. Thus, among other things, the present disclosure provides methods of screening for subjects having or at risk of having an iron-related disorder, such as those associated with iron overload or iron deficiency, as discussed herein and / or known in the art.

[0269] The assay methods may also involve assays for other markers and those discussed herein or known in the art. For example, the assay methods may include assays for, for example, hepcidin (discussed above), neogenin, growth differentiation factor 15 (GDF-15), neutrophil gelatinase-associated lipocalin (NGAL), interleukin 6 (IL-6) and / or BMP-6.

[0270] The methods described herein can also be used to determine whether a subject has or is at risk for developing an iron-related disorder, such as those discussed herein and known in the art. (a) determining the concentration or amount of RGMc (e.g., a membrane-bound RGMc peptide, a soluble RGMc peptide, a fragment of a membrane-bound RGMc peptide, a fragment of a soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof) in a test sample from the subject using a method described herein or known in the art; and (b) comparing the concentration or amount of RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, fragment of membrane-bound RGMc peptide, fragment of soluble RGMc, variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof) determined in step (a) with a predetermined level, wherein if the concentration or amount of RGMc determined in step (a) is favorable relative to the predetermined level, the subject is determined to not have or be at risk for an iron-related disorder as discussed herein and known in the art. However, if the concentration or amount of RGMc determined in step (a) is unfavorable relative to the predetermined level, the subject is determined to have or be at risk for an iron-related disorder as discussed herein and known in the art.

[0271] Further provided herein is a method for monitoring disease progression in a subject. Optimally, the method comprises: (a) determining the concentration or amount of RGMc (e.g., a membrane-bound RGMc peptide, a soluble RGMc peptide, a fragment of a membrane-bound RGMc peptide, a fragment of soluble RGMc, a variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof) in a test sample from the subject; (b) determining the concentration or amount of RGMc in the test sample after it has been obtained from the subject; and (c) comparing the concentration or amount of RGMc determined in step (b) with the concentration or amount of RGMc determined in step (a), wherein if the concentration or amount determined in step (b) is unchanged or unfavorable compared to the concentration or amount of RGMc determined in step (a), the disease in the subject is determined to continue, progress or worsen. In comparison, if the concentration or amount of RGMc determined in step (b) is favorable compared to the concentration or amount of RGMc determined in step (a), the disease in the subject is determined to disappear, regress or improve.

[0272] Optionally, the method further comprises comparing the concentration or amount of RGMc determined in step (b), e.g., with a predetermined level. Additionally, optionally, the method comprises treating the subject with one or more pharmaceutical compositions for a period of time if the comparison indicates that the concentration or amount of RGMc determined in step (b), e.g., has changed unfavorably relative to the predetermined level.

[0273] Still further, this method can be used to monitor treatment in a subject undergoing treatment with one or more pharmaceutical compositions. In particular, such a method includes providing a first test sample of the subject before the subject is administered one or more pharmaceutical compositions. The concentration or amount of RGMc in the first test sample from the subject is then determined (e.g., using a method described herein or a method known in the art). After the concentration or amount of RGMc is determined, optionally, the concentration or amount of RGMc is then compared to a predetermined level. If the concentration or amount of RGMc determined in the first test sample is lower than the predetermined level, the subject is not treated with one or more pharmaceutical compositions. However, if the concentration or amount of RGMc determined in the first test sample is higher than the predetermined level, the subject is treated with one or more pharmaceutical compositions for a period of time. The period of time that the subject is treated with one or more pharmaceutical compositions can be determined by a person skilled in the art (e.g., the period of time may be from about 7 days to about 2 years, preferably from about 14 days to about 1 year).

[0274] During the course of treatment with one or more pharmaceutical compositions, the second and subsequent test samples are then obtained from the subject.The number of test samples and the time when said test samples are obtained from the subject are not important.For example, the second test sample can be obtained 7 days after the subject is first administered one or more pharmaceutical compositions, the third test sample can be obtained 2 weeks after the subject is first administered one or more pharmaceutical compositions, the fourth test sample can be obtained 3 weeks after the subject is first administered one or more pharmaceutical compositions, the fifth test sample can be obtained 4 weeks after the subject is first administered one or more pharmaceutical compositions, etc.

[0275] After each of the second or subsequent test samples is obtained from the subject, the concentration or amount of RGMc (e.g., membrane-bound RGMc peptide, soluble RGMc peptide, fragment of membrane-bound RGMc peptide, fragment of soluble RGMc, variant of RGMc (membrane-bound or soluble RGMc), or any combination thereof) determined in the second and subsequent test samples is determined (e.g., using a method described herein or a method known in the art). The concentration or amount of RGMc determined in each of the second and subsequent test samples is then compared to the concentration or amount of RGMc determined in the first test sample (e.g., the test sample originally optionally compared to the predetermined level). If the concentration or amount of RGMc determined in step (c) is favorable compared to the concentration or amount of RGMc determined in step (a), it is determined that the disease in the subject has disappeared, regressed, or improved, and the subject should continue to be administered one or more pharmaceutical compositions of step (b). However, if the concentration or amount determined in step (c) is unchanged or unfavorable compared to the concentration or amount of RGMc determined in step (a), then it is determined that the disease in the subject is continuing, progressing or worsening, and the subject should be treated with one or more pharmaceutical compositions at higher concentrations than those administered to the subject in step (b) or the subject should be treated with one or more pharmaceutical compositions different from the one or more pharmaceutical compositions administered to the subject in step (b). In particular, the subject can be treated with one or more pharmaceutical compositions different from the one or more pharmaceutical compositions the subject has previously received to reduce or decrease the RGMc level in said subject.

[0276] Generally, for assays that can be tested repeatedly (e.g., monitoring disease progression and / or response to treatment), the second and subsequent test samples are obtained at a time after the first test sample is obtained from the subject. In particular, the second test sample of the subject can be obtained minutes, hours, days, weeks, or years after the first test sample is obtained from the subject. For example, the second test sample can be obtained about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours after the first test sample is obtained from the subject. 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 ​​weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks, 53 weeks, 54 weeks, 55 weeks, 56 weeks, 57 weeks, 58 weeks, 59 weeks, 60 weeks, 61 weeks, 62 weeks, 63 weeks, 64 weeks, 65 weeks, 66 weeks, 67 weeks, 68 weeks, 69 weeks, 70 weeks, 71 weeks, 72 weeks, 73 weeks, 74 weeks, 75 weeks, 76 weeks, 77 weeks, 78 weeks, 79 weeks, 80 weeks, 81 weeks, 82 weeks, 83 weeks, 84 weeks, 85 weeks, 86 weeks, 87 weeks, 88 weeks, 89 weeks, 90 weeks, 91 weeks, 92 weeks, 93 weeks, 94 weeks, 95 weeks, 96 weeks, 97 weeks, 98 weeks, 99 weeks, 100 weeks, 101 weeks, 102 weeks, 103 weeks, 104 weeks, 105 weeks, 106 weeks, 107 weeks, 108 weeks, 109 weeks, 109 weeks, 102 weeks, 105 weeks, 106 weeks, 107 weeks, 108 weeks, 109 weeks, 109 weeks, 110 weeks, 1 The subject can be obtained after 7 weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2 years, about 2.5 years, about 3.0 years, about 3.5 years, about 4.0 years, about 4.5 years, about 5.0 years, about 5.5 years, about 6.0 years, about 6....

Claims

1. (a) a heavy chain variable region (VH) comprising a complementarity determining region (CDR) 1 having the amino acid sequence of SEQ ID NO: 11, a CDR2 having the amino acid sequence of SEQ ID NO: 12, and a CDR3 having the amino acid sequence of SEQ ID NO: 13, and a light chain variable region (VL) comprising a complementarity determining region (CDR) 1 having the amino acid sequence of SEQ ID NO: 14, a CDR2 having the amino acid sequence of SEQ ID NO: 15, and a CDR3 having the amino acid sequence of SEQ ID NO: 16; (b) a VH comprising a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25, and a VL comprising a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28; or (c) a VH comprising a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 29, a CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 31; and a VL comprising a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR2 comprising the amino acid sequence of SEQ ID NO: 33, and a CDR3 comprising the amino acid sequence of SEQ ID NO:

34. Anti-Repulsive Guidance Molecule c (RGMc) antibodies, including (a) a VH comprising the amino acid sequence of SEQ ID NO: 3, and a VL comprising the amino acid sequence of SEQ ID NO: 4; (b) a VH comprising the amino acid sequence of SEQ ID NO: 7 and a VL comprising the amino acid sequence of SEQ ID NO: 8; or (c) a VH comprising the amino acid sequence of SEQ ID NO: 9, and a VL comprising the amino acid sequence of SEQ ID NO: 10; The anti-RGMc antibody of claim 1 .

3. An anti-RGMC antibody described in claim 1 or 2, which comprises a heavy chain constant domain.

4. An anti-RGMC antibody described in claim 3, wherein the heavy chain constant domain is a human IgG1 constant domain.

5. An anti-RGMC antibody described in any one of claims 1 to 4, comprising a light chain constant domain.

6. An anti-RGMC antibody described in any one of claims 1 to 5 that has been affinity matured.

7. An anti-RGMC antibody described in any one of claims 1 to 6, having improved human RGMC binding affinity relative to the h5F9.23 parent antibody.

8. A nucleic acid encoding the VH and / or VL of an anti-RGMC antibody described in any one of claims 1 to 7.

9. A nucleic acid encoding the heavy chain and / or light chain of an anti-RGMC antibody described in any one of claims 1 to 7.

10. A host cell containing the nucleic acid of claim 8 or 9.

11. A method for producing an anti-RGMc antibody described in any one of claims 1 to 7, comprising culturing a host cell described in claim 10 under conditions suitable for expressing the anti-RGMc antibody.

12. A pharmaceutical composition comprising an anti-RGMc antibody described in any one of claims 1 to 7, a nucleic acid described in claim 8 or 9, or a host cell described in claim 10.

13. The pharmaceutical composition of claim 12, in a lyophilized dosage form.

14. The pharmaceutical composition of claim 12, in a liquid dosage form.

15. A pharmaceutical composition for treating an iron metabolism disease in a subject, comprising an anti-RGMC antibody described in any one of claims 1 to 7.

16. The pharmaceutical composition described in claim 15, wherein the iron metabolism disease is anemia of chronic disease (ACD).

17. The pharmaceutical composition described in claim 15, wherein the iron metabolism disease is anemia of chronic kidney disease.

18. The pharmaceutical composition described in claim 15, wherein the iron metabolism disease is iron deficiency caused by elevated levels of hepcidin.

19. A pharmaceutical composition described in any one of claims 15 to 18, which inhibits RGMc activity in a subject.

20. The pharmaceutical composition described in claim 19, wherein inhibition of RGMc activity affects iron metabolism in a subject.

21. The pharmaceutical composition described in claim 19, wherein inhibition of RGMc activity results in a decrease in hepcidin expression in the subject.

22. A pharmaceutical composition described in any one of claims 15 to 21, wherein the subject has a serum hepcidin level higher than the serum hepcidin level of a normal subject.

23. A pharmaceutical composition described in any one of claims 15 to 22, wherein the subject has a serum hepcidin level greater than 300 mg / l prior to administration of the anti-RGMC antibody.

24. A pharmaceutical composition described in any one of claims 15 to 23, wherein the subject has a hemoglobin level lower than 15.5 g / dl prior to administration of the anti-RGMC antibody.

25. A pharmaceutical composition described in any one of claims 15 to 24, wherein the subject has a transferrin saturation rate of less than 25% prior to administration of the anti-RGMC antibody.

26. A pharmaceutical composition described in any one of claims 15 to 25, wherein the subject has a total iron binding capacity of less than 50% prior to administration of the anti-RGMC antibody.

27. ​​A pharmaceutical composition described in any one of claims 15 to 26, wherein the subject has a serum iron level lower than 60 μg / dl prior to administration of the anti-RGMC antibody.

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