Rgmc-selective inhibitors and use thereof

Abstract

To provide antibodies for the improved treatment of iron-related diseases, in particular, iron-restricted disorders caused by excessive hepcidin, e.g., anemia of chronic disease (ACD), iron-refractory iron deficiency anemia (IRIDA) and anemia of chronic kidney disease (CKD).SOLUTION: The present invention provides an isolated antibody that specifically binds human Repulsive Guidance Molecule C (RGMc), where: a) the antibody is a full-length antibody or antigen-binding fragment thereof; b) the antibody does not bind human Repulsive Guidance Molecule A (RGMa) and human Repulsive Guidance Molecule B (RGMb); and c) the antibody inhibits or reduces RGMc interaction with BMP6,.SELECTED DRAWING: None

Description

[Technical field]

[0001] [Related Applications] This international patent application claims the benefit of and priority to U.S. Provisional Application No. 62 / 749,469, filed October 23, 2018, the contents of which are incorporated herein by reference in their entirety.

[0002] [Field of the Invention] The present invention generally relates to RGMc binding agents (eg, molecules including antibodies and antigen-binding fragments) that specifically and selectively inhibit RGMc but not RGMa or RGMb. [Background technology]

[0003] [background] Abnormalities in iron homeostasis are associated with several diseases that can be difficult to treat. Such disorders can be broadly classified into two categories: i) iron overload diseases, including cirrhosis, cardiomyopathy, diabetes, etc.; and ii) iron deficiency diseases, including iron-restricted anemia, anemia of chronic disease ("ACD"), etc.

[0004] Hepcidin is the master regulator of systemic iron homeostasis, and its expression is mainly restricted to the liver. Hepcidin is produced as a propeptide and processed to the mature active peptide by furin or furin-like proteases. Hepcidin negatively regulates iron availability by binding to its receptor ferroportin (the only cellular iron efflux transporter), causing both internalization and degradation. Hepcidin thus blocks iron efflux from the primary cells for dietary iron absorption (enterocytes), regeneration of hemoglobin iron (macrophages) and release of iron stores from hepatocytes, resulting in reduced systemic iron availability. The central role of hepcidin in systemic iron homeostasis was soon clearly recognized by the finding that inactivation of its gene was associated with severe iron overload in the liver and pancreas.

[0005] It has been shown that BMP6 and BMP2 expression are required for the maintenance of iron homeostasis in mice. Both bone morphogenetic proteins 6 and 2 (BMP6 and BMP2, respectively) are members of the TGFβ superfamily of growth factors known to be involved in a variety of biological processes, including iron metabolism / homeostasis. It has been shown that BMP6 and BMP2 expression are required for the maintenance of iron homeostasis in mice. Consistent with the notion that it is involved in iron regulation, genetic dysfunction of BMP6 causes severe tissue iron overload. Similarly, BMP6 mutations have been found in patients with hereditary hemacromatosis, a heterogeneous group of genetic disorders characterized by parenchymal iron overload.

[0006] BMP6 binds to type I and type II serine-threonine kinase receptors (e.g., Alk2, Alk3, BMPR2, and ActRIIA). In addition, BMP6 has also been shown to directly bind to its co-receptor, repulsive guidance molecule C or RGMc (also known as hemojuvelin or HJV). Upon binding to its receptor, a larger multimeric complex containing HJV, HFE, TFR2, and neogenin then activates intracellular SMAD phosphorylation, thereby inducing nuclear translocation and increased hepcidin transcription. Thus, the BMP6 / HJV / SMAD axis is a major regulator of hepcidin expression in response to iron status.

[0007] The currently available treatments for the treatment of clinical indications including anemia, such as chemotherapy-induced anemia and anemia of chronic kidney disease, include intravenous iron (e.g., iron supplements and blood transfusions) and erythropoiesis stimulating agents (ESAs). Examples of ESAs include erythropoietin (Epo); epoetin alpha (Procrit / Epogen); epoetin beta (NeoRecormon); darbepoetin alpha (Aranesp); and methoxypolyethylene glycol-epoetin beta (Mircera). These treatments are suboptimal and may also be associated with unwanted side effects or adverse events, such as iron overload, cardiovascular and carcinogenic risks.

[0008] Iron overload in anemic patients who frequently take iron supplements, such as IV iron, is a dangerous side effect. Excess body iron can be very toxic and can affect multiple organs, resulting in a variety of serious diseases, such as liver disease, heart disease, diabetes, hormonal abnormalities, and a dysfunctional immune system. Similarly, patients who receive blood transfusions are at risk of toxicity associated with iron overload. For example, one unit of transfused blood contains approximately 250 mg of iron. In patients who receive a large number of transfusions, especially those with thalassemia major, sickle cell disease, myelodysplastic syndrome, aplastic anemia, hemolytic anemia, and refractory sideroblastic anemia, who may become transfusion-dependent, the excess iron from transfused red blood cells gradually accumulates in various tissues, causing morbidity and mortality. Thus, excess body iron induced by treatment can cause severe adverse reactions, including toxicity to the cardiovascular system, gastrointestinal system, immune system, bone / cartilage system, reproductive system, and renal system. As an additional or alternative treatment option to IV iron, ESA (e.g., EPO, such as Epogen® by Amgen) therapy is widely administered to a broad range of patient populations, including those suffering from cancer-related and chemotherapy-induced anemia in patients. Paradoxically, however, preclinical and clinical studies have shown that ESA (e.g., EPO) can potentially accelerate tumor growth and threaten survival in cancer patients.

[0009] In an attempt to avoid at least some of these unwanted side effects associated with exogenously administered ESAs and iron supplements (e.g., blood transfusions), hypoxia-inducible factor prolyl hydroxylase (HIF-PH) has gained considerable attention as a potential therapeutic target aimed at promoting endogenous EPO production. To this end, several HIF stabilizers (e.g., HIF-PH inhibitors) have been in development recently. These include, for example, roxadustat (Fibrogen), daprodustat (GSK), vadadustat (Akebia) and molidustat (Bayer). Although early efficacy data have shown superiority or non-inferiority to ESA treatment, long-term concerns remain over the risk of major negative cardiovascular events and increased cancer risk. In particular, since the HIF axis regulates a wide range of essential in vivo biological functions (e.g., angiogenesis), systemic intervention of this pathway may result in undesirable effects beyond the intended effects on erythropoiesis.

[0010] As an alternative approach, BMP6 inhibitors, including neutralizing antibodies, are being explored by several companies in an attempt to treat anemic patients (e.g., anti-BMP6 antibodies (e.g., WO2016 / 098079, Novartis; and, KY-1070, KyMab). However, these inhibitors run the risk of adversely modulating other aspects of BMP6 signaling, including bone and cartilage formation, ovarian function, and lipid metabolism.

[0011] Separately, in a 2015 paper (The AAPS Journal 17(4):930-938), Boser et al. (AbbVie) described the therapeutic use of two antibodies that bind both RGMa and RGMc (i.e., RGMa / c) in treating anemic conditions associated with hepcidin dysregulation using two preclinical models, demonstrating increased iron mobilization in vivo with a nonselective RGMa / c antibody. However, potential long-term risks of inhibiting the RGMa pathway were not discussed in the paper, although RGMa is speculated to function as a tumor suppressor, as well as an immunomodulator. Summary of the Invention [Problem to be solved by the invention]

[0012] Thus, there remains a significant unmet need to provide improved treatments for iron-related diseases, particularly iron-suppressive disorders caused by excess hepcidin, such as anemia of chronic disease (ACD), iron-refractory iron deficiency anemia (IRIDA), and anemia of chronic kidney disease (CKD). [Means for solving the problem]

[0013] The inventors of the present disclosure recognized the benefit of achieving a higher level of selectivity to more specifically inhibit the signaling pathways that regulate iron homeostasis while minimizing potential unwanted systemic effects. To that end, the inventors sought to inhibit BMP6 signaling in a liver-selective manner by specifically targeting repulsive guidance molecule c (RGMc) (also known as hemojuvelin (HJV) or hemacromatosis type 2 protein (HFE2)). The reason was that in this way, RGMc-selective inhibitors could provide efficacy for the treatment of iron-restricted disorders while achieving an improved safety profile compared to existing approaches that affect additional pathways required for other biological functions. It is therefore believed that RGMc-selective inhibitors according to the present disclosure may achieve an improved safety profile (e.g., reduced toxicity or adverse events / effects), for example, reduced risk of major negative cardiac events (e.g., stroke) and / or reduced risk of cancer progression. Advantageously, RGMc selective inhibitors may reduce the risk of iron overload associated with exogenously administered iron (e.g., IV iron or blood transfusions). RGMc selective inhibitors may allow iron-restricted anemic patients to require lower doses of ESA and / or iron supplement therapy, e.g., less frequent IV iron or blood transfusions.

[0014] RGMc is a member of the RGM class of proteins, namely RGMa, RGMb and RGMc. RGM has been reported to interact with multiple members of the BMP class of growth factors, including BMP6. RGMc is expressed as membrane-bound and soluble forms in mammals and plays a role in iron homeostasis / metabolism within the BMP6 axis.

[0015] Both RGMa and RGMb are found in the nervous and immune systems, while RGMc is found in skeletal muscle, but primarily in the liver. Each of these family members shares significant structural homology, especially across their BMP-binding domains, but their physiological roles are quite different. RGMa and RGMb have been reported to have roles in nervous system biology, immunity, inflammation, angiogenesis, and proliferation. Unlike RGMa and RGMb, the known functions of RGMc are primarily localized in hepatocytes. Thus, the identification of RGMc-selective antibodies that do not bind to RGMa or RGMb may provide potential for liver-specific regulation of BMP6 biology.

[0016] Thus, RGMc is an obligate co-receptor expressed in the liver for certain BMPs such as BMP6, and increases hepcidin expression, thereby inhibiting iron transport. In other words, by targeting RGMc, which has a more restricted expression than RGMa, RGMb, and BMP6, a more tissue-specific effect can be achieved while reducing off-target effects. This approach offers the potential to address both iron-restricted anemia and iron overload conditions without broadly inhibiting RGM and BMP6 function throughout the body.

[0017] Thus, the present invention encompasses the recognition that selective targeting of RGMc provides an advantageous approach to achieve both efficacy and safety (reduced toxicity) compared to conventional approaches such as direct BMP6 antagonists and non-selective RGMc / RGMa / RGMb inhibitors. Advantageously, unlike prior art antibodies that non-selectively bind to RGMc (see, e.g., PCT / US2012 / 069586), the antibodies of the present invention are selective for RGMc over RGMa and RGMb.

[0018] Thus, in one aspect, the invention provides an RGMc-specific antibody, or antigen-binding fragment thereof, characterized in that it selectively binds to RGMc. In one embodiment, the invention provides an isolated antibody, or antigen-binding fragment thereof, that selectively binds to human RGMc and does not bind or has reduced binding to human RGMa and human RGMb. In some embodiments, the antibody, or antigen-binding fragment thereof, inhibits or reduces the interaction of RGMc with BMP (e.g., BMP6). In some embodiments, the antibody, or antigen-binding fragment thereof, does not inhibit or reduce the interaction of RGMc with neogenin.

[0019] In other aspects, the invention provides RGMc-specific antibodies, or antigen-binding fragments thereof, that bind to a particular moiety or moieties on RGMc, and may thereby provide particularly advantageous selectivity for RGMc over RGMa / b. In some embodiments, RGMc-specific antibodies encompassed by the invention bind to a first and / or a second binding region of human RGMc, where the first binding region is YVSSTLSL (SEQ ID NO: 46) in the α1 helix domain and the second binding region is FHSAVHGIEDL (SEQ ID NO: 47) in the α3 helix domain. In some embodiments, the antibody, or antigen-binding fragment thereof, binds to at least one amino acid residue in the first binding region and / or binds to at least one amino acid residue in the second binding region.

[0020] Thus, the present disclosure provides an RGMc selective antibody that binds to an epitope that includes one or more amino acid residues of YVSSTLSL (SEQ ID NO: 46) and may further include one or more amino acid residues of FHSAVHGIEDL (SEQ ID NO: 47).

[0021] The present disclosure provides RGMc selective antibodies that bind to an epitope that includes one or more amino acid residues of FHSAVHGIEDL (SEQ ID NO: 47) and may further include one or more amino acid residues of YVSSTLSL (SEQ ID NO: 46).

[0022] In some embodiments, an RGMc selective antibody binds to an epitope comprising one or more amino acid residues of YVSSTLSL (SEQ ID NO: 46) and one or more amino acid residues of FHSAVHGIEDL (SEQ ID NO: 47).

[0023] In other aspects, the invention provides RGMc-specific antibodies, or antigen-binding fragments thereof, where the antibody is defined by the amino acid sequences and variants of its complementarity determining regions (CDRs). For example, the antibody may comprise one or more CDR sequences selected from SEQ ID NOs: 6-11, 14-19, 22-27, 30-35, and 38-43. In some embodiments, each CDR comprises variations of 1, 2, 3, 4, or up to 5 amino acid residues compared to the corresponding CDR sequence.

[0024] In another aspect, the present invention provides an RGMc-specific antibody, or an antigen-binding fragment thereof, wherein the antibody is defined by the sequence of its heavy and / or light chain variable region. For example, in some embodiments, the antibody, or an antigen-binding fragment thereof, comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 36, or SEQ ID NO: 44. In some embodiments, the antibody, or an antigen-binding fragment thereof, comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 29, SEQ ID NO: 37, or SEQ ID NO: 45. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region disclosed herein. In some embodiments, the heavy chain variable region and / or the light chain variable region have an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the heavy chain variable region and the light chain variable region disclosed herein.

[0025] In other aspects, the invention provides RGMc-specific antibodies, or antigen-binding fragments thereof, that compete for binding to human RGMc and / or bind to the same epitope as the antibodies disclosed herein.

[0026] In some embodiments, preferred antibodies according to the present disclosure have the following profile: i) the mechanism of action of such antibodies is that they bind to RGMc, thereby directly competing with BMP6 binding in the BMP6-hepcidin signaling axis; ii) such antibodies are selective binders of RGMc over RGMa or RGMb, preferably with no detectable cross-reactivity; iii) such antibodies show species cross-reactivity to human RGMc, rodents (mouse and rat) and non-human primates (e.g., cynomolgus monkeys (cyno)); iv) such antibodies have a K D (i.e., K D≦1 nM), more preferably ≦0.1 nM; v) such antibodies demonstrate in vivo efficacy in one or more appropriate preclinical models (e.g., rats) at a dose of 10 mg / kg or lower (preferably 5 mg / kg or less, e.g. 1 mg / kg or less) after 24-48 hours (preferably 24 hours); and / or vi) such antibodies are capable of being formulated with a solubility of at least 50 mg / mL, more preferably ≧100 mg / mL (for potential subcutaneous administration). In particularly preferred embodiments, all of the above criteria (i)-(vi) are met.

[0027] In other aspects, pharmaceutical compositions comprising the antibodies disclosed herein and therapeutic uses of such antibodies / compositions are provided. Such antibodies and compositions can be used in methods of treating diseases associated with RGMc in human subjects. In some embodiments, the disease associated with RGMc is anemia, such as iron-restricted anemia (or functional iron deficiency). In some embodiments, the anemia can be iron deficiency anemia (IRIDA), anemia of chronic disease (ACD), treatment-induced anemia (e.g. chemotherapy-induced anemia), cancer-related anemia, or anemia associated with chronic kidney disease (CKD). The CKD can be dialysis-dependent CKD. The CKD can be non-dialysis-dependent CKD.

[0028] In some embodiments, the pharmaceutical compositions / antibodies may be administered to patients who are receiving or have received other therapeutic agents (e.g., erythropoietin stimulating agents, HIF stabilizers, iron supplements, iron transfusions, anti-cancer agents, and / or anti-inflammatory agents). In some embodiments, the methods reduce toxicity associated with ESAs, iron supplements, or iron transfusions.

[0029] Thus, when used in conjunction with other therapies for anemia (e.g., erythropoietin stimulating agents, HIF stabilizers, iron supplements, iron transfusions, anti-cancer agents, and / or anti-inflammatory agents), the RGMc selective inhibitors disclosed herein may reduce the need for that therapy, and thus the dose and / or frequency of that therapy may be reduced.

[0030] In some embodiments, the pharmaceutical compositions / antibodies may be used to achieve faster relief of anemia in a subject when used in conjunction with other therapies for anemia (e.g., erythropoietin stimulators, HIF stabilizers, iron supplements, iron transfusions, anti-cancer agents, and / or anti-inflammatory agents).

[0031] In some embodiments, the pharmaceutical compositions / antibodies may be used to reduce iron overload associated with blood transfusions and iron therapy, such as IV iron, in patients.

[0032] In some embodiments, the pharmaceutical compositions / antibodies can be used to sensitize anemia of ESA hyporesponsiveness. For example, approximately 5-10% of patients with CKD who have received ESA therapy show hyporesponsiveness to ESA (ESA resistance). The RGMc selective inhibitors disclosed herein can be used to make this type of anemia more responsive to ESA therapy.

[0033] In accordance with the present invention, the RGMc selective inhibitors encompassed herein can be used in the treatment of anemia in an amount effective to achieve one or more of the following: increasing serum iron in a subject, down-regulating hepcidin expression in a subject, increasing transferrin saturation in a subject, increasing erythropoietin production in a subject, reducing unsaturated iron binding capacity (UIBC) in a subject, reducing total iron binding capacity (TIBC) in a subject, and / or increasing serum ferritin levels in a subject.

[0034] In another aspect, the present invention provides a method for making a pharmaceutical composition comprising an RGMc selective inhibitor, the method comprising the steps of: i) identifying an antibody or antigen-binding fragment thereof for its ability to selectively bind RGMc over RGMa and RGMb; ii) identifying the antibody or antigen-binding fragment thereof for its ability to inhibit / neutralize RGMc activity in vivo based on step (i); and iii) selecting the inhibiting / neutralizing antibody for formulation into a pharmaceutical composition based on steps (i) and (ii). In some embodiments, the method further comprises a step of positive selection and may comprise a step of negative selection. In some embodiments, the identifying step (ii) further comprises a step of measuring an iron parameter selected from the group consisting of serum iron, TIBC, UIBC, hepcidin expression, and transferrin saturation. [Brief description of the drawings]

[0035] [Figure 1] FIG. 1 shows the relationship between hepcidin and serum iron levels in anemia of inflammation / chronic disease. The figure also shows how an RGMc antagonist (e.g., an RGMc-specific antibody) would be expected to decrease hepcidin levels and increase serum iron. [Diagram 2] 1 is a graph showing Octet® binding affinity of first generation RGMc-specific antibodies to RGMa, RGMb, and RGMc. [Diagram 3] Figure 3A: In vitro BMP6 activity assay using HepG2 cells. Increase in BMP6 activity (mean ± SD) indicates antibody-mediated inhibition of BMP6 binding to RGMc. Figure 3B: Increase in BMP6 activity (mean ± SD) in the presence of SR-RC-AB3 and SR-RC-AB4. A high affinity non-specific RGMc-binding protein was used as a positive control along with a negative isotype control. [Figure 4]Figure 4A is a graph showing binding of affinity matured RGMc antibodies to RGMa-his (monovalent form) or RGMa-Fc (avid form). RGM01, a high affinity non-specific RGM binding protein, was used as a positive control. Figure 4B shows that cross-reactivity to both human and mouse RGMc antigens was maintained throughout the affinity maturation process. [Diagram 5] Figure 5A is a graph showing the activity (mean ± SD) of the SR-RC-AB3 family of affinity matured antibodies in an in vitro BMP6 assay. Figure 5B is a graph showing the activity (mean ± SD) of the SR-RC-AB4 family of affinity matured antibodies in an in vitro BMP6 assay. A high affinity nonspecific RGM-binding protein was used as a positive control along with a negative isotype control. [Figure 6] Figure 6A is a graph showing the effect of affinity matured antibodies on serum iron levels (mean ± SD) in rats. Figure 6B is a graph showing the effect of affinity matured antibodies on unbound iron binding capacity (UIBC) (mean ± SD) in rats. A high affinity nonspecific RGM-binding protein was used as a positive control along with a negative isotype control. Significance was determined by 1-way ANOVA against isotype control; *p value < 0.05, **p value < 0.01, ***p value < 0.001. [Figure 7]Figure 7A is a graph showing serum iron levels (mean ± SEM) in female Sprague-Dawley rats over time when treated with a single dose of SR-RC-AB8 or SR-RC-AB9 (20 mg / kg). Isotype antibodies were used as controls. Figure 7B shows serum iron (top) and UIBC (bottom) measurements (mean ± SEM) in a PK / PD study in which female Sprague-Dawley rats were administered a single IV dose of SR-RC-AB8 (0.6 mg / kg-20 mg / kg) (n=5-11 animals / group). Figure 7C shows antibody serum concentrations (mean ± SEM) in the same animals as Figure 7B. Figure 7D shows the relationship between antibody exposure (PK) and iron parameters (PD) at 20 mg / kg (all values ​​are mean ± SEM). [Figure 8A] FIG. 1 is a graph showing the reduction of deuterium uptake in peptides representing RGMc regions A and B in the presence of SR-RC-AB9 Fab. [Figure 8B] The amino acid sequences of deuterium-protected regions A and B aligned across RGM family proteins are shown. [Figure 8C] Schematic modeling the structure of RGMc in the presence of BMP2. H / D exchange protected regions A and B are highlighted. [Figure 8D] Schematic mapping of the SR-RC-AB9 fab epitope onto the BMP2 binding site and highlighting potential contact residues within H / D exchange protected region B. [Figure 8E] Schematic showing the structural homology of H / D exchange protected region A within α-helices 1 / 2 / 3 of RGMa / b / c and highlighting potential structural determinants of SR-RC-AB9 specificity for RGMc over RGMa and RGMb. [Figure 8F] FIG. 1 is a diagram showing a typical structure of an antibody Fab fragment, highlighting CDRs H1-H3 that can span the surface of binding regions A and B of RGMc. [Figure 9]Figure 9A shows serum iron parameters (serum iron and UIBC, mean ± SEM) over time following a single IV dose of SR-RC-AB8 (10 mg / kg) in female cynomolgus macaques. Figure 9B shows the mean serum concentrations of SR-RC-AB8 (mean ± SEM) up to 10 weeks after dosing. Figure 9C shows the relationship between antibody exposure (PK) and serum iron parameters (PD). All values ​​are mean ± SD. [Figure 10] FIG. 10A shows the change in serum iron (mean ± SEM) from baseline (day -3) following weekly infusion of SR-RC-AB8 (20 mg / kg) alone or in combination with epoietin alpha (EPO) (10 μg / kg) in a PGPS model of anemia of chronic disease (ACD) in female Lewis rats. FIG. 10B (mean ± SD) shows absolute serum iron levels at day 24. Significance was determined by 1-way ANOVA versus ACD isotype control; **p value <0.01. FIG. 10C shows the change in hemoglobin (mean ± SEM) from baseline measurements (day -3) in response to weekly administration of SR-RC-AB8 (20 mg / kg), EPO (10 μg / kg), or both. Absolute hemoglobin levels (mean ± SD) at day 24 are shown in FIG. 10D. Significance was determined by 1-way ANOVA versus ACD isotype control; **p-value <0.01. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] [Definition] In order to make this disclosure easier to understand, certain terms are defined first.These definitions should be read in light of the remainder of this disclosure as understood by those skilled in the art.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.Further definitions are provided throughout the detailed description.

[0037] Affinity:Affinity is the strength of binding of a molecule (e.g., an antibody) to its ligand (e.g., an antigen). It is typically measured as the equilibrium dissociation constant (K D ) is measured and reported by K D is the antibody association rate ("on rate" or K on ) (how quickly it binds to its antigen) versus its dissociation rate (the "off rate" or K off ) (how quickly an antibody dissociates from its antigen). For example, an antibody with an affinity of ≦5 nM has a K of 5 nM or less as determined by a suitable in vitro binding assay, such as a biolayer interferometry (BLI)-based assay (e.g., Octet®), a surface plasmon resonance (SPR)-based assay (e.g., Biacore), and a solution equilibrium titration-based assay (e.g., Meso Scale Discovery or MDS). D value (i.e., affinity of 5 nM or greater).

[0038] anemia: Anemia is a medical condition in which red blood cell count or hemoglobin is lower than normal. For men, anemia is typically defined as a hemoglobin level lower than 13.5 grams / 100 ml, and for women, as a hemoglobin level lower than 12.0 grams / 100 ml. Anemia can be caused by reduced production of red blood cells or hemoglobin, or increased loss (usually due to bleeding) or destruction of red blood cells. Anemia can be diagnosed, for example, by measuring serum iron parameters, which may indicate anemia if they deviate from the normal range. Useful iron parameters include serum iron, total iron binding capacity (TIBC), unsaturated iron binding capacity (UIBC), and transferrin saturation. Transferrin saturation can be calculated by dividing serum iron by total iron binding capacity (TIBC) and expressed as a percentage.

[0039] Anemia of chronic disease:As used herein, the term "anemia of chronic disease" or "ACD" refers to a form of anemia that is the result of another condition. Such a condition may be associated with chronic infection, chronic immune activation, and / or malignancy (e.g., chronic kidney disease or cancer).

[0040] antibody: The term "antibody" encompasses any naturally occurring, recombinant, modified or engineered immunoglobulin or immunoglobulin-like structure, or antigen-binding fragment or portion thereof, or derivatives thereof, as further described elsewhere herein. Thus, the term refers to an immunoglobulin molecule that specifically binds to a target antigen, including, for example, chimeric, humanized, fully human, and bispecific antibodies. An intact antibody generally contains at least two full-length heavy chains and two full-length light chains, but in some cases may contain fewer chains, such as naturally occurring antibodies of camelids, which may contain only heavy chains. An antibody may be derived from a single source alone, or may be "chimeric", i.e., different portions of the antibody may be derived from two different antibodies. An antibody, or an antigen-binding portion thereof, may be produced in a hybridoma, by recombinant DNA technology, or by enzymatic or chemical cleavage of an intact antibody. The term "antibody" as used herein includes monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, and antibody fusions (sometimes referred to herein as "antibody conjugates"). In some embodiments, the term also encompasses peptibodies.

[0041] antigen:The term "antigen" broadly includes any molecule that contains an antigenic determinant within the binding region(s) to which an antibody or fragment specifically binds. An antigen may be a single unit molecule (e.g., a protein monomer or fragment) or a complex composed of multiple components. An antigen provides an epitope, e.g., a molecule or a portion of a molecule, or a complex of molecules or portions of a molecule, that is capable of binding by a selective binding agent, e.g., an antigen binding protein (including, e.g., an antibody). Thus, a selective binding agent can specifically bind to an antigen formed by two or more components in a complex. In some embodiments, an antigen can be used in an animal to produce antibodies capable of binding to the antigen. An antigen may possess one or more epitopes capable of interacting with different antigen binding proteins, e.g., antibodies.

[0042] Antigen-binding portion / fragment:As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., RGMc / HJV). Antigen-binding portions include, but are not limited to, any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. In some embodiments, an antigen-binding portion of an antibody may be obtained from an intact antibody molecule using any suitable standard technique, such as proteolytic or recombinant genetic engineering techniques, including, for example, the manipulation and expression of DNA encoding the variable (and optionally constant) domains of the antibody. Non-limiting examples of antigen-binding portions include: (i) a Fab fragment (a monovalent fragment consisting of the VL, VH, CL, and CH1 domains); (ii) a F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region); (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a single-chain Fv (scFv) molecule (see, e.g., Bird et al. (1988) SCIENCE 242:423-426; and Huston et al. (1988) PROC. NAT'L. ACAD. SCI. USA 85:5879-5883); (vi) a dAb fragment (see, e.g., Ward et al. (1989) NATURE 341:544-546); and (vii) a minimal recognition unit consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR)). Other forms of single chain antibodies, such as diabodies, are also encompassed.The term antigen-binding portion of an antibody also includes a "single-chain Fab fragment", known as "scFab", containing an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have, from the N-terminal to the C-terminal direction, one of the following orders: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL; wherein said linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids.

[0043] Combined area: As used herein, a "binding region" is a portion of an antigen that, when bound to an antibody or fragment thereof, can form the antibody-antigen interaction interface. Upon antibody binding, the binding region is protected from surface exposure and can be detected by a suitable technique, such as hydrogen-deuterium exchange mass spectrometry (HDX-MS). Antibody-antigen interaction can be mediated through multiple (e.g., two or more) binding regions. The binding region may comprise an antigenic determinant, i.e., an epitope.

[0044] Biolayer Interferometry (BLI): BLI is a label-free technique for optically measuring biomolecular interactions, for example between ligands immobilized on a biosensor chip surface and analytes in solution, allowing real-time measurement of antibody affinity. BLI offers the ability to precisely and accurately monitor binding specificity, association rate (e.g., "on" rate) and dissociation rate (e.g., "off" rate), or concentration. BLI platform instruments are commercially available, for example, by ForteBio, and are commonly referred to as the Octet® System.

[0045] BMP6 / BMP-6:As used herein, the terms "bone morphogenetic protein 6," "BMP6 (or BMP-6)," "VGR," and "VGR1" all refer to proteins that are members of the bone morphogenetic family of proteins that interact with RGMc molecules as co-receptors, e.g., human BMP6 accession number NP_001709.

[0046] Cancer-related anemia: The term "cancer-related anemia" or CRA, which may also be referred to as "cancer-related anemia," refers to anemia associated with, caused by, and / or exacerbated by the presence of cancer.

[0047] Chemotherapy-induced anemia: The term "chemotherapy-induced anemia", or CIA, also referred to as "chemotherapy-related anemia", is a type of treatment-induced anemia and refers to anemia caused and / or exacerbated by chemotherapy. The term "chemotherapy" in the context of this disclosure encompasses any anti-cancer agent, drug, or treatment aimed at treating cancer in a patient (e.g., killing malignant cells), which reduces hematopoiesis.

[0048] Chronic kidney disease: The term "chronic kidney disease" (CKD) refers to kidney disease that causes gradual loss of kidney function over a period of months to years. For example, such kidney disease can be caused by diabetes, hypertension, glomerulonephritis, and polycystic kidney disease. CKD is associated with insufficient production of erythropoietin (EPO) by kidney cells, which causes fewer red blood cells to be produced in the bone marrow, eventually resulting in anemia.

[0049] Clinical benefit: As used herein, the term "clinical benefit" is intended to include both efficacy and safety of a therapy. Thus, a therapeutic treatment that achieves a desired clinical benefit is both effective and safe (e.g., has tolerable or acceptable toxicity or adverse events).

[0050] Combination therapy:"Combination therapy" refers to a treatment regimen for a clinical indication that includes two or more therapeutic agents. Thus, the term refers to a therapeutic regimen in which a first therapy, comprising a first composition (e.g., active ingredient), is administered to a subject together with a second therapy, comprising a second composition (active ingredient), intended to treat the same or overlapping disease or clinical condition. Both the first and second compositions may act on the same cellular target, or on separate cellular targets. The phrase "together" in the context of combination therapy means that the therapeutic effect of the first therapy overlaps in time and / or space with the therapeutic effect of the second therapy in the subject receiving the combination therapy. Thus, combination therapy may be formulated as a single formulation for simultaneous administration of the treatments, or as separate formulations for sequential administration.

[0051] Combinatory or combinatorial epitopes: A combinatorial epitope is an epitope recognized and bound by a combinatorial antibody at a site (i.e., antigenic determinants) formed by non-contiguous portions of the antigen component(s) that are in close proximity in the three-dimensional structure to form the epitope. Combinatorial epitopes are also called discontinuous epitopes. Thus, an antibody of the invention may bind to an epitope formed by two or more components (e.g., portions or segments) of RGMc. A combinatorial epitope may include amino acid residue(s) from a first portion / component of RGMc and amino acid residue(s) from a second portion / component of RGMc, etc. Each portion / component may be of a single protein or of two or more proteins of the antigenic complex. A combinatorial epitope is formed by structural contributions by two or more components (e.g., portions or segments, e.g., amino acid residues) of an antigen or antigen complex.

[0052] Conflict or cross-conflict:The term "compete" when used in the context of antigen-binding proteins (e.g., antibodies or antigen-binding fragments thereof) that compete for the same epitope means that the competition between the antigen-binding proteins, as determined by the assay in which the antigen-binding proteins are tested, prevents or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein to a common antigen (e.g., RGMc or a fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay; solid-phase direct biotin-avidin EIA; solid-phase direct labeling assay, and solid-phase direct labeling sandwich assay. Typically, when a competing antigen-binding protein is present in excess, it inhibits (e.g., reduces) specific binding of a reference antigen-binding protein to a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more. In some embodiments, the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof cross-block each other with respect to the same antigen, as assayed using standard test conditions, e.g., by Biacor or Octet, e.g., according to the manufacturer's instructions (e.g., binding is assayed at room temperature, about 20-25° C.). In some embodiments, the first antibody or fragment thereof and the second antibody or fragment thereof may have the same epitope. In other embodiments, the first antibody or fragment thereof and the second antibody or fragment thereof may have non-identical but overlapping epitopes. In further embodiments, the first antibody or fragment thereof and the second antibody or fragment thereof may have distinct (different) epitopes that are in close proximity in three-dimensional space, thereby cross-inhibiting antibody binding through steric hindrance."Cross-blocking" means that the binding of a first antibody to an antigen prevents the binding of a second antibody to the same antigen, and similarly, the binding of a second antibody to an antigen prevents the binding of the first antibody to the same antigen.

[0053] Complementarity determining regions:As used herein, the term "CDR" refers to the complementarity determining region in the antibody variable sequence. There are three CDRs in each of the heavy and light chain variable regions, and they 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 can bind to an antigen. The exact boundaries of these CDRs are defined differently by different systems. The system described by Kabat (Kabat et al. (1987; 1991) Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.) 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 may be referred to as Kabat CDRs. Chothia and coworkers (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; and Chothia et al. (1989) Nature 342:877-883) have developed a system for numbering the CDRs based on the Kabat CDRs. It has been found that certain sub-portions within the CDRs adopt nearly identical peptide backbone conformations, despite the high diversity at the amino acid sequence level. 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 may be called Chothia CDRs, and have overlapping boundaries with Kabat CDRs. Other boundaries that define CDRs that overlap with Kabat CDRs are described by Padlan (1995) FASEB J.9:133-139 and MacCallum (1996) J.Mol.Biol.262(5):732-45. Still other CDR boundary definitions may not strictly follow one of the systems herein, but still overlap with Kabat CDRs, but they may be shorter or longer in light of predictions or experimental findings that certain residues or groups of residues or even the entire CDR do not significantly affect antigen binding.The methods used herein may utilize CDRs defined according to any of these systems, although particular embodiments use the CDRs defined by Kabat or Chothia.

[0054] Conformational epitopes: A conformational epitope is an epitope that is recognized and bound by a conformational antibody in a three-dimensional conformation, but not by an unfolded peptide of the same amino acid sequence. A conformational epitope can be called a conformation-specific epitope, a conformation-dependent epitope, or a conformation-sensitive epitope. The corresponding antibody or a fragment thereof that specifically binds to such an epitope can be called a conformation-specific antibody, a conformation-selective antibody, or a conformation-dependent antibody. The binding of an antigen to a conformational epitope depends on the three-dimensional structure (conformation) of the antigen or antigen complex.

[0055] Constant region: Immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy and light chain constant domain amino acid sequences are known in the art.

[0056] Effective Dose: An "effective amount" (or therapeutically effective amount) is a dose or administration regimen that achieves a statistically significant clinical benefit in a patient population.

[0057] Epitope:The term "epitope", which may also be referred to as antigenic determinant, is a molecular determinant (e.g., a polypeptide determinant) that can be specifically bound by a binding agent, an immunoglobulin, or a T-cell receptor. Epitope determinants include chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and in certain embodiments may have specific three-dimensional structural characteristics, and / or specific charge characteristics. The epitope recognized by an antibody or an antigen-binding fragment of an antibody is a structural element of the antigen that interacts with the CDR (e.g., complementary site) of the antibody or fragment. An epitope may be formed by the contribution of several amino acid residues, which interact with the CDR of the antibody to generate specificity. An antigenic fragment may contain more than one epitope. In certain embodiments, an antibody is said to specifically bind to an antigen when it recognizes its target antigen in a complex mixture of proteins and / or macromolecules. For example, antibodies are said to "bind to the same epitope" if the antibodies cross-compete (one prevents the binding or modulatory effect of the other).

[0058] Erythropoiesis stimulators: The term "erythropoiesis stimulating agent" or "ESA" as used herein refers to a drug that stimulates bone marrow to produce red blood cells. Erythropoietin or EPO is an example of an ESA, and may include erythropoietin alpha, delta, omega, and zeta, including engineered forms of erythropoietin, such as darbepoetin alpha. The term "EPO therapy" may be used to broadly encompass therapy using one or more ESA drugs.

[0059] Human antibodies:The term "human antibody" (or "fully human antibody") as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include, for example, within the CDRs, particularly CDR3, amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted into human framework sequences.

[0060] Humanized antibodies:The term "humanized antibody" refers to an antibody that contains heavy and light chain variable region sequences 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 more "human-like", i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. Also, a "humanized antibody" is an antibody, or a variant, derivative, analog, or fragment thereof, that immunospecifically binds to an antigen of interest, and comprises a FR region having substantially the amino acid sequence of a human antibody and a CDR region having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of a CDR refers to a CDR that has an amino acid sequence that is 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 the non-human antibody CDR. A humanized antibody comprises substantially all of 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 those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the FR regions being of a human immunoglobulin consensus sequence. In one embodiment, the humanized antibody also comprises at least a portion of an immunoglobulin Fc region (typically that of a human immunoglobulin). In some embodiments, the humanized antibody comprises a light chain and at least the variable domain of a heavy chain. The antibody may also comprise the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody comprises only a humanized light chain. In some embodiments, the humanized antibody comprises only a humanized heavy chain. In certain embodiments, the humanized antibody comprises only a humanized variable domain of the light chain and / or only a humanized heavy chain.

[0061] Hydrogen / Deuterium Exchange Mass Spectrometry (HDX-MS):HDX-MS is a well-known technique used to confirm proteins in solution and to investigate protein-protein interactions by measuring the degree of solvent accessibility. See, e.g., Wei et al., (2014) Drug Discov Today 19(1):95-102. "Hydrogen / deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics: methodology and applications."

[0062] Hypoxia-inducible factor (HIF) stabilizers: HIF prolyl-hydroxylase (HIF-PH) inhibitors can stabilize HIF (e.g., HIF-2α), thereby increasing the endogenous production of erythropoietin (EPO) in vivo. These agents may be called "HIF activators". Examples of HIF stabilizers include, but are not limited to: Roxadustat (FG-4592); Vadadustat (AKB-6548), Daprodustat (GSK1278863), Dsidustat (ZYAN-1), Molidustat (Bay 85-3934); MK-8617, YC-1, IOX-2, 2-methoxyestradiol, GN-44028, AKB-4924, Bay 87-2243, FG-2216 and FG-4497.

[0063] Iron Therapy: As used herein, "iron therapy" refers to therapeutic treatment that involves iron supplements (e.g., oral or intravenous (iv) iron).

[0064] Isolated: As used herein, an "isolated" antibody refers to an antibody that is substantially free from other antibodies having different antigenic specificities. In some embodiments, an isolated antibody is substantially free from other unintended cellular material and / or chemicals.

[0065] Potency:As used herein, the term "potency" refers to the activity of a drug (e.g., an inhibitory antibody (or fragment) having inhibitory activity) in terms of the concentration or amount of drug that produces a defined effect. For example, an antibody capable of producing a particular effect at a given dose will be more potent than another antibody that requires twice the amount (dosage) to produce the same effect. Potent The potency can be measured by in vitro assays such as BMP6 activation / inhibition assays, as described herein. The potency can also be measured by in vivo assays such as those measuring hepcidin expression, serum iron, ferritin expression, and iron binding capacity, as described herein. Typically, antibodies with higher affinity tend to show higher potency than antibodies with lower affinity.

[0066] Protection (from solvent exposure): In the context of HDX-MS-based assessment of protein-protein interactions (e.g., antibody-antigen binding), the degree to which a protein (e.g., a region of a protein that contains an epitope) is exposed to solvent, thereby allowing proton exchange, is inversely correlated with the degree of binding / interaction. Thus, when an antibody described herein binds to a region of an antigen, the binding region is "protected" from exposure to solvent, since the protein-protein interaction prevents the surrounding solvent from accessing the binding region. Thus, the protected region represents the site of interaction. Typically, the appropriate solvent is a physiological buffer.

[0067] RGM: As used herein, "RGM" refers to a class of proteins that belong to the repulsive guidance molecule family.

[0068] RGMc:As used herein, the terms "repulsive guidance molecule C," "RGMc," "hemojuvelin," "HJV," "hemacromatosis type 2 protein," and "HFE2A" all refer to proteins that are members of the repulsive guidance molecule family of proteins that interact with BMP molecules as co-receptors, e.g., human RGMc Accession No. NP_998818; SEQ ID NO:1.

[0069] Disorders related to RGMc:"RGMc-associated disorder" means i) any disease or disorder whose etiology and / or progression can be attributable, at least in part, to RGMc signaling or dysregulation thereof, and / or ii) a condition in which inhibition of RGMc may provide clinical benefit. RGMc disorders include, for example: anemia associated with inflammation / chronic disease (e.g., Castleman syndrome, chronic kidney disease, diabetes, heart failure / heart disease, idiopathic autoimmune hemolytic anemia, idiopathic pulmonary arterial hypertension, inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus, systemic juvenile idiopathic arthritis), anemia associated with cancer / proliferative disorders (e.g., acute leukemia, diffuse large B-cell lymphoma, Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndromes, non-Hodgkin's lymphoma, Waldenström macroglobulinemia, bladder cancer, breast cancer, colorectal cancer, gastric cancer, head and neck cancer, hepatocellular carcinoma, lung cancer, ovarian cancer, prostate cancer, and renal cancer), anemia associated with infection (e.g., endocarditis, Helicobacter pylori infection, hepatitis, human immunodeficiency virus, malaria), anemia associated with neurological disorders (e.g., For example, acute ischemic stroke, amyotrophic lateral sclerosis (ALS), focal cerebral ischemia / reperfusion, intracranial cerebral hemorrhage, and subarachnoid hemorrhage), treatment-induced or treatment-related anemia (e.g., antibiotic-induced, bariatric surgery-related, chemotherapy-induced, secondary iron overload), anemia associated with a genetic disorder or other condition (e.g., age-related (anemia of the elderly), anemia of critical illness, anemia of surgical procedures, blood loss / hemorrhage, calciphylaxis, celiac disease, copper deficiency, Fanconi anemia, hereditary hemacromatosis (type 4), hereditary spherocytosis, IRIDA, obesity, organ transplant, paroxysmal nocturnal hemoglobinuria, sickle cell anemia, sports / exercise-induced anemia, thalassemia, thrombotic thrombocytopenic purpura).

[0070] RGM inhibitors:The term "RGM inhibitor" or "RGM antagonist" refers to any agent capable of antagonizing the biological activity or function of RGM (e.g., RGMa, RGMb, and / or RGMc). The term is not intended to be limiting in its mechanism of action and includes, for example, neutralizing antibodies, competitive inhibitors, receptor antagonists, furin and / or TMPRSS6 cleavage enhancers, and soluble RGM peptides.

[0071] RGMc selective inhibitors: The term "RGMc-selective" (or "RGMc-specific") inhibitor refers to agents (including small molecules and biologics, e.g., antibodies) capable of selectively antagonizing the biological activity or function of RGMc over RGMa and RGMb. For clarity, a monoclonal antibody that binds to and inhibits / neutralizes both RGMa and RGMc is not an RGMc-selective inhibitor.

[0072] Specific binding: As used herein, the terms "specific binding" or "specifically binds" mean that the interaction of an antibody or antigen-binding fragment thereof with an antigen is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope). For example, an antibody, or antigen-binding fragment thereof, binds to a specific protein, rather than to proteins in general.

[0073] In some embodiments, the antibody, or antigen-binding fragment thereof, has a K D But at least, about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13In some embodiments, the term "specific binding to an epitope of RGMc," "specifically binds to an epitope of RGMc," "specific binding to RGMc," or "specifically binds to RGMc" refers to binding to RGMc and having a binding affinity of 1.0×10 or less as determined by a suitable in vitro binding assay, such as surface plasmon resonance and biolayer interferometry (BLI). -7 M or lower dissociation constant (K D ) refers to an antibody, or antigen-binding fragment thereof, having a specific binding affinity to both human and non-human (e.g., mouse) orthologs of RGMc. For example, in some embodiments, the antibody, or antigen-binding fragment thereof, has less than a 5-fold difference in cross-reactivity to human, mouse, rat and / or cynomolgus RGMc.

[0074] The antibody, or antigen-binding fragment thereof, may have low cross-reactivity with other RGM family members (e.g., human RGMa and human RGMb). Thus, in some embodiments, the antibody, or antigen-binding fragment thereof, binds to human RGMc with higher affinity (i.e., has low cross-reactivity) than human RGMa and / or human RGMb. For example, in some embodiments, the antibody, or antigen-binding fragment thereof, binds to human RGMc with at least 1000-fold higher affinity (i.e., has a 1000-fold lower K) than human RGMa and / or human RGMb. D ) and binds to human RGMc.

[0075] subject:The term "subject" in the context of therapeutic applications refers to an individual who receives clinical care or intervention, such as treatment, diagnosis, etc. Suitable subjects include, but are not limited to, vertebrates, including mammals (e.g., human and non-human mammals). When the subject is a human subject, the term "patient" may be used interchangeably. In a clinical context, the term "patient population" or "patient subpopulation" is used to refer to a group of individuals that fall into a set of criteria, such as clinical criteria (e.g., disease presentations, disease stage, susceptibility to a particular condition, responsiveness to treatment, etc.), medical history, health status, sex, age group, genetic criteria (e.g., carriers of a particular mutation, polymorphism, gene duplication, DNA sequence repeat, etc.) and lifestyle factors (e.g., smoking, alcohol consumption, exercise, etc.).

[0076] Surface Plasmon Resonance (SPR): Surface plasmon resonance is an optical phenomenon that can detect unlabeled interactants in real time. SPR-based biosensors, such as those marketed by Biacore, can be used to measure biomolecular interactions, including protein-protein interactions, such as antigen-antibody binding. The technique is widely known in the art and is useful for determining parameters such as binding affinity, kinetic rate constants and thermodynamics.

[0077] Total iron binding capacity: As used herein, the term "total iron binding capacity", "TIBC", or "transferrin iron binding capacity" refers to a laboratory test that measures the blood's ability to bind iron to transferrin. TIBC is determined by measuring the maximum amount of iron that blood can carry, which is an indirect measurement of transferrin. Typically, TIBC is calculated by measuring serum iron and serum unsaturated iron binding capacity (UIBC) and adding the two values ​​together. Alternatively, TIBC can be measured directly by adding an excess of iron to a sample, removing unbound iron, and measuring the iron dissociated from transferrin. In general, the "normal" range for TIBC is, for example, 255-450ug / dL, although measurements may vary among populations.

[0078] toxicity: The term "toxicity" or "toxicity" as used herein refers to unwanted in vivo effects in a patient associated with a therapy administered to the patient, such as undesirable side effects and adverse events. "Tolerability" refers to the level of toxicity associated with a therapy or therapeutic regimen that can be reasonably tolerated by a patient without interrupting therapy due to toxicity. In the context of this disclosure, toxicity may include, but is not limited to: higher risk of death, adverse cardiovascular response and / or stroke (which may be associated with chronic diseases such as chronic kidney disease (CKD)), and reduced overall survival, increased risk of tumor progression or recurrence in cancer patients.

[0079] Treat / treatment: The term "treat" or "treatment" includes therapeutic treatment, prophylactic treatment, and applications that reduce the risk of a subject developing a disorder or other risk factor. Thus, the term is intended to broadly mean causing a therapeutic benefit in a patient, for example, by enhancing or boosting the body's immunity; by reducing or reversing immunosuppression; by reducing, removing, or eradicating harmful cells or substances from the body; by reducing disease burden (e.g., tumor burden); by preventing recurrence or relapse; by extending refractory periods, and / or otherwise improving survival. The term includes therapeutic treatment, prophylactic treatment, and applications that reduce the risk of a subject developing a disorder or other risk factor. Treatment does not require a complete cure of the disorder, but encompasses embodiments that reduce symptoms or underlying risk factors. In the context of combination therapy, the term may refer to i) the ability of the second therapy to reduce the effective dose of the first therapy, thereby reducing side effects and increasing tolerability; ii) the ability of the second therapy to make the patient more responsive (e.g., sensitive) to the first therapy; iii) the ability to achieve additive or synergistic clinical benefit; and / or iv) the ability to achieve faster onset of relief (e.g., therapeutic benefit).

[0080] Unbound iron-binding capacity:The term "unbound iron binding capacity" or "UIBC" as used herein refers to the amount of available iron binding capacity of transferrin. UIBC is often calculated by assessing the difference between TIBC and the amount of serum iron. Alternatively, UIBC can be calculated directly by adding a known amount of excess iron to a sample and subtracting the remaining unbound iron. In general, the "normal" range for UIBC is, for example, 120-470ug / dL (21-84umol / L), although measurements may vary between populations or individuals.

[0081] Variable region: The term "variable region" or "variable domain" refers to portions of the light and / or heavy chains of an antibody, typically comprising approximately the amino terminal 120-130 amino acids in the heavy chain and about the amino terminal 100-110 amino acids in the light chain. In certain embodiments, the variable regions of different antibodies differ extensively in amino acid sequence, even among antibodies of the same species. The variable regions of an antibody typically determine the specificity of a particular antibody for its target.

[0082] Other than in the examples, or unless otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as being modified in all instances by the term "about."

[0083] As used herein, in the specification and the claims, the indefinite articles "a" and "an" should be understood to mean "at least one," unless clearly dictated to the contrary.

[0084] In the specification and claims, the term "and / or" as used herein should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements, whether related or unrelated to those elements specifically identified, may be present other than the elements expressly identified by the "and / or" clause, unless expressly indicated to the contrary. Thus, as a non-limiting example, a reference to "A and / or B," when used with open-ended language such as "comprising," may, in one embodiment, refer to A without B (which may include elements other than B); in another embodiment, refer to B without A (which may include elements other than A); in yet another embodiment, refer to both A and B (which may include other elements); and so forth.

[0085] As used herein in the specification and claims, the phrase "at least one" in a reference to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows that elements other than those specifically identified in the list of elements to which the phrase "at least one" refers (whether related or unrelated to those elements specifically identified) may be present. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") may refer, in one embodiment, to at least one (which may include more than one) A with no B (which may include elements other than B); in another embodiment, to at least one (which may include more than one) B with no A (which may include elements other than A); in yet another embodiment, to at least one (which may include more than one) A and at least one (which may include more than one) B (which may include other elements); etc.

[0086] The use of ordinal terms in the claims to modify claim elements, such as "first," "second," "third," etc., does not, in and of itself, imply any priority, precedence, or ordering of one claim element over another, or the temporal order in which acts of a method are performed, but is merely used as a label to distinguish one claim element having a particular name from another element having the same name (where the claim elements would not be distinguishable without the use of ordinal terms).

[0087] Ranges provided herein are understood to be shorthand for all values ​​within the range. For example, a range of 1-50 is understood to include any number, combination of numbers, or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, such as 10-20, 1-10, 30-40, etc.

[0088] [Regulation of iron homeostasis via the BMP6 / hepcidin axis] Hepcidin is a small peptide secreted by the liver and is the central regulator of iron homeostasis in the body. Its main function is to inhibit the transport of iron from cells to the plasma compartment of blood from three main sources: dietary iron absorption through duodenal enterocytes, release of hemoglobin iron from regenerating red blood cells, and release of stored iron from hepatocytes. Iron regulation is achieved by hepcidin-mediated degradation of the iron efflux transporter ferroportin on the surface of duodenal enterocytes, reticuloendothelial macrophages resident in the liver and spleen, and hepatocytes. As ferroportin is degraded, macrophages and duodenal enterocytes can no longer release iron into the blood, and as a result, iron delivery to transferrin is reduced. Thus, as shown in Figure 1, increased expression of hepcidin reduces iron efflux. Conversely, decreased expression of hepcidin increases iron efflux. Thus, inherited and acquired disorders that disrupt normal hepcidin production can result in iron deficiency (high levels of hepcidin) or iron overload (hepcidin deficiency).

[0089] As the main factor maintaining iron homeostasis, hepcidin synthesis is transcriptionally regulated by several stimuli, including intracellular and extracellular iron concentrations, erythropoeitic demand, and inflammation. In healthy individuals, hepcidin is maintained at homeostatic levels due to the activity of a group of coordinating proteins within the iron regulatory complex. Genetic analysis of patients suffering from hereditary hemacromatosis (HH), in which genetic mutations cause excessive dietary iron absorption and storage in the periphery with associated tissue / organ toxicity, has identified key proteins regulating iron homeostasis. The most common type of HH is due to mutations in the HFE gene, which encodes an atypical MHC class protein that prevents binding of transferrin to the transferrin receptor. A rarer form of HH occurs in patients with mutations in TFR2, hepcidin, and HJV (hereafter referred to as "RGMc"). Each of these rare forms is characterized by inappropriately low hepcidin levels, supporting a role for RGMc, HFE, and TFR2 in the upstream regulation of hepcidin expression.Interestingly, mutations in RGMc are the most common cause of severe forms of juvenile HH, which can be fatal if left untreated.

[0090] Hepcidin expression is stimulated by extracellular stimuli, including the inflammatory signal IL-6 and the developmental signaling bone morphogenetic protein (BMP). Mice with targeted deletion of BMP signaling components (specific ligands, receptors, and co-receptors) are characterized by an iron-overload phenotype, highlighting the important role of BMP signaling in hepcidin expression. Regulation of BMP signaling occurs at multiple levels to provide signal specificity and fine-tuning; increasing co-receptors or disrupting ligand-receptor interactions are the main mechanisms for BMP signaling regulation. RGMc is thought to be a key co-receptor for BMP-mediated increased hepcidin expression in the liver, sensitizing hepatocytes to low BMP levels under normal physiological conditions, thereby increasing hepcidin expression and regulating iron homeostasis. Due to its potency and limited role in iron regulation, RGMc is an attractive therapeutic target for disorders in which hepcidin-associated iron restriction plays a key role, such as anemia of chronic disease.

[0091] BMPs are a widespread subfamily of growth factors within the TGFβ superfamily that were originally discovered for their ability to induce bone and cartilage formation. Besides their association with bone, like many other growth factors, BMPs are involved in a diverse set of biological processes. For example, BMP6 has roles in many different biological processes, including lipid metabolism and ovarian physiology, but in the liver it functions as a key control point in iron regulation in humans through the regulation of hepcidin, a central regulator of iron homeostasis.

[0092] Therefore, BMP6 has become an alternative therapeutic target for treating diseases and disorders associated with iron dysregulation. Several groups, including Eli Lilly and Novartis, have developed antibodies that specifically bind and neutralize BMP6, and have proposed the therapeutic use of such antibodies to treat conditions such as anemia. However, these approaches that aim to directly inhibit BMP6 signaling systemically may disrupt many different physiological processes in which BMP6 is involved, and there is a high risk of producing unintended and unwanted effects (e.g., toxicity).

[0093] Recently, repulsive guidance molecules (RGMs) have been identified as proteins that interact with BMPs. The RGM family of proteins includes at least three known members, namely RGMa, RGMb, and RGMc, and is so named due to the role of RGMa, the founding member, in guiding axon growth during embryogenesis.

[0094] Structurally, RGM is a glycosylphosphatidyl-inositol (GPI)-anchored protein consisting of an N-terminal signal sequence, an RGD motif (RGMa and RGMc), a partial von Willebrand type D (vWFD) domain, and an autocatalytic Gly-Asp-Pro-His (GDPH) cleavage site. RGM has been reported to interact as a coreceptor with multiple members of the BMP class of growth factors, including BMP6. Each of the RGM family members shares significant structural homology, particularly across their BMP-binding domains (see Table 1). RGMa and RGMc in particular share high sequence homology.

[0095] Despite considerable homology between family members, their expression and biological functions appear to be quite distinct: RGMa and RGMb have been reported to have roles in nervous system biology, immunity, inflammation, angiogenesis, and proliferation, whereas RGMc is predominantly expressed in the liver (e.g., hepatocytes), which is the main site of hepcidin synthesis and is involved in the regulation of iron metabolism.

[0096] Evidence supports the notion that inhibition of the BMP6 / RGMc axis may be an attractive therapeutic approach for treating iron dysregulation. For example, US2013 / 0330343A1 disclosed that monoclonal antibodies that bind and neutralize RGMa / c can 1) reduce hepcidin levels, 2) increase serum iron levels and transferrin saturation, and 3) reduce unsaturated iron binding capacity (UIBC) in treated animals.

[0097] The inventors of the present disclosure recognized that selective inhibition of RGMc may provide an improved safety profile, because such inhibitors should only target RGMc expressed by the liver, while leaving intact the normal biological functions of RGMa and RGMb. Therefore, the inventors sought to identify RGMc-selective antibodies that do not bind to RGMa or RGMb, in order to achieve liver-specific modulation of BMP6 activity.

[0098] JPEG2025004154000001.jpg54153

[0099] [RGMc / Hemojuvelin (HJV)] At least three isoforms of human RGMc exist, including a precursor protein (isoform a; SEQ ID NO: 1) and two splice variants (isoform b and isoform c; SEQ ID NO: 2 and 3, respectively). The major isoform, human RGMc isoform "a", consists of 426 amino acids and represents the longest transcript. Unless otherwise indicated herein, the term "RGMc" refers to the full-length protein, heterodimer, or cleavage product of RGMc isoform a. Below are the amino acid sequences of the RGMc isoforms, RGMa, and RGMb.

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[0105] RGMc is a coreceptor for BMP6, which is part of a larger multimeric protein complex that includes BMP6, BMP6-binding type I and type II serine-threonine kinase receptors (e.g., Alk2, Alk3, BMPR2, and ActRIIA), neogenin, RGMc (HJV), HFE, and TFR2. Binding of BMP6 to its receptor, then the larger multimeric complex, induces downstream SMAD phosphorylation and induces nuclear translocation of transcription factors that mediate increased hepcidin transcription. Thus, BMP6 signaling has been shown to directly regulate iron homeostasis through regulation of hepcidin expression.

[0106] RGMc is expressed in membrane-bound and soluble forms in mammals. Membrane-bound RGMc is found on the cell surface in two distinct forms, a single-chain full-length form and a disulfide-linked two-chain (heterodimer) form. The heterodimeric form of RGMc is the result of autoproteolysis occurring at the FGDPH motif, yielding a C-terminal fragment of approximately 35 kD and an N-terminal fragment of approximately 20 kD, which remain linked by disulfide bonds. Single-chain, full-length RGMc has been shown to interact with BMP2, but previous studies suggest that autoproteolysis is important for correct folding of the protein and that autocatalytic processing is a general and important feature of RGMc. For example, it has been shown that only the heterodimeric form of RGMc activates hepcidin expression in vitro and that autocatalytic processing of RGMa is required for its growth-inhibitory effect on axons. Furthermore, several JHH-associated mutations are located near the RGMc autocatalytic cleavage site, and these mutations have reduced signaling activity and are often retained in the ER.

[0107] In addition, at least two proteases, furin and TMPRSS6, have been shown to cleave RGMc to produce several soluble forms of RGMc (s-RGMc or s-HJV). In particular, furin cleavage produces an approximately 40 kD s-RGMc fragment. This fragment has been shown to act as a decoy receptor that competes with membrane-bound RGMc for binding to BMP ligands, thereby reducing hepcidin expression and increasing serum iron levels. Interestingly, furin activity has been shown to increase in iron deficiency and hypoxia. However, since furin is also involved in the processing of pro-hepcidin, the effect of furin on hepcidin expression may be complex and most likely depends on specific cellular conditions.

[0108] On the other hand, TMPRSS6 can cleave RGMc at multiple sites, resulting in cleavage fragments with various functions. The role of TMPRSS6 in regulating hepcidin expression was demonstrated by a study showing that its inactivation in mice results in increased hepcidin levels, blockage of iron absorption, and anemia. Furthermore, iron-refractory iron deficiency anemia (IRIDA) can be caused by mutations in the gene encoding TMPRSS6 / matriptase-2. However, it has also been shown that RGMc fragments shed by TMPRSS6 have a reduced ability to bind BMP.

[0109] As mentioned above, hepcidin is the main regulator of systemic iron mobilization and is regulated by bone morphogenetic protein (BMP)-mediated signaling (especially BMP6). RGMc binds to BMP6 to regulate Smad-mediated pathway, leading to the regulation of hepcidin expression, which then leads to the degradation of ferroportin (iron transporter protein), thereby blocking iron release from macrophages and enterocytes into circulation. Therefore, it is believed that increasing RGMc activity increases hepcidin expression and reduces iron release from cells. In this situation, RGMc activity can cause imbalance in iron homeostasis due to systemic iron depletion, resulting in iron-related diseases. In other words, increasing hepcidin levels mediated by RGMc can lead to anemia, while reducing its expression can be a causative feature in many hemochromatosis (iron overload) diseases.

[0110] Thus, the present application provides RGMc-selective monoclonal antibodies and fragments thereof that specifically bind and inhibit RGMc activity in vivo without cross-reacting with RGMa or RGMb. To the best of the applicant's knowledge, these antibodies and fragments represent a novel class of such inhibitors. The rationale for this approach includes the following. First, based on the recognition that hepcidin is a key regulator of iron homeostasis and BMP6 is required for that process, an inhibitor that blocks this signaling axis is desirable. Second, since BMP6 is widely expressed and plays a diverse range of biological roles, it is advantageous to avoid direct targeting of BMP6. Since the RGM family of proteins is known to interact with and be co-receptors for BMPs such as BMP6, BMP2 and BMP4, targeting the co-receptors may provide an alternative and attractive approach to block signaling and regulate hepcidin. Third, however, it is recognized that RGMa and RGMb exert distinct biological functions, and therefore such inhibitors should selectively inhibit RGMc without affecting RGMa / b. In this way, selective inhibition of hepatic RGMc can be achieved while minimizing potential unwanted side effects that may result from inadvertently and systemically blocking the normal functions of BMP6, RGMa and RGMb.

[0111] The present disclosure provides antibodies, and antigen-binding fragments thereof, that specifically bind and inhibit the function of RGMc or RGMc-containing complexes, but do not bind or inhibit the function of RGMa and RGMb. In some embodiments, the antibodies, and antigen-binding fragments thereof, described herein inhibit the interaction of BMPs (e.g., BMP6) with RGMc. In some embodiments, the antibodies or fragments described herein do not inhibit the interaction of neogenin with RGMc. In any embodiment, the antibodies and fragments encompassed by the present invention provide exquisite specificity and targeting of the RGMc pathway. Advantageously, such antibodies or fragments may achieve reduced toxicity. Importantly, because RGMc expression is restricted to a subset of tissues, inhibition of the RGMc-mediated BMP6 pathway should achieve a tissue-selective effect, unlike direct antagonism of BMP6 itself or broad inhibition of the RGM family, thus reducing toxicity from systemic effects.

[0112] Thus, the present invention encompasses the recognition that the use of selective inhibitors of RGMc provides an advantageous approach for the treatment of conditions involving iron dysregulation, as compared to non-selective inhibitors. To this end, the inventors of the present disclosure have sought to design and produce antibodies or fragments thereof that specifically and selectively target RGMc in vivo without targeting RGMa or RGMb. This is based, at least in part, on the concept that selectivity provides an improved safety profile (e.g., reduced toxicity or side effects) for the in vivo administration of such inhibitors.

[0113] For example, targeting RGMc together with RGMa and / or RGMb may increase the broad non-hepatic tissue expression of RGMa / b and their known roles in various physiological processes and associated potential toxicity. In particular, RGMa has been shown to be critical for embryonic development and is expressed in multiple tissues including the brain, skin, heart, liver, lung, kidney, testis, and gastrointestinal tract of adult mice. Consistent with these data, approximately half of mice lacking the RGMa gene die in utero with an exancephalic phenotype and major morphological and developmental defects. RGMa has also been shown to play a key role in neuronal regeneration, recovery, and survival after injury. Pathologically, elevated levels of RGMa are observed in brain lesions due to injury and ischemic stroke; Alzheimer's disease amyloid plaques; and the substantia nigra of Parkinson's disease patients. However, decreased RGMa expression and increased promoter methylation status have been shown to be closely associated with the origin and progression of colorectal cancer (Zhao et al., Oncol Rep 2012;27(5):1653-9). These evidences suggest that RGMa / c cross-reactive antibodies, for example, may cause or exacerbate cancer progression.

[0114] In addition to embryonic development and neuronal regeneration, RGMa has also been shown to regulate immune responses to injury; it is expressed by dendritic cells in brain and spinal cord lesions and coordinates T cell responses. In particular, inhibition of RGMa has been shown to suppress T cell responses in a mouse model of multiple sclerosis (experimental autoimmune encephalomyelitis; EAE). In humans, RGMa inhibition reduces T cell proliferation and the production of pro-inflammatory cytokines in peripheral blood mononuclear cells (PBMCs). Furthermore, among T cell subsets, RGMa has been shown to be highly expressed on Th17 cells. Th17 cells play a key role in host defense against infection by, among other things, recruiting neutrophils and macrophages to infected tissues, so pharmacological disruption of normal immune function is undesirable. Advantageously, by selectively targeting and inhibiting RGMc, but not RGMa, such side effects can be effectively avoided.

[0115] Although many of these functions are related to injury, RGMa may also regulate neuronal regeneration in healthy individuals. Given its important role in regulating the CNS response to injury, the immune response, and its unknown role in various organs, inhibition of RGMa activity is undesirable due to the risk of potential off-target side effects.

[0116] RGMb has also been shown to be expressed in multiple tissues, including brain, bone, heart, lung, liver, kidney, testis, ovary, uterus, epididymis, and pituitary in adult mice. RGMb knockout mice die by postnatal day 12 and are developmentally stunted. RGMb, like RGMa, also has a role in immune responses. Notably, RGMb is expressed at high levels in lung macrophages and dendritic cells. In its absence, IL-6 levels are elevated in these cell types, suggesting a role in regulating inflammation. RGMb has also been shown to promote neurite outgrowth. Recently, RGMb has been described as a binding partner for PD-L2 in the lung, and blocking this interaction prevents the development of respiratory tolerance, highlighting a role for RGMb in pulmonary immunity. This raises the possibility that inhibitors that interfere with RGMb function may cause dysregulation of immune responses.

[0117] Furthermore, it has been suggested that RGMb may act as a tumor suppressor, possibly by inhibiting SMAD activation. Indeed, decreased expression of RGMb is associated with poor prognosis in non-small cell lung carcinoma. The RGMb gene is inactivated in a subtype of colorectal cancer, and RGMb has been shown to be a negative regulator of breast cancer growth in vitro. Thus, not only may inhibition of RGMb have effects on the immune and nervous systems, but such inhibition may also increase cancer progression and / or development.

[0118] In summary, the broad expression patterns and pleiotropic functions in various biological processes of the related family members RGMa and RGMb contrast with the restricted expression pattern of RGMc and its focused role in maintaining iron homeostasis, highlighting the importance of taking an RGMc-specific approach toward discovering novel therapeutics for iron-restricted anemia without exacerbating off-target effects.

[0119] Thus, in one aspect, the present invention aims to inhibit RGMc function in vivo without causing potential side effects or toxicity associated with disruption of RGMa and RGMb activity. It is believed that liver-selective inhibition of BMP6 signaling by interfering with RGMc function may provide a method for targeting various iron-restricted anemias, including anemia of chronic kidney disease, anemia of cancer, and anemia of chronic inflammation.

[0120] According to the present invention, the selective inhibitor of RGMc does not inhibit other closely related factors such as RGMa and RGMb. Such inhibitors can be used to normalize iron homeostasis, including treating various forms of anemia or diseases associated with iron-restricted anemia. Indeed, as described herein, RGMc-specific antibodies have been shown to selectively suppress the bone morphogenetic protein 6 (BMP6)-hepcidin axis and increase iron availability for erythropoiesis.

[0121] [RGMc inhibitor] To practice the methods of the invention, any suitable inhibitor of RGM / HJV may be used, provided that such agent inhibits or antagonizes RGM with sufficient selectivity with respect to the RGMc isoform. Preferably, such RGMc inhibitors have no measurable inhibitory activity against RGMa and RGMb at doses that provide clinical benefit (e.g., therapeutic efficacy and an acceptable toxicity profile) when administered to a human subject.

[0122] The term "inhibitor" as used herein refers to any agent capable of blocking or antagonizing RGMc signaling. Suitable inhibitors include small molecules, nucleic acid-based agents, biologics (e.g., polypeptide-based agents, such as antibodies and other discovery agents), and any combination thereof.

[0123] In some embodiments, such agents can prevent or reduce RGMc activity by inhibiting the association of RGMc with the BMP receptor complex. In some embodiments, such agents can prevent or reduce RGMc activity by inhibiting the binding of RGMc to BMPs. In some embodiments, such agents can prevent or reduce RGMc activity by inhibiting the binding of RGMc to BMP6. In some embodiments, such agents can prevent or reduce RGMc activity by inhibiting the binding of RGMc to neogenin. In some embodiments, such agents are competitive inhibitors of RGMc / BMP6 binding. In some embodiments, such agents are competitive inhibitors of RGMc / neogenin binding.

[0124] In some embodiments, the inhibitor may be an antibody (including fragments thereof, such as domain antibodies (dAbs) as described in U.S. Pat. Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197; and 6,696,245), a small molecule inhibitor, an Adnectin, an Affibody, a DARPin, an Anticalin, an Avimer, a Versabody, or a gene therapy. The use of inhibitors encompassed by the present invention also includes antibody mimetics, such as monobodies and single domain antibodies. Monobodies are synthetic binding proteins that typically use the fibronectin type III domain (FN3) as a molecular scaffold. Monobodies include Adnectins™, which are based on the tenth fibronectin type III domain.

[0125] [RGMc specific antibody] Recently, a monoclonal antibody specifically binding to RGMa / RGMc was shown to increase serum iron by downregulating hepcidin in rats and cynomolgus monkeys (Boser et al. (2015) AAPS J. 2015 Jul;17(4):930-938). In this case, the researchers reported no obvious toxicity in healthy animals receiving RGMa / RGMc antibodies, and normalization of blood parameters was seen after the recovery period (about 12 weeks) following the administration period. However, potential risks associated with the concomitant inhibition of RGMa, for example to the nervous system, are not known from the published studies. Furthermore, numerous other toxicities, such as toxicities associated with non-hepatic expression of RGMa (e.g., toxicities associated with the brain, skin, heart, lungs, kidneys, testes, and gastrointestinal tract), are possible in humans, as outlined above. A reduction in RGMa expression has also been shown to be associated with the origin and progression of colorectal cancer. Furthermore, RGMa expression has been shown to regulate the immune response to injury. Thus, the present invention provides novel RGMc selective inhibitors that do not bind to and inhibit RGMa and / or RGMb activity / function.

[0126] In some embodiments of the invention, the RGMc selective inhibitor (e.g., an antibody) inhibits or reduces the interaction between BMP and RGMc. In other embodiments, the RGMc selective inhibitor (e.g., an antibody) inhibits or reduces the interaction between BMP6 and RGMc. In other embodiments, the RGMc selective inhibitor does not inhibit or reduce the interaction with neogenin.

[0127] In some embodiments, the RGMc selective inhibitor (e.g., an antibody) modulates the interaction of RGMc with one or more of the following interacting proteins: HFE, TFR2, ALK2, ALK3, BMPR2, ACVR2A, and TMPRSS6. In another embodiment, the RGMc selective inhibitor (e.g., an antibody) inhibits the interaction of RGMc with one or more of the following interacting proteins: HFE, TFR2, ALK2, ALK3, BMPR2, ACVR2A, and TMPRSS6. In another embodiment, the RGMc selective inhibitor (e.g., an antibody) inhibits the interaction of HFE and RGMc. In another embodiment, the RGMc selective inhibitor (e.g., an antibody) inhibits the interaction of TFR2 and RGMc. In another embodiment, the RGMc selective inhibitor (e.g., an antibody) inhibits the interaction of ALK2 and RGMc. In another embodiment, the RGMc selective inhibitor (e.g., an antibody) inhibits the interaction of ALK3 and RGMc. In another embodiment, the RGMc selective inhibitor (e.g., an antibody) inhibits the interaction between BMPR2 and RGMc. In another embodiment, the RGMc selective inhibitor (e.g., an antibody) inhibits the interaction between ACVR2A and RGMc. In another embodiment, the RGMc selective inhibitor (e.g., an antibody) inhibits the interaction between TMPRSS6 and RGMc.

[0128] In some embodiments, the RGMc comprises a naturally occurring mammalian amino acid sequence. In some embodiments, the RGMc comprises a naturally occurring human amino acid sequence. In some embodiments, the RGMc comprises a human, monkey, rat or mouse amino acid sequence. In some embodiments, the RGMc is the human RGMc sequence set forth in SEQ ID NO: 1, or a fragment thereof. In some embodiments, the RGMa is a soluble form of RGMc.

[0129] In some embodiments, the RGMc selective inhibitor has a K of ≦5 nM (e.g., ≦0.1 nM) in a suitable in vitro binding assay, such as Octet® or MSD. DHowever, unlike previously described RGMc antibodies, these RGMc-specific antibodies do not show any detectable binding to RGMa or RGMb under identical assay conditions. For example, RGMc-selective inhibitors have a K of ≦0.1 nM. D can bind to RGMc by MSD but does not show any detectable binding to RGMa or RGMb under identical assay conditions.

[0130] In some embodiments, RGMa comprises a naturally occurring mammalian amino acid sequence. In some embodiments, RGMa comprises a naturally occurring human amino acid sequence. In some embodiments, RGMa comprises a human, monkey, rat or mouse amino acid sequence. In some embodiments, RGMa is the human RGMa sequence set forth in SEQ ID NO:4, or a fragment thereof. In some embodiments, RGMa is a soluble form of RGMa.

[0131] In some embodiments, RGMb comprises a naturally occurring mammalian amino acid sequence. In some embodiments, RGMb comprises a naturally occurring human amino acid sequence. In some embodiments, RGMb comprises a human, monkey, rat or mouse amino acid sequence. In some embodiments, RGMb is the human RGMb sequence set forth in SEQ ID NO:5, or a fragment thereof. In some embodiments, RGMb is a soluble form of RGMb.

[0132] An embodiment of the present invention relates to an isolated monoclonal antibody, or an antigen-binding fragment thereof, that selectively binds to RGMc and comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3.

[0133] In one embodiment, an antibody, or antigen-binding fragment thereof, that selectively binds to RGMc has one or more CDR sequences that are substantially similar to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3, when compared to the corresponding CDR regions. For example, the antibody may comprise one or more CDR sequences (e.g., SEQ ID NOs: 6-11, 14-19, 22-27, 30-35, or 38-43) that each contain 1, 2, 3, 4, or up to 5 amino acid residue variations compared to the corresponding CDR region in any one of SEQ ID NOs: 6-11, 14-19, 22-27, 30-35, or 38-43.

[0134] As used herein, the phrase "amino acid variation" or "amino acid alteration" or "alteration of an amino acid residue" includes amino acid substitutions and / or deletions.

[0135] In one embodiment, the antibody, or antigen-binding fragment thereof, comprises at least three CDRs (which may contain up to three amino acid changes for each CDR, e.g., 1, 2, or 3 amino acid changes) selected from the following: CDR-H1: SEQ ID NO: 6; CDR-H2: SEQ ID NO: 7; CDR-H3: SEQ ID NO: 8; CDR-L1: SEQ ID NO: 9; CDR-L2: SEQ ID NO: 10; and CDR-L3: SEQ ID NO: 11.

[0136] In one embodiment, the antibody, or antigen-binding fragment thereof, comprises at least three CDRs (which may contain up to three amino acid changes for each CDR, e.g., 1, 2, or 3 amino acid changes) selected from the following: CDR-H1: SEQ ID NO: 14; CDR-H2: SEQ ID NO: 15; CDR-H3: SEQ ID NO: 16; CDR-L1: SEQ ID NO: 17; CDR-L2: SEQ ID NO: 18; and CDR-L3: SEQ ID NO: 19.

[0137] In one embodiment, the antibody, or antigen-binding fragment thereof, comprises at least three CDRs (which may contain up to three amino acid changes for each CDR, e.g., 1, 2, or 3 amino acid changes) selected from the following: CDR-H1: SEQ ID NO: 22; CDR-H2: SEQ ID NO: 23; CDR-H3: SEQ ID NO: 24; CDR-L1: SEQ ID NO: 25; CDR-L2: SEQ ID NO: 26; and CDR-L3: SEQ ID NO: 27.

[0138] In one embodiment, the antibody, or antigen-binding fragment thereof, comprises at least three (e.g., three, four, five, or preferably all six) CDRs (which may contain up to three amino acid changes for each CDR, e.g., one, two, or three amino acid changes) selected from the following: CDR-H1: SEQ ID NO: 30; CDR-H2: SEQ ID NO: 31; CDR-H3: SEQ ID NO: 32; CDR-L1: SEQ ID NO: 33; CDR-L2: SEQ ID NO: 34; and CDR-L3: SEQ ID NO: 35.

[0139] In one embodiment, the antibody, or antigen-binding fragment thereof, comprises at least three CDRs (which may contain up to three amino acid changes for each CDR, e.g., 1, 2, or 3 amino acid changes) selected from the following: CDR-H1: SEQ ID NO: 38; CDR-H2: SEQ ID NO: 39; CDR-H3: SEQ ID NO: 40; CDR-L1: SEQ ID NO: 41; CDR-L2: SEQ ID NO: 42; and CDR-L3: SEQ ID NO: 43.

[0140] In another aspect, the invention provides an antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region comprising CDR-H1, CDR-H2, and / or CDR-H3 with specific amino acid alterations.

[0141] In some embodiments, the antibody or antigen-binding fragment comprises the CDR-H1 shown in SEQ ID NO: 14, with the proviso that the serine residue at position 4 of SEQ ID NO: 14 may be substituted with arginine. In another embodiment, the antibody or antigen-binding fragment comprises the CDR-H1 shown in SEQ ID NO: 14, with the proviso that the alanine residue at position 7 of SEQ ID NO: 14 may be substituted with serine. In another embodiment, the antibody or antigen-binding fragment comprises the CDR-H1 shown in SEQ ID NO: 14, with the proviso that the serine residue at position 9 of SEQ ID NO: 14 may be substituted with glutamine. In some embodiments, the antibody or antigen-binding fragment comprises the CDR-H1 shown in SEQ ID NO: 14, with the proviso that (i) the serine residue at position 4 of SEQ ID NO: 14 may be substituted with arginine; (ii) the alanine residue at position 7 of SEQ ID NO: 14 may be substituted with serine; and / or (ii) the serine residue at position 9 of SEQ ID NO: 14 may be substituted with glutamine.

[0142] In some embodiments, the antibody or antigen-binding fragment comprises the CDR-H2 set forth in SEQ ID NO: 15, with the proviso that the threonine residue at position 8 of SEQ ID NO: 15 may be substituted with valine. In another embodiment, the antibody or antigen-binding fragment comprises the CDR-H2 set forth in SEQ ID NO: 15, with the proviso that the asparagine residue at position 10 of SEQ ID NO: 15 may be substituted with serine. In some embodiments, the antibody or antigen-binding fragment comprises the CDR-H2 set forth in SEQ ID NO: 15, with the proviso that (i) the threonine residue at position 8 of SEQ ID NO: 15 may be substituted with valine; and / or (ii) the asparagine residue at position 10 of SEQ ID NO: 15 may be substituted with serine.

[0143] In some embodiments, the antibody or antigen-binding fragment comprises the CDR-H3 set forth in SEQ ID NO: 16, with the proviso that the isoleucine residue at position 5 of SEQ ID NO: 16 may be substituted with tyrosine. In another embodiment, the antibody or antigen-binding fragment comprises the CDR-H3 set forth in SEQ ID NO: 16, with the proviso that the alanine residue at position 6 of SEQ ID NO: 16 may be substituted with valine. In some embodiments, the antibody or antigen-binding fragment comprises the CDR-H3 set forth in SEQ ID NO: 16, with the proviso that (i) the isoleucine residue at position 5 of SEQ ID NO: 16 may be substituted with tyrosine; and / or (ii) the alanine residue at position 6 of SEQ ID NO: 16 may be substituted with valine.

[0144] In some embodiments, the antibody or antigen-binding fragment comprises a CDR-L1 as set forth in SEQ ID NO: 17 and may comprise up to three amino acid alterations. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-L2 as set forth in SEQ ID NO: 18 and may comprise up to three amino acid alterations. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-L3 as set forth in SEQ ID NO: 19 and may comprise up to three amino acid alterations.

[0145] In a particular embodiment, the present invention provides an isolated antibody that specifically binds to human RGMc, wherein the antibody is a full-length antibody or an antigen-binding fragment thereof, wherein the antibody comprises at least three of the following six CDRs: a) CDR-H1: SEQ ID NO: 14: i) the serine residue at position 4 of SEQ ID NO: 14 may be substituted with arginine; ii) the alanine residue at position 7 of SEQ ID NO: 14 may be substituted with serine; and / or iii) the serine residue at position 9 of SEQ ID NO: 14 may be substituted with glutamine; b) CDR-H2: SEQ ID NO: 15: i) the threonine residue at position 8 of SEQ ID NO: 15 may be substituted with a valine; and / or ii) the asparagine residue at position 10 of SEQ ID NO: 15 may be substituted with serine; c) CDR-H3: SEQ ID NO: 16: i) the isoleucine residue at position 5 of SEQ ID NO: 16 may be substituted with a tyrosine; and / or ii) the alanine residue at position 6 of SEQ ID NO: 16 may be substituted with a valine; d) CDR-L1: SEQ ID NO: 17; e) CDR-L2: SEQ ID NO: 18; and f) CDR-L3: SEQ ID NO: 19.

[0146] In some embodiments, such antibodies have a K of <1 nM (preferably <0.1 nM) as measured in a suitable in vitro binding assay, such as Octet® or MSD. D In a preferred embodiment, such antibodies are also isoform specific, in that they selectively bind and inhibit the activity of RGMc, but do not bind and inhibit the activity of RGMa and RGMb.

[0147] In some embodiments, the heavy chain CDR and / or light chain CDR sequences do not contain any amino acid changes. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO:6. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-H2 comprising the sequence set forth in SEQ ID NO:7. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-H3 comprising the sequence set forth in SEQ ID NO:8. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-L1 comprising the sequence set forth in SEQ ID NO:9. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-L2 comprising the sequence set forth in SEQ ID NO:10. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-L3 comprising the sequence set forth in SEQ ID NO:11.

[0148] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a CDR-H3 having the amino acid sequence of SEQ ID NO:8 and a light chain variable region comprising a CDR-L3 having the amino acid sequence of SEQ ID NO:11. In some embodiments, the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region comprising a CDR-H2 having the amino acid sequence of SEQ ID NO:7 and a light chain variable region comprising a CDR-L2 having the amino acid sequence of SEQ ID NO:10. In some embodiments, the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region comprising a CDR-H1 having the amino acid sequence of SEQ ID NO:6 and a light chain variable region comprising a CDR-L1 having the amino acid sequence of SEQ ID NO:9.

[0149] In certain embodiments, the antibody or antigen-binding fragment comprises heavy chain CDR-H1, CDR-H2, and CDR-H3 sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively, and light chain CDR-L1, CDR-L2, and CDR-L3 sequences set forth in SEQ ID NOs: 9, 10, and 11, respectively.

[0150] In some embodiments, the antibody or antigen-binding fragment comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO: 14. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-H2 comprising the sequence set forth in SEQ ID NO: 15. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-H3 comprising the sequence set forth in SEQ ID NO: 16. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-L1 comprising the sequence set forth in SEQ ID NO: 17. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-L2 comprising the sequence set forth in SEQ ID NO: 18. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-L3 comprising the sequence set forth in SEQ ID NO: 19.

[0151] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a CDR-H3 having the amino acid sequence of SEQ ID NO: 16 and a light chain variable region comprising a CDR-L3 having the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region comprising a CDR-H2 having the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising a CDR-L2 having the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 14 and a light chain variable region comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 17.

[0152] In certain embodiments, the antibody or antigen-binding fragment comprises heavy chain CDR-H1, CDR-H2, and CDR-H3 sequences set forth in SEQ ID NOs: 14, 15, and 16, respectively, and light chain CDR-L1, CDR-L2, and CDR-L3 sequences set forth in SEQ ID NOs: 17, 18, and 19, respectively.

[0153] In some embodiments, the antibody or antigen-binding fragment comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO:22. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-H2 comprising the sequence set forth in SEQ ID NO:23. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-H3 comprising the sequence set forth in SEQ ID NO:24. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-L1 comprising the sequence set forth in SEQ ID NO:25. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-L2 comprising the sequence set forth in SEQ ID NO:26. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-L3 comprising the sequence set forth in SEQ ID NO:27.

[0154] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a CDR-H3 having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region comprising a CDR-L3 having the amino acid sequence of SEQ ID NO: 27. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region comprising a CDR-H2 having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising a CDR-L2 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 22 and a light chain variable region comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 25.

[0155] In certain embodiments, the antibody or antigen-binding fragment comprises heavy chain CDR-H1, CDR-H2, and CDR-H3 sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively, and light chain CDR-L1, CDR-L2, and CDR-L3 sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively.

[0156] In some embodiments, the antibody or antigen-binding fragment comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO: 30. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-H2 comprising the sequence set forth in SEQ ID NO: 31. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-H3 comprising the sequence set forth in SEQ ID NO: 32. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-L1 comprising the sequence set forth in SEQ ID NO: 33. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-L2 comprising the sequence set forth in SEQ ID NO: 34. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-L3 comprising the sequence set forth in SEQ ID NO: 35.

[0157] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a CDR-H3 having the amino acid sequence of SEQ ID NO: 32 and a light chain variable region comprising a CDR-L3 having the amino acid sequence of SEQ ID NO: 35. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region comprising a CDR-H2 having the amino acid sequence of SEQ ID NO: 31 and a light chain variable region comprising a CDR-L2 having the amino acid sequence of SEQ ID NO: 34. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 30 and a light chain variable region comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 33.

[0158] In certain embodiments, the antibody or antigen-binding fragment comprises heavy chain CDR-H1, CDR-H2, and CDR-H3 sequences set forth in SEQ ID NOs: 30, 31, and 32, respectively, and light chain CDR-L1, CDR-L2, and CDR-L3 sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively.

[0159] In some embodiments, the antibody or antigen-binding fragment comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO: 38. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-H2 comprising the sequence set forth in SEQ ID NO: 39. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-H3 comprising the sequence set forth in SEQ ID NO: 40. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-L1 comprising the sequence set forth in SEQ ID NO: 41. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-L2 comprising the sequence set forth in SEQ ID NO: 42. In some embodiments, the antibody or antigen-binding fragment comprises a CDR-L3 comprising the sequence set forth in SEQ ID NO: 43.

[0160] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a CDR-H3 having the amino acid sequence of SEQ ID NO: 40 and a light chain variable region comprising a CDR-L3 having the amino acid sequence of SEQ ID NO: 43. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region comprising a CDR-H2 having the amino acid sequence of SEQ ID NO: 39 and a light chain variable region comprising a CDR-L2 having the amino acid sequence of SEQ ID NO: 42. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 38 and a light chain variable region comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 41.

[0161] In certain embodiments, the antibody or antigen-binding fragment comprises heavy chain CDR-H1, CDR-H2, and CDR-H3 sequences set forth in SEQ ID NOs: 38, 39, and 40, respectively, and light chain CDR-L1, CDR-L2, and CDR-L3 sequences set forth in SEQ ID NOs: 41, 42, and 43, respectively.

[0162] An embodiment of the invention relates to a monoclonal antibody, or antigen-binding fragment thereof, that selectively binds to RGMc and comprises a heavy chain variable region sequence and a light chain variable region sequence.

[0163] In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 36, or SEQ ID NO: 44. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a light chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 29, SEQ ID NO: 37, or SEQ ID NO: 45.

[0164] In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:12 and / or a light chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:13. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 12, or a light chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 12, and a light chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13.

[0165] In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:20, and / or a light chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:21. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:20, or a light chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:21. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:20, and a light chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:21.

[0166] In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28 and / or a light chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28, or a light chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28, and a light chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29.

[0167] In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:36, and / or a light chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:37. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:36, or a light chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:37. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:36, and a light chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:37.

[0168] In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:44 and / or a light chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:45. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:44, or a light chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:45. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:44, and a light chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:45.

[0169] In some embodiments, the heavy and / or light chain variable region sequences do not vary in any of the CDR sequences provided herein. For example, in some embodiments, a degree of sequence variation (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) may occur within the heavy and / or light chain variable amino acid sequences, excluding any of the CDR sequences provided herein.

[0170] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 12 and / or a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the antibody, or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 12 or a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the antibody, or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 12 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 13.

[0171] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 20 and / or a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the antibody, or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 20 or a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the antibody, or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 20 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 21.

[0172] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 28 and / or a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 29. In some embodiments, the antibody, or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 28 or a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 29. In some embodiments, the antibody, or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 28 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 29.

[0173] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 36 and / or a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, the antibody, or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 36 or a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, the antibody, or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 36 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 37.

[0174] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 44 and / or a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 45. In some embodiments, the antibody, or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 44 or a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 45. In some embodiments, the antibody, or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 44 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 45.

[0175] In some embodiments, the "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990 (modified by Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993). Such an algorithm has been incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of interest. When gaps exist between the two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0176] In any antibody or antigen-binding fragment described herein, one or more conservative mutations may be introduced into the CDR or framework sequences at positions where the residues are unlikely to be involved in antibody-antigen interactions. In some embodiments, such conservative mutation(s) may be introduced into the CDR or framework sequences at positions where the residues are unlikely to be involved in interactions with RGMc, as determined based on crystal structures. In some embodiments, similar interfaces (e.g., residues involved in antigen-antibody interactions) may be predicted from known structural information about other antigens that share structural similarities.

[0177] As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Mutations can be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, such as those found in references that collect such methods (e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FMAusubel, et al., eds., John Wiley & Sons, Inc., New York). Conservative amino acid substitutions include those made between amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0178] In some embodiments, the antibodies provided herein contain mutations that confer desirable properties to the antibody. For example, to avoid potential complications due to Fab arm exchange (known to occur in native IgG4 mAbs), the antibodies provided herein may contain a stabilizing "Adair" mutation (Angal et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody", Mol Immunol 30, 105-108; 1993), in which serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline to provide an IgG1-like (CPPCP (SEQ ID NO: 48)) hinge sequence. Thus, any antibody may contain the stabilizing "Adair" mutation or the amino acid sequence CPPCP (SEQ ID NO: 48). In one embodiment, the antibodies described herein contain a heavy chain immunoglobulin constant domain of human IgG4 with a backbone substitution of Ser to Pro that creates an IgG1-like hinge and allows for the formation of interchain disulfide bonds.

[0179] The antibodies of the present disclosure that selectively bind to RGMc may comprise an antibody constant region or a portion thereof. L The domain may be attached at its C-terminus to a light chain constant domain such as Cκ or Cλ. H The domain or fragment thereof may be attached to all or a portion of a heavy chain, such as IgA, IgD, IgE, IgG, and IgM, and any isotype subclass. The antibody may include an appropriate constant region (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Thus, antibodies within the scope of the present disclosure may include any of the V H and V LThe antibody or antigen-binding fragment thereof may comprise a heavy chain immunoglobulin constant domain comprising all or a fragment of a human IgG1 or human IgG4 constant domain. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain immunoglobulin constant domain comprising all or a fragment of a human Igλ constant domain or a human Igκ constant domain.

[0180] In some embodiments, the antibody that selectively binds to RGMc may or may not include a framework region of an antibody disclosed herein. In some embodiments, the antibody that selectively binds to RGMc is a murine antibody and includes a murine framework region sequence. In other embodiments, the antibody is a chimeric antibody, or an antigen-binding fragment thereof. In another embodiment, the antibody is a humanized antibody, or an antigen-binding fragment thereof. In another embodiment, the antibody is a fully human antibody, or an antigen-binding fragment thereof. In one embodiment, the antibody includes a framework region that includes a human germline amino acid sequence.

[0181] The antibodies, and antigen-binding fragments thereof, described herein may have any structure suitable for binding to an antigen. For example, in one embodiment, the antibody, or antigen-binding fragment thereof, comprises four polypeptide chains, including two heavy chain variable regions and two light chain variable regions. In another embodiment, the antibody, or antigen-binding fragment thereof, comprises one heavy chain variable region and one light chain variable region. In exemplary embodiments, the antibody, or antigen-binding fragment thereof, is a Fab fragment, a F(ab')2 fragment, a scFab fragment, a scFv, or a diabody.

[0182] In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain immunoglobulin constant domain of a human IgG1 constant domain or a human IgG4 constant domain. In an exemplary embodiment, the heavy chain immunoglobulin constant domain is a human IgG4 constant domain. In one embodiment, the antibody, or antigen-binding fragment thereof, binds to a conformational epitope. In one embodiment, the antibody, or antigen-binding fragment thereof, binds to a combinatorial or discontinuous epitope.

[0183] In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain immunoglobulin constant domain of a human IgG4 constant domain with a Ser to Pro backbone substitution creating an IgG1-like hinge and allowing interchain disulfide bond formation, In one embodiment, the antibody, or antigen-binding fragment thereof, further comprises a light chain immunoglobulin constant domain comprising a human Igλ constant domain or a human Igκ constant domain.

[0184] In one embodiment, the antibody is an IgG having four polypeptide chains, two heavy chains and two light chains. In an exemplary embodiment, the antibody may be a humanized antibody, a human antibody, or a chimeric antibody. In one embodiment, the antibody comprises a framework having a human germline amino acid sequence.

[0185] In one embodiment, the invention provides an antibody or antigen-binding fragment thereof that competes for binding with an antibody or antigen-binding fragment thereof described herein. In one embodiment, the invention provides an antibody or antigen-binding fragment thereof that binds to the same epitope as an antibody or antigen-binding fragment thereof described herein.

[0186] In another aspect of the invention, a class of RGMc-specific antibodies is provided that do not bind to RGMa and / or RGMb, respectively. Antibodies that specifically bind to RGMc (e.g., antibodies described herein) have a K of ≦5 nM (e.g., ≦5 nM, ≦4 nM, ≦3 nM, ≦2 nM, ≦1 nM, ≦0.5 nM, ≦0.1 nM, and ≦0.05 nM).D In one embodiment, the antibody binds with a K of ≦5 nM. D In another embodiment, the antibody binds to human RGMc with a K of ≦4 nM. D In another embodiment, the antibody binds to human RGMc with a K of ≦3 nM. D In another embodiment, the antibody binds to human RGMc with a K of ≦2 nM. D In another embodiment, the antibody binds to human RGMc with a K of ≦1 nM. D In another embodiment, the antibody binds to human RGMc with a K of ≦0.5 nM. D In another embodiment, the antibody binds to human RGMc with a K of ≦0.1 nM. D In another embodiment, the antibody binds to human RGMc with a K of ≦0.05 nM. D Those skilled in the art are familiar with suitable techniques and assays which may be used to determine binding activity in vitro.

[0187] In a preferred embodiment, suitable means for measuring the affinity of an antibody to its antigen(s) include, but are not limited to, the so-called Biolayer Interferometry (BLI) technology and Surface Plasmon Resonance (SPR) based assays. The former includes the Octet® system and the latter includes the Biacore system. In another embodiment, suitable means for measuring the affinity of an antibody to its antigen(s) include Meso Scale Discovery (MSD).

[0188] Aspects of the disclosure relate to specific antibodies, or antigen-binding fragments thereof, provided herein, such as antibodies that compete or cross-compete with any of the antibodies having one or more CDR sequences (1, 2, 3, 4, 5, or 6 CDR sequences) described above. In one embodiment, the invention provides antibodies, or antigen-binding fragments thereof, that compete or cross-compete with an antibody having heavy chain CDR sequences set forth in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, and / or light chain CDR sequences set forth in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11. In one embodiment, the invention provides antibodies that compete or cross-compete with an antibody, or antigen-binding fragments thereof, that has a heavy chain variable region sequence comprising SEQ ID NO:12, and / or a light chain variable region sequence comprising SEQ ID NO:13.

[0189] In one embodiment, the invention provides antibodies, and antigen-binding fragments thereof, that compete or cross-compete with an antibody having the heavy chain CDR sequences set forth in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, and / or the light chain CDR sequences set forth in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. In one embodiment, the invention provides antibodies that compete or cross-compete with an antibody, or antigen-binding fragments thereof, having a heavy chain variable region sequence comprising SEQ ID NO: 20, and / or a light chain variable region sequence comprising SEQ ID NO: 21.

[0190] In one embodiment, the invention provides antibodies, and antigen-binding fragments thereof, that compete or cross-compete with an antibody having the heavy chain CDR sequences set forth in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, and / or the light chain CDR sequences set forth in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27. In one embodiment, the invention provides antibodies that compete or cross-compete with an antibody, or antigen-binding fragments thereof, having a heavy chain variable region sequence comprising SEQ ID NO:28, and / or a light chain variable region sequence comprising SEQ ID NO:29.

[0191] In one embodiment, the invention provides antibodies, and antigen-binding fragments thereof, that compete or cross-compete with an antibody having the heavy chain CDR sequences set forth in SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, and / or the light chain CDR sequences set forth in SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35. In one embodiment, the invention provides antibodies that compete or cross-compete with an antibody, or antigen-binding fragments thereof, having a heavy chain variable region sequence comprising SEQ ID NO: 36, and / or a light chain variable region sequence comprising SEQ ID NO: 37.

[0192] In one embodiment, the invention provides antibodies, and antigen-binding fragments thereof, that compete or cross-compete with an antibody having the heavy chain CDR sequences set forth in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, and / or the light chain CDR sequences set forth in SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43. In one embodiment, the invention provides antibodies that compete or cross-compete with an antibody, or antigen-binding fragments thereof, having a heavy chain variable region sequence comprising SEQ ID NO: 44, and / or a light chain variable region sequence comprising SEQ ID NO: 45.

[0193] In some embodiments, an antibody, or antigen-binding fragment thereof, binds at or near the same epitope as any of the antibodies provided herein. In some embodiments, an antibody, or antigen-binding fragment thereof, binds near an epitope if it binds within 15 amino acid residues of the epitope. In some embodiments, any antibody, or antigen-binding fragment thereof, provided herein binds within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues of the epitope bound by any of the antibodies provided herein.

[0194] In another embodiment, the equilibrium dissociation constant (K D ) is 10 -8Provided herein are antibodies, or antigen-binding fragments thereof, that compete or cross-compete for binding to any of the antigens provided herein (e.g., human RGMc) with less than 10 M. In other embodiments, the antibodies are -11 M~10 -8 K within M D In some embodiments, provided herein is an RGMc-specific antibody or antigen-binding fragment thereof that competes for binding with an antibody or antigen-binding fragment thereof described herein. In some embodiments, provided herein is an RGMc-specific antibody or antigen-binding fragment thereof that binds to the same epitope as an antibody or antigen-binding fragment thereof described herein.

[0195] The antibody provided herein can be characterized using any suitable method. For example, one method is to identify the epitope that an antigen binds, i.e. "epitope mapping". There are many suitable methods for mapping and characterizing the location of an epitope on a protein, including crystal structure analysis of antibody-antigen complexes, competitive assays, gene fragment expression assays, and synthetic peptide-based assays (e.g., as described in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999). In a further example, epitope mapping can be used to determine the sequence that an antibody binds to. An epitope can be a linear epitope, i.e., comprised of a stretch of amino acids, or a conformational epitope formed by the three-dimensional interaction of amino acids, which does not necessarily comprise a stretch (linear sequence of primary structure).

[0196] Peptides of different lengths (e.g., at least 4-6 amino acids long) may be isolated or synthesized (e.g., recombinantly) and used in binding assays with antibodies. In another example, the epitope to which an antibody binds may be determined in a systematic screen by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody. According to gene fragment expression assays, an open reading frame encoding a target antigen is fragmented either randomly or by specific gene construction and the degree of reactivity of the expressed antigen fragments with the antibody to be tested is determined. Gene fragments may be produced, for example, by PCR and then transcribed and translated into protein in vitro in the presence of radioactive amino acids. Binding of the antibody to the radiolabeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis. Specific epitopes may be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, defined libraries of overlapping peptide fragments may be tested for binding to a test antibody in a simple binding assay. In further examples, mutagenesis of antigen-binding domains, domain swapping experiments and alanine scanning mutagenesis can be performed to identify residues necessary, sufficient and / or essential for epitope binding.For example, domain swapping experiments can be performed using mutants of target antigens in which various fragments of RGMc are replaced (swapped) with sequences from closely related but antigenically different proteins, such as another member of the RGMc protein family (e.g., RGMa or RGMb).The importance of specific antigen fragments to antibody binding can be evaluated by evaluating the binding of the antibody to mutant RGMc.

[0197] Alternatively, a competition assay can be performed with another antibody known to bind to the same antigen to determine if one antibody binds to the same epitope as the other antibody. Competition assays are well known to those of skill in the art.

[0198] Furthermore, the interaction of any antibody provided herein with one or more residues in RGMc can be determined by routine techniques. For example, a crystal structure can be determined, thereby determining the distance between a residue in RGMc and one or more residues in an antibody (or antigen-binding fragment). Based on such distance, it can be determined whether a particular residue in RGMc interacts with one or more residues in an antibody. Furthermore, the preferential binding of a candidate antibody can be determined using suitable methods such as competition assays and targeted mutagenesis assays.

[0199] In some embodiments, the antibodies or antigen-binding fragments thereof of the invention that selectively bind to RGMc comprise one or more of the complementarity determining regions (CDRs) described herein. In some embodiments, the invention provides nucleic acid molecules encoding antibodies or antigen-binding fragments thereof that selectively bind to RGMc as described herein. In one embodiment, the nucleic acid molecule encodes one or more of the CDR sequences described herein.

[0200] [RGMc-specific antibody binding region by H / DX-MS] In the context of this disclosure, the "binding region(s)" of an antigen provides the structural basis for antibody-antigen interaction. As used herein, "binding region" refers to the interface region between the antibody and antigen where the antibody or fragment protects the binding region from solvent exposure when bound to RGMc in physiological solution (as determined by a suitable technique such as hydrogen / deuterium exchange mass spectrometry (H / DX-MS)).

[0201] The art is familiar with H / DX-MS, a technique that is widely used to investigate protein conformation or protein-protein interactions in solution. This method relies on the exchange of hydrogens in protein backbone amides with deuterium present in solution. By measuring the rate of hydrogen-deuterium exchange, information about protein dynamics and conformation can be obtained (reviewed in Wei et al. (2014) "Hydrogen / deuteriumex change mass spectrometry for probing higher order structure of protein therapeutics: methodology and applications." Drug Disco Today. 19(1):95-102; incorporated by reference). The application of this technique is based on the premise that upon formation of an antibody-antigen complex, the interface between the binding partners may occlude the solvent, thereby reducing or preventing the rate of H / D exchange due to steric hindrance of the solvent.

[0202] Using this technique, the binding region of RGMc by the antibody (or fragment such as Fab) can be determined. In some embodiments, the portion on human RGMc identified as important for binding to an antibody or fragment that meets the selection criteria set forth herein comprises at least a portion of the amino acid stretch YVSSTLSL (SEQ ID NO: 46) present within the α-1 helix domain of RGMc. The same region may be identified as amino acid residues 46-53 on full-length RGMc (SEQ ID NO: 1). As shown in FIG. 8B, this binding region shows sequence diversity among RGM family members. Thus, without being bound by a particular theory, it is believed that this first binding region (region A) may contribute to the specificity of RGMc over RGMa and RGMb. In some embodiments, the epitope for the antibody (or antigen-binding fragment) comprises at least one amino acid residue of the binding region, YVSSTLSL. In some embodiments, the epitope for the antibody (or antigen-binding fragment) comprises two or more amino acid residues of the binding region.

[0203] In some embodiments, the portion on human RGMc identified as important for binding to an antibody or fragment that meets the selection criteria set forth herein comprises at least a portion of the amino acid stretch FHSAVHGIEDL (SEQ ID NO: 47) present within the alpha-3 helix domain of RGMc. As shown in the RGMc / BMP2 structural model (FIG. 8C), the alpha-3 helix is ​​in close proximity to BMP2. Thus, without being bound by any particular theory, it is believed that this second binding region (region B) may contribute to the functional (e.g., inhibitory) effect of the BMP competitive antibodies described herein. In some embodiments, the epitope for the antibody (or antigen-binding fragment) comprises at least one amino acid residue of the binding region, FHSAVHGIEDL. In some embodiments, the epitope for the antibody (or antigen-binding fragment) comprises two or more amino acid residues of the binding region.

[0204] Several observations support the notion that SR-RC-AB3-derived antibodies (eg, SR-RC-AB9) bind to at least one amino acid within region A and / or region B of RGMc.

[0205] First, based on the HDX-MS data of SR-RC-AB9 Fab and RGMc, regions A and B are relatively flexible and solvent accessible. Therefore, they may be available for binding to antibodies. The relative deuterium uptake of these regions in the unbound form of RGMc showed a relative deuterium uptake of about 50-60%. Less accessible regions usually have a relative deuterium uptake of less than 30%. Given the high conformational dynamics of the N-terminal domain of RGMc, it is conceivable that the domain could adopt several conformations that would increase the possibility of antibody binding (see, for example, Figure 8C, PDB ID 4UI1, RGMc bound to BMP2).

[0206] Second, regions A and B also have several contact points for BMP2 (see Figures 8D and 8E), suggesting that these regions are "hot spots" for binding interactions. Furthermore, looking more closely at regions A and B, several residues may serve as structural determinants of RGMc specificity over RGMa and RGMb (see Figure 8E).

[0207] Third, the CDRs (H1-H3) of the antibody can span the surface of regions A and B of the N-terminal domain of RGMc (see FIG. 8F). This may suggest that at least one (e.g., one or more) residues in each region participate as an epitope for antibody binding. Furthermore, the shape of the deuterium uptake curve (FIG. 8A) showed significant H / D exchange over the time points, suggesting that the antibody-antigen interaction within regions A and B is fairly stable.

[0208] Finally, the binding data for RGMc SR-RC-AB9 fab and the N-terminus of RGMc may also shed light on the idea that both helices (1&3) may be necessary for antibody binding. In particular, the high binding affinity of SR-RC-AB9 to RGMc suggests that there should be significant interfacial buried surfaces of the antigen / antibody interaction. These highly buried surface regions may be possible if both regions A (helix 1) and B (helix 3) are involved in binding to the antibody.

[0209] Thus, in one embodiment, an RGMc-specific antibody of the invention binds to at least one amino acid residue of YVSSTLSL (SEQ ID NO: 46). In another embodiment, an RGMc-specific antibody of the invention binds to at least one amino acid residue of FHSAVHGIEDL (SEQ ID NO: 47). In another embodiment, an RGMc-specific antibody of the invention binds to at least one amino acid residue of YVSSTLSL (SEQ ID NO: 46) and / or at least one amino acid residue of FHSAVHGIEDL (SEQ ID NO: 47). In another embodiment, an RGMc-specific antibody of the invention binds to at least one amino acid residue of YVSSTLSL (SEQ ID NO: 46) and at least one amino acid residue of FHSAVHGIEDL (SEQ ID NO: 47). In another embodiment, an RGMc-specific antibody binds to a discontinuous epitope comprising at least one amino acid of each of a first and a second binding region in human RGMc, wherein the first binding region comprises the amino acid sequence set forth in SEQ ID NO: 46; and / or the second binding region comprises the amino acid sequence set forth in SEQ ID NO: 47. In a particular embodiment, the RGMc-specific antibody binds to a discontinuous epitope comprising at least one amino acid from each of a first and second binding region in human RGMc, wherein the first binding region comprises the amino acid sequence set forth in SEQ ID NO: 46; and the second binding region comprises the amino acid sequence set forth in SEQ ID NO: 47.

[0210] In some embodiments, the human RGMc-specific antibodies provided herein bind to at least a portion of the binding region (region A) having the amino acid sequence YVSSTLSL (SEQ ID NO: 46). In another embodiment, the RGMc-specific antibodies provided herein bind to at least a portion of the binding region (region B) having the amino acid sequence FHSAVHGIEDL (SEQ ID NO: 47). In another embodiment, the RGMc-specific antibodies provided herein bind to at least a portion of the binding region having the amino acid sequence YVSSTLSL (SEQ ID NO: 46) and / or at least a portion of the binding region having the amino acid sequence FHSAVHGIEDL (SEQ ID NO: 47). In another embodiment, the RGMc-specific antibodies provided herein bind to at least a portion of the binding region having the amino acid sequence YVSSTLSL (SEQ ID NO: 46) and at least a portion of the binding region having the amino acid sequence FHSAVHGIEDL (SEQ ID NO: 47), or portions thereof. In another embodiment, the RGMc-specific antibody binds to a discontinuous epitope comprising at least a portion of a first and a second binding region in human RGMc, wherein the first binding region comprises the amino acid sequence set forth in SEQ ID NO: 46; and / or the second binding region comprises the amino acid sequence set forth in SEQ ID NO: 47. In a particular embodiment, the RGMc-specific antibody binds to a discontinuous epitope comprising at least a portion of a first and a second binding region in human RGMc, wherein the first binding region comprises the amino acid sequence set forth in SEQ ID NO: 46; and the second binding region comprises the amino acid sequence set forth in SEQ ID NO: 47.

[0211] In some embodiments, the RGMc-specific antibody of the invention binds to a discontinuous epitope in human RGMc, where a) the antibody is a full-length antibody or an antigen-binding fragment thereof; b) the antibody binds to a first binding region comprising at least one amino acid residue of YVSSTLSL (SEQ ID NO: 46). In another embodiment, the RGMc-specific antibody of the invention binds to a discontinuous epitope in human RGMc, where a) the antibody is a full-length antibody or an antigen-binding fragment thereof; b) the antibody binds to a second binding region comprising at least one amino acid residue of FHSAVHGIEDL (SEQ ID NO: 47). In another embodiment, the RGMc-specific antibody of the invention binds to a discontinuous epitope in human RGMc, where a) the antibody is a full-length antibody or an antigen-binding fragment thereof; b) the antibody binds to a first binding region comprising at least one amino acid residue of YVSSTLSL (SEQ ID NO: 46); and c) the antibody binds to a second binding region comprising at least one amino acid residue of FHSAVHGIEDL (SEQ ID NO: 47).

[0212] [Nucleic acid] In some embodiments, the antibodies, antigen-binding fragments thereof, and / or compositions of the present disclosure may be encoded by a nucleic acid molecule. Such nucleic acid molecules include, but are not limited to, DNA molecules, RNA molecules, polynucleotides, oligonucleotides, mRNA molecules, vectors, plasmids, etc. In some embodiments, the present disclosure may include cells that are programmed or produced to express nucleic acid molecules encoding the compounds and / or compositions of the present disclosure.

[0213] In some embodiments, the present invention provides a nucleic acid molecule encoding the aforementioned antibody, or an antigen-binding fragment thereof. For example, in one embodiment, the present invention provides a nucleic acid molecule encoding a polypeptide comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or a combination thereof, as described herein. The nucleic acid molecule may, in some embodiments, encode a polypeptide comprising CDRH1, CDRH2, and CDRH3 as described herein. In some embodiments, the nucleic acid molecule may encode a polypeptide comprising CDRL1, CDRL2, and CDRL3 as described herein. In some embodiments, the nucleic acid molecule encodes a polypeptide that may comprise up to 5, 4, 3, 2, or 1 amino acid residue variation compared to the corresponding CDR region in any one of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or a combination thereof, as described herein.

[0214] In one embodiment, the nucleic acid molecule encodes a polypeptide comprising a heavy chain variable domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:12 and / or a light chain variable domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:13. In one embodiment, the nucleic acid molecule encodes a polypeptide comprising a heavy chain variable domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:20 and / or a light chain variable domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:21. In one embodiment, the nucleic acid molecule encodes a polypeptide comprising a heavy chain variable domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:28 and / or a light chain variable domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:29. In one embodiment, the nucleic acid molecule encodes a polypeptide comprising a heavy chain variable domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:36 and / or a light chain variable domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:37.In one embodiment, the nucleic acid molecule encodes a polypeptide comprising a heavy chain variable domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:44 and / or a light chain variable domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:45.

[0215] In some embodiments, the nucleic acid molecule encodes an antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 36, SEQ ID NO: 44, and a light chain variable domain amino acid sequence set forth in SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 29, SEQ ID NO: 37, or SEQ ID NO: 45. In some embodiments, the nucleic acid molecule encodes an antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain amino acid sequence set forth in SEQ ID NO: 12, and a light chain variable domain amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the nucleic acid molecule encodes an antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain amino acid sequence set forth in SEQ ID NO: 20, and a light chain variable domain amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the nucleic acid molecule encodes an antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain amino acid sequence set forth in SEQ ID NO: 28, and a light chain variable domain amino acid sequence set forth in SEQ ID NO: 29. In some embodiments, the nucleic acid molecule encodes an antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain amino acid sequence set forth in SEQ ID NO: 36, and a light chain variable domain amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, the nucleic acid molecule encodes an antibody, or an antigen-binding fragment thereof, comprising the heavy chain variable domain amino acid sequence set forth in SEQ ID NO:44 and the light chain variable domain amino acid sequence set forth in SEQ ID NO:45.

[0216] In some cases, the nucleic acids of the present disclosure include codon-optimized nucleic acids. Methods for making codon-optimized nucleic acids are known in the art and may include, but are not limited to, those described in U.S. Patent Nos. 5,786,464 and 6,114,148, the contents of each of which are incorporated herein by reference in their entirety.

[0217] [Production of antibodies that bind to RGMc] The present invention encompasses methods of screening for, producing and manufacturing processes for antibodies or fragments thereof that bind with high affinity to RGMc and not to RGMa and / or RGMb, as well as pharmaceutical compositions and related kits containing same. The art is familiar with various techniques and methods that can be used to obtain the antibodies of the present disclosure, or antigen-binding fragments thereof. For example, antibodies can be produced using recombinant DNA methods, hybridoma technology, phage or yeast display technology, transgenic animals (e.g., XenoMouse®), or any combination thereof.

[0218] i. Immunization and Hybridomas In some methods described herein, a particular antigen (e.g., an RGMc peptide or protein, e.g., a soluble RGMc peptide or protein) can be used to immunize a non-human animal ("host"), e.g., a rodent, e.g., a mouse, hamster, or rat. In one embodiment, the non-human animal is a mouse. In another embodiment, the host can be a camelid. In a further embodiment, the host can be a shark.

[0219] After immunization, which may include single or multiple steps of antigen exposure (e.g., injection), splenocytes are harvested from the animal and relevant B cells are fused with immortalized myeloma cells to form hybridomas for antibody production. Hybridomas can be produced according to known methods (see, e.g., Kohler and Milstein (1975) Nature, 256:495-499). Hybridomas formed in this manner are then screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA), Bio-Layer Interferometry (BLI) technology (e.g., OCTET) and surface plasmon resonance (e.g., BIACORE) analysis, to identify one or more hybridomas that produce antibodies that specifically bind to the particular antigen. Any form of a particular antigen may be used as an immunogen, such as recombinant antigens, naturally occurring forms, any variants or fragments thereof, as well as antigenic peptides thereof (e.g., any epitope described herein as a linear epitope or within a scaffold as a conformational epitope).

[0220] ii. Screening library(s) In some embodiments, a method or process for generating or identifying an antibody includes screening a protein expression library, such as a phage, yeast, or ribosome display library, that expresses antibodies or fragments thereof (e.g., scFvs). For example, a library of human combinatorial antibodies or scFv fragments can be synthesized on phage or yeast, and the library can then be screened with an antigen of interest or an antibody-binding fragment thereof to isolate phage or yeast that bind the antigen from which an antibody or immunoreactive fragment can be obtained (Vaughan et al., 1996, PMID:9630891; Sheets et al., 1998, PMID:9600934; Boder et al., 1997, PMID:9181578; Pepper et al., 2008, PMID:18336206).

[0221] Phage display is further described, for example, in Ladner et al., U.S. Patent No. 5,223,409; Smith (1985) Science 228:1315-1317; Clackson et al. (1991) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol., 222:581-597; WO92 / 18619; WO91 / 17271; WO92 / 20791; WO92 / 15679; WO93 / 01288; WO92 / 01047; WO92 / 09690; and WO90 / 02809. Yeast display is further described, for example, in US 7,700,302 and US 8,877,688. In a particular method, a yeast display library expresses full-length antibodies.

[0222] Kits for generating phage or yeast display libraries are commercially available. There are other methods and reagents that can be used in generating and screening antibody display libraries (see US 5,223,409; WO 92 / 18619, WO 91 / 17271, WO 92 / 20791, WO 92 / 15679, WO 93 / 01288, WO 92 / 01047, WO 92 / 09690; and Barbas et al., 1991, PMID: 1896445). Such techniques advantageously allow for the screening of large numbers of candidate antibodies.

[0223] Regardless of how obtained, antibody-producing cells (e.g., yeast colonies, hybridomas, etc.) may be selected, cloned, and further screened for desirable characteristics, including, for example, robust growth, high antibody production, and desirable antibody characteristics / properties such as high affinity and specific binding to the antigen of interest. Preferably, such antibodies have a K of ≦5 nM, preferably ≦1 nM, more preferably ≦0.1 nM. D Thus, RGMc antibodies encompassed by the invention herein exhibit an in vitro binding activity with a K value for recombinant RGMc of ≦5 nM, preferably ≦1 nM, more preferably ≦0.1 nM. D It has a value.

[0224] Other selection criteria may include polyspecific reagent (PSR) score and / or hydrophobic interaction chromatography (HIC) retention time. PSR score is one way of determining the extent / degree of general binding (i.e., non-specific binding) to a protein. For example, in some cases, the polyspecific reagent (PSR) is a preparation of soluble membranes from 293 and / or CHO cells. Low (0.1-0.33) or zero (0-0.10) PSR scores are the most desirable profiles, with moderate (0.33-0.66) or high (0.66-1.00) scores being undesirable. Methods for determining PSR scores are well known to those of skill in the art. In some embodiments, the antibodies or anti-binding fragments thereof described herein have a PSR score of <0.1. In other embodiments, the antibodies or anti-binding fragments thereof described herein have a PSR score of 0.

[0225] On the other hand, HIC retention time is one way to determine the success of an antibody in self-interaction, which predicts whether the antibody will remain soluble / monodisperse in solution (also called aggregation tendency). A high HIC retention time indicates a greater tendency to aggregate. This property may be due to patches of hydrophobic residues / amino acids in the antibody. Alternatively, the presence of post-translational modifications (especially sugars) may result in an antibody having a low HIC retention time. Thus, in some embodiments, the antibodies or antigen-binding fragments described herein have a low HIC retention time (e.g., less than 10.5 minutes). In some embodiments, the antibodies or antigen-binding fragments described herein have a medium HIC retention time (e.g., 10.5 minutes or more and less than 11.5 minutes). In some embodiments, the antibodies or antigen-binding fragments described herein have a high HIC retention time (e.g., 11.5 minutes or more).

[0226] Methods for selecting, cloning and expanding colonies and / or hybridomas are well known to those of skill in the art. Once a desired antibody is identified, the relevant genetic material can be isolated, manipulated and expressed using common, art-recognized molecular biology and biochemical techniques.

[0227] iii. Humanization The antibody or fragment of the present invention is preferably a fully human antibody or a humanized antibody. Therefore, whatever the origin, the method may include a step of humanizing one or more antibodies or fragments thereof, where it is understood that human antibody sequences can be generated using molecular engineering techniques known in the art and introduced into the expression systems and host cells described herein. Such non-natural recombinantly produced human antibodies (and subject compositions) are fully compatible with the teachings of the present disclosure and are expressly included within the scope of the present invention. In certain selected embodiments, the RGMc antibody of the present invention comprises a recombinantly produced human antibody.

[0228] iv. Affinity Maturation and Optimization In some embodiments, the antibodies produced by the above-described methods have intermediate affinity (e.g., about 10 6 ~10 7 M -1 Ka or about 10 -6 ~10 -7 K of M D). Thus, the antibody or fragment thereof may be subjected to a process of affinity maturation as part of the optimization, if desired. The term "affinity maturation" has a meaning readily understood by a person skilled in the art. Briefly, it refers to further modifying the amino acid sequence of a candidate antibody or fragment (often called "parent" or "parental") to achieve an improved binding profile for a particular antigen. Typically, the parent antibody and its affinity matured counterpart (often called "progeny" or "offspring") retain the same epitope. A suitable in vitro binding assay may be performed at the appropriate step(s) during the affinity maturation process to screen for improved binders. Optionally, in some embodiments, a functional assay (e.g., a cell-based titer assay, an in vitro functional assay, etc.) may be performed to confirm the desired function.

[0229] Affinity maturation typically involves sequence diversification and / or mutagenesis, although the exact means of introducing or making the mutations or sequence changes is not limited. In some embodiments, mutagenesis involves the introduction of one or more changes (e.g., substitutions or deletions) in the amino acid residues of one or more CDRs. Thus, in some embodiments, the VR or CDR sequences described herein may contain up to 1, 2, 3, or 4 amino acid changes compared to a reference sequence (e.g., that of a parent sequence). In some embodiments, the VR or CDR sequences described herein may contain up to 1, 2, 3, or 4 amino acid substitutions. In some embodiments, the VR or CDR sequences described herein may contain up to 1, 2, 3, or 4 deletions. Additionally or alternatively, the process of mutagenesis may involve so-called oligo-walking of the variable regions or CDRs.

[0230] For example, affinity maturation of antibodies can be achieved by a variety of techniques, including random mutagenesis (Gram H., et al. Proc. Natl. Acad. Sci. USA (1992) 89, 3576-3580, and Hawkins RE, et al. J. Mol. Biol. (1992) 226, 889-896), random mutagenesis of CDR sequences, e.g., CDR walking (Yang WP, et al., J. Mol. Biol. (1995) 254, 392-403), directed mutagenesis of residues (Ho M., et al., J. Biol. Chem. (2005) 280, 607-617, and Ho M., et al. This can be achieved by several methods, including in vitro techniques to mimic somatic hypermutation (SHM) (Bowers PM, et al., Proc. Natl. Acad. Sci. USA (2006) 103, 9637-9642) and techniques to mimic somatic hypermutation (SHM) in vitro (Bowers PM, et al., Proc. Natl. Acad. Sci. USA (2011) 108, 20455-20460).

[0231] In one embodiment, an antibody may be affinity matured by mutagenesis on the variable heavy or variable light chain region. In another embodiment, an antibody may be affinity matured by mutagenesis on either the variable heavy or variable light chain CDRs. In another embodiment, an antibody may be affinity matured by mutagenesis on the variable heavy chain CDR3. In another embodiment, an antibody may be affinity matured by mutagenesis on the variable heavy chain CDR2. In another embodiment, an antibody may be affinity matured by mutagenesis on the variable heavy chain CDR1. In another embodiment, an antibody may be affinity matured by mutagenesis on the variable light chain CDR3. In another embodiment, an antibody may be affinity matured by mutagenesis on the variable light chain CDR2. In another embodiment, an antibody may be affinity matured by mutagenesis on the variable light chain CDR1.

[0232] In some embodiments, affinity maturation may involve screening an antibody library ("repertoire") containing variants of one or more CDRs, which may be combined with at least one CDR of a parent antibody. This process is often referred to as CDR shuffling or CDR diversification. In some embodiments, a variable heavy chain CDR3 (e.g., CDR-H3) may be used to screen a library containing a variable heavy chain CDR1 and CDR2 (e.g., CDR-H1 and CDR-H2) variant repertoire (also referred to as CDR-H1 / H2 diversification). In some embodiments, a variable light chain CDR3 (e.g., CDR-L3) may be used to screen a library containing a variable light chain CDR1 and CDR2 (e.g., CDR-L1 and CDR-L2) variant repertoire (also referred to as CDR-L1 / L2 diversification).

[0233] In some embodiments, affinity maturation involves screening an antibody library ("repertoire") containing light chain variants, which may be combined with the heavy chain of a parent antibody (light chain shuffling). For example, in some embodiments, selected heavy chains are introduced into an antibody library containing light chain variants, thereby creating a new antibody library that can be screened for improved affinity. In some embodiments, a repertoire of naturally occurring variable region variants may be obtained from a non-immunized donor. Examples of heavy or light chain shuffling are described in the following references: Marks et al., (1992) Nature Biotech 10:779-78; Schier et al., (1996) J. Mol. Biol. 255, 28-43; Park et al., (2000) BBRC. 275. 553-557; and Chames et al., (2002) J. Immunol 1110-1118. In some embodiments, the light chain library comprises variants of lambda light chains. In some embodiments, the light chain library comprises variants of kappa light chains. In some embodiments, the light chain library comprises variants of both lambda and kappa light chains.

[0234] It should be understood that the various methods for affinity maturation can be combined in any order. For example, in one embodiment, the selected antibody can be heavy chain CDR diversified, followed by CDR-H3 mutagenesis. In another embodiment, the selected antibody can be heavy chain CDR diversified, followed by CDR-H3 mutagenesis, followed by light chain shuffling. In another embodiment, the selected antibody can be heavy chain CDR shuffled, followed by light chain shuffled, followed by CDR-H3 mutagenesis. In a preferred embodiment, the selected antibody can be light chain shuffled, followed by CDR-H1 / H2 diversification, followed by CDR-H3 mutagenesis.

[0235] In any of the methods of affinity maturation described above, the resulting novel antibodies can be selected for binding to the target antigen (e.g., soluble RGMc) using known techniques (e.g., FACS). Using FACS, binding specificity and affinity can be tested by competition with different antigen concentrations and / or unlabeled (cold) antigen. Binding affinity can be further evaluated using other techniques known in the art, such as ELISA, BLI (e.g., OCTET), and SPR (e.g., BIACORE).

[0236] In addition to the above-mentioned affinity maturation process, further optimization can be performed to achieve the desired product profile. Thus, antibodies can be further subjected to optimization steps and selected based on certain favorable physicochemical properties. For therapeutic antibodies (biologics), physicochemical criteria for potential development that can be evaluated include, but are not limited to: solubility, stability, immunogenicity, no self-association or aggregation, Fc function, internalization profile, pH sensitivity, glycosylation and manufacturability, such as cell viability and / or gene expression. In some embodiments, the optimization process includes mutagenesis of one or more amino acid sequences in the constant region.

[0237] In one aspect, the invention provides a method of making a pharmaceutical composition comprising an antibody, or antigen-binding fragment thereof, that specifically binds to human RGMc and not to human RGMa or human RGMb; wherein the antibody, or antigen-binding fragment thereof, inhibits RGMc but not RGMa or RGMb, the method comprising the steps of: i) providing at least one antigen comprising human RGMc (e.g., soluble human RGMc); ii) selecting a first pool of antibodies, or antigen-binding fragments thereof, that specifically bind to human RGMc, thereby providing a specific binder of human RGMc; iii) selecting a second pool of antibodies, or antigen-binding fragments thereof, that inhibit BMP6 signaling associated with RGMc, thereby producing a specific inhibitor of RGMc / BMP6 activity; iv) formulating the antibodies, or antigen-binding fragments thereof present in the first pool of antibodies and the second pool of antibodies into a pharmaceutical composition, thereby making a composition comprising the antibody, or antigen-binding fragment thereof.

[0238] In a preferred embodiment, the method further comprises removing any antibodies or antigen-binding fragments thereof that bind to human RGMa and / or human RGMb from the first pool of antibodies or antigen-binding fragments thereof. In one embodiment, the method further comprises determining or confirming the specificity of the antibodies or antigen-binding fragments thereof selected in step (ii) and / or (iii). In one embodiment, the method further comprises selecting for antibodies or antigen-binding fragments thereof that are cross-reactive with human and rodent antigens. In one embodiment, the method further comprises generating fully human or humanized antibodies or antigen-binding fragments thereof of the antibodies or antigen-binding fragments thereof present in the first pool of antibodies and the second pool of antibodies. In one embodiment, the method further comprises subjecting the antibodies or antigen-binding fragments thereof present in the first pool of antibodies and the second pool of antibodies to affinity maturation and / or optimization, thereby providing affinity matured and / or optimized antibodies or fragments thereof.

[0239] In some embodiments, the method of making a pharmaceutical composition further comprises testing one or more candidate RGMc inhibitor(s) in at least one in vivo model to evaluate or confirm efficacy in achieving or correcting iron regulation. In some embodiments, the method of making a pharmaceutical composition further comprises testing one or more candidate RGMc inhibitor(s) (e.g., an antibody that shows efficacy in vivo) in at least one in vivo model to evaluate safety / tolerability at or above a therapeutically effective dose. A suitable therapeutic antibody should show acceptable toxicity at doses sufficiently higher than those effective in animal models. In some embodiments, testing of in vivo efficacy and / or safety may be performed by using an antibody(s) that includes a non-CDR portion(s) that more closely matches the particular species used in preclinical trials (e.g., a murine counterpart, etc.).

[0240] In another embodiment, monoclonal antibodies are obtained from non-human animals and then modified using suitable recombinant DNA techniques (e.g., chimeras are produced). Various techniques for producing chimeric antibodies have been described. See, for example, Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851, 1985; Takeda et al., Nature 314:452, 1985; Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al., European Patent Publication EP 171496; European Patent Publication 0173494, British Patent GB 2177096B.

[0241] For further antibody production techniques, see, e.g., Antibodies: A Laboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988. The present disclosure is not necessarily limited to any particular origin, method of production, or other particular characteristics of the antibodies.

[0242] Some aspects of the present disclosure relate to a host cell transformed by a polynucleotide or vector. The host cell may be a prokaryotic or eukaryotic cell. The polynucleotide or vector present in the host cell may be integrated into the genome of the host cell or may be maintained extrachromosomally. The host cell may be any prokaryotic or eukaryotic cell, for example, a bacterial, insect, fungal, plant, animal or human cell. In some embodiments, the fungal cell is, for example, of the genus Saccharomyces, in particular of the species S. cerevisiae. The term "prokaryote" includes all bacteria that can be transformed or transfected with DNA or RNA molecules for the expression of an antibody or a corresponding immunoglobulin chain. Prokaryotic hosts may include gram-negative and gram-positive bacteria, for example, Escherichia coli, Salmonella typhimurium, Serratia marcescens and Bacillus subtilis. The term "eukaryote" includes yeast, higher plants, insects and vertebrate cells, for example, mammalian cells such as NSO and CHO cells. Depending on the host employed in a recombinant production procedure, the antibody or immunoglobulin chain encoded by the polynucleotide may be glycosylated or may be non-glycosylated. The antibody or the corresponding immunoglobulin chain may include an initial methionine amino acid residue.

[0243] In some embodiments, once the vector has been incorporated into a suitable host, the host may be maintained under conditions suitable for high level expression of the nucleotide sequence, followed, as desired, by recovery and purification of the immunoglobulin light chain, heavy chain, light / heavy chain dimer or intact antibody, antigen-binding fragment or other immunoglobulin form; see Beychok, Cells of Immunoglobulin Synthesis, Academic Press, NY, (1979). Thus, the polynucleotide or vector is introduced into a cell, which then produces the antibody or antigen-binding fragment. Furthermore, transgenic animals, preferably mammals, containing the host cells described above may be used for large-scale production of antibodies or antibody fragments.

[0244] The transformed host cells may be grown in fermenters and cultured using any suitable technique to achieve optimal cell growth. Once expressed, whole antibodies, their dimers, individual light and heavy chains, other immunoglobulin forms, or antigen-binding fragments may be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like; see Scopes, "Protein Purification", Springer Verlag, NY (1982). The antibodies or antigen-binding fragments may then be isolated from the growth medium, cell lysates, or cell membrane fractions. Isolation and purification of antibodies or antigen-binding fragments expressed, for example, by microorganisms, may be by any conventional means, such as preliminary chromatographic separation and immunological separation (e.g., involving the use of monoclonal or polyclonal antibodies directed against the constant regions of the antibodies).

[0245] An embodiment of the present disclosure relates to hybridomas that provide indefinitely long-term sources of monoclonal antibodies. As an alternative to obtaining immunoglobulins directly from hybridoma cultures, immortalized hybridoma cells can be used as sources of rearranged heavy and light chain loci for subsequent expression and / or genetic engineering. Rearranged antibody genes can be reverse transcribed from appropriate mRNA to produce cDNA. In some embodiments, the heavy chain constant region can be exchanged for that of a different isotype or removed entirely. Variable regions can be combined to encode a single chain Fv region. Multiple Fv regions can be combined to confer binding capacity to more than one target, or chimeric heavy and light chain combinations can be used. Any suitable method can be used for cloning antibody variable regions and producing recombinant antibodies.

[0246] In some embodiments, suitable nucleic acids encoding the variable regions of the heavy and / or light chains are obtained and inserted into expression vectors that can be transfected into standard recombinant host cells. A variety of such host cells can be used. In some embodiments, mammalian host cells can be advantageous for efficient processing and production. Exemplary mammalian cell lines useful for this purpose include CHO cells, 293 cells, or NSO cells. Production of antibodies or antigen-binding fragments can be carried out by culturing the modified recombinant host under culture conditions suitable for host cell growth and expression of the coding sequence. Antibodies or antigen-binding fragments can be recovered by isolating them from the culture. Expression systems can be designed to include a signal peptide so that the resulting antibody is secreted into the medium; however, intracellular production is also possible.

[0247] The present disclosure also includes polynucleotides encoding at least the variable regions of the immunoglobulin chains of the antibodies described herein. In some embodiments, the variable regions encoded by the polynucleotides comprise at least one complementarity determining region (CDR) of the VH and / or VL variable regions of the antibodies produced by any one of the hybridomas described above.

[0248] The polynucleotide encoding the antibody or antigen-binding fragment may be, for example, DNA, cDNA, RNA, or synthetically produced DNA or RNA, or a recombinantly produced chimeric nucleic acid molecule, including any of these polynucleotides, either alone or in combination. In some embodiments, the polynucleotide is part of a vector. Such vectors may contain additional genes, such as marker genes, that enable the β vector to be expressed in a suitable host cell and under suitable conditions.

[0249] In some embodiments, the polynucleotide is operably linked to an expression control sequence that allows expression in prokaryotic or eukaryotic cells. Expression of the polynucleotide includes transcription of the polynucleotide into a translatable mRNA. Regulatory elements that ensure expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They may include regulatory sequences that facilitate transcription initiation, and may include poly-A signals that facilitate transcription termination and transcription stabilization. Further regulatory elements may include transcriptional and translational enhancers, and / or naturally associated or heterologous promoter regions. Possible regulatory elements that allow expression in prokaryotic host cells include, for example, PL, Lac, Trp or Tac promoters in E. coli, and examples of regulatory elements that allow expression in eukaryotic host cells are AOX1 or GAL1 promoters (yeast), or CMV promoter, SV40 promoter, RSV promoter (Rous sarcoma virus), CMV enhancer, SV40 enhancer or globin introns (mammalian and other animal cells).

[0250] Besides elements responsible for transcription initiation, such regulatory elements may also contain transcription termination signals downstream of the polynucleotide, such as the SV40-poly-A site or the tk-poly-A site. Furthermore, depending on the expression system used, leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the polynucleotide, as previously described. The leader sequence(s) are assembled in the appropriate phase with the translation, initiation and termination sequences, and preferably the leader sequence is capable of directing the secretion of the translated protein, or a part thereof, for example, into the extracellular medium. Heterologous polynucleotide sequences may also be used that code for fusion proteins containing C- or N-terminal identification peptides that confer desired properties, for example stabilization or simplified purification of the expressed recombinant product.

[0251] In some embodiments, the polynucleotides encoding at least the variable domains of the light and / or heavy chains may encode the variable domains of both or only one of the immunoglobulin chains. Similarly, the polynucleotides may be under the control of the same promoter and may be separately controlled for expression. Furthermore, some aspects relate to vectors, particularly plasmids, cosmids, viruses and bacteriophages, conventionally used in genetic engineering, that contain polynucleotides encoding the variable domains of the immunoglobulin chains of an antibody or antigen-binding fragment; they may be combined with a polynucleotide encoding the variable domain of the other immunoglobulin chain of the antibody.

[0252] In some embodiments, the expression control sequences are provided as eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, although control sequences for prokaryotic hosts may also be used. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, or bovine papilloma viruses may be used for delivery of polynucleotides or vectors to targeted cell populations (e.g., to engineer cells to express antibodies or antigen-binding fragments). Recombinant viral vectors may be constructed using a variety of suitable methods. In some embodiments, polynucleotides and vectors may be reconstituted into liposomes for delivery to target cells. Vectors containing polynucleotides (e.g., sequences encoding heavy and / or light variable domain(s) of immunoglobulin chains and expression control sequences) may be transferred into host cells by suitable methods that vary depending on the type of cellular host.

[0253] Thus, in one aspect of the invention, there is provided a method of making a pharmaceutical composition comprising an RGMc selective inhibitor (e.g., a neutralizing antibody), the method comprising the steps of: i) identifying an antibody or antigen-binding fragment for its ability to selectively bind RGMc over RGMa and RGMb; ii) identifying an antibody or antigen-binding fragment for its ability to inhibit / neutralize RGMc activity in vivo based on step (i); iii) selecting an inhibitory / neutralizing antibody for formulation into a pharmaceutical composition based on steps (i) and (ii). In one embodiment, step (i) comprises positive selection and may further comprise negative selection. In some embodiments, the identification step (i) comprises screening a library. In some embodiments, the library is a phage library or a yeast library. In some embodiments, the identification step (ii) comprises measuring an iron parameter selected from the group consisting of serum iron, total iron binding capacity (TIBC), unsaturated iron binding capacity (UIBC), and transferrin saturation. In some embodiments, step (ii) comprises measuring hepcidin expression. In some embodiments, hepcidin expression is liver hepcidin levels and / or serum hepcidin levels. In some embodiments, step (ii) comprises identifying neutralizing antibodies or antigen-binding fragments capable of increasing serum iron levels and / or suppressing hepcidin expression in vivo. In some embodiments, step (i) comprises selectively identifying RGMc over RGMa and RGMb.

[0254] In another embodiment, the antibody or antigen-binding fragment produced by the methods disclosed herein binds to an epitope other than the BMP6 binding site. In some embodiments, the epitope is other than the neogenin binding site. In some embodiments, the epitope is other than both the BMP6 binding site and the neogenin binding site. In some embodiments, the antibody or antigen-binding fragment preferentially binds to membrane-bound RGMc over soluble RGMc. In some embodiments, the antibody induces internalization of the antibody-antigen complex.

[0255] In another embodiment, the pharmaceutical compositions produced by the methods disclosed herein are formulated for intravenous or subcutaneous administration.

[0256] [Modification] The antibodies of the disclosure, or antigen-binding fragments thereof, may be modified with a detectable label or moiety, including, but not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, non-radioactive paramagnetic metal ions, and affinity labels for detection and isolation of RGMc. The detectable substance or moiety may be linked or conjugated directly to the polypeptides of the disclosure, or indirectly through an intermediate, such as a linker (e.g., a cleavable linker), using appropriate techniques. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, or acetylcholinesterase; non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; non-limiting examples of suitable fluorescent materials include biotin, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; non-limiting examples of bioluminescent materials include luciferase, luciferin, and aequorin; an example of a suitable radioactive material includes a radioactive metal ion, e.g., an α-emitter or other Radioisotopes, such as iodine (131I, 125I, 123I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115mIn, 113mIn, 112In, 111In), and technetium (99Tc, 99mTc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), and molybdenum (99Mo) , xenon (133Xe), fluorine (18F), 153Sm, Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 86R, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, and tin (113Sn, 117Sn).

[0257] The detectable substance may be linked or conjugated directly to an antibody of the present disclosure that specifically binds RGMc, or indirectly via an intermediate (such as a linker) using suitable techniques. Any of the antibodies provided herein that are conjugated to a detectable substance may be used in any suitable diagnostic assay, such as those described herein.

[0258] Additionally, the antibodies, or antigen-binding fragments thereof, of the present disclosure may be modified with a drug. The drug may be linked or conjugated directly to the polypeptides of the present disclosure or indirectly via an intermediate, such as a linker (e.g., a cleavable linker), using appropriate techniques.

[0259] [Pharmaceutical compositions and preparations] Further provided are pharmaceutical compositions / formulations for use as medicines suitable for administration in human and non-human subjects. For example, one or more RGMc-specific antagonists (e.g., antibodies) encompassed by the present invention can be formulated or mixed with a pharma- ceutically acceptable carrier (excipient), including, for example, a buffer, to form a pharmaceutical composition. Such formulations can be used for the treatment of diseases or disorders involving RGMc signaling. In a particularly preferred embodiment, the formulations can be used for the treatment of anemia as described herein.

[0260] The pharmaceutical composition of the present invention can be administered to patients to alleviate RGMc-related symptoms.For example, the RGMc-related symptoms can be iron-restricted anemia.The examples of iron-restricted anemia encompassed by the present invention include, but are not limited to, anemia of chronic disease (e.g., CKD), iron-refractory iron deficiency anemia (IRIDA), anemia induced by cancer, and / or anemia induced by chemotherapy.

[0261] "Acceptable" means that the carrier is compatible with (and preferably capable of stabilizing) the active ingredient of the composition and is not harmful to the subject being treated. Examples of pharma-ceutically acceptable excipients (carriers), including buffers, will be apparent to those skilled in the art and have been previously described. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover.

[0262] The pharmaceutical compositions used in this method may contain pharma- ceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover).

[0263] As used herein, "pharmaceutical acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound (e.g., antibody, immunoconjugate, or bispecific molecule) may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0264] The pharmaceutical compounds described herein may include one or more pharma- ceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see, for example, Berge, SM, et al. (1977) J.Pharm.Sci.66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids, such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like, and those derived from non-toxic organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, and the like, as well as those derived from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[0265] The pharmaceutical compositions described herein may contain pharma- ceutically acceptable antioxidants. Examples of pharma-ceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium hydrogen sulfate, sodium disulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0266] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0267] These compositions may contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured both by sterilization procedures (described above) and by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like, in the compositions. In addition, prolonged absorption of injectable formulations can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0268] Other acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin, gelatin, etc. The excipients may include proteins such as riboflavin, riboflavins, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

[0269] In some examples, the pharmaceutical compositions described herein include liposomes comprising antibodies that specifically bind to RGMc, which can be prepared by any suitable method, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al. Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with long circulation times are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be produced by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to produce liposomes with the desired diameter.

[0270] The antibodies described herein may be entrapped in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions, in microcapsules (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively), prepared, for example, by coacervation techniques or interfacial polymerization. Exemplary techniques have been previously described, see, e.g., Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[0271] In other examples, the pharmaceutical compositions described herein can be formulated in sustained release form.Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies, the matrices being in the form of shaped articles, such as films, or microcapsules.Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT™ (injectable microspheres made of lactic acid-glycolic acid copolymers and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0272] Pharmaceutical compositions used for in vivo administration must be sterile, which is readily accomplished, for example, by filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0273] The pharmaceutical compositions described herein may be in unit dosage form, such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.

[0274] To prepare solid compositions such as tablets, the main active ingredient may be mixed with pharmaceutical carriers, such as conventional tableting ingredients, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents (e.g., water) to form a solid preformulation composition containing a homogenous mixture of the compounds of the present disclosure, or their non-toxic pharmaceutically acceptable salts. When these preformulation compositions are referred to as homogenous, it is meant that the active ingredient is evenly distributed throughout the composition so that the composition can be easily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing 0.1 mg to about 500 mg of the active ingredient of the present disclosure. The tablets or pills of the novel composition may be coated or otherwise compounded to provide a dosage form that provides the benefit of extended action. For example, a tablet or pill may comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components may be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0275] Suitable surfactants include, inter alia, non-ionic agents such as polyoxyethylene sorbitans (e.g., Tween™ 20, 40, 60, 80 or 85) and other sorbitans (e.g., Span™ 20, 40, 60, 80 or 85). Compositions having a surfactant suitably contain 0.05-5% surfactant, and may be 0.1-2.5%. It will be appreciated that other ingredients, such as mannitol or other pharma- ceutically acceptable vehicles, may be added if necessary.

[0276] Suitable emulsions can be prepared using commercially available fat emulsions such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipiphysan™. The active ingredient may be dissolved in a premixed emulsion composition or alternatively in an oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and in an emulsion formed upon mixing of phospholipids (e.g. egg phospholipids, soybean phospholipids or soybean lecithin) with water. It is understood that other ingredients such as glycerol or glucose can be added to adjust the tonicity of the emulsion. Suitable emulsions typically contain up to 20% oil (e.g. 5-20%).

[0277] An emulsion composition may be prepared by mixing an antibody of the invention with Intralipid™ or its components (soybean oil, egg phospholipids, glycerol and water).

[0278] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharma- ceutically acceptable, aqueous or organic solvents, or mixtures thereof, as well as powders. The liquid or solid compositions may contain suitable pharma- ceutically acceptable excipients as set out above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

[0279] Compositions in preferably sterile pharma- ceutically acceptable solvents may be nebulized by the use of gases. Nebulized solutions may be directly inhaled from the nebulizing device, or the nebulizing device may be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from a device that delivers the formulation in an appropriate manner.

[0280] [Therapeutic use, treatment methods, patient populations] Abnormalities in iron homeostasis are associated with multiple diseases that can be difficult to diagnose and treat. Such disorders can be broadly classified into two categories: i) iron overload diseases, including cirrhosis, cardiomyopathy, diabetes, etc.; and ii) iron deficiency diseases, including anemia, anemia of chronic disease ("ACD"), etc. Iron deficiency anemia (IDA) can be classified into two main forms: absolute iron deficiency (AID) and functional iron deficiency (FID). AID is defined by reduced body iron stores, while FID is a disorder in which total body iron stores are normal or increased, but iron supply to the bone marrow (e.g., iron mobilization) is dysregulated or insufficient.

[0281] Current treatment options for iron-related diseases include external iron supplementation, such as IV iron, and ESA therapy (e.g., EPO and similar drugs). However, these treatments are associated with unwanted side effects. For example, it has been shown that chronically anemic patients who take frequent iron supplements, such as IV iron, can develop iron overload. Excess body iron can be highly toxic and can affect multiple organs, resulting in a variety of serious conditions, such as liver disease, heart disease, diabetes, hormonal abnormalities, and a dysfunctional immune system. Similarly, patients who receive blood transfusions are at risk for toxicity associated with iron overload. For example, in patients who receive a large number of transfusions, especially those with thalassemia major, sickle cell disease, myelodysplastic syndrome, aplastic anemia, hemolytic anemia, and refractory sideroblastic anemia, who may become transfusion-dependent, excess iron from transfused red blood cells (approximately 250 mg / transfusion) gradually accumulates in various tissues, causing morbidity and mortality. Thus, treatment-induced excess iron in the body can cause severe adverse reactions, including toxicity to the cardiovascular, gastrointestinal, immune, bone / cartilage, reproductive, and renal systems.

[0282] As an additional or alternative treatment option to IV iron, ESA therapy (e.g., EPO) is widely administered to a broad range of patient populations, including those suffering from cancer-related and chemotherapy-induced anemia. Paradoxically, however, recent preclinical and clinical studies have shown that ESAs can potentially accelerate tumor growth and threaten survival in cancer patients.

[0283] HIF stabilizers aim to stimulate the body's ability to produce endogenous erythropoietin to increase red blood cells through the HIF signaling cascade, instead of exogenously added erythropoietin or its equivalent. It is hypothesized that this approach could potentially reduce risk factors associated with ESA treatment, such as increased cancer progression and major negative cardiac events (e.g., stroke). However, to date, improved safety has not yet been clearly established.

[0284] Recently, a monoclonal antibody specifically binding to RGMa / RGMc was shown to increase serum iron in rats and cynomolgus monkeys by downregulating hepcidin (Boser et al. (2015) AAPS J. 2015 Jul;17(4):930-938). In this case, the researchers did not observe any obvious toxicity in healthy animals receiving RGMa / RGMc antibodies, and normalization of blood parameters was seen after a recovery period (approximately 12 weeks) following the administration period. However, potential risks associated with the simultaneous inhibition of RGMa on, for example, the nervous system and immunomodulatory effects are not known from the published study.

[0285] Thus, there is a need for improved therapies for achieving iron homeostasis that can be used effectively and safely to treat patients with diseases and disorders associated with an imbalance in iron homeostasis, such as anemia.

[0286] Thus, in one aspect, the present invention provides improved methods for treating and / or preventing conditions associated with disruptions in iron metabolism, particularly conditions associated with iron deficiency (e.g., anemia). RGMc selective inhibitors (e.g., monoclonal antibodies and antigen-binding fragments thereof that selectively bind and inhibit RGMc but not RGMa / b) can be used in treating conditions associated with functional iron deficiency in a subject. Therapeutic uses include administration of one or more RGMc selective inhibitors according to the present disclosure in an amount effective to treat the subject. Treatment may include reduction, alleviation, normalization or maintenance of one or more symptoms / parameters of anemia, such as serum iron level, transferrin saturation (TAST), reticulocyte hemoglobin content (CHr), reticulocyte count, red blood cell count, hemoglobin, and hematocrit. RGMc selective inhibitors may provide clinical benefit in terms of the extent of therapeutic effect and / or the kinetics or timing of such effect (e.g., faster relief, longer effect, etc.) RGMc selective inhibitors may provide clinical benefit in terms of reducing toxicity or adverse events associated with therapies aimed at treating anemia or the underlying disease causing anemia.

[0287] The condition that can be treated and / or prevented by using the method of the present invention includes any disease, disorder, or syndrome associated with iron metabolism disorder.Iron metabolism disorder can be associated with one or more of the following disorders: iron uptake, iron absorption, iron transport, iron storage, iron processing, iron mobilization, and iron utilization.Generally, iron metabolism disorder leads to iron overload or iron deficiency.

[0288] The disease or disorder of iron metabolism can be any disease or disorder in which iron homeostasis is disrupted in a subject. This homeostasis depends on the proper regulation of appropriate plasma iron levels. Iron circulates in plasma bound to transferrin, which is a vehicle for iron delivery to cells. 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.

[0289] Diseases associated with iron deficiency include, but are not limited to, anemia of chronic disease, iron deficiency anemia, absolute iron deficiency, functional iron deficiency, and microcytic anemia. Disruption of this iron homeostasis can also result in anemia of chronic disease, where diseased subjects show high levels of blood hepcidin. The term "anemia of chronic disease" (ACD) refers to any anemia that develops as a result of, for example, prolonged infection, inflammation, neoplastic disorder, etc. The anemia that develops is often characterized by short red blood cell life span and iron sequestration in macrophages, thereby reducing the amount of iron available to make new red blood cells. Conditions associated with anemia of chronic disease include, but are not limited to, chronic bacterial endocarditis, osteomyelitis, rheumatic fever, ulcerative colitis, chronic kidney disease, and neoplastic disorder.

[0290] Additionally, a subject having a disease associated with iron deficiency (e.g., ACD) may have or be at risk for a disease or disorder such as fatigue, joint pain, bone or joint disease (osteoarthritis, osteoporosis), rheumatoid arthritis, inflammatory bowel disease, shortness of breath, cardiac arrhythmias, liver disease, diabetes, infertility, frailty, depression, mood or mental disorders, poor cognitive ability or neurodegenerative diseases, iron-refractory iron deficiency anemia, anemia of chronic kidney disease, resistance to erythropoiesis stimulating agents, aplastic anemia, cancer, hypoplastic anemia, paroxysmal nocturnal hemoglobinuria, von Willebrand disease, and hemophilia hereditary hemorrhagic telangiectasia.

[0291] Further conditions include diseases and disorders associated with infection, inflammation, and neoplasia, including, for example, inflammatory infections (e.g., lung abscess, tuberculosis, etc.), inflammatory non-infectious disorders (e.g., rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, hepatitis, inflammatory bowel disease, etc.), and various cancers, tumors, and malignancies (e.g., carcinomas, sarcomas, lymphomas, etc.). Iron deficiency anemia can also result from chemotherapy treatments, or conditions such as blood loss due to, for example, pregnancy, menstruation, infancy and infancy, injury, etc.

[0292] Conditions associated with iron overload include both primary and secondary iron overload diseases, syndromes, or disorders, including, but not limited to, hereditary hemacromatosis, porphyria cutanea tarda, hereditary spherocytosis, hypochromic anemia, hysererythropoietic anemia (CDAI), congenital dyserythroid anemia (CDAII), faciogenital dysplasia (FGDY), Aarskog syndrome, atransferrinemia, sideroblastic anemia (SA), pyridoxine-responsive sideroblastic anemia, red blood cell enzymopathies, such as glucose-6-phosphate dehydrogenase (G6PD) or pyruvate kinase deficiency (PKD), and hemoglobinopathies, such as thalassemia and sickle cell. Several studies have suggested an association between iron metabolism disorders such as thalassemia and hemacromatosis and several disease conditions such as type II (non-insulin-dependent) diabetes and atherosclerosis (AJ Matthews et al., J. Surg. Res., 1997, 73:3540: TP Tuomainen et al., Diabetes Care, 1997, 20:426-428).

[0293] Research also suggests that iron metabolism plays a role in several other disease states, including cardiovascular disease (see, e.g., P. Tuo mainen et al., Circulation, 1997, 97:1461-1466; JM McCord, Circulation, 1991, 83:1112-1114; JL Sullivan, J. Clin. Epidemiol., 1996, 49:1345-1352).

[0294] A role for hepcidin and iron in neurological conditions has been proposed recently. For example, increased hepcidin levels have been shown in a variety of neurological diseases, including focal cerebral ischemia / reperfusion, intracerebral hemorrhage, subarachnoid hemorrhage, acute ischemic stroke, ischemic stroke, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and chronic mild stress (Vela et al., J Transl Med 2018;16:25).

[0295] Furthermore, iron metabolism may also play an important role in restless legs syndrome (Daubian-Nose P.et al.,Sleep Sci 2014;7(4):234-7 and Connor et al.,Sleep Medicine 2017;31:61-70). Indeed, increased pro-hepcidin levels have been shown in brain tissue of patients with restless legs syndrome, while reduced pro-hepcidin levels have been observed in cerebrospinal fluid in early-onset disease (Clardy et al.,J Neurol Sci 2006;247:173-9). Low iron depots in the brain of RLS patients are associated with higher expression of brain hepcidin (Rizzo G.et al,Mov Disord 2013;28:1886-90 and Clardy et al.,J Neurol Sci 2006;247:173-9). Low levels of iron in the brain of RLS patients persist even in the presence of HH (Haba-Rubio J.et al.,J Neurol Neurosurg Psychiatry 2005;76:1009-10). Furthermore, transferrin receptor expression in the brain microvasculature of RLS patients is low, suggesting low iron transport across the BBB (Connor JR et al.,Brain 2011;134:959-68). Interestingly, restless legs syndrome has been suggested as a comorbidity in rheumatoid arthritis, both diseases that are associated with iron dysregulation (John A.Gjevre and Regina M.Taylor Gjevre,Autoimmune Diseases 2013;Article ID 352782).

[0296] i. Iron refractory iron deficiency anemia (IRIDA) Iron-refractory iron deficiency anemia is an autosomal recessive disorder caused by mutations in Tmprss6. TMPRSS6 is a negative regulator of hepcidin by cleaving membrane-bound RGMc to produce soluble RGMc (s-RGMc), which acts as a decoy receptor for BMP6. Decreased BMP6 signaling leads to decreased hepcidin expression. Thus, mutations that inhibit TMPRSS6 function promote the accumulation of membrane-bound RGMc, thereby leading to increased hepcidin expression and iron deficiency anemia. Patients with IRIDA have too little iron in their blood, which causes their red blood cells to be smaller in size (microcytes) and paler in color (hypochromic). Other phenotypes of the disease may include low transferrin saturation, low serum ferritin, and low serum iron levels. Unfortunately, the high levels of hepcidin in these patients make them refractory to oral iron administration.

[0297] Given the well-defined mechanism of IRIDA that results in an increase in membrane-bound RGMc (and increased hepcidin levels), an agent that specifically binds to RGMc and reduces hepcidin levels while limiting potential side effects from targeting related RGM proteins (e.g., RGMa and RGMb) would be beneficial.

[0298] Thus, RGMc-selective inhibitors (e.g., monoclonal antibodies disclosed herein, derivatives thereof, monoclonal antibodies competing therewith, and engineered molecules including antigen-binding fragments thereof) can be used for the treatment of IRIDA in a subject (e.g., a human patient). Thus, in one aspect, a method for treating, preventing, or ameliorating symptoms associated with IRIDA is disclosed. In one embodiment, the method comprises administering to a patient with IRIDA an RGMc antagonist. In another embodiment, the method comprises administering to a patient with IRIDA an antibody that specifically binds to RGMc and inhibits its function. In another embodiment, the RGMc-specific antibody does not bind (or binds less) to RGMa and / or RGMb. In another embodiment, the RGMc-specific antibody is any one of the RGMc-specific antibodies described herein or any derivative thereof. In some embodiments, the RGMc-specific (RGMc-selective) antibody competes (e.g., cross-competes or cross-inhibits) for antigen binding with one or more of the RGMc antibodies disclosed herein.

[0299] ii. Anemia of Chronic Disease (ACD) Anemia of chronic disease (e.g., anemia of chronic inflammation) is a form of anemia that is the result of chronic infection, chronic immune activation (e.g., inflammation), and / or malignancy, and is the most common form of anemia in hospitalized patients.

[0300] Typically, ACD begins with an inflammatory response followed by inflammatory cytokine release that mediates disease progression. Cytokines reduce red blood cell production, promote red blood cell lysis, and stimulate macrophages to store and retain iron as ferritin, ultimately resulting in insufficient iron availability. In addition, there is a large increase in interleukin-6 (IL-6), which stimulates hepcidin expression, thereby inducing the degradation of ferroportin, thereby blocking iron release from macrophages and enterocytes to the environment. In addition to the inflammatory response, other mechanisms may also play a role in ACD, such as reduced red blood cell production by reducing the ability of bone marrow to respond to erythropoietin.

[0301] As suggested above, there are many conditions that can cause inflammation leading to anemia, including autoimmune diseases such as rheumatoid arthritis (RA) or lupus, cancer, chronic infections such as HIV / AIDS and tuberculosis, chronic kidney disease (CKD), gastrointestinal inflammatory conditions such as inflammatory bowel disease (IBD) including Crohn's disease and ulcerative colitis, diabetes, and heart failure.Inflammatory anemia generally progresses slowly, but anemia of severe disease can progress quickly in patients who are hospitalized for severe acute infection, trauma, or other conditions that can cause inflammation.In addition, certain drug treatments such as chemotherapy can rapidly induce inflammation and anemia.

[0302] Other anemia-associated inflammatory diseases may include, for example, Castleman syndrome, chronic kidney disease, diabetes, heart failure / heart disease, idiopathic autoimmune hemolytic anemia, idiopathic pulmonary arterial hypertension, inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus, and systemic juvenile idiopathic arthritis. Infectious diseases causing anemia may include, for example, endocarditis, Helicobacter pylori infection, hepatitis, human immunodeficiency virus, and malaria. Neurological diseases associated with anemia may include, for example, acute ischemic stroke, amyotrophic lateral sclerosis, focal cerebral ischemia / reperfusion, intracranial cerebral hemorrhage, and subarachnoid hemorrhage.

[0303] Thus, RGMc-selective inhibitors (e.g., monoclonal antibodies disclosed herein, derivatives thereof, monoclonal antibodies competing therewith, and engineered molecules including antigen-binding fragments thereof) can be used for the treatment of ACD (e.g., one or more of the ACD symptoms included herein) in a subject (e.g., a human patient). Thus, in one aspect, a method of treating (e.g., preventing or ameliorating symptoms associated therewith) anemia of chronic disease (ACD) in a human patient is disclosed. In some embodiments, the method comprises administering to a patient having ACD an RGMc antagonist. In some embodiments, the method comprises administering to a patient having ACD an antibody that specifically binds to RGMc and inhibits its function. In some embodiments, the RGMc-specific (RGMc-selective) antibody does not bind (or binds less to) RGMa and / or RGMb. In some embodiments, the RGMc-specific antibody is any one of the RGMc-specific antibodies described herein, or any derivative thereof. In some embodiments, an RGMc-specific (RGMc-selective) antibody competes (eg, cross-competes or cross-blocks) for antigen binding with one or more of the RGMc antibodies disclosed herein.

[0304] iii. Cancer-related anemia Approximately 30-90% of cancer patients are affected by anemia (Knight et al., Am J Med 2004;116 Supp l7A:11S-26S). For example, approximately 32% of non-Hodgkin's lymphoma patients and 49% of gynecologic cancer patients have anemia at diagnosis (Moullet et al., Ann Oncol 1998;8:1109-1115 and Ludwig et al., Eur J Cancer 2004;40:2293-2306). Anemia can be caused by factors related to the cancer itself (cancer-induced anemia) or can be caused as a result of chemotherapy (chemotherapy-induced anemia). Furthermore, radiation therapy to the skeleton has been shown to be associated with hematological toxicity (Jefferies et al., Radiother Oncol 1998;48:23-27). The pathology of anemia can be divided into three categories: 1) reduced production of functional red blood cells (RBCs); 2) increased destruction of RBCs; and 3) blood loss. However, the causes of anemia can be multifactorial, complicating evaluation and treatment. Anemia can result from bleeding, hemolysis, nutritional disorders, genetic diseases, renal failure, hormonal dysfunction, and / or combinations thereof. In addition, cancer cells themselves can cause and / or exacerbate anemia by infiltrating the bone marrow and producing cytokines that result in iron sequestration (resulting in reduced RBC production), shortened RBC survival, and / or altered coagulation.

[0305] In addition, cancer patients may also experience chronic blood loss at the tumor site due to vascular or organ damage. Other contributing factors to anemia in cancer patients may include appetite loss, immune-mediated antibody-induced hemolysis, or altered clotting ability.

[0306] Finally, anemia has also been identified as an adverse prognostic factor in cancer patients, for example, it is predicted that there is a 65% increased risk of death and a nearly four-fold higher average annual health care cost per patient. Therefore, the goal of treating anemia in cancer patients is to improve the patient's quality of life (QoL) and reduce reliance on blood transfusions. Blood transfusions are associated with potential risks of infectious disease transmission, transfusion reactions, lung injury, and alloimmunization. Transfusions can also increase the risk of mortality and morbidity, including stroke, myocardial infarction, acute renal failure, and cancer recurrence (Annals of Oncology 21(Supplement 7):vii167-vii172, 2010; PLOS ONE, DOI:10.1371 / journal.pone.0163817(Sept 28, 2016); Annals of Oncology 23:1954-1962, 2012; and Annals of Clinical & Laboratory Science, vol. 47, no. 2, 2017).

[0307] The term "cancer" as used herein refers to a physiological condition in multicellular eukaryotic organisms that is typically characterized by unregulated cell proliferation and malignant tumors.The term broadly encompasses tumors, solid and liquid malignancies, including hematological cancers (e.g., leukemia, lymphoma and myeloma), and myelofibrosis. Cancers that cause anemia may include, for example, acute leukemia, diffuse large B-cell lymphoma, Hodgkin's lymphoma, lymphocytic leukemia, multiple myeloma, myelodysplastic syndromes, myeloproliferative disorders (e.g., myelofibrosis), non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, bladder cancer, breast cancer, colorectal cancer, gastric cancer, head and neck cancer, hepatocellular carcinoma, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, brain tumors, hepatocellular carcinoma, renal cell carcinoma, testicular cancer, and thyroid cancer (Maccio et al., Haematologica 2015;100(1):124-132 and Vela et al., Exp&Mol Medicine 2018;50:e436.

[0308] Thus, RGMc selective inhibitors (e.g., monoclonal antibodies disclosed herein, derivatives thereof, competing monoclonal antibodies therewith, and engineered molecules including antigen-binding fragments thereof) can be used for the treatment of cancer-induced or cancer-related anemia in a subject (e.g., a human patient). Thus, in one aspect, a method for treating (e.g., preventing or ameliorating symptoms associated with) cancer-induced anemia. In some embodiments, the method comprises administering an RGMc antagonist to a patient having cancer-induced anemia. In some embodiments, the cancer is acute leukemia, diffuse large B-cell lymphoma, Hodgkin's lymphoma, lymphocytic leukemia, multiple myeloma, myelodysplastic syndrome, non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, bladder cancer, breast cancer, colorectal cancer, gastric cancer, head and neck cancer, hepatocellular carcinoma, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, brain cancer, renal cell carcinoma, testicular cancer, thyroid cancer, or myelofibrosis. In some embodiments, the method comprises administering to the patient an antibody that specifically binds to RGMc and inhibits its function. In some embodiments, the RGMc-specific antibody does not bind (or binds less) to RGMa and / or RGMb. In some embodiments, the RGMc-specific antibody is any one of the RGMc-specific antibodies described herein or any derivative thereof. In some embodiments, an RGMc-specific (RGMc-selective) antibody competes (eg, cross-competes or cross-blocks) for antigen binding with one or more of the RGMc antibodies disclosed herein.

[0309] iv. Treatment-induced anemia Various treatments administered to patients are associated with unwanted side effects, including anemia in the treated patient. In some cases, the patient may already suffer from anemia, but the therapy received may further exacerbate the condition.

[0310] A large proportion of patients undergoing cancer treatments, such as chemotherapy and radiation therapy, experience treatment-induced anemia. Typically, these therapies aim to kill cancerous cells in the body, but also affect healthy cells, including blood cells (e.g., hematopoietic cells and hematopoietic stem cells), thereby causing anemia.

[0311] Chemotherapeutic agents have been shown to induce anemia by directly impairing hematopoiesis in bone marrow. In addition, cytotoxic effects on renal function can cause anemia through reduced erythropoietin production. In particular, platinum-based regimens commonly used in lung, ovarian, and head and neck cancers are known to induce anemia through bone marrow and renal toxicity (Groopman et al., J Natl Cancer Inst 1999;91:1616-1634). Furthermore, repeated cycles of chemotherapy have also been shown to increase the severity of anemia in these patients (Ludwig et al., Eur J Cancer 2004;40:2293-2306).

[0312] Interestingly, recent data suggest that immunotherapy may also cause (and / or increase the risk of) anemia in cancer patients. For example, a recent study identified hemolytic anemia as a potential complication of treatment with nivolumab (anti-PD-1 antibody) (Palla et al., Case Rep Oncol 2016;9:691-697). Other cases of autoimmune hemolytic anemia have been reported following the use of nivolumab, including cases of anemia in patients receiving ipilimumab (anti-CTLA-4 antibody) and pembrolizumab (anti-PD-1 antibody). Thus, anemia caused by or with a high chance / risk of occurring due to the administration of anticancer drugs / drugs (including immunotherapy) is encompassed by the term "chemotherapy-induced anemia."

[0313] Finally, as mentioned above, the presence of anemia has been associated as a prognostic factor for cancer survival (Caro JJ et al., Cancer 2001 15;91(12):2214-21). Therefore, it makes sense to treat anemia in cancer patients or those undergoing chemotherapy, as it may result in improved outcomes. Furthermore, given the well-described relationship between cancer, chemotherapy, and anemia, the National Comprehensive Cancer Network (NCCN) regularly releases guidelines for the diagnosis, evaluation, and treatment of cancer- and chemotherapy-induced anemia (see, for example, Rodgers et al, Cancer-and Chemotherapy Induced Anemia, JNCCN 2012;10:628-53).

[0314] Thus, RGMc selective inhibitors (e.g., the monoclonal antibodies disclosed herein, their derivatives, competing monoclonal antibodies, and engineered molecules including antigen-binding fragments thereof) can be used for the treatment of therapy-induced anemia (e.g., chemotherapy-induced anemia) in a subject (e.g., a human patient).

[0315] Thus, in one aspect, a method is provided for treating, preventing, or ameliorating symptoms associated with chemotherapy-induced anemia, hi one embodiment, the method comprises administering an RGMc antagonist to a patient having chemotherapy-induced anemia.

[0316] In another embodiment, the method comprises administering to the patient an antibody that specifically binds to RGMc and inhibits its function. In another embodiment, the RGMc-specific antibody does not bind (or binds poorly) to RGMa and / or RGMb. In another embodiment, the RGMc-specific antibody is any one of the RGMc-specific antibodies or any derivatives thereof described herein. In some embodiments, the RGMc-specific (RGMc-selective) antibody competes for antigen binding (e.g., cross-competes or cross-inhibits) with one or more of the RGMc antibodies disclosed herein.

[0317] v. Chronic kidney disease (CKD) Anemia is a common complication associated with chronic kidney disease (CKD). In fact, approximately 14% of the US adult population had CKD in 2007-2010, and anemia was nearly twice as prevalent in people with CKD, 15.4% of CKD patients compared with 8.4% in the general population (Stauffer and Fan, PLoS One 2014;9:e84943).

[0318] While the kidneys are well known to act as blood filters, removing waste products, and controlling fluid and electrolyte balance, a lesser known role of the kidney is to produce erythropoietin, which stimulates red blood cell production. Thus, disruption of renal function has the potential to produce anemia. Furthermore, underlying inflammation in CKD patients may also produce and / or exacerbate anemia in these patients.

[0319] CKD is characterized by the gradual loss of kidney function over an extended period of time (months or years). Causes of CKD include, for example, diabetes, hypertension, glomerulonephritis, and polycystic kidney disease. However, idiopathic cases (i.e., the cause is unknown) also exist, which are often associated with small kidneys.

[0320] Treatment to slow or stop the progression of CKD usually entails treating the original disease, but blood pressure control with angiotensin-converting enzyme inhibitors or angiotensin II receptor antagonists is a common treatment, as it has been found to slow the progression of the disease.However, in severe cases (e.g., end-stage renal disease), patients must undergo dialysis or even kidney transplantation.Patients may be dialysis-dependent (e.g., chronic hemodialysis patients); patients may be non-dialysis-dependent.

[0321] CKD patients with anemia can be treated with oral or intravenous iron. In certain cases, treatment may include infusion and / or blood transfusion of a genetically engineered form of erythropoietin (EPO). However, iron replacement is not always effective; for example, some patients are or become hyporesponsive to ESA treatment. Furthermore, ESAs, such as EPO treatment, are associated with an increased chance of cardiovascular events, such as heart attacks and strokes. Therefore, improved treatments that can regulate iron levels with fewer side effects are needed.

[0322] Thus, RGMc selective inhibitors (e.g., monoclonal antibodies disclosed herein, derivatives thereof, competitive monoclonal antibodies, and engineered molecules including antigen-binding fragments thereof) may be used for the treatment of cancer-induced or cancer-related anemia in a subject (e.g., a human patient). Thus, in one aspect, a method is provided for treating, preventing, or ameliorating symptoms associated with chronic kidney disease (e.g., anemia). In one embodiment, the method comprises administering to a patient having chronic kidney disease an RGMc antagonist. In another embodiment, the method comprises administering to the patient an antibody that specifically binds to RGMc and inhibits its function. In another embodiment, the RGMc-specific antibody does not bind (or binds less to) RGMa and / or RGMb. In another embodiment, the RGMc-specific antibody is any one of the RGMc-specific antibodies described herein or any derivatives thereof. In some embodiments, an RGMc-specific (RGMc-selective) antibody competes (eg, cross-competes or cross-blocks) for antigen binding with one or more of the RGMc antibodies disclosed herein.

[0323] In any of the above embodiments in the treatment of anemia or a disease characterized by or associated with anemia, the RGMc inhibitor therapy described herein may partially or completely replace the current or standard therapy(s) administered to the patient, such as ESA, HIF stabilizer, iron supplement (e.g., IV iron) and blood transfusion.In some embodiments, the addition of RGMc inhibitor therapy in a therapeutic regimen according to the present disclosure allows the patient to receive such therapy at a reduced dose or frequency of administration.This may mitigate or avoid unwanted side effects or other risks, such as iron overload, in the patient.In some embodiments, the RGMc inhibitor therapy may completely replace other therapies for treating anemia in the patient.

[0324] In any of the above embodiments in the treatment of anemia or a disease characterized by or associated with anemia, the RGMc inhibitor therapy described herein can be used with another therapy as an add-on or adjuvant therapy.In certain embodiments, patients may receive or continue to receive other therapies to treat anemia, but at lower doses or less frequently.In some embodiments, patients who are resistant or poorly responsive to such therapies may become more responsive (e.g., sensitive) to the therapy when treated with an RGMc inhibitor together with the therapy.

[0325] Selection of Subjects for Treatment with RGMc Inhibitors The methods of treatment described herein may include measuring one or more factors to determine the presence and / or severity of a disease (e.g., anemia) in a subject. Generally, the definition of anemia is Hb<12g / dl in women and Hb<13g / dl in men. Although the measurement of hemoglobin is accepted as one measurement of anemia, determining the type (AID or FID) and cause of anemia may involve many different tests. For example, there are several well-known tests that may help determine the type and severity of anemia in a subject, including, but not limited to, the measurement of hemoglobin (Hb), serum ferritin (SF), transferrin saturation (TSAT), soluble transferrin receptor (sTfR), ferritin index (FI=sTfR / logSF), hypochromic reticulocytes (CHR), and C-reactive protein (CRP). Mean corpuscular volume (MCV) or mean corpuscular hemoglobin concentration (MCHC) are also measurements that can be correlated with the type of anemia.

[0326] The exact criteria for defining AID and FID are subject to debate and vary by study, disease, gender and authority. For example, without being bound by any particular theory, FID is defined as: 1) serum transferrin saturation (TSAT) < 20% and ferritin ≥ 100ng / mL (Ludwig et al., Ann Oncol 2013;24(7):1886-1892); 2) TSAT < 16% and ferritin > 100ng / ml (mild) or serum ferritin 30-100ng / ml (moderate) (Bach et al., Clinical Interventions In Aging 2014;9:1187-1196); 3) TSAT 20-50% and ferritin 30-800ng / ml (Gilreath et al., AmJ Hematol 2014;89(2):203-212); 4) TSAT < 20% and ferritin > 30ng / ml and > 100ng / ml (cancer patients) (Ludwig et al., Wien Klin Wochenschr 2015;127:907-919) or 5) TSAT<20% and ferritin>30ng / ml (female) or>40ng / ml (male) (Hedenus et al., Med Oncol 2014;31(12):302). In other examples, FID can be detected using hypochromic red blood cells (%) (see, e.g., Bovy et al., Nephrol Dial Tranplant 2007;22(4):1156-1162 and Murphy et al., Ann Hematol 2006;85(7):455-457). In another example, FID is defined as when a patient has Hb<110g / l, iron-limited red blood cell production (hypochromia (%)>5), and systemic inflammation (CRP>10) (they may have ferritin levels between 30-800ng / ml) (Neoh et al., Support Care Cancer 2017;25:1209-1214).

[0327] While not being bound by any particular theory, AID may be defined, for example, as 1) TSAT<20% and ferritin<30ng / ml (Ludwig et al., Ann Oncol 2013;24(7):1886-1892); 2) TSAT<20% and ferritin<100ng / ml (cancer patients) (Ludwig et al., Wien Klin Wochenschr 2015;127:907-919); or 3) TSAT<20% and ferritin levels<40ug / L (Hashemi et al., Int J Hematol Oncol Stem Cell Res 2017;11(3):192-198).

[0328] Therapeutic methods may involve the measurement of other markers described herein and known in the art, such as hepcidin (see, e.g., US20150202224A1 and Kroot et al. Clinical Chemistry 2011;57(12):1650-1669), neogenin, growth differentiation factor 15 (GDF-15), neutrophil gelatinase-binding lipocalin (NGAL), interleukin 6 (IL-6), and / or BMP6.

[0329] Additionally, identification of other risk factors and / or diseases may help determine the presence and type of anemia, for example, a patient may have an infection, cancer, or chronic kidney disease. Genetic testing may also be useful to determine certain iron-related diseases, such as iron-refractory iron deficiency anemia (IRIDA), thalassemia, hereditary spherocytosis, hereditary elliptocytosis, abetalipoproteinemia, sickle cell anemia, congenital dyserythroid anemia, hereditary hemacromatosis (HH), and juvenile hemacromatosis.

[0330] The subject in any of the methods described herein may be a subject who has, has been diagnosed with, is suspected of having, or is at risk of developing iron deficiency anemia or a disease, disorder, or condition associated with iron deficiency anemia. The subject may be a mammal, and may be human or non-human. In certain embodiments, the subject is a human subject. In another embodiment, the subject may be a human male subject. In another embodiment, the subject may be a human female subject. In another embodiment, the subject may be a critical care patient undergoing chemotherapy, recovering from a surgical procedure, or may be at risk for 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 or solid tumor. The autoimmune disease can 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 can be chronic kidney disease, and the subject can be or is not undergoing dialysis.The viral infection can be hepatitis B or C infection, or human immunodeficiency virus infection.Any disease and disorder can be the underlying cause of ACD.The surgical procedure can be perioperative or postoperative.The surgical procedure can be tumor surgical procedure.

[0331] Subjects in need of treatment are typically evaluated by a medical history and physical examination consistent with the disease or condition in question. Diagnosis of various conditions that can be treated by the methods described herein is within the skill of the art. The RGMc antagonists described herein can be administered to subjects in clinical need of iron delivery or to subjects with functional iron deficiency, such as subjects on erythropoietin therapy. The determination of the need for parenteral or iv iron treatment is within the ability of those skilled in the art. For example, the need can be assessed by monitoring the iron status of the subject. Diagnosis of iron deficiency can be based on appropriate laboratory tests as described above, such as Hg levels according to Table 2 or >2g / dl below baseline, and / or red blood cells with MCV less than 80 femtoliters (fL). Other measurements to diagnose iron deficiency may be based on the measurement of serum ferritin (SF), transferrin saturation (TSAT), soluble transferrin receptor (sTfR), ferritin index (FI=sTfR / logSF), hypopigmented reticulocytes (CHR), C-reactive protein (CRP), mean corpuscular volume (MCV) and / or mean corpuscular hemoglobin concentration (MCHC).

[0332] JPEG2025004154000007.jpg80153

[0333] The subject who needs treatment may be determined through the diagnosis of the subject who suffers from the disease, disorder or condition associated with iron deficiency anemia.For example, many chronic renal insufficiency patients who receive erythropoietin require iron supplements to maintain target iron levels.As another example, most hemodialysis patients require iron supplements due to the blood loss associated with dialysis and the resulting negative iron balance.

[0334] The frequency of monitoring can depend on the disease, disorder or condition that the subject suffers from or is at risk of.For example, in the subject who starts erythropoietin therapy, iron index can be monitored monthly.As another example, in the subject who achieves target Hb range or the subject who receives intravenous iron therapy, TSAT and ferritin level can be monitored every 3 months.

[0335] The iron status of a subject may represent absolute iron deficiency (AID) or functional iron deficiency (FID), both of which can be treated using the compositions and therapeutic methods described herein. Absolute iron deficiency occurs when the amount of available iron is insufficient to meet the body's needs. The insufficiency may be due to insufficient iron intake, reduced dietary iron bioavailability, increased iron utilization, or chronic blood loss. Prolonged iron deficiency may result in iron deficiency anemia-microcytosis, hypochromia (insufficient iron stores). AID generally refers to cases where TSAT<20% and ferritin<100ng / mL.

[0336] TSAT is the ratio of serum iron to total iron binding capacity multiplied by 100. The TSAT value tells the clinician how much serum iron is actually bound to the available transferrin for iron binding. For example, a value of 15% means that 15% of the iron-binding sites on transferrin are occupied by iron.

[0337] FID can occur when iron cannot be released rapidly enough to meet the bone marrow demand for red blood cell production despite adequate total body iron stores. In these instances, ferritin levels may be normal or high, but the supply of iron to the erythroid system is limited as indicated by low transferrin saturation and an increased number of microcytic hypochromic red blood cells. FID can be characterized by the following features: inadequate hemoglobin response to erythropoietin; serum ferritin may be normal or high; transferrin saturation (TSAT) is usually <20%; and / or reduced mean corpuscular volume (MCV; e.g., less than 80 fL) or mean corpuscular hemoglobin concentration (MCHC; e.g., less than 34±2 g / dl) (severe cases). Functional iron deficiency (i.e., iron stores are considered adequate but not available for iron delivery) generally refers to cases with TSAT<20% and ferritin>100 ng / mL.

[0338] Assessment of the need for iron therapy may follow, for example, the National Kidney Foundation's Kidney Disease Outcomes Quality Initiative. See NKF-K / DOQI, Clinical Practice Guidelines for Anemia of Chronic Kidney Disease (2000); Am J Kidney Dis (2001) 37(supp 1), S182-S238. The DOQI provides optimal practice for the treatment of anemia in chronic renal insufficiency. The DOQI guidelines specify intravenous iron therapy for renal disease based on hemoglobin, transferrin saturation (TSAT), and ferritin levels.

[0339] Further guidelines for iron management and for determining AID vs. FID in cancer and / or chemotherapy patients have also been suggested, e.g., by Steinmetz et al., Ther Adv Hematol 2012;3(3):177-91, Mikhael et al., Curr Oncol 2007;14:209-217, the European Organization for Research and Treatment of Cancer (EORTC; Bokemeyer et al., Eur J Cancer 2007;43:258-270; and Aapro and Link, Oncologist 2008;13(Suppl.3):33-36), the American Society of Hematology (ASH) and the American Society of Clinical Oncology (ASCO) (see, e.g., Rizzo et al., Blood 2010;100:2303-2320), the National Comprehensive Cancer Network (NCCN) guidelines version 3. See 2018 Cancer-and Chemotherapy-induced Anemia, European Society for Medical Oncology (ESMO) (see, e.g., Aepro et al., Annals of Oncology 2018;0:1-15), and National Institute for Health and Care Excellence (NICE) (see, e.g., Technology appraisal guidance published 26 Nov 2014; nice.org.uk / guidance / ta323).

[0340] The subject in need of treatment can be determined by the target iron level of the subject. For example, the target hemoglobin level of the subject can be selected as 11.0g / dL-12.0g / dL (hematocrit approximately 33%-36%). To achieve the target hemoglobin at the optimal erythropoietin dose, sufficient iron can be provided to maintain TSAT≧20% and ferritin 100ng / mL. Achieving the target hemoglobin level at the optimal erythropoietin dose is associated with providing sufficient iron to maintain TSAT higher than 20%.

[0341] Iron therapy can be given to maintain target hemoglobin while preventing iron deficiency and also preventing iron overload. Adjustment of iron dosage to maintain target levels of hemoglobin, hematocrit, and iron store laboratory parameters is within the ordinary skill of the art. For example, if a subject is anemic or iron deficient, iron supplements can be administered when the patient has ferritin<800ng / mL, TSAT<50%, and / or hemoglobin<12g / dL. Iron overload can be avoided by withholding iron for TSAT>50% and / or ferritin>800ng / mL.

[0342] If a subject is not anemic or iron deficient but still requires an RGMc antagonist, for example if the subject has restless legs syndrome, hemoglobin and TSAT levels are not necessarily relevant, but ferritin >800 may provide a general cut-off point for dosing.

[0343] [Administration] To carry out the methods disclosed herein, an effective amount of the above-mentioned pharmaceutical composition can be administered to a subject (e.g., a human) in need of treatment via an appropriate route (e.g., intravenous administration), e.g., as a bolus, or by continuous infusion over many years, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, inhalation or topical routes. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be directly nebulized, and lyophilized powders can be nebulized after reconstitution. Alternatively, an antibody that specifically binds to RGMc, or an antigen-binding fragment thereof, can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.

[0344] The subject treated by the methods described herein may be a mammal, more preferably a human. Mammals include, but are not limited to, livestock, sport animals, pets, primates, horses, dogs, cats, mice and rats. The human subject in need of treatment may be a human patient who has, is at risk of, or is suspected of having an RGMc-related symptom, such as those described above (e.g., anemia). A subject who has an RGMc-related symptom may be identified by routine medical examination, such as laboratory tests, organ function tests, CT scans, or ultrasound. A subject who is suspected of having any such symptom shows one or more symptoms of the symptom. A subject who is at risk for a symptom may be a subject who has one or more risk factors for the symptom.

[0345] The terms "effective amount" and "effective dose" as used herein refer to any amount or dose of a compound or composition sufficient to meet its intended purpose(s), i.e., a desired biological or pharmaceutical (clinical) response in a tissue or subject with an acceptable benefit / risk ratio. For example, in certain embodiments of the present invention, the intended purpose may be to inhibit RGMc activity in vivo to achieve a clinically meaningful outcome associated with RGMc inhibition. For example, clinically meaningful effects may include increased serum iron, restoration or normalization of hemoglobin or red blood cell count, increased transferrin saturation, and the like. In some embodiments, clinically meaningful effects may include reduced toxicity or cardiovascular risk (e.g., stroke or heart failure) and risk of harm such as cancer. In some embodiments, clinically meaningful effects may include reduced need for other therapies such as iron supplements. In some embodiments, clinically meaningful effects may include improved quality of life or general well-being as perceived by the patient. In some embodiments, a clinically meaningful effect may include an accelerated or faster alleviation of one or more parameters indicative of anemia. Such alleviation may be achieved by an add-on or combination therapy that includes an RGMc inhibitor and another therapy (e.g., ESA, HIF stabilizer, etc.). For example, typically, when EPO therapy (e.g., monotherapy) is initiated, it takes 1-2 months (e.g., 6-8 weeks) for the treatment to show effect. For this reason, patients who experience severe anemia may further require IV iron or blood transfusions during that period. However, when used with an RGMc selective inhibitor such as those disclosed herein, the onset of therapeutic effect may be significantly more rapid (e.g., 1-4 weeks), which may reduce the need for further IV iron or blood transfusions.

[0346] As recognized by those skilled in the art, the effective amount varies depending on the specific condition being treated, the severity of the condition, individual patient parameters including age, health, size, sex and weight, duration of treatment, nature of concurrent therapy (if any), specific route of administration, and similar factors within the knowledge and experience of health professionals. These factors are well known to those skilled in the art and can be addressed within the scope of routine experimentation. It is generally preferred that the maximum dosage of each component or combination thereof is used, i.e., the highest safe dosage according to sound medical judgment. However, those skilled in the art understand that a patient may require a lower dosage or a tolerable dosage for medical reasons, psychological reasons, or virtually any other reason.

[0347] Empirical considerations such as half-life generally contribute to the determination of dosage. For example, antibodies compatible with the human immune system, such as humanized or fully human antibodies, can be used to extend the half-life of the antibody and prevent it from being attacked by the host's immune system. The frequency of administration can be determined and adjusted over the course of treatment, and is generally, but not necessarily, based on the treatment and / or suppression and / or remission and / or delay of RGMc-related symptoms. Alternatively, sustained continuous release formulations of antibodies that specifically bind to RGMc may be appropriate. Various formulations and devices for achieving sustained release will be apparent to those skilled in the art and are within the scope of this disclosure.

[0348] In one example, dosages for antibodies specifically binding to RGMc described herein may be empirically determined in individuals who have been given one or more administrations (singular or multiple) of the antibody. Individuals are given incremental doses of an RGMc antagonist. Indicators of RGMc-related symptoms may be followed to assess efficacy. For example, methods for measuring anemia are well known in the art and are further described herein.

[0349] The present invention encompasses the recognition that agents capable of specifically modulating RGMc signaling as opposed to RGMa and / or RGMb may offer an improved safety profile when used as pharmaceuticals. Thus, the present invention includes antibodies and antigen-binding fragments thereof that specifically bind to and inhibit the activation of RGMc, but not RGMa and / or RGMb, thereby providing specific inhibition of RGMc signaling in vivo while minimizing unwanted side effects from affecting RGMa and / or RGMb signaling.

[0350] In some embodiments, the antibodies, or antigen-binding fragments thereof, described herein are not toxic when administered to a subject. In some embodiments, the antibodies, or antigen-binding fragments thereof, described herein exhibit reduced toxicity when administered to a subject compared to antibodies that specifically bind to both RGMc and RGMa. In some embodiments, the antibodies, or antigen-binding fragments thereof, described herein exhibit reduced toxicity when administered to a subject compared to antibodies that specifically bind to both RGMc and RGMb. In some embodiments, the antibodies, or antigen-binding fragments thereof, described herein exhibit reduced toxicity when administered to a subject compared to antibodies that specifically bind to RGMa, RGMb and RGMc.

[0351] Generally, for administration of any of the antibodies described herein, the initial candidate dose may be about 0.1-20 mg / kg, e.g., 1-20 mg / kg per administration, e.g., weekly, every two weeks, every three weeks, monthly, every three months, etc. For example, a patient may receive an infusion of about 1-10 mg / kg per week, every two weeks, every three weeks, or every four weeks, etc., in an amount effective to treat the disease (e.g., cancer) and that is well tolerated (within acceptable toxicity or adverse events).

[0352] For purposes of this disclosure, a typical dosage (per administration, e.g., injection and infusion) may range anywhere from about 0.1 mg / kg to 1 mg / kg, 2 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, or more, depending on the factors mentioned above. For repeated administration over several days or longer, depending on the condition, treatment is sustained until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved to alleviate the RGMc-related signs, or symptoms thereof.

[0353] An exemplary dosing regimen includes administration of an initial dose followed by one or more maintenance doses, where the latter is typically less than the former. For example, the initial dose may be, for example, about 2-30 mg / kg, once a week or twice a week. This may be followed by a maintenance dose(s), for example, about 0.1-20 mg / kg (e.g., 1, 2, 3, 5, 10, 15 mg / kg), for example, weekly, biweekly, monthly, etc. However, other dosing regimens may be useful depending on the pharmacokinetic decay pattern the practitioner wishes to achieve. Pharmacodynamic (PD) experiments have shown that PD effects (e.g., increased serum iron levels) persist for at least 3 weeks after administration of the antibodies disclosed herein (e.g., Ab8; 20 mg / kg) to preclinical animal models (e.g., rat models). Without wishing to be bound by any particular theory, this sustained effect after administration may be advantageous, since the antibody may be administered less frequently while maintaining a clinically effective serum concentration in the subject (e.g., human subject) to which the antibody is administered. In some embodiments, the administration frequency is once every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, or every ten weeks; or once every month, every two months, or every three months, or once for a longer period of time. The progress of this therapy is easily monitored by conventional techniques and assays. The administration regimen (including the antibody used) may vary over time.

[0354] In some embodiments, for a normal weight adult patient, a dosage in the range of about 0.3 to 5.00 mg / kg may be administered. The particular dosing regimen, e.g., dosage, timing and repetition, will depend on the particular individual and that individual's medical history, as well as the characteristics of the individual drug (e.g., the drug's half-life, and other relevant considerations).

[0355] For purposes of this disclosure, the appropriate dose of an antibody that specifically binds to RGMc depends on the specific antibody (or composition thereof) used, the type and severity of the symptoms, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's medical history and response to antagonists, and the discretion of the attending physician. In some embodiments, the clinician administers an antibody that specifically binds to RGMc until a dose is reached that achieves the desired result. Administration of an antibody that specifically binds to RGMc may be continuous or intermittent, depending, for example, on the physiological state of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to the skilled practitioner. Administration of an antibody that specifically binds to RGMc may be essentially continuous over a preselected period of time, or may be in a series of spaced doses, such as before, during, or after the onset of an RGMc-related symptom (e.g., anemia).

[0356] As used herein, the term "treating" refers to the application or administration of a composition comprising one or more active agents to a subject having an RGMc-related sign, a symptom of the sign, or a predisposition toward the sign, for the purpose of curing, heal, alleviating, relieving, altering, remedying, ameliorating, improving, or affecting the sign, a symptom of the sign, or a predisposition toward the sign.

[0357] Alleviating an RGMc-associated symptom with an antibody that specifically binds to RGMc includes delaying the onset or progression of the symptom or reducing the severity of the symptom. Alleviating the symptom does not necessarily require a therapeutic outcome. As used herein, "delaying" the onset of a symptom associated with an RGMc-associated symptom means deferring, hindering, slowing, retarding, stabilizing, and / or postponing the progression of the symptom. The delay may vary in length of time depending on the symptom being treated and / or the history of the individual. A method of "delaying" or alleviating the onset of a symptom or delaying the onset of a symptom is a method that reduces the likelihood of one or more symptoms of the symptom occurring within a given time frame and / or reduces the severity of the symptom within a given time frame compared to not using the method. Such comparisons are typically based on clinical trials with a large enough number of subjects to provide statistically significant results.

[0358] [Combination therapy] The present disclosure further encompasses pharmaceutical compositions and related methods used as combination therapy to treat subjects who may benefit from RGMc inhibition in vivo. In any of these embodiments, such subjects may receive combination therapy comprising a first composition comprising at least one RGMc antagonist, such as an antibody or antigen-binding fragment thereof as described herein, together with a second composition comprising at least one additional treatment intended to treat the same or overlapping disease or clinical condition. Both the first and second compositions may act on the same cellular target, or distinct cellular targets. In some embodiments, the first and second compositions may treat or alleviate the same or overlapping set of symptoms or aspects of a disease or clinical condition. In some embodiments, the first and second compositions may treat or alleviate distinct sets of symptoms or aspects of a disease or clinical condition. By way of example, the first composition may treat a disease or condition (e.g., CKD or cancer), while the second composition may treat inflammation, reduced red blood cell production, anemia, etc. associated with the same disease. Such combination therapy can be administered together.The phrase "together" in the context of combination therapy means that the therapeutic effect of the first therapy overlaps with the therapeutic effect of the second therapy in the subject who receives combination therapy in time and / or space.Therefore, combination therapy can be formulated as a single preparation for simultaneous administration, or as separate preparations for sequential administration of the therapy.

[0359] In a preferred embodiment, the combination therapy produces a synergistic effect in treating a disease. The term "synergy" refers to a greater than additive effect (e.g., greater efficacy) of each monotherapy combined.

[0360] In some embodiments, combination therapy may achieve a faster therapeutic effect than monotherapy.

[0361] In some embodiments, the combination therapy comprising the pharmaceutical compositions described herein produces the overall equivalent efficacy produced by another therapy (e.g., monotherapy of the second drug), but produces fewer unwanted side effects associated with the second drug or less severe toxicity compared to monotherapy of the second drug.In some embodiments, such combination therapy allows for a lower dose of the second drug, but maintains overall efficacy.Such combination therapy may be particularly suitable for patient populations where long-term treatment is warranted and / or includes pediatric patients.

[0362] Thus, in one aspect, the present invention provides pharmaceutical compositions and methods for use in combination therapy for reducing RGMc activity, as described herein, and treating or preventing a disease or condition associated with RGMc signaling. Thus, the method or pharmaceutical composition further comprises a second therapy. In some embodiments, the second therapy may be useful for treating or preventing a disease or condition associated with RGMc signaling. The second therapy may reduce or treat at least one symptom or symptoms associated with the target disease. The first and second therapies may exert their biological effects by similar or unrelated mechanisms of action; or, either or both of the first and second therapies may exert their biological effects by multiple mechanisms of action. For example, in the context of anemia, an RGMc inhibitor may be administered in combination (e.g., sequentially or simultaneously) with a second therapy that increases red blood cell production (e.g., EPO therapy, blood transfusion, etc.). Alternatively, in the context of anemia of chronic disease, RGMc inhibitors may be administered in combination (e.g., sequentially or simultaneously) with a second therapy (e.g., chemotherapy, radiation therapy, immunotherapy, etc.) to treat the underlying disease, such as cancer and immune disorders.

[0363] It should be understood that the pharmaceutical compositions described herein may have the first and second therapies in the same pharma- ceutically acceptable carrier or in different pharma- ceutically acceptable carriers for each described embodiment. It should be further understood that the first and second therapies may be administered simultaneously or sequentially in the described embodiments.

[0364] One or more RGMc inhibitors of the invention (e.g., antibodies or antigen-binding fragments thereof) may be administered to a patient who has received or is receiving one or more additional therapeutic agents. Examples of additional therapeutic agents that can be used with the RGMc antibodies or the invention include, but are not limited to, erythropoietin stimulating agents (ESAs), hypoxia inducible factor (HIF) stabilizers, hepcidin antagonists, iron supplements (e.g., administered orally or intravenously), anti-inflammatory and / or anti-cancer drugs.

[0365] ESAs are drugs that stimulate the bone marrow to make red blood cells and are useful in treating various types of anemia in patients. However, even in the presence of increased red blood cell production, anemia persists in the absence of sufficient iron. This is because red blood cells require hemoglobin to transport oxygen, and iron is important for hemoglobin synthesis. Thus, without sufficient iron, newly produced red blood cells will lack hemoglobin and will be hypochromic (lacking the red hemoglobin pigment) and microcytic (smaller than normal).

[0366] Thus, in one embodiment, an RGMc antagonist (eg, an RGMc antibody) described herein may be administered to a patient who has received or is receiving an ESA to treat an iron-related disorder.Examples of ESAs include, but are not limited to, epoetin alfa, epoetin beta, epoetin delta (e.g., Dynepo®), epoetin omega (e.g., Epomax®), darbepoetin alfa (Aranesp®), Epocept® (Lupin pharma), Nanokine® (Nanogen Pharmaceutical biotechnology), Epofit® (Intas pharma), Epogen® (manufactured by Amgen), Epogin® (Chugai Pharma), Eprex® (manufactured by Janssen-Cilag), Binocrit® (manufactured by Sandoz), PDpoetin® (manufactured by Pooyeshdarou Biopharmaceutical), Procrit®, Bioyetin® (manufactured by Probiomed), NeoRecormon® (Hoffmann-La Roche), Silapo® / Retacrit® (Stada / Hospira), EPOTrust® (Panacea Biotec Ltd), Erypro Safe™ (Biocon Ltd.), Repoitin® (Serum Institute of India Limited), Vintor® (Emcure Pharmaceuticals), Erykine (Intas Biopharmaceutica), Wepox® (Wockhardt Biotech), Espogen™ (LG life sciences), ReliPoietin™ (Reliance Life Sciences), Shanpoietin™ (Shantha Biotechnics Ltd), Zyrop® (Cadila Healthcare Ltd.), EPIAO® (rHuEPO) (Shenyang Sunshine Pharmaceutical Co.LTD.), and China Cinnapoietin® (CinnaGen Includes biopharmaceutical products manufactured in Iran.

[0367] Although ESAs are generally effective, there is evidence that a subset of patients do not respond to treatment, even at high doses (Solak Y, et al. Blood Purification. 2016; 42(2): 160-7). Furthermore, some studies have shown that the use of ESAs may result in higher cerebrovascular and cardiovascular (CV) event rates and mortality (Biggar P. et al., Kidney Research and Clinical Practice 2017 Sep; 36(3): 209-223). ESAs may also be associated with increased cancer risk (and possibly poorer response to anticancer treatment) in some patients. Thus, without being bound by any particular theory, the use of RGMc inhibitors in patients who have received or are receiving ESAs may improve response rates and reduce the dose of ESA required to be effective, thereby reducing the occurrence of adverse toxicity.

[0368] Hypoxia-inducible factor (HIF) prolyl hydroxylase (PH) enzyme inhibitors (also known as HIF-PHI or HIF stabilizers) are a class of drugs for the treatment of anemia. These drugs act by stabilizing the HIF complex to stimulate endogenous erythropoietin production. Thus, HIF-PH inhibitors that increase erythropoietin production can be complimented by drugs that increase serum iron levels (e.g., RGMc inhibitors). Thus, in some embodiments, RGMc inhibitors (e.g., RGMc antibodies) described herein can be administered to patients who have received or are receiving HIF stabilizers or prolyl hydroxylase (PH) enzyme inhibitors to treat iron-related diseases. Examples of HIF stabilizers or HIF-PH inhibitors include, but are not limited to, Roxadustat (FG-4592), Vadadustat (AKB-6548), Daprodustat (GSK-1278863), Molidustat (BAY 85-3934), Desidustat (ZYAN1), DMOG (N-(2-methoxy-2-oxoacetyl)glycine methyl ester), IOX2 (N-[[1,2-dihydro-4-hydroxy-2-oxo-1-(phenylmethyl)-3-quinolinyl]carbonyl]glycine), JNJ-42041935 (1-[6-chloro-5-(trifluoromethoxy)-1H-benzimidazol-2-yl]-1H-pyrazole-4-carboxylic acid), N-oxalylglycine (2-(carboxymethylamino)-2-glyoxylic acid), 1,4-dihydrophenothrin-4-one-3-carboxylic acid, and Adaptaquin (7-[(4-chlorophenyl)[(3-hydroxy-2-pyridinyl)amino]methyl]-8-quinolinol).

[0369] In some embodiments, the RGMc-specific inhibitors described herein may be administered to patients who have received or are receiving iron supplements (e.g., oral or intravenous (iv) iron). Examples of iv iron preparations include, but are not limited to, iron dextran, ferric hydroxide-dextran complex, ferric hydroxide-sucrose complex, ferric gluconate, sodium ferric gluconate, iron sucrose, Fe saccharate, ferumoxytol, ferric carboxymaltose, and iron isomaltoside. Examples of oral iron supplements may include, but are not limited to, ferric pyrophosphate, ferrous gluconate, ferrous sulfate, ferrous fumarate, ferrous carbonate, and ferric carbonyl.

[0370] Although intravenous and oral iron supplementation is generally effective, studies have shown various toxicities or adverse events associated with iron. Examples of adverse events include cytotoxicity, renal tubular failure, altered neutrophil function, promotion of atherosclerosis, promotion of tumor growth, and generation of free radicals (Cancado and Munoz, Rev Bras Hematol Hemoter 2011;33(6):461-9). Therefore, without being bound by a particular theory, using an RGMc-specific inhibitor with iv or oral iron can improve response rate and reduce the dose of iron required to have a therapeutic effect, thereby reducing the occurrence and / or severity of adverse events / toxicity.

[0371] It is recognized that certain drugs designed to treat various disease states often induce or exacerbate anemia in the treated patient (e.g., treatment or drug induced anemia, e.g., chemotherapy induced anemia and radiation therapy induced anemia). In some embodiments, the patient is treated with myelosuppressive drugs that may cause side effects including anemia. Such patients may benefit from the RGMc inhibitors of the present disclosure used in conjunction with myelosuppressive therapy.Examples of myelosuppressive therapies include, but are not limited to: peginterferon alfa-2a, interferon alfa-n3, peginterferon alfa-2b, aldesleukin, gemtuzumab ozogamicin, interferon alfacon-1, rituximab, ibritumomab Tiuxetan, tositumomab, alemtuzumab, bevacizumab, L-phenylalanine, bortezomib, cladribine, carmustine, amsacrine, chlorambucil, raltitrexed, mitomycin, bexarotene, vindesine, floxuridine, thioguanine, vinorelbine, dexrazoxane, sorafenib, streptozocin, gemcitabine, teniposide, epirubicin, chloramphenicol, lenalidomide, altretamine, zidovudine, cisplatin, oxaliplatin, cyclophosphamide, fluorouracil, propylthiouracil, pentostatin, methotrexate, carbamazepine, vinblastine, linezolid, imatinib, clofarabine ... Methotrexed, daunorubicin, irinotecan, methimazole, etoposide, dacarbazine, temozolomide, tacrolimus, sirolimus, mechlorethamine, azacitidine, carboplatin, dactinomycin, cytarabine, doxorubicin, hydroxyurea, busulfan, topotecan, mercaptopurine, thalidomide, melphalan, fludarabine, flucytosine, capecitabine, procarbazine, arsenic trioxide, idarubicin, ifosfamide, mitoxantrone, lomustine, paclitaxel, docetaxel, dasatinib, decitabine, nelarabine, everolimus, vorinostat, thiotepa, ixabepilone, nilotinib, belinostat, trabectedin, trastuzumab These include emtansine, temsirolimus, bosutinib, bendamustine, cabazitaxel, eribulin, ruxolitinib, carfilzomib, tofacitinib, ponatinib, pomalidomide, obinutuzumab, tedizolid phosphate, blinatumomab, ibrutinib, palbociclib, olaparib, dinutuximab, and colchicine.

[0372] In some embodiments, the RGMc-specific inhibitors described herein may be administered to patients who have undergone or are undergoing cancer therapy. Such cancer therapy may include, but is not limited to, chemotherapy, radiation therapy / irradiation therapy, small molecule drugs, or therapeutic antibodies, such as cancer vaccines; engineered immune cell therapy; chemotherapy; radiation therapy(s); VEGF agonists; IGF1 agonists; FXR agonists; CCR2 inhibitors; CCR5 inhibitors; dual CCR2 / CCR5 inhibitors; lysyl oxidase-like-2 inhibitors; ASK1 inhibitors; acetyl-CoA carboxylase (ACC) inhibitors; p38 kinase inhibitors; pirfenidone; nintedanib; M-CSF inhibitors (e.g., M-CSF receptor antagonists and M-CSF neutralizers); MAPK inhibitors (e.g., Erk inhibitors), immune checkpoint agonists or antagonists (e.g., anti-PD-1 and anti-PD-L1); IL-11 antagonists; and IL-6 antagonists. Other examples of additional therapeutic agents that can be used together with RGMc specific inhibitors include, but are not limited to, indoleamine 2,3-dioxygenase (IDO) inhibitors, tyrosine kinase inhibitors, Ser / Thr kinase inhibitors, dual specific kinase inhibitors. In some embodiments, such agents can be PI3K inhibitors, PKC inhibitors, or JAK inhibitors. In some embodiments, JAK inhibitors can be JAK1 inhibitors and / or JAK2 inhibitors, which can be used in the treatment of myeloproliferative disorders, such as primary myelofibrosis.

[0373] It is also understood that other cancer treatments, such as radiation therapy, can cause anemia in cancer patients. Thus, in one embodiment, the RGMc-specific inhibitors described herein can be used in conjunction with radiation therapy.

[0374] In some embodiments, the additional agent administered as a combination therapy or used with the RGMc inhibitor is or includes an inhibitor of a member of the TGFβ superfamily of growth factors or a regulator thereof. In some embodiments, such an inhibitor is or includes a TGFβ inhibitor, such as a TGFβ neutralizing antibody. In some embodiments, the TGFβ inhibitor is an antibody that inhibits TGFβ1 and TGFβ2. In some embodiments, the TGFβ inhibitor is an antibody that inhibits TGFβ1 and TGFβ3. In some embodiments, the TGFβ inhibitor is an isoform-selective inhibitor of TGFβ1, such as a TGFβ1 activation inhibitor. In some embodiments, the TGFβ inhibitor is an isoform-selective inhibitor of TGFβ3, such as a TGFβ3 activation inhibitor.

[0375] In some embodiments, the combination therapy used in treating cancer in a subject comprises a cancer immunotherapy (e.g., a checkpoint inhibitor), a TGFβ1-selective inhibitor, and an RGMc-selective inhibitor disclosed herein, where the checkpoint inhibitor can be a PD-(L)1 antibody and further, the TGFβ1-selective inhibitor can be a TGFβ1-selective activation inhibitor.

[0376] In some embodiments, the RGMc-specific inhibitors described herein can be used in conjunction with other compounds, drugs, and / or agents used to treat diseases associated with inflammation or chronic kidney disease. Such compounds, drugs, and / or agents include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs), angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs), diuretics, beta-blockers, calcium channel blockers, central sympatholytic agents, bicarbonate supplements, insulin, vitamin D supplements, uric acid reducing agents (e.g., allopurinol), HMG-CaA reductase inhibitors (statins), pirfenidone, TGF-β pathway inhibitors, anti-inflammatory agents (including nonsteroidal anti-inflammatory drugs), antioxidant inflammation modifiers (e.g., bardoxolone methyl), endothelin-1 antagonists (e.g., avosentan and atrasentan), advanced glycation end products (ADs), and / or anti-inflammatory agents (e.g., erythropoietin ... product inhibitors, aspirin, metformin, aminoguanidine, calcitriol, erythropoietin, androgens, phosphate binders, vitamin B12, and vitamin B6 and its derivatives (e.g., pyridoxamine).

[0377] In some embodiments, the RGMc-specific inhibitors described herein can be used with granulocyte colony-stimulating factor (G-CSF) or its derivatives, such as Neupogen®, Granix®, or Zarxio®. G-CSF is a glycoprotein used to stimulate the production of blood cells (e.g., white blood cells or hematopoietic stem cells) to promote their ability to function. Thus, G-CSF is useful in reducing the risk of patients developing infections, anemia, and bleeding problems after undergoing myelosuppressive treatments (e.g., chemotherapy). However, there are side effects to the use of G-CSF, including: thrombocytopenia, nausea, fever, bone pain, elevated lactate dehydrogenase, elevated alkaline phosphatase, petechiae, back pain, epistaxis (nosebleeds), cough, and / or dyspnea (shortness of breath). Thus, it is contemplated that the RGMc-specific inhibitors described herein may complement the pharmacological effects of G-CSF (e.g., reducing anemia and promoting erythropoiesis) and / or reduce the effective dose of G-CSF, thereby reducing side effects and increasing tolerability. Thus, in one embodiment, the RGMc-specific inhibitors described herein may be administered to patients who have undergone or are undergoing G-CSF therapy. In another embodiment, the RGMc-specific inhibitors described herein reduce the effective dose of G-CSF required to achieve a favorable pharmacological effect, e.g., required to promote blood cell production and / or required to reduce anemia. In another embodiment, the RGMc-specific inhibitors described herein complement the effects of G-CSF, e.g., further increasing erythropoiesis and / or reducing anemia.

[0378] The combination therapy discussed herein can advantageously utilize a lower dose of administered therapeutic agent, thus avoiding possible toxicity or complications associated with various monotherapy. In some embodiments, the use of the RGMc-specific inhibitors described herein can make poor or non-responders to therapy (e.g., standard therapy) more responsive. In some embodiments, the use of the RGMc-specific inhibitors described herein can reduce the dose of therapy (e.g., standard therapy), still resulting in comparable clinical efficacy in patients, but with less or less drug-related toxicity or adverse events.

[0379] [Inhibition of RGMc activity] Methods of the present disclosure include methods of inhibiting RGMc activity in one or more biological systems. Such methods may include contacting one or more biological systems with an antibody and / or composition of the present disclosure. Inhibitors (e.g., antibodies) and / or compositions according to such methods may include, but are not limited to, biological molecules, including, but not limited to, recombinant proteins, protein complexes and / or antibodies described herein, or antigenic fragments thereof.

[0380] The inhibitors described herein that selectively bind to RGMc, e.g., antibodies and antigen-binding fragments thereof, can be used in a wide variety of applications in which modulation of RGMc activity is desired. In one embodiment, the present invention provides a method of inhibiting RGMc activation by exposing an RGMc peptide or protein to an inhibitor, e.g., an antibody, or antigen-binding fragment thereof, that selectively binds to RGMc. The above-mentioned method can be performed in vitro, e.g., to inhibit RGMc activity in cultured cells. The above-mentioned method can also be performed in vivo, e.g., in a subject in need of RGMc inhibition or in an animal model in which the effect of RGMc inhibition is evaluated.

[0381] Any of the inhibitors described herein that selectively bind to RGMc, e.g., antibodies, or antigen-binding fragments thereof, and any pharmaceutical compositions containing such inhibitors or antibodies, are suitable for use in the methods of the present invention.

[0382] Thus, in one aspect of the present invention, a method is provided for inhibiting RGMc activity by exposing RGMc to an inhibitor, e.g., an antibody, or there antigen-binding fragment, that selectively binds to RGMc. In some embodiments, the inhibitor (e.g., an antibody or there antigen-binding fragment) competes with BMP for binding to RGMc. In some embodiments, the BMP is BMP2 and / or BMP6. In some embodiments, the BMP is BMP6.

[0383] In another embodiment, the inhibitor (e.g., an antibody or antigen-binding fragment thereof) inhibits binding of neogenin to RGMc. In some embodiments, the inhibitor (e.g., an antibody or antigen-binding fragment thereof) competes with neogenin for binding to RGMc.

[0384] In some embodiments, the inhibitor (e.g., an antibody or antigen-binding fragment thereof) does not inhibit binding of neogenin to RGMc. In some embodiments, the inhibitor (e.g., an antibody or antigen-binding fragment thereof) does not compete with neogenin for binding to RGMc.

[0385] In another embodiment, the inhibitor (e.g., an antibody or antigen-binding fragment thereof) inhibits binding of RGMc to other binding partners or proteins that complex with RGMc. For example, other binding partners that complex with RGMc, BMP, and / or neogenin include HFE, TFR2, ALK2, ALK3, BMPR2, ACVR2A, and TMPRSS6.

[0386] In another embodiment, the antibody or antigen-binding fragment thereof that inhibits RGMc activity can also modulate other biological processes (e.g., downstream effects). For example, the antibody or antigen-binding fragment thereof that inhibits RGMc activity reduces hepcidin expression in a subject. In some embodiments, the antibody or antigen-binding fragment thereof that inhibits RGMc activity increases ferroportin expression in a subject. In some embodiments, the antibody or antigen-binding fragment thereof that inhibits RGMc activity increases transferrin saturation in a subject. In some embodiments, the antibody or antigen-binding fragment thereof that inhibits RGMc activity increases red blood cell production in a subject. In some embodiments, the antibody or antigen-binding fragment thereof that inhibits RGMc activity reduces unsaturated iron binding capacity (UIBC) in a subject. In some embodiments, the antibody or antigen-binding fragment thereof that inhibits RGMc activity reduces total iron binding capacity (TIBC) in a subject. In some embodiments, the antibody or antigen-binding fragment thereof that inhibits RGMc activity increases serum ferritin levels in a subject.

[0387] In some embodiments, the methods described herein can be performed in a subject. The subject treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. The human subject in need of treatment can be a human patient having, at risk of, or suspected of having an RGMc-related symptom, such as those described above (e.g., anemia).

[0388] Thus, in one embodiment, a method of increasing serum iron levels in a subject is provided, the method comprising administering to the subject a pharmaceutical composition of the invention (e.g., an RGMc-specific inhibitor disclosed herein) in an amount effective to downregulate hepcidin in the subject. In some embodiments, a method for downregulating hepcidin in a subject is provided, the method comprising administering to the subject a pharmaceutical composition of the invention (e.g., an RGMc-specific inhibitor disclosed herein) in an amount effective to downregulate hepcidin in the subject. In some embodiments, the pharmaceutical composition reduces liver hepcidin mRNA levels. In some embodiments, the pharmaceutical composition reduces liver hepcidin protein levels, serum (circulating) hepcidin protein levels, or both.

[0389] In one embodiment, a method of increasing serum iron levels in a subject is provided, the method comprising administering to the subject a pharmaceutical composition of the invention (e.g., an RGMc-specific inhibitor disclosed herein) in an amount effective to increase ferroportin expression in the subject compared to baseline. In some embodiments, a method for increasing ferroportin expression in a subject is provided, the method comprising administering to the subject a pharmaceutical composition of the invention (e.g., an RGMc-specific inhibitor disclosed herein) in an amount effective to increase ferroportin expression in the subject. In some embodiments, the pharmaceutical composition increases enterocyte (intestine) ferroportin protein levels. In some embodiments, the pharmaceutical composition increases hepatocyte (liver) ferroportin protein levels. In some embodiments, the pharmaceutical composition increases macrophage ferroportin protein levels. In some embodiments, the pharmaceutical composition increases adipocyte ferroportin protein levels.

[0390] In some embodiments, a method for increasing transferrin saturation in a subject is provided, the method comprising administering to the subject a pharmaceutical composition of the invention (e.g., an RGMc-specific inhibitor disclosed herein) in an amount effective to increase transferrin saturation (TSAT) in the subject. In some embodiments, the method increases transferrin saturation by ≧1%, ≧2%, ≧3%, ≧4%, ≧5%, ≧6%, ≧7%, ≧8%, ≧9%, or ≧10% compared to baseline. In some embodiments, the method increases transferrin saturation by ≧1%. In some embodiments, the method increases transferrin saturation by ≧2% compared to baseline. In some embodiments, the method increases transferrin saturation by ≧3% compared to baseline. In some embodiments, the method increases transferrin saturation by ≧4% compared to baseline. In some embodiments, the method increases transferrin saturation by ≧5% compared to baseline. In some embodiments, the method increases transferrin saturation by ≧6% compared to baseline. In some embodiments, the method increases transferrin saturation by ≧7% compared to baseline. In some embodiments, the method increases transferrin saturation by ≧8% compared to baseline. In some embodiments, the method increases transferrin saturation by ≧9% compared to baseline. In some embodiments, the method increases transferrin saturation by ≧10% compared to baseline.

[0391] In some embodiments, the method increases transferrin saturation in the subject to a level of ≧15%, ≧16%, ≧17%, ≧18%, ≧19%, ≧20%, ≧21%, ≧22%, ≧23%, ≧24%, or ≧25%. In some embodiments, the method increases transferrin saturation in the subject to a level of ≧15%. In some embodiments, the method increases transferrin saturation in the subject to a level of ≧16%. In some embodiments, the method increases transferrin saturation in the subject to a level of ≧17%. In some embodiments, the method increases transferrin saturation in the subject to a level of ≧18%. In some embodiments, the method increases transferrin saturation in the subject to a level of ≧19%. In some embodiments, the method increases transferrin saturation in the subject to a level of ≧20%. In some embodiments, the method increases transferrin saturation in a subject to a level of ≧21%. In some embodiments, the method increases transferrin saturation in a subject to a level of ≧22%. In some embodiments, the method increases transferrin saturation in a subject to a level of ≧23%. In some embodiments, the method increases transferrin saturation in a subject to a level of ≧24%. In some embodiments, the method increases transferrin saturation in a subject to a level of ≧25%. .

[0392] In some embodiments, a method for increasing red blood cell production in a subject is provided, the method comprising administering to the subject a pharmaceutical composition of the present invention (e.g., an RGMc-specific inhibitor disclosed herein) in an amount effective to increase red blood cell production in the subject.

[0393] In some embodiments, a method for increasing hemoglobin (Hb) levels in a subject is provided, the method comprising administering to the subject a pharmaceutical composition of the invention (e.g., an RGMc-specific inhibitor disclosed herein) in an amount effective to increase hemoglobin levels. In some embodiments, the method increases Hb levels by ≧1, ≧2, or ≧3 g / dl compared to baseline. In some embodiments, the method increases Hb levels by ≧1 g / dl compared to baseline. In some embodiments, the method increases Hb levels by ≧2 g / dl compared to baseline. In some embodiments, the method increases Hb levels by ≧3 g / dl compared to baseline. In some embodiments, the method increases Hb in the subject to a level of ≧10 g / dl. In some embodiments, the method increases Hb to a level of ≧11 g / dl. In some embodiments, the method increases Hb to a level of ≧12 g / dl. In some embodiments, the methods increase Hb to a level of ≧13 g / dl.

[0394] In some embodiments, a method for reducing unsaturated iron binding capacity (UIBC) in a subject is provided, the method comprising administering to the subject a pharmaceutical composition of the present invention (e.g., an RGMc-specific inhibitor disclosed herein) in an amount effective to reduce unsaturated iron binding capacity in the subject. A typical range for UIBC in a subject is 120-470 ug / dL (21-84 umol / L), although levels may vary between populations. Thus, in some embodiments, the subject has a UIBC of ≧400, ≧450, ≧500, ≧550, ≧600, ≧650, ≧700, ≧750, or ≧800 ug / dL. In other embodiments, an effective amount of an RGMc-specific inhibitor reduces UIBC in a subject by ≧50, ≧100, ≧150, ≧200, ≧250, ≧300, ≧350, or ≧450 ug / dL.

[0395] In some embodiments, a method for reducing total iron binding capacity (TIBC) in a subject is provided, the method comprising administering to the subject a pharmaceutical composition of the invention (e.g., an RGMc-specific inhibitor disclosed herein) in an amount effective to reduce total iron binding capacity in the subject. A typical range for TIBC is 255-450 ug / dL, although levels may vary between populations. Thus, in some embodiments, the subject has a TIBC of ≧400, ≧450, ≧500, ≧550, ≧600, ≧650, ≧700, ≧750, or ≧800 ug / dL. In other embodiments, an effective amount of an RGMc-specific inhibitor reduces TIBC in the subject by ≧50, ≧100, ≧150, ≧200, ≧250, ≧300, ≧350, or ≧450 ug / dL.

[0396] In some embodiments, a method for increasing serum ferritin levels in a subject is provided, the method comprising administering to the subject a pharmaceutical composition of the present invention (e.g., an RGMc-specific inhibitor disclosed herein) in an amount effective to increase serum ferritin levels in the subject. In some embodiments, the method increases serum ferritin levels in the subject by ≧5, ≧10, ≧15, ≧20, ≧30, ≧40, ≧50, ≧60, ≧70, ≧80, ≧90, or ≧100 ng / ml compared to baseline. In some embodiments, the method increases serum ferritin levels in the subject by ≧5 ng / ml compared to baseline. In some embodiments, the method increases serum ferritin levels in the subject by ≧10 ng / ml compared to baseline. In some embodiments, the method increases serum ferritin levels in the subject by ≧15 ng / ml compared to baseline. In some embodiments, the method increases serum ferritin levels in the subject by ≧20 ng / ml compared to baseline. In some embodiments, the method increases serum ferritin levels in the subject by ≧30 ng / ml compared to baseline. In some embodiments, the method increases serum ferritin levels in the subject by ≧40 ng / ml compared to baseline. In some embodiments, the method increases serum ferritin levels in the subject by ≧50 ng / ml compared to baseline. In some embodiments, the method increases serum ferritin levels in the subject by ≧60 ng / ml compared to baseline. In some embodiments, the method increases serum ferritin levels in the subject by ≧70 ng / ml compared to baseline. In some embodiments, the method increases serum ferritin levels in the subject by ≧80 ng / ml compared to baseline. In some embodiments, the method increases serum ferritin levels in the subject by ≧90 ng / ml compared to baseline. In some embodiments, the method increases serum ferritin levels in the subject by ≧100 ng / ml compared to baseline.

[0397] In some embodiments, the method increases serum ferritin in the subject to a level of ≧30 ng / ml. In some embodiments, the method increases serum ferritin in the subject to a level of ≧40 ng / ml. In some embodiments, the method increases serum ferritin in the subject to a level of ≧50 ng / ml. In some embodiments, the method increases serum ferritin in the subject to a level of ≧60 ng / ml. In some embodiments, the method increases serum ferritin in the subject to a level of ≧70 ng / ml. In some embodiments, the method increases serum ferritin in the subject to a level of ≧80 ng / ml. In some embodiments, the method increases serum ferritin in the subject to a level of ≧90 ng / ml. In some embodiments, the method increases serum ferritin in the subject to a level of ≧100 ng / ml.

[0398] In some embodiments, a method for treating a disease associated with RGMc in a subject is provided, the method comprising administering to the subject a pharmaceutical composition of the present invention (e.g., an RGMc-specific inhibitor disclosed herein) in an amount effective to treat the disease. In some embodiments, the disease associated with RGMc is anemia. In some embodiments, the anemia is iron-refractory iron deficiency anemia (IRIDA). In some embodiments, the anemia is anemia of chronic disease (ACD). In some embodiments, the ACD is cancer-related anemia. In another embodiment, the ACD is anemia associated with chronic kidney disease (CKD). In some embodiments, the anemia is chemotherapy-induced anemia.

[0399] [Kits for use in alleviating RGMc-related symptoms and associated diseases / disorders] The present disclosure also provides kits for use in alleviating a disease / disorder associated with an RGMc-related symptom (e.g., anemia). Such kits may include one or more containers containing an inhibitor that selectively binds to RGMc, e.g., an antibody, or antigen-binding fragment thereof.

[0400] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include instructions for administering an inhibitor, such as an antibody, or antigen-binding fragment thereof, that selectively binds to RGMc to treat, delay onset, or alleviate a target disease, such as those described herein. The kit may further include instructions for selecting an individual appropriate for treatment based on identifying whether the individual has the target disease. In yet other embodiments, the instructions include instructions for administering the antibody, or antigen-binding fragment thereof, to an individual at risk for the target disease.

[0401] Instructions for use of an inhibitor, e.g., an antibody, or antigen-binding fragment thereof, that selectively binds to RGMc, generally include information regarding the dosage, administration schedule, and route of administration for the intended treatment. The container may be a unit dose, bulk package (e.g., a multi-dose package), or sub-unit dose. Instructions provided in the kits of the present disclosure are typically instructions written on a label or insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions carried on a magnetic or optical disk storage device) are also acceptable. The label or insert may indicate that the composition is used to treat, delay the onset of, and / or alleviate a disease or disorder associated with a TGFβ-related symptom. Instructions may be provided for carrying out any of the methods described herein.

[0402] The kit of the present disclosure may be provided in a suitable package. Suitable packages include, but are not limited to, vials, bottles, jars, flexible packages (e.g., sealed Mylar or plastic bags), and the like. Packages for use in combination with a particular device, such as an inhaler, a nasal administration device (e.g., a sprayer), or an infusion device, such as a minipump, are also contemplated. The kit may have a sterile access port (e.g., the container may be a vial with a stopper that can be pierced by a hypodermic injection needle or a solution bag for intravenous administration). The container may have a sterile access port (e.g., the container may be a vial with a stopper that can be pierced by a hypodermic injection needle or a solution bag for intravenous administration). At least one active agent in the composition is an inhibitor that selectively binds to RGMc as described herein, such as an antibody, or an antigen-binding fragment thereof.

[0403] The kit may provide additional components, such as buffers and interpretive information. Typically, the kit includes a container and a label or package insert(s) on or associated with the container. In some embodiments, the disclosure provides an article of manufacture that includes the contents of the kit described above.

[0404] Although several embodiments of the present disclosure have been described and illustrated herein, those skilled in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein, and each such variation and / or modification is deemed to be within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and structures described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or structures will depend on the particular application(s) for which the teachings of the present disclosure are used. Those skilled in the art will recognize, or be able to ascertain, within the bounds of routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Thus, it should be understood that the foregoing embodiments are provided by way of example only, and that, within the scope of the appended claims and their equivalents, the present disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure relates to each individual feature, system, article, material, and / or method described herein. Furthermore, any combination of two or more of such features, systems, articles, materials, and / or methods is within the scope of the present disclosure, if such features, systems, articles, materials, and / or methods are not mutually inconsistent.

[0405] [List of preferred embodiments] 1. An isolated antibody that specifically binds to human repulsive guidance molecule C (RGMc), a) the antibody is a full length antibody or an antigen-binding fragment thereof; b) the antibody does not bind to human repulsive guidance molecule A (RGMa) and human repulsive guidance molecule B (RGMb); and c) The antibody inhibits or reduces the interaction of BMP6 with RGMc.

[0406] 3. An antibody according to embodiment 1 or embodiment 2, wherein the antibody binds to a discontinuous epitope comprising at least a fragment of each of the first and second binding regions in human RGMc, wherein the first binding region may comprise the amino acid sequence set forth in SEQ ID NO: 46 and the second binding region may comprise the amino acid sequence set forth in SEQ ID NO: 47.

[0407] 4. An isolated antibody that binds to an epitope in human RGMc, a) the antibody is a full-length antibody or an antigen-binding fragment thereof; and b) the antibody binds to an epitope comprising at least one amino acid residue of YVSSTLSL (SEQ ID NO: 46) and / or at least one amino acid residue of FHSAVHGIEDL (SEQ ID NO: 47).

[0408] 5. The antibody of embodiment 4, wherein the epitope comprises at least one amino acid residue of YVSSTLSL (SEQ ID NO: 46) and at least one amino acid residue of FHSAVHGIEDL (SEQ ID NO: 47).

[0409] 6. The antibody of embodiment 4 or 5, wherein the antibody does not bind to human RGMa and human RGMb.

[0410] 7. The antibody of any one of embodiments 4 to 76, wherein the antibody inhibits or reduces the interaction of BMP6 with RGMc.

[0411] 8. The antibody of any one of embodiments 4 to 7, wherein the antibody does not inhibit or reduce the interaction of neogenin with RGMc.

[0412] 9. An isolated antibody that specifically binds to human RGMc, wherein the antibody is a full-length antibody or an antigen-binding fragment thereof, wherein the antibody comprises at least three of the following six CDRs: a) CDR-H1: SEQ ID NO: 14: i. the...

Claims

1. An antibody that selectively binds to human repulsive guidance molecule C (RGMc), The antibody is a fully human or humanized antibody, wherein: a) the antibody is a full-length antibody or an antigen-binding fragment thereof; b) the antibody does not bind to human repulsive guidance molecule A (RGMa) and human repulsive guidance molecule B (RGMb); and c) the antibody inhibits or reduces the interaction of BMP6 with RGMc; d) the antibody is i) binds to a first and a second binding region in human RGMc, wherein the first binding region is YVSSTLSL (SEQ ID NO: 46) and the second binding region is FHSAVHGIEDL (SEQ ID NO: 47), and binding to the first and second binding regions is determined by detecting protection of the first and second binding regions from surface exposure by antibody binding by hydrogen deuterium exchange mass spectrometry (HDX-MS); and / or ii) cross-competes for binding to human RGMc with an antibody comprising a heavy chain variable region sequence comprising SEQ ID NO:36 and a light chain variable region sequence comprising SEQ ID NO:37; and, e) the antibody comprises a heavy chain variable region having an amino acid sequence at least 90% identical to SEQ ID NO: 36, and a light chain variable region having an amino acid sequence at least 90% identical to SEQ ID NO: 37; antibody.

2. 2. The antibody of claim 1, comprising: a) a heavy chain variable region having an amino acid sequence at least 95% identical to SEQ ID NO: 36; and / or b) a light chain variable region having an amino acid sequence at least 95% identical to SEQ ID NO:37; Including, antibody.

3. 3. The antibody of claim 1 or 2, the antibody is a human IgG1, IgG2, or IgG4 antibody; The antibody may be a human IgG4 antibody, and the human IgG4 antibody may be an IgG １ Optionally, the amino acid sequence may include a Ser to Pro backbone substitution resulting in a cytosine-like hinge. antibody.

4. The antibody according to any one of claims 1 to 3, The antibody has a K Ｄ and binding to RGMc at a value, wherein the antibody may induce internalization of the antibody-antigen complex. antibody.

5. The antibody according to any one of claims 1 to 4, A heavy chain variable region sequence as set forth in SEQ ID NO: 36, and a light chain variable region sequence as set forth in SEQ ID NO:

37. antibody.

6. A method comprising administering to a subject an antibody according to any one of claims 1 to 4, and a pharma- ceutically acceptable carrier. Pharmaceutical compositions.

7. 7. A pharmaceutical composition according to claim 6 for the treatment of anemia in a human subject, comprising: the pharmaceutical composition is administered to the subject in an amount effective to treat the anemia; Wherein the anemia may be iron-refractory iron deficiency anemia (IRIDA), or anemia associated with chronic disease (ACD), iron deficiency anemia, cancer-related anemia, and chemotherapy-induced anemia; Pharmaceutical compositions.

8. 8. The pharmaceutical composition according to claim 7, The anemia is cancer-related anemia or chemotherapy-induced anemia. Pharmaceutical compositions.

9. 8. The pharmaceutical composition according to claim 7, The ACD is anemia associated with chronic kidney disease (CKD); The subject may be dialysis-dependent or non-dialysis-dependent. Pharmaceutical compositions.

10. 8. The pharmaceutical composition according to claim 7, The ACD is associated with one or more conditions selected from the group consisting of chronic bacterial endocarditis, osteomyelitis, rheumatic fever, ulcerative colitis, chronic kidney disease, and neoplastic disorders; Pharmaceutical compositions.

11. The pharmaceutical composition according to any one of claims 7 to 10, The composition is administered in combination with an additional therapeutic agent, wherein the additional therapeutic agent may be an erythropoietin stimulating agent (ESA), a HIF stabilizer, an iron supplement, or a blood transfusion. Pharmaceutical compositions.

12. 12. The pharmaceutical composition according to claim 11, The treatment reduces toxicity associated with ESAs, HIF stabilizers, iron supplements, or blood transfusions, where the toxicity may include iron overload or cancer risk. Pharmaceutical compositions.

13. The pharmaceutical composition according to any one of claims 7 to 12, The subject is a) currently on iron therapy but would benefit from a reduced dose of iron therapy; b) had received iron therapy but discontinued it due to toxicity; c) have received iron therapy and would benefit from a dose reduction in iron therapy; d) at risk for cancer; and / or e) Have been diagnosed with cancer; Pharmaceutical compositions.

14. 14. The pharmaceutical composition of claim 13, the cancer is a myeloproliferative disorder; The myeloproliferative disorder may be myelofibrosis. Pharmaceutical compositions.

15. 15. The pharmaceutical composition of claim 14, the myelofibrosis is primary myelofibrosis, the subject has received or may be receiving a Janus kinase (JAK) inhibitor, and further, the JAK inhibitor may be a JAK1 inhibitor and / or a JAK2 inhibitor; Pharmaceutical compositions.

16. 7. The pharmaceutical composition according to claim 6 for increasing serum iron levels in a subject, comprising: The pharmaceutical composition comprises: i) increasing serum iron levels in said subject; ii) downregulating hepcidin in said subject; iii) reducing hepatic hepcidin mRNA levels; iv) decreasing hepatic hepcidin protein levels, serum (circulating) hepcidin protein levels, or both; v) increasing transferrin saturation in said subject; vi) increasing red blood cell production in said subject; vii) reducing unsaturated iron binding capacity in said subject; viii) reducing the total iron binding capacity in said subject; and / or ix) increasing serum ferritin levels in said subject. administered to the subject in an amount effective to wherein the amount may be an amount effective to achieve a serum ferritin level of ≧20 nanograms per milliliter of blood. method.

17. The pharmaceutical composition according to any one of claims 7 to 16, the subject has received erythropoietin (EPO / ESA) therapy, HIF stabilizer therapy, IV iron supplementation, or a combination thereof; wherein the subject may have experienced or be at risk for an adverse event associated with EPO therapy, HIF stabilizer therapy, or IV iron supplementation; Pharmaceutical compositions.

18. 7. The pharmaceutical composition according to claim 6 for treating an iron disorder in a subject, comprising: the iron disorder results in anemia in the subject; The subject may have or be at risk for fatigue, joint pain, bone or joint disease, rheumatoid arthritis, inflammatory bowel disease, shortness of breath, arrhythmia, liver disease, diabetes, infertility, frailty, depression, mood or heart disorders, poor cognitive ability or neurodegenerative disease, iron refractory iron deficiency anemia, anemia associated with chronic kidney disease, resistance to erythropoiesis stimulating agents, aplastic anemia, hypoplastic anemia, paroxysmal nocturnal hemoglobinuria, von Willebrand disease, and hemophilia hereditary hemorrhagic telangiectasia, Pharmaceutical compositions.

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