RGMc selective inhibitors and uses thereof

Abstract

Selective inhibitors of repulsive targeting molecule C (RGMc) are described. Also provided are related methods, including methods for preparing these inhibitors, and therapeutic uses of these inhibitors in the treatment of disorders such as anemia.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This international patent application claims the benefit of and priority to U.S. Provisional Patent Application Serial No. 62 / 749,469, filed on October 23, 2018, the entire contents of which are incorporated herein by reference. Field of the Invention

[0003] The present invention generally relates to RGMc binding agents (eg, antibodies and molecules comprising antigen binding fragments) that specifically and selectively inhibit RGMc without inhibiting RGMa or RGMb. Background of the Invention

[0005] Abnormalities in iron homeostasis are associated with a number of diseases that can be difficult to treat. Such disorders can be broadly divided into two categories: i) iron overload diseases, which include cirrhosis, cardiomyopathy, diabetes, etc.; and ii) iron deficiency diseases, which include iron-restrictive anemia, anemia of chronic disease ("ACD"), etc.

[0006] Hepcidin is a key regulator of systemic iron homeostasis, and its expression is mainly confined to the liver. Hepcidin is produced as a propeptide and processed into a mature active peptide by furin or furin-like proteases. Hepcidin negatively regulates iron utilization by binding to its receptor ferroportin (the only cellular iron exporter) and causing internalization and degradation of both. Thus, hepcidin blocks the export of iron from key cells for dietary iron absorption (enterothelial cells), hemoglobin iron recycling (macrophages), and storage iron release from hepatocytes, resulting in reduced systemic iron utilization. The important role of hepcidin in systemic iron homeostasis was soon clearly recognized by the discovery that inactivation of the hepcidin gene was associated with severe iron overload in the liver and pancreas.

[0007] It has been shown that BMP6 and BMP2 expression are required to maintain the iron homeostasis of mice. Bone morphogenetic proteins 6 and 2 (respectively BMP6 and BMP2) are members of the growth factor TGFβ superfamily, which are known to participate in a variety of biological processes, including iron metabolism / homeostasis. It has been shown that BMP6 and BMP2 expression are required to maintain the iron homeostasis of mice. Consistent with the viewpoint that it participates in iron regulation, the genetic damage of BMP6 causes serious tissue iron overload. Similarly, BMP6 mutations have been found in patients suffering from hereditary hemochromatosis, which is a heterogeneous genetic disorder group characterized by substantial iron overload.

[0008] 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 hepcidin regulatory protein or HJV. BMP6 binds to its receptor, and then binds to a larger multimeric complex (including HJV, HFE, TFR2, and neogenin), activating intracellular SMAD phosphorylation, which induces nuclear translocation and increased hepcidin transcription. Therefore, the BMP6 / HJV / SMAD axis is the main regulator of hepcidin expression in response to iron status.

[0009] Currently available therapies for the treatment of clinical indications involving anemia (e.g., 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 α (Procrit / Epogen); Epoetin β (NeoRecormon); Darbepoetin α (Aranesp); and methoxypolyethylene glycol-epoetin β (Mircera). These therapies are suboptimal and may also be associated with adverse side effects or adverse events (e.g., iron overload, cardiovascular disease, and carcinogenic risk).

[0010] Iron overload is a dangerous side effect in anemic patients who frequently receive iron supplements (e.g., IV iron). Excess body iron can be highly toxic, which can affect many organs, leading to a variety of serious conditions, such as liver disease, heart disease, diabetes, hormonal abnormalities, and immune system dysfunction. Similarly, patients who receive blood transfusions are also at risk for toxicity associated with iron overload. For example, one unit of transfused blood contains approximately 250 mg of iron. In patients who receive many 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 gradually accumulates in various tissues, leading to morbidity and mortality. Therefore, treatment-induced excess body iron can lead to serious adverse reactions, including toxicity to the cardiovascular, gastrointestinal, immune, bone / cartilage, reproductive, and renal systems. As an additional or alternative treatment option to IV iron, ESAs (e.g., EPO, such as Amgen's ) treatment has been widely used in numerous patient populations, including those with cancer-related and chemotherapy-induced anemia. However, paradoxically, preclinical and clinical studies have shown that ESAs (e.g., EPO) can potentially accelerate tumor growth and compromise the survival of cancer patients.

[0011] In an attempt to avoid at least some of these adverse side effects associated with topical ESAs and iron supplements (such as blood transfusions), hypoxia-inducible factor prolyl hydroxylases (HIF-PH) have received widespread attention as a potential therapeutic target that aims to promote endogenous EPO production. For this reason, many HIF stabilizers (e.g., HIF-PH inhibitors) have recently been developed. These include, for example, rosuvastatin (Fibrogen), daptostat (GSK), valdustat (Akebia) and molistat (Bayer). Although early efficacy data show superiority or non-inferiority to ESA treatment, long-term concerns remain about the risk of major adverse cardiovascular events and increased cancer risk. In particular, since the HIF axis regulates a wide range of key biological functions (e.g., angiogenesis) in vivo, systemic intervention of this pathway may result in adverse effects on erythropoiesis that exceed expectations.

[0012] As an alternative approach, some companies are exploring BMP6 inhibitors (including neutralizing antibodies) in an attempt to treat anemic patients (e.g., anti-BMP6 antibodies, e.g., WO 2016 / 098079, Novartis; and KY-1070, KyMab). However, these inhibitors risk adversely regulating other aspects of BMP6 signaling, including bone and cartilage formation, ovarian function, and fat metabolism.

[0013] Additionally, in a 2015 paper (The AAPS Journal 17(4):930-938), et al. (AbbVie) described the therapeutic use of two antibodies that bind to both RGMa and RGMc (i.e., RGMa / c) in the treatment of anemia conditions associated with hepcidin dysregulation using two preclinical models, which showed that the non-selective RGMa / c antibody enhanced iron mobilization in vivo. However, despite the speculation that RGMa could function as both a tumor suppressor and an immunomodulator, the paper did not discuss the potential long-term risks of inhibiting the RGMa pathway.

[0014] Therefore, there remains a significant unmet need to provide improved treatments for iron-related diseases, particularly iron-restrictive 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). SUMMARY OF THE INVENTION

[0016] The inventors of the present disclosure recognize the benefits of achieving higher levels of selectivity to more specifically inhibit the signaling pathways regulating iron homeostasis while minimizing potential undesirable systemic effects. To this end, the inventors attempted to inhibit BMP6 signaling in a liver-selective manner by specifically targeting repulsive guidance molecule c (RGMc), also known as hepcidin regulatory protein (HJV) or type 2 hemochromatosis protein (HFE2). It is reasonable to believe that, compared with existing methods that affect other pathways required for other biological functions, in this way, RGMc-selective inhibitors can provide efficacy for treating iron-restricted obstacles while obtaining improved safety characteristics. Therefore, it can be expected that RGMc-selective inhibitors according to the present invention can achieve improved safety characteristics (e.g., reduced toxicity or adverse events / effects), such as reducing the risk of major adverse cardiac events (e.g., stroke) and / or reducing the risk of cancer development. Advantageously, RGMc-selective inhibitors can reduce the risk of iron overload associated with exogenously applied iron (e.g., IV iron or blood transfusion). RGMc-selective inhibitors may allow patients with iron-restricted anemia to require lower doses of ESAs and / or iron replacement therapy, eg, less frequent IV iron or blood transfusions.

[0017] RGMc is a member of the RGM class of proteins (i.e., 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 in mammals in both membrane-bound and soluble forms and plays a role in iron homeostasis / metabolism within the BMP6 axis.

[0018] Both RGMa and RGMb are found in the nervous and immune systems, while RGMc is found in skeletal muscle but is primarily found in the liver. Although each of these family members shares significant structural homology, especially in their BMP binding domains, their physiological roles are quite different. RGMa and RGMb are reported to play a role in nervous system biology, immunity, inflammation, angiogenesis, and growth. Unlike RGMa and RGMb, the known functions of RGMc are primarily restricted to hepatocytes. Thus, selective antibodies that recognize RGMc that do not bind to RGMa or RGMb may provide the potential for liver-specific modulation of BMP6 biology.

[0019] Therefore, RGMc is an obligate co-receptor expressed by the liver for certain BMPs (e.g., BMP6) that enhance the expression of hepcidin and thus inhibit the transport of iron. In other words, by targeting RGMc, whose expression is more restrictive than RGMa, RGMb, and BMP6, more tissue-specific effects can be achieved while reducing off-target effects. This approach presents the possibility of simultaneously addressing iron-restricted anemias and iron overload conditions without extensively inhibiting systemic RGM and BMP6 function.

[0020] Thus, the present invention encompasses the recognition that selective targeting of RGMc provides an advantageous approach to achieving efficacy and safety (reduced toxicity) compared to conventional approaches (e.g., direct BMP6 antagonists and non-selective inhibitors of RGMc / RGMa / RGMb). 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 relative to RGMa and RGMb.

[0021] Therefore, in one aspect, the present invention provides RGMc-specific antibodies or antigen-binding fragments thereof, characterized in that they selectively bind to RGMc. In one embodiment, the present invention provides a separated antibody or antigen-binding fragment thereof, which selectively binds to human RGMc and does not bind to human RGMa and human RGMb or reduces their binding. In some embodiments, the antibody or antigen-binding fragment thereof inhibits or reduces the integration 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 regeneration protein.

[0022] In other aspects, the present invention provides RGMc-specific antibodies or antigen-binding fragments thereof that bind to specific portions on RGMc, which can provide particularly favorable selectivity for RGMc relative to RGMa / b. In some embodiments, the RGMc-specific antibodies encompassed by the present invention bind to the first and / or second binding regions of human RGMc, wherein the first binding region is YVSSTLSL (SEQ ID NO: 46) within the α1 helical domain, and the second binding region is FHSAVHGIEDL (SEQ ID NO: 47) within the α3 helical domain. In some embodiments, the antibody or antigen-binding fragment thereof binds to at least one amino acid residue within the first binding region and / or binds to at least one amino acid residue within the second binding region.

[0023] Thus, the present disclosure provides RGMc selective antibodies that bind to an epitope comprising one or more amino acid residues of YVSSTLSL (SEQ ID NO: 46), optionally further comprising one or more amino acid residues of FHSAVHGIEDL (SEQ ID NO: 47).

[0024] The present disclosure provides RGMc selective antibodies that bind to an epitope comprising one or more amino acid residues of FHSAVHGIEDL (SEQ ID NO: 47), optionally further comprising one or more amino acid residues of YVSSTLSL (SEQ ID NO: 46).

[0025] In some embodiments, the 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).

[0026] In other aspects, the present invention provides RGMc-specific antibodies or antigen-binding fragments thereof, wherein the antibodies are defined by the amino acid sequences and variants of their complementary determining regions (CDRs). For example, the antibodies may comprise one or more CDR sequences selected from SEQ ID NO: 6-11, SEQ ID NO: 14-19, SEQ ID NO: 22-27, SEQ ID NO: 30-35, and SEQ ID NO: 38-43. In some embodiments, each CDR comprises a maximum of 1, 2, 3, 4, or 5 amino acid residue variations compared to the corresponding CDR sequence.

[0027] In other aspects, the present invention provides RGMc-specific antibodies or antigen-binding fragments thereof, wherein the antibodies are defined by their heavy chain variable region and / or light chain variable region sequences. For example, in some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having amino acids as shown 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 antigen-binding fragment thereof comprises a light chain variable region having an amino acid sequence as shown 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 that is 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.

[0028] In other aspects, the present invention provides RGMc-specific antibodies or antigen-binding fragments thereof that compete with the antibodies disclosed herein for binding to human RGMc and / or bind to the same epitope.

[0029] In some embodiments, preferred antibodies according to the present disclosure have the following characteristics: i) the mechanism of action of such antibodies is such that the antibodies bind to RGMc, thereby directly competing for BMP6 binding in the BMP6-hepcidin signaling axis; ii) such antibodies are selective binders of RGMc relative to RGMa or RGMb, preferably with no detectable cross-reactivity thereto; iii) such antibodies show species cross-reactivity to human RGMc, rodents (mouse and rat) and non-human primates (e.g., cynomolgus monkeys); iv) such antibodies have affinity for human and preferably also rodent RGMc, with a K D Equal to or less than 1 nM (i.e., K D ≤1 nM), more preferably ≤0.1 nM; v) such antibodies show in vivo efficacy in one or more suitable preclinical models (e.g., rats) at a dose of 10 mg / kg or less (preferably 5 mg / kg or less, e.g., 1 mg / kg or less) after 24-48 hours (preferably after 24 hours); and / or vi) such antibodies allow formulations 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.

[0030] In other aspects, pharmaceutical compositions comprising antibodies disclosed herein, and therapeutic uses of such antibodies / compositions are provided. Such antibodies and compositions can be used in methods for treating diseases associated with RGMc in human subjects. In some embodiments, the disease associated with RGMc is anemia, for example, iron-restricted anemia (or functional iron deficiency). In some embodiments, 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). CKD can be dialysis-dependent CKD. CKD can be non-dialysis-dependent CKD.

[0031] In some embodiments, the pharmaceutical composition / antibody can be administered to a patient who is receiving or has received another therapeutic agent (e.g., an erythropoietin stimulator, a HIF stabilizer, an iron supplement, an iron infusion, an anticancer agent, and / or an anti-inflammatory agent). In some embodiments, the method reduces the toxicity associated with ESAs, iron supplements, or iron infusions.

[0032] Thus, when used in combination with another therapy for anemia (e.g., erythropoietin stimulators, HIF stabilizers, iron supplements, iron infusions, anticancer agents, and / or anti-inflammatory drugs), the RGMc selective inhibitors disclosed herein can reduce the need for treatment, thereby reducing the dose and / or frequency of treatment.

[0033] In some embodiments, the pharmaceutical composition / antibody can be used to achieve faster relief of anemia when used in combination with another anemia therapy (e.g., an erythropoietin stimulator, a HIF stabilizer, iron supplementation, iron infusion, an anti-cancer agent, and / or an anti-inflammatory drug).

[0034] In some embodiments, the pharmaceutical compositions / antibodies can be used to reduce iron overload associated with iron therapy, such as blood transfusions and IV iron, in patients.

[0035] In some embodiments, the pharmaceutical composition / antibody can be used to sensitize ESA hyporesponsive anemia. For example, approximately 5-10% of CKD patients receiving ESA treatment show hyporesponsiveness to ESA (ESA resistance). The RGMc-selective inhibitors disclosed herein can be used to make this type of anemia more sensitive to ESA therapy.

[0036] According to the present invention, the RGMc-selective inhibitors encompassed herein can be used to treat anemia in an amount effective to achieve one or more of the following: increase serum iron in a subject, downregulate hepcidin expression in a subject, increase transferrin saturation in a subject, increase erythropoiesis in a subject, reduce unsaturated iron binding capacity (UIBC) in a subject, reduce total iron binding capacity (TIBC) in a subject, and / or increase serum ferritin levels in a subject.

[0037] On the other hand, the present invention provides a method for preparing a pharmaceutical composition comprising a selective inhibitor of RGMc, the method comprising the following steps: i) identifying the ability of an antibody or an antigen-binding fragment thereof to selectively bind to RGMc relative to RGMa and RGMb; ii) identifying the ability of the antibody or antigen-binding fragment to inhibit / neutralize RGMc activity in vivo based on step (i); iii) selecting an inhibitory / neutralizing antibody based on steps (i) and (ii) to be formulated into a pharmaceutical composition. In some embodiments, the preparation method further comprises a positive selection step and optionally a negative selection step. In some embodiments, the identification step (ii) also comprises measuring an iron parameter selected from the following: serum iron, TIBC, UIBC, hepcidin expression, and transferrin saturation. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Depicts the relationship between hepcidin and serum iron levels in inflammation / anemia of chronic disease. The figure also shows how RGMc antagonists (e.g., RGMc-specific antibodies) are expected to reduce hepcidin levels and increase serum iron.

[0040] Figure 2 The first generation of RGMc-specific antibodies showed the efficacy against RGMa, RGMb and RGMc. Plot of binding affinity.

[0041] Figure 3A Depicted is an in vitro BMP6 activity assay using HepG2 cells. An increase in BMP6 activity (mean ± SD) indicates antibody-mediated inhibition of BMP6 binding to RGMc. Figure 3B is a graph showing the increase in BMP6 activity (mean ± SD) in the presence of SR-RC-AB3 and SR-RC-AB4. Along with the negative isotype control, a high affinity nonspecific RGM binding protein was used as a positive control.

[0042] Figure 4A is a graph showing the binding of affinity matured RGMc antibodies to RGMa-his (monovalent format) or RGMa-Fc (avid format). RGM01 (high affinity non-specific RGM binding protein) was used as a positive control. Figure 4B Cross-reactivity with both human and mouse RGMc antigens was shown to be maintained throughout the affinity maturation process.

[0043] 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. Along with a negative isotype control, a high affinity non-specific RGM binding protein was used as a positive control.

[0044] Fig. 6A is a graph showing the effect of affinity matured antibodies on rat serum iron levels (mean ± SD). Figure 6B It is a figure showing the effect of affinity-matured antibodies on the unbound iron binding capacity (UIBC) (mean ± SD) of rats. Together with negative isotype controls, high-affinity nonspecific RGM binding proteins were used as positive controls. Significance was determined relative to isotype controls by one-way ANOVA; *p value < 0.05, **p value < 0.01, ***p value < 0.001.

[0045] Fig. 7A is a graph showing serum iron levels (mean ± SEM) over time in female Sprague-Dawley rats when treated with a single dose of 20 mg / kg of SR-RC-AB8 or SR-RC-AB9. Isotype antibodies were used as controls. Figure 7BShown are serum iron (top) and UIBC (bottom) measurements (mean ± SEM) from a PK / PD study in which female Sprague-Dawley rats were administered a single IV dose of SR-RC-AB8 at 0.6 mg / kg to 20 mg / kg (n=5-11 animals / group). Figure 7C Shows the Figure 7B Antibody serum concentrations in the same animals (mean ± SEM). Fig.7D Shown is the relationship between antibody exposure (PK) and iron parameters (PD) at 20 mg / kg (all values ​​are mean ± SEM).

[0046] Fig. 8A is a graph showing the reduction in deuterium uptake in peptides representing regions A and B of RGMc in the presence of SR-RC-AB9 Fab.

[0047] Figure 8B The amino acid sequences of the deuterium protected regions A and B of the RGM family protein alignment are depicted.

[0048] Figure 8C Figure 2 is a modeled diagram of the structure of RGMc in the presence of BMP2. Highlighted are regions A and B protected by H / D exchange.

[0049] Fig.8D is a diagram mapping the SR-RC-AB9 fab epitope onto the BMP2 binding site and highlighting potential contact residues within region B that are protected by H / D exchange.

[0050] Fig. 8E is a diagram showing the structural homology of region A protected by H / D exchange within alpha helices 1 / 2 / 3 of RGMa / b / c and highlighting potential structural determinants of the specificity of SR-RC-AB9 for RGMc relative to RGMa and RGMb.

[0051] Fig.8F is a diagram depicting a typical structure of a Fab fragment of an antibody highlighting CDRs H1-H3, which may span the surface of binding regions A and B of RGMc.

[0052] Fig.9A Shown are serum iron parameters (serum iron and UIBC, mean ± SEM) over time following a single IV dose of 10 mg / kg of SR-RC-AB8 in female cynomolgus monkeys. Fig. 9B Mean serum concentrations of SR-RC-AB8 up to 10 weeks after dosing are shown (mean ± SEM). Fig. 9C The relationship between antibody exposure (PK) and serum iron parameters (PD) is shown. All values ​​are mean ± SD.

[0053] Fig. 10A Shown are changes in serum iron from baseline on day -3 following weekly injections of 20 mg / kg of SR-RC-AB8 alone or in combination with epoietin alpha (EPO) (10 μg / kg) in the PGPS model of anemia of chronic disease (ACD) in female Lewis rats. Fig. 10B (Mean ± SD) shows absolute serum iron levels on day 24. Significance determined by one-way ANOVA versus ACD isotype control; **p value < 0.01. Fig. 10C Shown are changes in hemoglobin relative to baseline measurements on Day -3 in response to weekly dosing with SR-RC-AB8 (20 mg / kg), EPO (10 μg / kg), or both (mean ± SEM). Fig. 10D Absolute hemoglobin levels (mean ± SD) on day 24 are shown in . Significance was determined by one-way ANOVA versus ACD isotype control; **p value < 0.01. DETAILED DESCRIPTION OF THE INVENTION

[0055] definition

[0056] In order to more easily understand the present disclosure, some terms are first defined. These definitions should be read in light of the remainder of the present disclosure and as understood by those of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art are generally understood. Additional definitions are set forth throughout the detailed description.

[0057] Affinity: Affinity is the strength with which a molecule (e.g., an antibody) binds to its ligand (e.g., an antigen). It is usually measured by the equilibrium dissociation constant (K D ) to measure and report. D is the antibody dissociation rate (“dissociation rate” or K) of the antibody off ) (how quickly the antibody dissociates from its antigen) and the rate at which the antibody binds (the “on rate” or K on ) (how quickly the antibody binds to the antigen). For example, an antibody with an affinity ≤ 5 nM has a K D A value of 5 nM or less (i.e., an affinity of 5 nM or greater) is determined by a suitable in vitro binding assay (e.g., a biolayer interferometry (BLI)-based assay (e.g., ), surface plasmon resonance (SPR)-based assays (e.g., Biacore), and solution equilibrium titration-based assays (e.g., Meso Scale Discovery or MDS) assays.

[0058] Anemia: Anemia is a medical condition in which red blood cell count or hemoglobin is lower than normal. For men, anemia is generally defined as a hemoglobin level less than 13.5 grams / 100ml, and for women, it is defined as a hemoglobin less than 12.0 grams / 100ml. Anemia may be caused by a reduction in red blood cell or hemoglobin production or an increase in red blood cell loss (usually due to bleeding) or destruction. Anemia can be diagnosed by, for example, measuring serum iron parameters, wherein when it deviates from the normal range, serum iron parameters can indicate anemia. 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 serum iron divided by total iron binding capacity (TIBC) (expressed as a percentage).

[0059] Anemia of chronic disease: As used herein, the term "anemia of chronic disease" or "ACD" refers to a form of anemia that is caused by another condition. Such conditions may be associated with chronic infection, chronic immune activation, and / or malignancy (eg, chronic kidney disease or cancer).

[0060] 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 derivative thereof, as further described elsewhere herein. Thus, the term refers to an immunoglobulin molecule that specifically binds to a target antigen, and includes, for example, chimeric, humanized, fully human and bispecific antibodies. A complete antibody typically comprises at least two full-length heavy chains and two full-length light chains, but may comprise fewer chains in certain cases, such as antibodies naturally occurring in camelids, which may comprise only heavy chains. An antibody may be derived from only a single source, or it may be "chimeric", i.e., different parts of the antibody may be derived from two different antibodies. Antibodies or their antigen-binding portions may be produced in hybridomas, produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. As used herein, the term antibody includes monoclonal antibodies, bispecific antibodies, small antibodies, domain antibodies, synthetic antibodies (sometimes referred to as "antibody mimics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to as "antibody conjugates" herein), respectively. In some embodiments, the term also encompasses peptibodies.

[0061] Antigen: The term "antigen" broadly includes any molecule containing an antigenic determinant within a binding region to which an antibody or fragment specifically binds. An antigen can be a single subunit molecule (e.g., a protein monomer or fragment) or a complex composed of multiple components. An antigen provides an epitope, such as a molecule or a portion of a molecule, or a complex of molecules or portions of molecules, that can be bound by a selective binding agent such as an antigen binding protein (including, for example, 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 an antibody that can bind to the antigen. An antigen can have one or more epitopes that can interact with different antigen binding proteins (e.g., antibodies).

[0062] 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, the antigen binding portion of an antibody can be derived from a whole antibody molecule, for example, using any suitable standard technique such as proteolytic digestion or recombinant genetic engineering techniques (involving manipulation and expression of DNA encoding antibody variable domains and optionally constant domains). Non-limiting examples of antigen binding portions include: (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bond in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody; (v) single-chain Fv (scFv) molecules (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) dAb fragments (see, e.g., Ward et al., (1989) NATURE 341:544-546); and (vii) a minimum recognition unit consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementary determining region (CDR)). Other forms of single-chain antibodies, such as diabodies, are also included. The antigen-binding portion of the term antibody includes a "single-chain Fab fragment", otherwise known as "scFab", which comprises an antibody heavy chain variable region (VH), an antibody constant region 1 (CH1), an antibody light chain variable region (VL), an antibody light chain constant region (CL), and a linker, wherein the antibody domain and the linker have one of the following orders along the N-terminal to C-terminal direction: 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; and wherein the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids.

[0063] Binding region: As used herein, a "binding region" is a portion of an antigen that, when bound to an antibody or fragment thereof, can form an interface for antibody-antigen interactions. Upon antibody binding, the binding region becomes protected from surface exposure, which can be detected by appropriate techniques, such as hydrogen deuterium exchange mass spectrometry (HDX-MS). Antibody-antigen interactions can be mediated by multiple (e.g., two or more) binding regions. A binding region can comprise an antigenic determinant or epitope.

[0064] Biolayer Interferometry (BLI): BLI is a label-free technique for optically measuring biomolecular interactions, such as between a ligand immobilized on the surface of a biosensor tip and an analyte in solution, which enables real-time measurement of antibody affinity. BLI provides the ability to monitor binding specificity, rates or concentrations of association (e.g., "on" rate), and dissociation (e.g., "off" rate) with precision and accuracy. BLI platform instruments are commercially available from, for example, ForteBio and are generally referred to as system.

[0065] 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 the RGMc molecule as a co-receptor, for example, human BMP6 accession number NP_001709.

[0066] Cancer-related anemia: The term "cancer-related anemia" or CRA, which may also be referred to as "cancer-associated anemia," refers to anemia that is related to, caused by, and / or exacerbated by the presence of cancer.

[0067] Chemotherapy-induced anemia: The term "chemotherapy-induced anemia" or CIA, also known as "chemotherapy-associated anemia", is a type of treatment-induced anemia and refers to anemia caused and / or exacerbated by chemotherapy. In the context of the present disclosure, the term "chemotherapy" shall encompass any anticancer agent, drug, and therapy intended to treat a patient's cancer (e.g., kill malignant cells) that impairs hematopoiesis.

[0068] Chronic Kidney Disease: The term "chronic kidney disease" (CKD) refers to a kidney disease in which kidney function is gradually lost over a period of months to years. For example, such kidney disease can be caused by diabetes, high blood pressure, glomerulonephritis, and polycystic kidney disease. CKD is associated with insufficient production of erythropoietin (EPO) by kidney cells, which results in fewer red blood cells being produced in the bone marrow, ultimately leading to anemia.

[0069] Clinical benefit: As used herein, the term "clinical benefit" is intended to include both the efficacy and safety of a treatment. Thus, a treatment method that achieves a desired clinical benefit is both effective and safe (eg, has tolerable or acceptable toxicity or adverse events).

[0070] 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 treatment regimen in which a first therapy comprising a first composition (e.g., an active ingredient) is administered to a subject in combination with a second therapy comprising a second composition (active ingredient) for the purpose of treating the same or overlapping diseases or clinical conditions. The first and second compositions may act on the same cellular target or on discrete cellular targets. In the case of combination therapy, the phrase "in combination with" means that the therapeutic effect of the first therapy overlaps temporally and / or spatially with the therapeutic effect of the second therapy in the subject receiving the combination therapy. Thus, combination therapy can be formulated as a single formulation for simultaneous administration, or as separate formulations for sequential administration of therapies.

[0071] Combinatory or combinatorial epitope: A combinatorial epitope is an epitope recognized and bound by a combinatorial antibody at a site (i.e., antigenic determinant) formed by non-continuous parts of one or more components of an antigen, which are tightly bound together in a three-dimensional structure to form an epitope. A combinatorial epitope is also called a discontinuous epitope. Therefore, the antibodies of the present invention can bind to an epitope formed by two or more components (e.g., parts or fragments) of RGMc. A combinatorial epitope may comprise amino acid residues from a first part / component of RGMc and amino acid residues from a second part / component of RGMc, and so on. Each part / component may be a part / component of a single protein or a part / component of two or more proteins of an antigen complex. A combinatorial epitope is formed by the structural contribution of two or more components (e.g., parts or segments, such as amino acid residues) of an antigen or an antigen complex.

[0072] Competition or cross-competition: In the context of antigen binding proteins (e.g., antibodies or antigen binding fragments thereof) that compete for the same epitope, the term "competition" refers to competition between antigen-binding proteins as determined by an assay in which the antigen binding protein being tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein to a common antigen (e.g., RGMc or a fragment thereof). Various types of competitive binding assays can be used to determine whether one antigen binding protein competes with another, e.g., solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays; solid phase direct biotin-avidin EIA; solid phase direct label assays, and solid phase direct label sandwich assays. Typically, when a competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) 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, e.g., as assayed using standard assay conditions, e.g., according to the manufacturer's instructions (e.g., at room temperature, In some embodiments, the first antibody or its fragment and the second antibody or its fragment may have the same epitope. In other embodiments, the first antibody or its fragment and the second antibody or its fragment may have different but overlapping epitopes. In yet another embodiment, the first antibody or its fragment and the second antibody or its fragment may have a separation (different) epitope that is in close proximity in three-dimensional space, so that the antibody binding is cross-blocked by steric hindrance. "Cross-blocking" refers to that the combination of the first antibody and the antigen prevents the combination of the second antibody and the same antigen, and similarly, the combination of the second antibody and the antigen prevents the combination of the first antibody and the same antigen.

[0073] Complementarity determining region: As used herein, the term "CDR" refers to the complementarity determining region within an antibody variable sequence. There are three CDRs in each variable region of the heavy and light chains, which are referred to as CDR1, CDR2, and CDR3 for each variable region. As used herein, the term "CDR set" refers to the group of three CDRs present in a single variable region that can bind an antigen. The exact boundaries of these CDRs have been defined differently according to 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 colleagues (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; and Chothia et al. (1989) Nature 342:877-883) found that, despite great diversity at the amino acid sequence level, certain subportions within the Kabat CDRs adopt nearly identical peptide backbone conformations. These subportions are designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" represent the light chain and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, whose boundaries overlap with the Kabat CDRs and whose definitions coincide with the Kabat CDRs. Other boundaries of CDRs where CDRs overlap have been 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 will still overlap with Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental findings that particular residues or groups of residues, or even entire CDRs, will not significantly affect antigen binding. Although certain embodiments use CDRs defined by Kabat or Chothia, the methods used herein can utilize CDRs defined according to any of these systems.

[0074] Conformational epitope: A conformational epitope is an epitope that is recognized and bound by a conformational antibody in a three-dimensional conformation, but is not recognized in an unfolded peptide of the same amino acid sequence. A conformational epitope may be referred to as a conformation-specific epitope, a conformation-dependent epitope, or a conformation-sensitive epitope. The corresponding antibody or fragment thereof that specifically binds to such an epitope may be referred to as 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.

[0075] Constant region: An immunoglobulin constant domain refers to a heavy chain or light chain constant domain. The amino acid sequences of human IgG heavy and light chain constant domains are known in the art.

[0076] Effective Amount: An "effective amount" (or therapeutically effective amount) is a dosage or dosing regimen that achieves a statistically significant clinical benefit in a patient population.

[0077] Epitope: The term "epitope", which may also be referred to as an antigenic determinant, is a molecular determinant (e.g., a polypeptide determinant) that can be specifically bound by a binding agent, immunoglobulin, or T-cell receptor. Epitope determinants include chemically active surface clusters of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope recognized by an antibody or antigen-binding fragment of an antibody is a structural element of an antigen that interacts with the CDRs (e.g., complementary sites) of the antibody or fragment. An epitope may be formed by components from several amino acid residues that interact with the CDRs of the antibody to produce specificity. An antigenic fragment may contain more than one epitope. In certain embodiments, an antibody is specifically bound 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 they cross-compete (one prevents the binding or modulatory effect of another).

[0078] Erythropoiesis stimulating agent: As used herein, the term "erythropoiesis stimulating agent" or "ESA" refers to a drug that stimulates the bone marrow to produce red blood cells. Erythropoietin or EPO is an example of an ESA, and can include erythropoietin alpha, delta, omega, and zeta, including engineered forms of erythropoietin, e.g., darbepoetin alfa. The term "EPO therapy" may be used to broadly encompass therapy employing one or more ESA agents.

[0079] Human antibody: As used herein, the term "human antibody" (or "fully human antibody") 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, in the CDRs and in particular in the CDR3, amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed 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 onto human framework sequences.

[0080] Humanized antibody: The term "humanized antibody" refers to an antibody that comprises heavy and light chain variable region sequences from non-human species (e.g., mice), but in which 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. Similarly, a "humanized antibody" is an antibody or a variant, derivative, analog, or fragment thereof that immunospecifically binds to an antigen of interest, and that comprises a FR region having a substantially human antibody amino acid sequence and a CDR region having a substantially non-human antibody amino acid sequence. As used herein, the term "substantially" in the case of a CDR refers to a CDR having 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 a non-human antibody CDR. Humanized antibodies include substantially all (Fab, Fab', F(ab')2, FabC, Fv) of at least one and usually two variable domains, wherein all or substantially all CDR regions correspond to those of non-human immunoglobulins (i.e., donor antibodies), and all or substantially all FR regions are those of human immunoglobulin consensus sequences. In one embodiment, the humanized antibody also includes at least a portion of an immunoglobulin Fc region, typically an Fc region of a human immunoglobulin. In some embodiments, the humanized antibody includes a light chain and at least a heavy chain variable domain. The antibody may also include a heavy chain CH1, hinge, CH2, CH3, and CH4 region. In some embodiments, the humanized antibody includes only a humanized light chain. In some embodiments, the humanized antibody includes only a humanized heavy chain. In a specific embodiment, the humanized antibody includes only a humanized variable domain of a light chain and / or a humanized heavy chain.

[0081] Hydrogen / deuterium exchange mass spectrometry (HDX-MS): HDX-MS is a well-known technique used to interrogate protein identification and protein-protein interactions in solution by measuring the degree of solvent accessibility. See, e.g., Wei et al., (2014) Drug Discov Today 19(1):95-102. "Hydrogen / deuterium exchange massspectrometry for probing higher order structure of protein therapeutics: methodology and applications".

[0082] Hypoxia-inducible factor (HIF) stabilizers: HIF prolyl hydroxylase (HIF-PH) inhibitors can stabilize HIF (e.g., HIF-2α), which can lead to increased endogenous production of erythropoietin (EPO) in the body. These agents may also be referred to as "HIF activators." Examples of HIF stabilizers include, but are not limited to: Rosuvastat (FG-4592); Valdustat (AKB-6548), Dapostat (GSK1278863), Dsidustat (ZYAN-1), Morifocal (Bay 85-3934); MK-8617, YC-1, IOX-2, 2-methoxyestradiol, GN-44028, AKB-4924, Bay 87-2243, FG-2216, and FG-4497.

[0083] Iron therapy: As used herein, "iron therapy" refers to therapeutic therapy comprising iron supplementation (eg, oral or intravenous (iv) iron).

[0084] Isolated: As used herein, an "isolated" antibody refers to an antibody that is substantially free of other antibodies with different antigenic specificities. In some embodiments, an isolated antibody is substantially free of other unintended cellular material and / or chemicals.

[0085] Efficacy: The term "efficacy" as used herein refers to the activity of a drug, e.g., an inhibitory antibody (or fragment) having inhibitory activity, relative to the concentration or amount of the drug that produces a defined effect. For example, an antibody that is able to produce certain effects at a given dose has a higher efficacy than another antibody that requires twice the amount (dose) to produce the same effect. Efficacy can be measured, for example, by in vitro assays, e.g., the BMP6 activation / inhibition assay described herein. Efficacy can also be measured by in vivo assays, e.g., those measuring hepcidin expression, serum iron, ferritin expression, and iron binding capacity as described herein. In general, antibodies with higher affinity tend to show higher efficacy than antibodies with lower affinity.

[0086] Protection (from solvent exposure): In the context of HDX-MS-based assessment of protein-protein interactions (e.g., antibody-antigen binding), the extent to which a protein (e.g., a region of a protein containing an epitope) is exposed to solvent to allow proton exchange to occur is inversely correlated with the extent 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 because protein-protein interactions prevent the binding region from being accessible to the surrounding solvent. Thus, the protected region indicates the site of interaction. Typically, a suitable solvent is a physiological buffer.

[0087] RGM: As used herein, "RGM" refers to a class of proteins belonging to the family of repulsive guidance molecules.

[0088] RGMc: As used herein, the terms "repulsive guidance molecule C", "RGMc", "hepcidin regulatory protein", "HJV", "hemochromatosis 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, for example, human RGMc Accession No. NP_998818; SEQ ID NO:1.

[0089] RGMc-related disorders: "RGMc-related disorders" refers to any disease or condition i) in which at least part of the pathogenesis and / or progression is attributed to RGMc signaling or its dysregulation, and / or ii) in which inhibition of RGMc may provide clinical benefit. RGMc disorders may include, for example: anemia associated with inflammatory / chronic diseases (e.g., Castleman syndrome, chronic kidney disease, diabetes, heart failure / heart disease, idiopathic autoimmune hemolytic anemia, idiopathic pulmonary hypertension, inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus, systemic juvenile idiopathic arthritis), anemia associated with cancer / proliferative diseases (e.g., acute leukemia, diffuse large B-cell lymphoma, Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome, non-Hodgkin's lymphoma, anemia associated with cerebral ischemia, cerebral macroglobulinemia, bladder cancer, breast cancer, colorectal cancer, gastric cancer, head and neck cancer, hepatocellular carcinoma, lung cancer, ovarian cancer, prostate cancer, and kidney cancer), anemia associated with infection (e.g., endocarditis, Helicobacter pylori infection, hepatitis, human immunodeficiency virus, malaria), anemia associated with neurologic disorders (e.g., acute ischemic stroke, amyotrophic lateral sclerosis (ALS), focal cerebral ischemia / reperfusion, intracranial hemorrhage, and subarachnoid hemorrhage), treatment-induced or treatment-related anemia (e.g., antibiotic-induced Anemias related to iron overload include anemia due to age-related causes (e.g., iron overload, induced by steroids, iron overload, iron insufficiency, anaemia due to chemotherapy, secondary iron overload), anemias related to inherited disorders or other conditions (e.g., age-related (anemia of the elderly), severe anemia, anemia of surgery, blood loss / hemorrhage, calciphylaxis, celiac disease, copper deficiency, Fanconi anemia, hereditary hemochromatosis (type 4), hereditary spherocytosis, IRIDA, obesity, organ transplantation, paroxysmal nocturnal hemoglobinuria, sickle cell anemia, exercise / exercise-induced anemia, thalassemia, thrombotic thrombocytopenic purpura).

[0090] RGM inhibitor: The term "RGM inhibitor" or "RGM antagonist" refers to any agent that can antagonize the biological activity or function of RGM (e.g., RGMa, RGMb and / or RGMc). The term is not intended to limit its mechanism of action and includes, for example, neutralizing antibodies, competitive inhibitors, receptor antagonists, furin and / or TMPRSS6 cleavage enhancers, and soluble RGM peptides.

[0091] RGMc selective inhibitor: The term "RGMc selective" (or "RGMc specific") inhibitor refers to an agent (including small molecules and biologics, e.g., antibodies) that is able to selectively antagonize the biological activity or function of RGMc relative to RGMa and RGMb. For clarity, a monoclonal antibody that binds and inhibits / neutralizes both RGMa and RGMc is not a RGMc selective inhibitor.

[0092] Specific binding: As used herein, the term "specific binding" or "specifically binds" refers to an interaction of an antibody or antigen-binding fragment thereof with an antigen that is dependent on the presence of a specific 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.

[0093] In some embodiments, if the K of the antibody for the target D is at least about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M or less, the antibody or antigen-binding fragment thereof specifically binds to a target, such as RGMc. In some embodiments, the terms "specifically bind to an epitope of RGMc", "specifically bind to an epitope of RGMc", "specifically bind to RGMc" or "specifically binds to RGMc" as used herein refer to binding to RGMc and having a specific affinity of 1.0×10 -7 M or less dissociation constant (K D ) as determined by a suitable in vitro binding assay such as surface plasmon resonance and biolayer interferometry (BLI). In one embodiment, the antibody or antigen binding fragment thereof can specifically bind to human and non-human (e.g., mouse) orthologs of RGMc. For example, in some embodiments, the cross-reactivity of the antibody or antigen binding fragment thereof to human, mouse, rat and / or cynomolgus monkey RGMc differs by less than 5-fold.

[0094] The antibody or antigen-binding fragment thereof may also 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 a higher affinity than human RGMa and / or human RGMb (i.e., has low cross-reactivity). 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., 1000-fold lower K) than to human RGMa and / or human RGMb. D ) binds to human RGMc.

[0095] Subject: The term "subject" in the context of therapeutic applications refers to an individual who is receiving clinical care or intervention (e.g., treatment, diagnosis, etc.). Suitable subjects include vertebrates, including but not limited to mammals (e.g., humans and non-human mammals). When the subject is a human subject, the term "patient" can 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 manifestation, disease stage, susceptibility to certain conditions, responsiveness to treatment, etc.), medical history, health status, gender, age group, genetic criteria (e.g., carriers of certain mutations, polymorphisms, gene duplications, DNA sequence duplications, etc.) and lifestyle factors (e.g., smoking, drinking, exercise, etc.).

[0096] Surface plasmon resonance (SPR): Surface plasmon resonance is an optical phenomenon that enables real-time detection of unlabeled interactors. SPR-based biosensors, such as those commercially available from Biacore, can be used to measure biomolecular interactions, including protein-protein interactions, such as antigen-antibody binding. The technology is well known in the art and can be used to determine parameters such as binding affinity, kinetic rate constants, and thermodynamics.

[0097] 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 ability of blood to bind iron to transferrin. TIBC is determined by measuring the maximum amount of iron that the 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 these two values ​​together. Alternatively, TIBC can be directly determined by adding excess iron to the sample, removing unbound iron, and measuring the iron that dissociates from transferrin. Typically, the "normal" range for TIBC is, for example, 255-450 ug / dL, although measurements can vary between different populations.

[0098] Toxicity: As used herein, the term "toxicity" or "toxicity" refers to adverse in vivo effects in a patient associated with a therapy administered to the patient, e.g., undesirable side effects and adverse events. "Tolerability" refers to a level of toxicity associated with a therapy or treatment regimen that can be reasonably tolerated by a patient without requiring discontinuation of treatment due to toxicity. In the context of the present disclosure, toxicity may include, but is not limited to, a greater risk of death, adverse cardiovascular reactions, and / or stroke (which may be associated with chronic diseases such as chronic kidney disease (CKD)), and shortened overall survival, increased risk of tumor progression or recurrence in cancer patients.

[0099] Treatment / treatment: The term "treatment" or "treatment" includes therapeutic treatment, preventive treatment, and applications that reduce the risk of a subject developing a disorder or other risk factor. Therefore, the term is intended to broadly represent: for example, by enhancing or improving the body's immunity; reducing or reversing immunosuppression; reducing, removing or eliminating harmful cells or substances in the body; reducing the burden of disease (e.g., tumor load); preventing recurrence or relapse; prolonging the refractory period and / or otherwise improving survival. The term includes therapeutic treatment, preventive treatment, and applications in which the risk of a subject developing a disorder or other risk factor is reduced. Treatment does not require a complete cure of the disorder, and covers embodiments in which symptoms or underlying risk factors are alleviated. In the case of combination therapy, the term may also refer to: i) the ability of the second treatment to reduce the effective dose of the first treatment, thereby reducing side effects and improving tolerability; ii) the ability of the second treatment to make the patient more sensitive (e.g., sensitization) to the first treatment; iii) the ability to achieve additive or synergistic clinical benefits; and / or iv) the ability to obtain faster relief (e.g., therapeutic benefit).

[0100] Unbound iron binding capacity: As used herein, the term "unbound iron binding capacity" or "UIBC" refers to the amount of available iron binding capacity of transferrin. UIBC is usually calculated by assessing the difference between the amount of TIBC and serum iron. Alternatively, UIBC can be directly calculated by adding a known amount of excess iron to the sample and subtracting the remaining unbound iron. Typically, although the measured value may vary between populations or individuals, the "normal" range of UIBC is, for example, 120-470ug / dL (21-84umol / L).

[0101] Variable region: The term "variable region" or "variable domain" refers to a portion of the light and / or heavy chain of an antibody, typically including approximately the amino-terminal 120 to 130 amino acids in the heavy chain and approximately 100 to 110 amino-terminal amino acids in the light chain. In certain embodiments, the variable regions of different antibodies vary greatly in amino acid sequence, even between antibodies of the same species. The variable region of an antibody generally determines the specificity of a particular antibody for its target.

[0102] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term "about."

[0103] Unless explicitly stated to the contrary, the indefinite articles "a" and "an" as used in the specification and claims should be understood to mean "at least one".

[0104] As used herein in the specification and claims, the phrase "and / or" should be understood to mean "either or both" of the elements so combined, i.e., elements that are present in connection in some cases and separately in other cases. Unless expressly indicated to the contrary, other elements may optionally be present in addition to the elements specifically identified by the "and / or" clause, whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, in one embodiment, when used in conjunction with open language such as "comprising," a reference to "A and / or B" may refer to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); and so on.

[0105] As used herein in the specification and claims, when referring to a list of one or more elements, the phrase "at least one" should be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each and every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows that elements other than the elements specifically indicated in the list of elements to which the phrase "at least one" refers may optionally be present, whether related to or unrelated to those specifically indicated elements. Thus, as a non-limiting example, in one embodiment, "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 to at least one, optionally including more than one, A, without B (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, without A (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); and so on.

[0106] The use of ordinal terms such as "first", "second", "third" and the like in the claims to modify claim elements does not itself imply any priority, rank or order of one claim element relative to another claim element or the temporal order of performing method actions, but is merely used as a mark to distinguish one claim element with a certain name from another element with the same name (but used in ordinal numbers) to distinguish the claim elements.

[0107] The ranges provided herein are understood to be shorthand for all values ​​within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange 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, e.g., 10-20, 1-10, 30-40, etc.

[0108] Regulation of iron homeostasis via the BMP6 / hepcidin axis

[0109] Hepcidin is a small, liver-secreted peptide and a central regulator of iron homeostasis in the body. Its primary function is to inhibit the transport of iron from the cellular to the plasma compartment of the blood from three major sources: absorption of dietary iron across duodenal enterocytes, release of hemoglobin iron from recycled erythrocytes, and release of stored iron from hepatocytes. Iron regulation is achieved through hepcidin-mediated degradation of the iron exporting protein ferroportin located on the surface of duodenal enterocytes, liver and spleen resident reticuloendothelial macrophages, and hepatocytes. As ferroportin is degraded, macrophages and duodenal enterocytes are no longer able to release iron into the blood, thereby reducing the transfer of iron to transferrin. Thus, as Figure 1 As shown, as hepcidin expression increases, iron export decreases. Conversely, as hepcidin expression decreases, iron export increases. Therefore, genetic and acquired disorders that disrupt normal hepcidin production can lead to iron deficiency (high hepcidin levels) or iron overload (hepcidin deficiency).

[0110] As a major player in maintaining iron homeostasis, hepcidin synthesis is transcriptionally regulated by a variety of stimuli, including intracellular and extracellular iron concentrations, erythropoietic requirements, and inflammation. In healthy individuals, hepcidin is maintained at steady-state levels due to the activity of a group of coordinated proteins in the iron regulatory complex. Genetic analysis of patients with hereditary hemochromatosis (HH), in which genetic mutations lead to the absorption and peripheral storage of excess dietary iron and tissue / organ toxicity, has led to the identification of key proteins that regulate iron homeostasis. The most common type of HH is caused by mutations in the HFE gene, which encodes an atypical MHC-like protein that interferes with transferrin-transferrin receptor binding. Rare forms of HH occur in patients with mutations in TFR2, hepcidin, and HJV (hereinafter referred to as "RGMc"). Each of these rare forms is characterized by inappropriately low hepcidin levels, supporting the role of RGMc, HFE, and TFR2 in the upstream regulation of hepcidin expression. Interestingly, mutations in RGMc are the most common cause of a severe form of juvenile HH, which can be fatal if left untreated.

[0111] Hepcidin expression is stimulated by extracellular stimuli, including the inflammatory signal IL-6 and the developmental signaling bone morphogenetic protein (BMP). Mice in which BMP signaling components (specific ligands, receptors, and co-receptors) are targeted for deletion are characterized by an iron overload phenotype, highlighting the key role of BMP signaling in hepcidin expression. Regulation of BMP signaling occurs at multiple levels to produce specificity and fine-tuning of the signal; co-receptor enhancement or interference with ligand-receptor interactions is the main mechanism of BMP signaling regulation. RGMc is a key co-receptor for BMP-mediated enhancement of hepcidin expression in the liver, and is believed to sensitize hepatocytes to low BMP levels under normal physiological conditions, thereby enhancing 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-related iron restriction plays a key role, such as anemia of chronic disease.

[0112] BMPs are a broad subfamily of growth factors in the TGFβ superfamily that were originally discovered through their ability to induce bone and cartilage formation. Like many other growth factors, BMPs are involved in diverse biological processes in addition to their association with bone. For example, while BMP6 plays a role in many different biological processes, including fat metabolism and ovarian physiology, in the liver, BMP6 plays a role as a key control point in human iron regulation by regulating hepcidin, a master regulator of iron homeostasis.

[0113] Therefore, BMP6 has become an optional therapeutic target for treating diseases and disorders involving iron imbalance. Multiple groups including Eli Lilly and Novartis have developed antibodies that specifically bind and neutralize BMP6, and suggest that such antibodies be used for the treatment of conditions such as anemia. However, these methods intended to directly and systematically inhibit BMP6 signaling are likely to disrupt many different physiological processes involving BMP6, thereby increasing the risk of causing unexpected and undesirable effects (e.g., toxicity).

[0114] Recently, repulsive guidance molecules (RGMs) have been identified as BMP-interacting proteins. The RGM protein family contains at least three known members, RGMa, RGMb, and RGMc, and is so named due to the role of the founder member, RGMa, in directing axon growth during embryogenesis.

[0115] Structurally, RGMs are glycosylphosphatidylinositol (GPI)-anchored proteins 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. RGMs have been reported to interact with multiple members of the BMP class of growth factors, including BMP6 as a co-receptor. Each RGM family member shares significant structural homology (see Table 1), especially in its BMP binding domain. RGMa and RGMc in particular share high sequence homology.

[0116] Despite considerable homology among family members, their expression and biological functions appear to be different. RGMa and RGMb have been reported to have roles in nervous system biology, immunity, inflammation, angiogenesis, and growth, whereas RGMc is primarily expressed in the liver (e.g., hepatocytes), which is the major site of hepcidin synthesis, and is involved in regulating iron metabolism.

[0117] There is evidence to support the view that inhibition of the BMP6 / RGMc axis may be an attractive therapeutic approach for treating iron disorders. For example, US2013 / 0330343A1 discloses that a monoclonal antibody that binds and neutralizes RGMa / c is able to: 1) reduce hepcidin levels, 2) increase serum iron levels and transferrin saturation, and 3) reduce unsaturated iron binding capacity (UIBC) in treated animals.

[0118] The inventors of the present disclosure recognize that RGMc selective inhibition can provide an improved safety profile. It is reasonable to believe that such inhibitors should only target liver-expressed RGMc, while keeping the normal biological functions of RGMa and RGMb intact. In this way, the inventors attempted to identify RGMc selective antibodies that do not bind RGMa or RGMb to achieve liver-specific regulation of BMP6 activity.

[0119] Table 1:

[0120]

[0121] RGMc / Hepcidin (HJV)

[0122] There are at least three isoforms of human RGMc, including a precursor protein (isoform a; SEQ ID NO: 1) and two splice variants (isoform b and isoform c; SEQ ID NOs: 2 and 3, respectively). Human RGMc isoform "a" is the major isoform, consisting of 426 amino acids and representing the longest transcript. Unless otherwise specified herein, the term "RGMc" refers to the full-length protein, heterodimer, or cleavage product of RGMc isoform a. The following are the amino acid sequences of the RGMc isoforms, RGMa and RGMb.

[0123] Human RGMc subtype a (Accession No. NP_998818; SEQ ID NO: 1):

[0124]

[0125] Human RGMc subtype b (Accession No. NP_660320; SEQ ID NO: 2):

[0126]

[0127] Human RGMc subtype c (Accession No. NP_998817; SEQ ID NO: 3):

[0128]

[0129] Human RGMa (Accession No. NP_001159755; SEQ ID NO: 4):

[0130]

[0131]

[0132] Human RGMb (Accession No. NP_001012779; SEQ ID NO: 5):

[0133]

[0134] RGMc is a co-receptor for BMP6, which is part of a larger multimeric protein complex comprising BMP6, BMP6 binding type I and type II serine threonine kinase receptors (e.g., Alk2, Alk3, BMPR2, and ActRIIA), regeneration protein, RGMc (HJV), HFE, and TFR2. BMP6 binds to its receptor, and in turn to the larger multimeric complex, inducing downstream SMAD phosphorylation, thereby inducing nuclear translocation of transcription factors that mediate increased hepcidin transcription. Therefore, BMP6 signaling has been shown to directly regulate iron homeostasis through regulation of hepcidin expression.

[0135] RGMc is expressed in mammals in membrane-bound and soluble forms. Membrane-bound RGMc is present on the cell surface in two different forms, namely a single-chain full-length form and a two-chain (heterodimer) form connected by disulfide bonds. The heterodimer form of RGMc is the result of autoproteolysis at the FGDPH motif to produce a C-terminal fragment of about 35kD and an N-terminal fragment of about 20kD, which is still connected by a disulfide bond. Although it has been shown that single-chain full-length RGMc interacts with BMP2, previous studies have shown that autoproteolysis is very important for the correct folding of proteins, and autocatalytic processing is a common and important feature of RGM. For example, it has been shown that only the heterodimer form of RGMc activates hepcidin expression in vitro, and RGMa autocatalytic processing is necessary for its growth inhibition of axons. In addition, several JHH-related mutations are located near the RGMc autocatalytic cleavage site, and these mutants have low signaling activity and are usually retained in the ER.

[0136] In addition, it has been shown that at least two proteases, furin and TMPRSS6, cleave RGMc to produce several soluble forms of RGMc (s-RGMc or s-HJV). Specifically, furin cleaves to produce 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 be enhanced in response to iron deficiency and hypoxia. However, since furin is also involved in the processing of prohepcidin, the effects of furin on hepcidin expression may be complex and most likely dependent on specific cellular conditions.

[0137] On the other hand, TMPRSS6 can cleave RGMc at multiple sites, resulting in cleavage fragments with different functions. Studies have shown that TMPRSS6 plays a role in regulating hepcidin expression, as evidenced by studies showing that its inactivation in mice leads to increased hepcidin levels, blockade of iron absorption, and anemia. In addition, iron-refractory iron deficiency anemia (IRIDA) may 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.

[0138] As mentioned above, hepcidin is a key regulator of systemic iron mobilization and is subsequently regulated by bone morphogenetic protein (BMP)-mediated signaling, particularly BMP6. RGMc binds to BMP6 and modulates Smad-mediated pathways, leading to the regulation of hepcidin expression, which in turn degrades ferroportin (iron transport protein), thereby preventing the release of iron from macrophages and enterocytes into the circulation. Therefore, increased RGMc activity may increase hepcidin expression, leading to a decrease in iron release from cells. In this case, RGMc activity may lead to an imbalance in iron homeostasis due to systemic iron depletion, causing iron-related diseases. In other words, RGMc-mediated increases in hepcidin levels may lead to anemia, while decreased expression of hepcidin may be the etiological feature of many hemochromatosis (iron overload) diseases.

[0139] Therefore, the present application provides RGMc selective monoclonal antibodies and fragments thereof, which specifically bind and inhibit RGMc activity in vivo without cross-reacting with RGMa or RGMb. To the knowledge of the applicant, these antibodies and fragments represent a new category of such inhibitors. The basic principle of this approach includes the following. First, based on the recognition that hepcidin is a key regulator of iron homeostasis and BMP6 is required in the process, what is needed is an inhibitor that can block the signal transduction axis. Second, since BMP6 is widely expressed and plays a wide variety of biological effects, it is advantageous to avoid directly targeting BMP6. Since it is known that the RGM protein family interacts with the co-receptors of BMP (such as BMP6, BMP2 and BMP4), targeting co-receptors can provide an optional and attractive approach to block the signal transduction that regulates hepcidin. Third, however, it is recognized that RGMa and RGMb play unique biological functions, and such inhibitors should selectively inhibit RGMc without affecting RGMa / b. In this way, selective inhibition of liver RGMc can be achieved while minimizing potential harmful side effects that may be caused by inadvertently and systematically blocking the normal functions of BMP6, RGMa and RGMb.

[0140] The present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to RGMc or a complex containing RGMc and inhibit its function but do not bind to or inhibit the function of RGMa and RGMb. In some embodiments, the antibodies and antigen-binding fragments thereof described herein inhibit the interaction of RGMc with BMP (e.g., BMP6). In some embodiments, the antibodies or fragments described herein do not inhibit the interaction of RGMc with regeneration proteins. In any embodiment, the antibodies and fragments encompassed by the present invention provide precise specificity and targeting of the RGMc pathway. Advantageously, such antibodies or fragments can achieve reduced toxicity. Importantly, since RGMc expression is confined to a subset of tissues, the inhibition of the RGMc-mediated BMP6 pathway should achieve tissue-selective effects, thereby reducing toxicity due to systemic effects.

[0141] Thus, the present invention encompasses the recognition that the use of selective inhibitors of RGMc provides an advantageous approach to treating conditions involving iron imbalance compared to non-selective inhibitors. To this end, the inventors of the present disclosure have attempted to design and generate 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 idea that selectivity provides an improved safety profile (e.g., reduced toxicity or adverse reactions) of administering such inhibitors in vivo.

[0142] For example, targeting RGMc as well as RGMa and / or RGMb may increase potential toxicity associated with the widespread non-hepatic tissue expression of RGMa / b and its known roles in various physiological processes. In particular, RGMa has been shown to be essential for embryonic development and is expressed in multiple tissues, including the brain, skin, heart, liver, lung, kidney, testis, and intestine of adult mice. Consistent with these data, approximately half of mice genetically deficient in RGMa 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 of injury and ischemic stroke; in Alzheimer's disease amyloid plaques; and in the substantia nigra of Parkinson's disease patients. However, reduced RGMa expression and increased promoter methylation status have been shown to be closely associated with the development and progression of colorectal cancer (Zhao et al., Oncol Rep 2012; 27(5): 1653-9). These lines of evidence suggest that RGMa / c cross-reactive antibodies, for example, may cause or exacerbate cancer progression.

[0143] In addition to embryonic development and neuronal regeneration, RGMa has been shown to regulate immune responses to injury; it is expressed by dendritic cells in brain and spinal cord lesions and regulates T cell responses. Specifically, in a mouse model of multiple sclerosis (experimental autoimmune encephalomyelitis; EAE), inhibition of RGMa has been shown to inhibit T cell responses. In humans, RGMa inhibition reduces T cell proliferation and proinflammatory cytokine production in peripheral blood mononuclear cells (PBMCs). Moreover, in T cell subsets, RGMa has been shown to be highly expressed on Th17 cells. Since Th17 cells play an important role in host defense against infection by recruiting neutrophils and macrophages to infected tissues, pharmacological interference with normal immune function is not desired. Advantageously, this side effect can be effectively avoided by selectively targeting and inhibiting RGMc instead of RGMa.

[0144] Although many of these functions are injury-related, RGMa can also regulate neuronal regeneration in healthy individuals. Given its important role in regulating the central nervous system response to injury, immune responses, and unknown roles in various organs, inhibiting RGMa activity is undesirable due to the risk of potential off-target side effects.

[0145] RGMb has also been shown to be expressed in multiple tissues including the brain, bone, heart, lung, liver, kidney, testis, ovary, uterus, epididymis, and pituitary of adult mice. RGMb knockout mice die at day 12 after birth and have retarded growth and development. Like RGMa, RGMb also has a role in immune responses. Specifically, 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 was described as a binding partner of PD-L2 in the lung, and blocking this interaction prevented the development of respiratory tolerance, highlighting the role of RGMb in lung immunity. It raises the possibility that inhibitors that interfere with RGMb function may lead to dysregulated immune responses.

[0146] Furthermore, it has been proposed that RGMb can act as a tumor suppressor, possibly by inhibiting SMAD activation. Indeed, in non-small cell lung cancer, reduced expression of RGMb is associated with a poor prognosis. The RGMb gene is inactivated in a subtype of colorectal cancer, and RGMb has been shown to be a negative regulator of breast cancer proliferation in vitro. Therefore, inhibition of RGMb may not only have effects on the immune and nervous systems, but this inhibition may also enhance the progression and / or development of cancer.

[0147] In conclusion, the broad expression profiles and pleiotropic functions of the related family members RGMa and RGMb in multiple biological processes contrast with the restricted expression pattern and focused role of RGMc in maintaining iron homeostasis. This underscores the importance of adopting an RGMc-specific approach to discover new therapeutics for iron-restricted anemias without complicating off-target effects.

[0148] Thus, in one aspect, the present invention aims to inhibit RGMc function without causing potential side effects or toxicity associated with interfering with RGMa and RGMb activity in vivo. It is expected that liver-selective inhibition of BMP6 signaling through intervention of RGMc function may provide a way to target a variety of iron-restricted anemias, including anemia of chronic kidney disease, anemia of cancer, and anemia of chronic inflammation.

[0149] According to the present invention, selective inhibitors of RGMc do not inhibit other closely related factors, such as RGMa and RGMb. Such inhibitors can be used to normalize iron homeostasis, including for the treatment of various forms of anemia or diseases involving iron-restricted anemia. In fact, as demonstrated herein, RGMc-specific antibodies were shown to selectively inhibit the bone morphogenetic protein 6 (BMP6)-hepcidin axis and improve the availability of iron for erythropoiesis.

[0150] RGMc inhibitors

[0151] To implement the methods of the present invention, any suitable inhibitory agent of RGM / HJV can be used, as long as these agents inhibit or antagonize RGM with sufficient selectivity for the RGMc subtype. Preferably, such RGMc inhibitory agents have no measurable inhibitory activity against RGMa and RGMb at a dose that provides clinical benefit (e.g., therapeutic efficacy and acceptable toxicity profile) when administered to human subjects.

[0152] As used herein, the term "inhibitor" refers to any agent capable of blocking or antagonizing RGMc signaling. Suitable inhibitory agents include small molecules, nucleic acid-based agents, biologics (e.g., polypeptide-based agents, such as antibodies and other finding agents), and any combination thereof.

[0153] In some embodiments, such agents can prevent or reduce RGMc activity by inhibiting the binding of RGMc to the BMP receptor complex. In some embodiments, such agents can prevent or reduce RGMc activity by inhibiting the binding of RGMc to BMP. 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 regeneration protein. In some embodiments, such agents are competitive inhibitors of RGMc / BMP6 binding. In some embodiments, such agents are competitive inhibitors of RGMc / regeneration protein binding.

[0154] In some embodiments, the inhibitor can be an antibody (including fragments thereof, e.g., domain antibodies (dAbs), as described, e.g., 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, e.g., monobodies and single domain antibodies. Monobodies are synthetic binding proteins that typically employ the fibronectin type III domain (FN3) as a molecular scaffold. Monobodies include Adnectins based on the tenth fibronectin type III domain. TM .

[0155] RGMc-specific antibodies

[0156] Recently, a monoclonal antibody that specifically binds to RGMa / RGMc was shown to increase serum iron in rats and cynomolgus monkeys by downregulating hepcidin ( et al. (2015) AAPS J. 2015 Jul; 17(4): 930–938). In this case, the researchers reported no apparent toxicity in healthy animals receiving the RGMa / RGMc antibody, and normalization of blood parameters was observed following a recovery period (approximately 12 weeks) following the dosing period. However, potential risks associated with simultaneous inhibition of RGMa, such as for the nervous system, could not be discerned from the published work. In addition, as described above, there may be many other toxicities in humans, such as those associated with non-hepatocyte expression of RGMa (e.g., those associated with the brain, skin, heart, lungs, kidneys, testes, and intestine). Reduced RGMa expression has also been shown to be associated with the development and progression of colorectal cancer. In addition, RGMa expression has been shown to modulate 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.

[0157] In some embodiments of the invention, a RGMc selective inhibitor (e.g., an antibody) inhibits or reduces the interaction of RGMc with BMP. In another embodiment, a RGMc selective inhibitor (e.g., an antibody) inhibits or reduces the interaction of RGMc with BMP6. In another embodiment, a RGMc-selective inhibitor does not inhibit or reduce the interaction with regeneration protein.

[0158] 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 RGMc with HFE. In another embodiment, the RGMc selective inhibitor (e.g., an antibody) inhibits the interaction of RGMc with TFR2. In another embodiment, the RGMc selective inhibitor (e.g., an antibody) inhibits the interaction of RGMc with ALK2. In another embodiment, the RGMc selective inhibitor (e.g., an antibody) inhibits the interaction of RGMc with ALK3. In another embodiment, the RGMc selective inhibitor (e.g., an antibody) inhibits the interaction of RGMc with BMPR2. In another embodiment, a RGMc selective inhibitor (eg, an antibody) inhibits the interaction of RGMc with ACVR2A. In another embodiment, a RGMc selective inhibitor (eg, an antibody) inhibits the interaction of RGMc with TMPRSS6.

[0159] In some embodiments, RGMc comprises a naturally occurring mammalian amino acid sequence. In some embodiments, RGMc comprises a naturally occurring human amino acid sequence. In some embodiments, RGMc comprises a human, monkey, rat or mouse amino acid sequence. In some embodiments, RGMc is a human RGMc sequence or a fragment thereof, as shown in SEQ ID NO: 1. In some embodiments, RGMa is a soluble form of RGMc.

[0160] In some embodiments, in a suitable in vitro binding assay (e.g. or MSD), RGMc-selective inhibitors have a K of ≤5 nM (e.g., ≤0.1 nM). D On the other hand, unlike the previously described RGMc antibodies, these RGMc-specific antibodies did not show any detectable binding to RGMa or RGMb under the same assay conditions. For example, RGMc-selective inhibitors can be detected with a K of ≤ 0.1 nM. D It binds to RGMc but does not show any detectable binding to RGMa or RGMb by MSD under the same assay conditions.

[0161] 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 a human RGMa sequence or a fragment thereof, as shown in SEQ ID NO:4. In some embodiments, RGMa is a soluble form of RGMa.

[0162] 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 a human RGMb sequence or a fragment thereof, as shown in SEQ ID NO:5. In some embodiments, RGMb is a soluble form of RGMb.

[0163] Aspects of the invention relate to isolated monoclonal antibodies or antigen-binding fragments thereof that selectively bind to RGMc and comprise six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3.

[0164] In one aspect, the antibody or antigen-binding fragment thereof selectively binds to RGMc and has one or more CDR sequences substantially similar to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and / or CDRL3 compared to the corresponding CDR regions. For example, the antibody can comprise one or more CDR sequences (e.g., SEQ ID NO:6-11, SEQ ID NO:14-19, SEQ ID NO:22-27, SEQ ID NO:30-35 or SEQ ID NO:38-43), each of which contains up to 1, 2, 3, 4 or 5 amino acid residue variations compared to the corresponding CDR regions in any one of SEQ ID NO:6-11, SEQ ID NO:14-19, SEQ ID NO:22-27, SEQ ID NO:30-35 or SEQ ID NO:38-43.

[0165] As used herein, the phrase "amino acid variation" or "amino acid change" or "change of an amino acid residue" encompasses amino acid substitutions and / or deletions.

[0166] In one embodiment, the antibody or antigen-binding fragment thereof comprises at least three CDRs selected from the following, optionally comprising up to 3 amino acid changes, for example, 1, 2 or 3 amino acid changes for each of the following CDRs: 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.

[0167] In one embodiment, the antibody or antigen-binding fragment thereof comprises at least three CDRs selected from the following, optionally comprising up to 3 amino acid changes, for example, 1, 2 or 3 amino acid changes for each of the following CDRs: 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.

[0168] In one embodiment, the antibody or antigen-binding fragment thereof comprises at least three CDRs selected from the following, optionally comprising up to 3 amino acid changes, for example, 1, 2 or 3 amino acid changes for each of the following CDRs: 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.

[0169] In one embodiment, the antibody or antigen-binding fragment thereof comprises at least three (e.g., 3, 4, 5 or preferably all 6) CDRs selected from the following, optionally comprising up to 3 amino acid changes, for example, 1, 2 or 3 amino acid changes for each of the following CDRs: 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.

[0170] In one embodiment, the antibody or antigen-binding fragment thereof comprises at least three CDRs selected from the following, optionally comprising up to 3 amino acid changes, for example, 1, 2 or 3 amino acid changes for each of the following CDRs: 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.

[0171] In another aspect, the present invention provides an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region, wherein the heavy chain variable region comprises CDR-H1, CDR-H2 and / or CDR-H3 having specific amino acid changes.

[0172] In some embodiments, the antibody or antigen-binding fragment thereof comprises the CDR-H1 set forth in SEQ ID NO: 14, provided that the serine residue at position 4 of SEQ ID NO: 14 can be replaced by arginine. In another embodiment, the antibody or antigen-binding fragment thereof comprises the CDR-H1 set forth in SEQ ID NO: 14, provided that the alanine residue at position 7 of SEQ ID NO: 14 can be replaced by serine. In another embodiment, the antibody or antigen-binding fragment thereof comprises the CDR-H1 set forth in SEQ ID NO: 14, provided that the serine residue at position 9 of SEQ ID NO: 14 can be replaced by glutamine. In some embodiments, the antibody or antigen-binding fragment thereof comprises the CDR-H1 set forth in SEQ ID NO: 14, provided that (i) the serine residue at position 4 of SEQ ID NO: 14 can be replaced by arginine; (ii) the alanine residue at position 7 of SEQ ID NO: 14 can be replaced by serine; and / or (iii) the serine residue at position 9 of SEQ ID NO: 14 can be replaced by glutamine.

[0173] In some embodiments, the antibody or antigen-binding fragment thereof comprises the CDR-H2 set forth in SEQ ID NO: 15, provided that the threonine residue at position 8 of SEQ ID NO: 15 can be replaced by valine. In another embodiment, the antibody or antigen-binding fragment thereof comprises the CDR-H2 set forth in SEQ ID NO: 15, provided that the asparagine residue at position 10 of SEQ ID NO: 15 can be replaced by serine. In some embodiments, the antibody or antigen-binding fragment thereof comprises the CDR-H2 set forth in SEQ ID NO: 15, provided that (i) the threonine residue at position 8 of SEQ ID NO: 15 can be replaced by valine; and / or (ii) the asparagine residue at position 10 of SEQ ID NO: 15 can be replaced by serine.

[0174] In some embodiments, the antibody or antigen-binding fragment thereof comprises the CDR-H3 set forth in SEQ ID NO: 16, provided that the isoleucine residue at position 5 of SEQ ID NO: 16 can be replaced by tyrosine. In another embodiment, the antibody or antigen-binding fragment thereof comprises the CDR-H3 set forth in SEQ ID NO: 16, provided that the alanine residue at position 6 of SEQ ID NO: 16 can be replaced by valine. In some embodiments, the antibody or antigen-binding fragment thereof comprises the CDR-H3 set forth in SEQ ID NO: 16, provided that (i) the isoleucine residue at position 5 of SEQ ID NO: 16 can be replaced by tyrosine, and / or (ii) the alanine residue at position 6 of SEQ ID NO: 16 can be replaced by valine.

[0175] In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-L1 as set forth in SEQ ID NO: 17, which optionally comprises up to 3 amino acid changes. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-L2 as set forth in SEQ ID NO: 18, which optionally comprises up to 3 amino acid changes. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-L3 as set forth in SEQ ID NO: 19, which optionally comprises up to 3 amino acid changes.

[0176] In a specific 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, and wherein the antibody comprises at least three of the following six CDRs:

[0177] a) CDR-H1: SEQ ID NO: 14, provided that:

[0178] i) the serine residue at position 4 of SEQ ID NO: 14 may be replaced by arginine;

[0179] ii) the alanine residue at position 7 of SEQ ID NO: 14 may be replaced by serine; and / or

[0180] iii) the serine residue at position 9 of SEQ ID NO: 14 may be replaced by glutamine;

[0181] b) CDR-H2: SEQ ID NO: 15, provided that:

[0182] i) the threonine residue at position 8 of SEQ ID NO: 15 may be replaced by valine; and / or

[0183] ii) the asparagine residue at position 10 of SEQ ID NO: 15 may be replaced by serine;

[0184] c) CDR-H3: SEQ ID NO: 16, provided that:

[0185] i) the isoleucine residue at position 5 of SEQ ID NO: 16 may be replaced by tyrosine; and / or

[0186] ii) the alanine residue at position 6 of SEQ ID NO: 16 may be replaced by valine;

[0187] d) CDR-L1: SEQ ID NO: 17;

[0188] e) CDR-L2: SEQ ID NO: 18; and

[0189] f) CDR-L3: SEQ ID NO:19.

[0190] In some embodiments, as in a suitable in vitro binding assay such as Such antibodies have a K of <1 nM (preferably <0.1 nM) as measured in MSD or D Binds to human RGMc. In a preferred embodiment, such an antibody is also subtype specific in that it selectively binds to and inhibits the activity of RGMc and does not bind to and inhibit the activity of RGMa and RGMb.

[0191] In some embodiments, the heavy chain CDR and / or light chain CDR sequence does not contain any amino acid changes. In some embodiments, the antibody or its antigen-binding fragment comprises a CDR-H1 comprising the sequence shown in SEQ ID NO:6. In some embodiments, the antibody or its antigen-binding fragment comprises a CDR-H2 comprising the sequence shown in SEQ ID NO:7. In some embodiments, the antibody or its antigen-binding fragment comprises a CDR-H3 comprising the sequence shown in SEQ ID NO:8. In some embodiments, the antibody or its antigen-binding fragment comprises a CDR-L1 comprising the sequence shown in SEQ ID NO:9. In some embodiments, the antibody or its antigen-binding fragment comprises a CDR-L2 comprising the sequence shown in SEQ ID NO:10. In some embodiments, the antibody or its antigen-binding fragment comprises a CDR-L3 comprising the sequence shown in SEQ ID NO:11.

[0192] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a CDR-H3 having an amino acid sequence of SEQ ID NO: 8 and a light chain variable region comprising a CDR-L3 having an 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 an amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising a CDR-L2 having an 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 an amino acid sequence of SEQ ID NO: 6 and a light chain variable region comprising a CDR-L1 having an amino acid sequence of SEQ ID NO: 9.

[0193] In a specific embodiment, the antibody or antigen-binding fragment thereof comprises the heavy chain CDR-H1, CDR-H2 and CDR-H3 sequences shown as SEQ ID NOs: 6, 7 and 8, respectively, and the light chain CDR-L1, CDR-L2 and CDR-L3 sequences shown as SEQ ID NOs: 9, 10 and 11, respectively.

[0194] In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-H1 comprising the sequence shown in SEQ ID NO: 14. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-H2 comprising the sequence shown in SEQ ID NO: 15. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-H3 comprising the sequence shown in SEQ ID NO: 16. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-L1 comprising the sequence shown in SEQ ID NO: 17. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-L2 comprising the sequence shown in SEQ ID NO: 18. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-L3 comprising the sequence shown in SEQ ID NO: 19.

[0195] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a CDR-H3 having an amino acid sequence of SEQ ID NO: 16 and a light chain variable region comprising a CDR-L3 having an 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 an amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising a CDR-L2 having an 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 an amino acid sequence of SEQ ID NO: 14 and a light chain variable region comprising a CDR-L1 having an amino acid sequence of SEQ ID NO: 17.

[0196] In a specific embodiment, the antibody or antigen-binding fragment thereof comprises the heavy chain CDR-H1, CDR-H2 and CDR-H3 sequences shown in SEQ ID NOs: 14, 15 and 16, respectively, and the light chain CDR-L1, CDR-L2 and CDR-L3 sequences shown in SEQ ID NOs: 17, 18 and 19, respectively.

[0197] In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-H1 comprising the sequence shown in SEQ ID NO:22. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-H2 comprising the sequence shown in SEQ ID NO:23. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-H3 comprising the sequence shown in SEQ ID NO:24. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-L1 comprising the sequence shown in SEQ ID NO:25. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-L2 comprising the sequence shown in SEQ ID NO:26. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-L3 comprising the sequence shown in SEQ ID NO:27.

[0198] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a CDR-H3 having an amino acid sequence of SEQ ID NO: 24 and a light chain variable region comprising a CDR-L3 having an 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 an amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising a CDR-L2 having an 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 an amino acid sequence of SEQ ID NO: 22 and a light chain variable region comprising a CDR-L1 having an amino acid sequence of SEQ ID NO: 25.

[0199] In a specific embodiment, the antibody or antigen-binding fragment thereof comprises the heavy chain CDR-H1, CDR-H2 and CDR-H3 sequences shown as SEQ ID NOs: 22, 23 and 24, respectively, and the light chain CDR-L1, CDR-L2 and CDR-L3 sequences shown as SEQ ID NOs: 25, 26 and 27, respectively.

[0200] In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-H1 comprising the sequence shown in SEQ ID NO:30. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-H2 comprising the sequence shown in SEQ ID NO:31. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-H3 comprising the sequence shown in SEQ ID NO:32. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-L1 comprising the sequence shown in SEQ ID NO:33. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-L2 comprising the sequence shown in SEQ ID NO:34. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-L3 comprising the sequence shown in SEQ ID NO:35.

[0201] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a CDR-H3 having an amino acid sequence of SEQ ID NO: 32 and a light chain variable region comprising a CDR-L3 having an 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 an amino acid sequence of SEQ ID NO: 31 and a light chain variable region comprising a CDR-L2 having an 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 an amino acid sequence of SEQ ID NO: 30 and a light chain variable region comprising a CDR-L1 having an amino acid sequence of SEQ ID NO: 33.

[0202] In a specific embodiment, the antibody or antigen-binding fragment thereof comprises the heavy chain CDR-H1, CDR-H2 and CDR-H3 sequences shown as SEQ ID NOs: 30, 31 and 32, respectively, and the light chain CDR-L1, CDR-L2 and CDR-L3 sequences shown as SEQ ID NOs: 33, 34 and 35, respectively.

[0203] In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-H1 comprising the sequence shown in SEQ ID NO:38. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-H2 comprising the sequence shown in SEQ ID NO:39. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-H3 comprising the sequence shown in SEQ ID NO:40. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-L1 comprising the sequence shown in SEQ ID NO:41. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-L2 comprising the sequence shown in SEQ ID NO:42. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-L3 comprising the sequence shown in SEQ ID NO:43.

[0204] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a CDR-H3 having an amino acid sequence of SEQ ID NO: 40 and a light chain variable region comprising a CDR-L3 having an 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 an amino acid sequence of SEQ ID NO: 39 and a light chain variable region comprising a CDR-L2 having an 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 an amino acid sequence of SEQ ID NO: 38 and a light chain variable region comprising a CDR-L1 having an amino acid sequence of SEQ ID NO: 41.

[0205] In a specific embodiment, the antibody or antigen-binding fragment thereof comprises the heavy chain CDR-H1, CDR-H2 and CDR-H3 sequences shown as SEQ ID NOs:38, 39 and 40, respectively, and the light chain CDR-L1, CDR-L2 and CDR-L3 sequences shown as SEQ ID NOs:41, 42 and 43, respectively.

[0206] Aspects of the present invention relate to a monoclonal antibody or an antigen-binding fragment thereof that selectively binds to RGMc and comprises a heavy chain variable region sequence and a light chain variable region sequence.

[0207] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence that is 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 that is 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.

[0208] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:12, and the light chain variable region has an amino acid sequence that is 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 or a light chain variable region, wherein the heavy chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:12, and the light chain variable region has an amino acid sequence that is 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 and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:12, and the light chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:13.

[0209] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:20, and the light chain variable region has an amino acid sequence that is 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 or a light chain variable region, wherein the heavy chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:20, and the light chain variable region has an amino acid sequence that is 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 and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:20, and the light chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:21.

[0210] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28, and the light chain variable region has an amino acid sequence that is 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 or a light chain variable region, wherein the heavy chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28, and the light chain variable region has an amino acid sequence that is 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 and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28, and the light chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29.

[0211] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:36, and the light chain variable region has an amino acid sequence that is 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 or a light chain variable region, wherein the heavy chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:36, and the light chain variable region has an amino acid sequence that is 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 and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:36, and the light chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:37.

[0212] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:44, and the light chain variable region has an amino acid sequence that is 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 or a light chain variable region, wherein the heavy chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:44, and the light chain variable region has an amino acid sequence that is 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 and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:44, and the light chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:45.

[0213] In some embodiments, the heavy chain variable region and / or light chain variable region sequence does not change in any CDR sequence provided herein. For example, in some embodiments, the 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%) can appear in a heavy chain variable and / or light chain variable amino acid sequence in addition to any CDR sequence provided herein.

[0214] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 12 and / or a light chain variable domain comprising the amino acid sequence shown 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 shown in SEQ ID NO: 12 or a light chain variable domain comprising the amino acid sequence shown 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 shown in SEQ ID NO: 12 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 13.

[0215] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 20 and / or a light chain variable domain comprising the amino acid sequence shown 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 shown in SEQ ID NO: 20 or a light chain variable domain comprising the amino acid sequence shown 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 shown in SEQ ID NO: 20 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 21.

[0216] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 28 and / or a light chain variable domain comprising the amino acid sequence shown 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 shown in SEQ ID NO: 28 or a light chain variable domain comprising the amino acid sequence shown 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 shown in SEQ ID NO: 28 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 29.

[0217] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 36 and / or a light chain variable domain comprising the amino acid sequence shown 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 shown in SEQ ID NO: 36 or a light chain variable domain comprising the amino acid sequence shown 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 shown in SEQ ID NO: 36 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 37.

[0218] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 44 and / or a light chain variable domain comprising the amino acid sequence shown 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 shown in SEQ ID NO: 44 or a light chain variable domain comprising the amino acid sequence shown 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 shown in SEQ ID NO: 44 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 45.

[0219] In some embodiments, the "percent identity" of two amino acid sequences is determined using an algorithm modified from Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, such as that described in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is 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, word length = 3, to obtain amino acid sequences homologous to the protein molecule of interest. In the event that there is a gap 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 (eg, XBLAST and NBLAST) can be used.

[0220] In any antibody or antigen-binding fragment described herein, one or more conservative mutations may be introduced into a CDR or framework sequence at a position where the residue is unlikely to participate in antibody-antigen interactions. In some embodiments, such conservative mutations may be introduced into a CDR or framework sequence at a position where the residue is unlikely to participate in interactions with RGMc, as determined based on the crystal structure. In some embodiments, possible interfaces (e.g., residues involved in antigen-antibody interactions) may be derived from known structural information about other antigens that have a common structural similarity.

[0221] 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. Variants can be prepared according to methods known to those of ordinary skill in the art for altering polypeptide sequences, such as those found in references compiling such methods, for example, Molecular Cloning: A Laboratory Manual, edited by J. Sambrook et al., 2nd edition, published by Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 or Current Protocols in Molecular Biology, edited by FM Ausubel et al., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions between amino acids within 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.

[0222] In some embodiments, the antibodies provided herein include mutations that confer desired properties on the antibodies. For example, to avoid potential complications due to Fab-arm exchange (which is known to occur for natural IgG4 mAbs), the antibodies provided herein may include a stable '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, thereby generating an IgG1-like (CPPCP (SEQ ID ID NO: 48)) hinge sequence. Therefore, any antibody may include a stable "Adair" mutation or amino acid sequence CPPCP (SEQ ID NO: 48). In one embodiment, the antibodies described herein include a heavy chain immunoglobulin constant domain of a human IgG4 with a Ser to Pro backbone replacement, which generates an IgG1-like hinge and allows the formation of interchain disulfide bonds.

[0223] The antibodies of the present disclosure that selectively bind to RGMc may optionally comprise an antibody constant region or a portion thereof. L The domain may be linked at its C-terminus to a light chain constant domain, such as C κ or C λ Similarly, V H The domain or fragment thereof can be linked to all or part of a heavy chain, such as IgA, IgD, IgE, IgG, and IgM, and any isotype subclass. The antibody may comprise a suitable 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 comprise a V domain combined with any suitable constant region. H and V L In an exemplary embodiment, the antibody or its antigen-binding fragment comprises a heavy chain immunoglobulin constant domain, which comprises all or a fragment of a human IgG1 or human IgG4 constant domain. In some embodiments, the antibody or its antigen-binding fragment comprises a light chain immunoglobulin constant domain, which comprises all or a fragment of a human Igλ constant domain or a human Igκ constant domain.

[0224] In some embodiments, the antibody that selectively binds to RGMc may or may not contain the framework regions of the antibodies disclosed herein. In some embodiments, the antibody that selectively binds to RGMc is a murine antibody and contains murine framework region sequences. 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 contains a framework region containing human germline amino acid sequences.

[0225] The antibodies and antigen-binding fragments thereof described herein can have any configuration suitable for binding antigens. For example, in one embodiment, the antibody or its antigen-binding fragment comprises four polypeptide chains, including two heavy chain variable regions and two light chain variable regions. In another embodiment, the antibody or its antigen-binding fragment comprises one heavy chain variable region and one light chain variable region. In an exemplary embodiment, the antibody or its antigen-binding fragment is a Fab fragment, a F(ab')2 fragment, a scFab fragment, a scFv or a diabody.

[0226] In one embodiment, the antibody or its antigen binding fragment 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 its antigen binding fragment binds a conformational epitope. In one embodiment, the antibody or its antigen binding fragment binds a combination or discontinuous epitope.

[0227] 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 that creates an IgG1-like hinge and allows for the formation of interchain disulfide bonds. In one embodiment, the antibody or antigen-binding fragment thereof further comprises a light chain immunoglobulin constant domain containing a human Igλ constant domain or a human Igκ constant domain.

[0228] In one embodiment, the antibody is an IgG with four polypeptide chains, which are two heavy chains and two light chains. In an exemplary embodiment, the antibody can be a humanized antibody, a human antibody, or a chimeric antibody. In one embodiment, the antibody comprises a framework with a human germline amino acid sequence.

[0229] 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.

[0230] In another aspect of the invention, a class of RGMc-specific antibodies is provided, each of which does not bind to RGMa and / or RGMb. Such 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 Binding to Human RGMc. Those skilled in the art are familiar with suitable techniques and assays that can be used to determine in vitro binding activity.

[0231] In a preferred embodiment, suitable means for measuring the affinity of an antibody for its antigen include, but are not limited to, the so-called biolayer interferometry (BLI) technique and surface plasmon resonance (SPR) based analysis. The former includes Systems, and the latter include Biacore systems. In another embodiment, suitable formats for measuring the affinity of an antibody for its antigen include Meso Scale Discovery (MSD).

[0232] Aspects of the present disclosure relate to antibodies that compete or cross-compete with any specific antibody or antigen-binding fragment thereof provided herein, e.g., antibodies having one or more CDR sequences (1, 2, 3, 4, 5 or 6 CDR sequences) as described above. In one embodiment, the present invention provides antibodies and antigen-binding fragments thereof that compete or cross-compete with antibodies having heavy chain CDR sequences as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 and / or light chain CDR sequences as shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11. In one embodiment, the present invention provides antibodies that compete or cross-compete with antibodies or antigen-binding fragments thereof having heavy chain variable region sequences comprising SEQ ID NO: 12 and / or light chain variable region sequences comprising SEQ ID NO: 13.

[0233] In one embodiment, the present invention provides antibodies and antigen-binding fragments thereof that compete or cross-compete with antibodies having heavy chain CDR sequences as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and / or light chain CDR sequences as shown in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19. In one embodiment, the present invention provides antibodies that compete or cross-compete with antibodies or antigen-binding fragments thereof having heavy chain variable region sequences comprising SEQ ID NO: 20 and / or light chain variable region sequences comprising SEQ ID NO: 21.

[0234] In one embodiment, the present invention provides antibodies and antigen-binding fragments thereof that compete or cross-compete with antibodies having heavy chain CDR sequences as shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 and / or light chain CDR sequences as shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27. In one embodiment, the present invention provides antibodies that compete or cross-compete with antibodies or antigen-binding fragments thereof having heavy chain variable region sequences comprising SEQ ID NO: 28 and / or light chain variable region sequences comprising SEQ ID NO: 29.

[0235] In one embodiment, the present invention provides antibodies and antigen-binding fragments thereof that compete or cross-compete with antibodies having heavy chain CDR sequences as shown in SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 and / or light chain CDR sequences as shown in SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35. In one embodiment, the present invention provides antibodies that compete or cross-compete with antibodies or antigen-binding fragments thereof having heavy chain variable region sequences comprising SEQ ID NO: 36 and / or light chain variable region sequences comprising SEQ ID NO: 37.

[0236] In one embodiment, the present invention provides antibodies and antigen-binding fragments thereof that compete or cross-compete with antibodies having heavy chain CDR sequences as shown in SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40 and / or light chain CDR sequences as shown in SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43. In one embodiment, the present invention provides antibodies that compete or cross-compete with antibodies or antigen-binding fragments thereof having heavy chain variable region sequences comprising SEQ ID NO: 44 and / or light chain variable region sequences comprising SEQ ID NO: 45.

[0237] In some embodiments, the antibody or antigen binding fragment thereof binds at or near the same epitope as any antibody provided herein. In some embodiments, if the antibody or antigen binding fragment thereof binds within 15 or less amino acid residues of the epitope, it binds near 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 to which any antibody provided herein binds.

[0238] In another embodiment, provided herein is an antibody or antigen-binding fragment thereof with a distance between the antibody and the protein of less than 10 -8 The equilibrium dissociation constant K of M D In other embodiments, the antibodies are present at 10 -11 M to 10 -8 K in the M range DCompetition or cross-competition for binding to RGMc. In some embodiments, provided herein are RGMc-specific antibodies or antigen-binding fragments thereof that compete for binding with an antibody or antigen-binding fragment thereof described herein. In some embodiments, provided herein are RGMc-specific antibodies or antigen-binding fragments thereof that bind to the same epitope as an antibody or antigen-binding fragment thereof described herein.

[0239] The antibodies provided herein can be characterized using any suitable method. For example, one method is to identify the epitope or "epitope mapping" to which the antigen binds. There are many suitable methods for locating and characterizing the position of the epitope on the protein, including crystal structure analysis, competition analysis, gene fragment expression analysis and analysis based on synthetic peptides of the antibody-antigen complex, for example, 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 another example, epitope mapping can be used to measure the sequence to which the antibody binds. The epitope can be a linear epitope, i.e., a conformational epitope formed by the three-dimensional interaction of the amino acids contained in a single amino acid fragment, or a single fragment (primary structure linear sequence) that may not necessarily be contained.

[0240] Peptides of different lengths (e.g., at least 4-6 amino acids long) can be separated or synthesized (e.g., recombinantly) and used for the binding analysis of antibodies. In another example, the epitope to which the antibody binds can be determined in a systematic screening by using overlapping peptides derived from the target antigen sequence and measuring the combination of the antibody. According to gene fragment expression analysis, the open reading frame encoding the target antigen is randomly or fragmented by a specific genetic construction, and the expression fragment of the antigen is measured to be reactive with the antibody to be tested. For example, gene fragments can be produced by PCR, and then transcribed and translated into proteins in vitro in the presence of radioactive amino acids. The combination of the antibody and the radiolabeled antigen fragment is then measured by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified by using a large random peptide sequence library (phage library) displayed on the surface of a phage particle. Alternatively, the combination of a limited overlapping peptide fragment library and a test antibody can be tested in a simple binding analysis. In another example, mutagenesis, domain exchange experiments, and alanine scanning mutagenesis of the antigen binding domain can be performed to identify the residues required, sufficient, and / or necessary for the epitope to bind. For example, domain swap experiments can be performed using mutants of the target antigen, in which individual fragments of RGMc are replaced (swapped) with sequences from a closely related but antigenically distinct protein (e.g., another member of the RGMc protein family, such as RGMa or RGMb). By assessing antibody binding to mutant RGMc, the importance of specific antigen fragments for antibody binding can be assessed.

[0241] Alternatively, competition assays can be performed using other antibodies known to bind to the same antigen to determine whether the antibody binds to the same epitope as the other antibodies. Competition assays are well known to those skilled in the art.

[0242] In addition, the interaction of any antibody provided herein with one or more residues in RGMc can be determined by conventional techniques. For example, the crystal structure can be determined, and the distance between the residues in RGMc and one or more residues in the antibody (or antigen binding fragment) can be determined accordingly. Based on such distances, it can be determined whether a specific residue in RGMc interacts with one or more residues in the antibody. In addition, suitable methods such as competition assays and targeted mutagenesis assays can be used to determine the preferential binding of candidate antibodies.

[0243] In some embodiments, the antibodies or antigen-binding fragments thereof of the invention that selectively bind to RGMc comprise one or more complementarity determining regions (CDRs) as 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 CDR sequences as described herein.

[0244] RGMc-specific antibody binding regions determined by H / DX-MS

[0245] In the context of the present disclosure, the "binding region" of an antigen provides the structural basis for antibody-antigen interactions. As used herein, "binding region" refers to the interface region between an antibody and an antigen such that when bound to RGMc in a physiological solution, the antibody or fragment protects the binding region from solvent exposure, as determined by suitable techniques such as hydrogen / deuterium exchange mass spectrometry (H / DX-MS).

[0246] The art is familiar with H / DX-MS, which is a technique widely used to explore protein conformation or protein-protein interactions in solution. The method relies on the exchange of hydrogen in the protein backbone amide with deuterium present in solution. By measuring the hydrogen-deuterium exchange rate, one can obtain information about protein dynamics and conformation (reviewed in: Wei et al. (2014) "Hydrogen / deuterium exchange 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 technology is based on the premise that when the antibody-antigen complex is formed, the interface between the binding partners may block the solvent, thereby reducing or preventing the H / D exchange rate due to the spatial exclusion of the solvent.

[0247] Using this technique, the binding region of an antibody (or fragment, such as a Fab) to RGMc can be determined. In some embodiments, the portion of human RGMc identified as being important for binding to an antibody or fragment that meets the selection criteria described herein comprises at least a portion of the amino acid fragment YVSSTLSL (SEQ ID NO: 46), which is present within the α-1 helical domain of RGMc. The same region on full-length RGMc (SEQ ID NO: 1) can be identified as amino acid residues 46-53. Figure 8B As shown, this binding region shows sequence diversity between RGM family members. Therefore, without being bound by a particular theory, it can be expected that this first binding region (region A) can contribute to the specificity of RGMc relative to 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.

[0248] In some embodiments, the portion of human RGMc identified as being important for binding to an antibody or fragment that meets the selection criteria described herein comprises at least a portion of the amino acid fragment FHSAVHGIEDL (SEQ ID NO: 47), which is present within the α-3 helical domain of RGMc. As shown in the RGMc / BMP2 structural model ( Figure 8C ), the α-3 helix is ​​in close proximity to BMP2. Therefore, without being bound by a particular theory, it is expected that this second binding region (region B) can contribute to the functional (e.g., inhibitory) effects of the BMP competitive antibodies described herein. In some embodiments, the epitope for an antibody (or antigen binding fragment) comprises at least one amino acid residue of the binding region FHSAVHGIEDL. In some embodiments, the epitope for an antibody (or antigen binding fragment) comprises two or more amino acid residues of the binding region.

[0249] Several observations support the notion that antibodies derived from SR-RC-AB3 (eg, SR-RC-AB9) bind to at least one amino acid in region A and / or region B of RGMc.

[0250] First, based on the HDX-MS data of RGMc with SR-RC-AB9 Fab, regions A and B are relatively flexible and solvent accessible. Therefore, they are available for binding to antibodies. The relative deuterium uptake of these regions in the unbound form of RGMc shows Relative deuterium uptake. Relative deuterium uptake in less accessible regions is typically less than 30%. Considering the high conformational dynamics of the RGMc N-terminal domain, it is likely that this domain can adopt several conformations that increase the likelihood of antibody binding (see, e.g., Figure 8C , PDB ID 4UI1, RGMc bound to BMP2).

[0251] Second, regions A and B also have several contact points for BMP2 (see Fig.8D and 8E ), suggesting that these regions are "hot spots" for binding interactions. Moreover, upon closer inspection of regions A and B, some residues may serve as structural determinants of RGMc specificity relative to RGMa and RGMb (see Fig. 8E ).

[0252] Third, the CDRs (H1-H3) of the antibody can span the surface of regions A and B of the RGMc N-terminal domain (see Fig.8F ). This can indicate that at least one (e.g., one or more) residues in each region participate in the epitope that the antibody binds. In addition, the shape of the deuterium absorption curve ( Fig. 8A ) showed significant H / D exchange across time points, indicating that the antibody-antigen interaction is quite stable within regions A and B.

[0253] And finally, the binding data of the RGMc N-terminus to the RGMc SR-RC-AB9 fab may also illustrate the concept that both helices (1 & 3) may be required for antibody binding. In particular, the high binding affinity of SR-RC-AB9 to RGMc suggests that there must be significant interfacial buried surfaces for antigen / antibody interactions. These highly buried surface regions are possible when both region A (helix 1) and region B (helix 3) are involved in binding to the antibody.

[0254] Thus, in one embodiment, the RGMc-specific antibody of the present invention binds to at least one amino acid residue of YVSSTLSL (SEQ ID NO: 46). In another embodiment, the RGMc-specific antibody of the present invention binds to at least one amino acid residue of FHSAVHGIEDL (SEQ ID NO: 47). In another embodiment, the RGMc-specific antibody of the present 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, the RGMc-specific antibody of the present 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, the RGMc-specific antibody binds to a discontinuous epitope comprising at least one amino acid of each of the first and second binding regions within human RGMc, wherein the first binding region comprises the amino acid sequence as shown in SEQ ID NO: 46; and / or wherein the second binding region comprises the amino acid sequence as shown in SEQ ID NO: 47. In a specific embodiment, the RGMc-specific antibody binds to an epitope that comprises at least one discontinuous amino acid sequence of each of the first and second binding regions within human RGMc, wherein the first binding region comprises the amino acid sequence shown in SEQ ID NO:46; and wherein the second binding region comprises the amino acid sequence shown in SEQ ID NO:47.

[0255] 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 (SEQID NO: 46) and / or 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 a portion of the binding region having the amino acid sequence FHSAVHGIEDL (SEQ ID NO: 47), or a portion 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 within human RGMc, wherein the first binding region comprises the amino acid sequence set forth in SEQ ID NO: 46; and / or wherein 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 within human RGMc, wherein the first binding region comprises the amino acid sequence set forth in SEQ ID NO: 46; and wherein the second binding region comprises the amino acid sequence set forth in SEQ ID NO: 47.

[0256] In some embodiments, the RGMc-specific antibodies of the present invention bind to a discontinuous epitope within human RGMc, wherein a) the antibody is a full-length antibody or an antigen-binding fragment thereof; and 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 antibodies of the present invention bind to a discontinuous epitope within human RGMc, wherein a) the antibody is a full-length antibody or an antigen-binding fragment thereof; and 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 antibodies of the present invention bind to a discontinuous epitope within human RGMc, wherein 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).

[0257] Nucleic Acids

[0258] In some embodiments, the antibodies, antigen-binding fragments thereof and / or compositions of the present disclosure may be encoded by nucleic acid molecules. 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 programmed or generated to express nucleic acid molecules encoding the compounds and / or compositions of the present disclosure.

[0259] In some embodiments, the present invention provides nucleic acid molecules encoding the aforementioned antibodies or antigen-binding fragments thereof. For example, in one embodiment, the present invention provides nucleic acid molecules encoding polypeptides comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 or CDRL3 as described herein, or a combination thereof. In some embodiments, nucleic acid molecules can encode polypeptides comprising CDRH1, CDRH2 and CDRH3 as described herein. In some embodiments, nucleic acid molecules can encode polypeptides comprising CDRL1, CDRL2 and CDRL3 as described herein. In some embodiments, the polypeptide encoded by the nucleic acid molecule may contain up to 5, 4, 3, 2 or 1 amino acid residue variations compared to the corresponding CDR regions in any one of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 or CDRL3 as described herein, or a combination thereof.

[0260] In one embodiment, the nucleic acid molecule encodes a polypeptide comprising a heavy chain variable domain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:12, and / or a light chain variable domain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:13. In one embodiment, the nucleic acid molecule encodes a polypeptide comprising a heavy chain variable domain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:20, and / or a light chain variable domain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:21. In one embodiment, the nucleic acid molecule encodes a polypeptide comprising a heavy chain variable domain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:28, and / or a light chain variable domain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:29. In one embodiment, the nucleic acid molecule encodes a polypeptide comprising a heavy chain variable domain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:36, and / or a light chain variable domain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:37. In one embodiment, the nucleic acid molecule encodes a polypeptide comprising a heavy chain variable domain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:44, and / or a light chain variable domain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:45.

[0261] In some embodiments, the nucleic acid molecule encodes an antibody or antigen-binding fragment thereof comprising a heavy chain variable domain amino acid sequence as shown 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 as shown 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 antigen-binding fragment thereof comprising a heavy chain variable domain amino acid sequence as shown in SEQ ID NO: 12 and a light chain variable domain amino acid sequence as shown in SEQ ID NO: 13. In some embodiments, the nucleic acid molecule encodes an antibody or antigen-binding fragment thereof comprising a heavy chain variable domain amino acid sequence as shown in SEQ ID NO: 20 and a light chain variable domain amino acid sequence as shown in SEQ ID NO: 21. In some embodiments, the nucleic acid molecule encodes an antibody or antigen-binding fragment thereof comprising a heavy chain variable domain amino acid sequence as shown in SEQ ID NO: 28 and a light chain variable domain amino acid sequence as shown in SEQ ID NO: 29. In some embodiments, the nucleic acid molecule encodes an antibody or antigen-binding fragment thereof comprising a heavy chain variable domain amino acid sequence as shown in SEQ ID NO: 36 and a light chain variable domain amino acid sequence as shown in SEQ ID NO: 37. In some embodiments, the nucleic acid molecule encodes an antibody or antigen-binding fragment thereof comprising a heavy chain variable domain amino acid sequence as shown in SEQ ID NO: 44 and a light chain variable domain amino acid sequence as shown in SEQ ID NO: 45.

[0262] In some cases, nucleic acids of the invention include codon-optimized nucleic acids. Methods for producing codon-optimized nucleic acids are known in the art and may include, but are not limited to, the methods 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.

[0263] Generation of antibodies that bind to RGMc

[0264] The present invention includes methods for screening, generating and preparing antibodies or fragments thereof that bind to RGMc with high affinity and do not bind to RGMa and / or RGMb, as well as pharmaceutical compositions and related kits containing them. The art is familiar with various techniques and methods that can be used to obtain the antibodies or antigen-binding fragments thereof of the present disclosure. For example, recombinant DNA methods, hybridoma technology, phage or yeast display technology, transgenic animals (e.g., ) or some combination thereof to produce antibodies.

[0265] i. Immunization and hybridomas

[0266] In some methods described herein, a particular antigen (e.g., a 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, such as 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.

[0267] After immunization (which may include a single or multiple steps of antigen exposure (e.g., injection), spleen cells are harvested from the animal and the 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 (e.g., enzyme-linked immunosorbent assay (ELISA), biofilm 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 a given antigen. Any form of a given antigen can be used as an immunogen, e.g., a recombinant antigen, a naturally occurring form, any variant or fragment thereof, and an antigenic peptide thereof (e.g., any epitope described herein as a linear epitope or as a conformational epitope within a scaffold).

[0268] ii. Screening library / multiple libraries

[0269] In some embodiments, the method or process of preparing or identifying an antibody includes the step of screening a protein expression library (e.g., a phage, yeast or ribosome display library) expressing an antibody or a fragment thereof (e.g., scFv). For example, a library of human combinatorial antibodies or scFv fragments can be synthesized on phage or yeast, and then the library can be screened with the target antigen or its antibody binding fragment, and the phage or yeast that binds to the antigen can be isolated, from which antibodies or immunoreactive fragments 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).

[0270] Phage display is further described in, e.g., Ladner et al., U.S. Pat. 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; WO 92 / 18619; WO 91 / 17271; WO 92 / 20791; WO 92 / 15679; WO 93 / 01288; WO 92 / 01047; WO 92 / 09690; and WO 90 / 02809. Yeast display is further described in, e.g., US 7,700,302 and US 8,877,688. In a particular method, the yeast display library expresses full-length antibodies.

[0271] Kits for producing phage or yeast display libraries are commercially available. There are also other methods and reagents that can be used to produce and screen 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 technology advantageously allows screening of a large number of candidate antibodies.

[0272] Regardless of how it is obtained, the antibody-producing cells (e.g., yeast colonies, hybridomas, etc.) can be selected, cloned, and further screened for desirable characteristics, including, for example, robust growth, high antibody production, and desirable antibody properties / properties, such as high affinity and specific binding to the target antigen. Preferably, such antibodies exhibit a K of ≤ 5 nM, preferably ≤ 1 nM, and more preferably ≤ 0.1 nM. D Therefore, the RGMc antibodies encompassed by the present invention herein have a K of ≤5 nM, preferably ≤1 nM, and more preferably ≤0.1 nM for recombinant RGMc. D value.

[0273] Other selection criteria may include a polyspecific reagent (PSR) score and / or a hydrophobic interaction chromatography (HIC) retention time. The PSR score is a method of determining the level / extent 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 of 293 and / or CHO cells. A low (0.1-0.33) or zero (0-0.10) PSR score is the most ideal distribution, while a medium (0.33-0.66) or high (0.66-1.00) score is less ideal. Methods for determining PSR scores are well known to those of ordinary skill in the art. In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a PSR score of <0.1. In other embodiments, the antibodies or antigen-binding fragments thereof described herein have a PSR score of 0.

[0274] On the other hand, HIC retention time is a method of determining the propensity of antibodies to self-interact, which predicts whether they remain soluble / monodisperse in solution (also known as aggregation propensity). 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, post-translational modifications, particularly the presence of sugars, can cause antibodies to have low HIC retention times. Therefore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein have a low HIC retention time (e.g., <10.5 minutes). In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a moderate HIC retention time (e.g., ≥10.5 min and ≤11.5 min). In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a high HIC retention time (e.g., ≥11.5 minutes).

[0275] Methods for selecting, cloning and amplifying colonies and / or hybridomas are well known to those of ordinary skill in the art.Once the desired antibody is determined, common molecular biology and biochemical techniques recognized in the art can be used to isolate, manipulate and express the relevant genetic material.

[0276] iii. Humanization

[0277] Antibodies or fragments of the present invention are preferably fully human antibodies or humanized antibodies. Therefore, no matter how the source is, it should be understood that the method may include humanization of one or more antibodies or their fragments, wherein molecular engineering techniques known in the art can be used to prepare human antibody sequences, and introduced into expression systems and host cells as described herein. Such non-natural recombinantly produced human antibodies (and subject compositions) are fully compatible with the teachings of the present disclosure, and clearly remain within the scope of the present invention. In some selected aspects, RGMc antibodies of the present invention will comprise recombinantly produced human antibodies.

[0278] iv. Affinity maturation and optimization

[0279] In some embodiments, the antibodies produced by the above methods can have a medium affinity (e.g., a Ka of about 10 6 Up to 10 7 M -1 or K D About 10 -6 Up to 10 -7 M). Therefore, if necessary, the antibody or its fragment can be subjected to the process of affinity maturation as the optimized part. The term "affinity maturation" should have a meaning that is easily understood by the technician. In short, it refers to further modifying the amino acid sequence of the candidate antibody or fragment (usually referred to as the "parent" or "parent's") to achieve an improvement in the binding characteristics with a specific antigen. Typically, the parent antibody and the affinity-matured counterpart (sometimes referred to as the "offspring" or "offspring") retain the same epitope. Suitable in vitro binding assays can be performed at appropriate steps in the affinity maturation process to screen for improved binding entities. Optionally, in some embodiments, functional assays (e.g., cell-based potency assays, in vitro functional assays, etc.) can also be performed to confirm the desired function.

[0280] Affinity maturation generally involves sequence diversification and / or mutagenesis, while the exact method of introducing or generating mutations or sequence changes is not limited. In some embodiments, mutagenesis comprises introducing 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 comprise up to 1, 2, 3, or 4 amino acid changes relative to a reference sequence (e.g., a reference sequence of a parent sequence). In some embodiments, the VR or CDR sequences described herein may comprise up to 1, 2, 3, or 4 amino acid substitutions. In some embodiments, the VR or CDR sequences described herein may comprise up to 1, 2, 3, or 4 deletions. Additionally or alternatively, the process of mutagenesis may comprise so-called oligo-walking of the variable region or CDR.

[0281] For example, affinity maturation of antibodies can be accomplished by a variety of methods, including random mutagenesis (Gram H. et al., Proc. Natl. Acad. Sci. USA (1992) 89, 3576-3580 and Hawkins R. E. et al., J. Mol. Biol. (1992) 226, 889-896), random mutagenesis of CDR sequences, such as CDR walking (Yang WP et al., J. Mol. Biol. (1995) 254, 392-403), residue-directed mutagenesis (Ho M. et al., J. Biol. Chem. (2005) 280, 607-617 and Ho M. et al., Proc. Natl. Acad. Sci. USA (2006) 103, 9637-9642), and methods for reproducing somatic hypermutation (SHM) in vitro (Bowers et al., Proc. Natl. Acad. Sci. USA (2006) 103, 9637-9642). PM et al., Proc. Natl. Acad. Sci. USA (2011) 108, 20455-20460).

[0282] In one embodiment, the antibody affinity can be matured by mutagenesis on the variable heavy chain or variable light chain region. In another embodiment, the antibody affinity can be matured by mutagenesis on any one of the variable heavy chain CDR or the variable light chain CDR. In another embodiment, the antibody affinity can be matured by mutagenesis on the variable heavy chain CDR3. In another embodiment, the antibody affinity can be matured by mutagenesis on the variable heavy chain CDR2. In another embodiment, the antibody affinity can be matured by mutagenesis on the variable heavy chain CDR1. In another embodiment, the antibody affinity can be matured by mutagenesis on the variable light chain CDR3. In another embodiment, the antibody affinity can be matured by mutagenesis on the variable light chain CDR2. In another embodiment, the antibody affinity can be matured by mutagenesis on the variable light chain CDR1.

[0283] In some embodiments, affinity maturation involves screening an antibody library ("library") comprising variants of one or more CDRs, which can be combined with at least one CDR of a parent antibody. This process is sometimes referred to as CDR shuffling or CDR diversification. In some embodiments, a variable heavy chain CDR3 (e.g., CDR-H3) can be used to screen a library of variable heavy chain CDR1 and CDR2 (e.g., CDR-H1 and CDR-H2) libraries comprising variants (also referred to as CDR-H1 / H2 diversification). In some embodiments, a variable light chain CDR3 (e.g., CDR-L3) can be used to screen a library of variable light chain CDR1 and CDR2 (e.g., CDR-L1 and CDR-L2) libraries comprising variants (also referred to as CDR-L1 / L2 diversification).

[0284] In some embodiments, affinity maturation involves screening an antibody library ("library") comprising light chain variants, which can be combined with the heavy chain of a parent antibody (light chain shuffling). For example, in some embodiments, a selected heavy chain is introduced into an antibody library comprising light chain variants, thereby generating a new antibody library that can be screened for improved affinity. In some embodiments, a library of naturally occurring variable region variants can be obtained from an unimmunized donor. Examples of heavy or light chain shuffling are described in the following documents: 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 λ light chain variants. In some embodiments, the light chain library comprises κ light chain variants. In some embodiments, the light chain library comprises both lambda and kappa light chain variants.

[0285] It should be understood that the various methods for affinity maturation can be combined in any order. For example, in one embodiment, the antibody selected can undergo heavy chain CDR diversification, followed by CDR-H3 mutagenesis. In another embodiment, the antibody selected can undergo heavy chain CDR diversification, followed by CDR-H3 mutagenesis, followed by light chain reorganization. In another embodiment, the antibody selected can undergo heavy chain CDR reorganization, followed by light chain reorganization, followed by CDR-H3 mutagenesis. In a preferred embodiment, the antibody selected can undergo light chain reorganization, followed by CDR-H1 / H2 diversification, followed by CDR-H3 mutagenesis.

[0286] In any of the above methods for affinity maturation, the resulting new antibodies can be selected for binding to a target antigen (e.g., soluble RGMc) using known techniques (e.g., FACS). Binding specificity and affinity using FACS can be tested by varying antigen concentrations and / or competition with unlabeled (cold) antigens. Binding affinity can be further assessed using other techniques known in the art, such as ELISA, BLI (e.g., OCTET), and SPR (e.g., BIACORE).

[0287] In addition to the affinity maturation process discussed above, further optimization can be performed to achieve the desired product spectrum. Therefore, the antibody can be further optimized and selected according to some favorable physicochemical properties. For therapeutic antibodies (biological preparations), the physicochemical standards for developability that can be assessed include but are not limited to: solubility, stability, immunogenicity, lack of self-association or aggregation, Fc function, internalization characteristics, pH sensitivity, glycosylation and manufacturability, for example, cell viability and / or gene expression. In some embodiments, the optimization process involves the mutagenesis of one or more amino acid sequences in the constant region.

[0288] In one aspect, the present invention provides a method for preparing a pharmaceutical composition, which comprises an antibody or an antigen-binding fragment thereof that specifically binds to human RGMc but does not bind to human RGMa or human RGMb; wherein the antibody or the antigen-binding fragment thereof inhibits RGMc but does not inhibit RGMa or RGMb, and the method comprises the following steps: i) providing at least one antigen comprising human RGMc (e.g., soluble human RGMc), ii) selecting a first group of antibodies or antigen-binding fragments thereof that specifically bind to human RGMc to provide a specific binding body of human RGMc; iii) selecting a second group of antibodies or antigen-binding fragments thereof that inhibit RGMc-related BMP6 signaling, thereby producing a specific inhibitor of RGMc / BMP6 activity; iv) formulating the antibodies or antigen-binding fragments thereof present in the first group of antibodies and the second group of antibodies into a pharmaceutical composition, thereby preparing a composition comprising the antibodies or antigen-binding fragments thereof.

[0289] In a preferred embodiment, the method further comprises the step of removing any antibodies or antigen-binding fragments thereof that bind to human RGMa and / or human RGMb from the first group of antibodies or antigen-binding fragments thereof. In one embodiment, the method further comprises the step of determining or confirming the specificity of the antibodies or antigen-binding fragments thereof selected in steps (ii) and / or (iii). In one embodiment, the method further comprises the step of selecting antibodies or antigen-binding fragments thereof that cross-react with human and rodent antigens. In one embodiment, the method further comprises the step of producing fully human or humanized antibodies or antigen-binding fragments thereof present in the first group of antibodies and the second group of antibodies. In one embodiment, the method further comprises the step of subjecting the antibodies or antigen-binding fragments thereof present in the first group of antibodies and the second group of antibodies to affinity maturation and / or optimization to provide affinity matured and / or optimized antibodies or fragments thereof.

[0290] In some embodiments, the method for preparing a pharmaceutical composition further comprises a step of testing one or more candidate RGMc inhibitors in at least one in vivo model to evaluate or confirm the efficacy for achieving or correcting iron regulation. In some embodiments, the method for preparing a pharmaceutical composition further comprises a step of testing one or more candidate RGMc inhibitors (e.g., such antibodies that show efficacy in vivo) in at least one in vivo model to evaluate safety / tolerability at a dose equal to or higher than a therapeutically effective dose. Suitable therapeutic antibodies should show acceptable toxicity at a dose sufficiently greater than the effective dose in an animal model. In some embodiments, one or more antibodies can be used for in vivo efficacy and / or safety studies, the antibodies comprising non-CDR portions or portions that more closely match a specific species (e.g., a mouse counterpart, etc.) used in preclinical studies.

[0291] In another embodiment, a monoclonal antibody is obtained from a non-human animal and then modified using appropriate recombinant DNA technology, such as to make it chimeric. A variety of methods for preparing 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. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., European Patent Publication No. EP171496; European Patent Publication No. 0173494, British Patent GB 2177096B.

[0292] For other antibody production techniques, see, e.g., Antibodies: A Laboratory Manual, ed. Harlow et al., Cold Spring Harbor Laboratory, 1988. The present disclosure is not necessarily limited to any particular source, production method or other special features of the antibodies.

[0293] Some aspects of the present disclosure relate to host cells transformed with polynucleotides or vectors. The host cell can be a prokaryotic or eukaryotic cell. The polynucleotide or vector present in the host cell can be integrated into the genome of the host cell, or can be maintained outside the chromosome. The host cell can be any prokaryotic or eukaryotic cell, for example, bacteria, insects, fungi, plants, animals or human cells. In some embodiments, fungal cells are, for example, those of the genus Saccharomyces, particularly those of the species Saccharomyces cerevisiae. The term "prokaryotic" includes all bacteria that can be transformed or transfected with DNA or RNA molecules to express antibodies or corresponding immunoglobulin chains. Prokaryotic hosts can include gram-negative bacteria and gram-positive bacteria, such as, for example, Escherichia coli, Salmonella typhimurium, Serratia marcescens and Bacillus subtilis. The term "eukaryotic" includes yeast, higher plants, insects and vertebrate cells, for example, mammalian cells, such as NSO and CHO cells. Depending on the host used in the recombinant production procedure, the antibody or immunoglobulin chain encoded by the polynucleotide can be glycosylated or can be non-glycosylated. The antibody or corresponding immunoglobulin chain may also comprise an initial methionine amino acid residue.

[0294] In some embodiments, once the vector is incorporated into a suitable host, the host can be maintained under conditions suitable for high-level expression of the nucleotide sequence, and immunoglobulin light chains, heavy chains, light / heavy chain dimers or complete antibodies, antigen-binding fragments or other immunoglobulin forms can then be collected and purified as desired; see, Beychok, Cells of Immunoglobulin Synthesis, Academic Press, NY, (1979). Thus, a polynucleotide or vector is introduced into a cell, which then produces an antibody or antigen-binding fragment. In addition, transgenic animals, preferably mammals, comprising the above-described host cells can be used to mass-produce antibodies or antibody fragments.

[0295] The transformed host cells can be grown in a fermenter and cultured using any suitable technique to achieve optimal cell growth. Once expressed, full antibodies, dimers thereof, single light and heavy chains, other immunoglobulin forms or antigen binding fragments can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, etc.; see Scopes, "Protein Purification", Springer Verlag, NY (1982). Antibodies or antigen binding fragments can then be separated from growth medium, cell lysates or cell membrane fractions. For example, separation and purification of microbially expressed antibodies or antigen binding fragments can be carried out by any conventional means, for example, preparative chromatographic separations and immunological separations, such as those involving the use of monoclonal antibodies or polyclonal antibodies for example, for the constant region of the antibody.

[0296] Aspects of the present disclosure relate to hybridomas, which provide a monoclonal antibody source that is infinitely extended. As an alternative to obtaining immunoglobulins directly from the culture of hybridomas, immortalized hybridoma cells can be used as a source of rearranged heavy chain and light chain loci for subsequent expression and / or genetic manipulation. The antibody gene of the rearrangement can be reverse transcribed from appropriate mRNA to produce cDNA. In some embodiments, the constant region of the heavy chain can be exchanged for the constant region of different isotypes or eliminated completely. The variable region can be connected to encode a single-chain Fv region. Multiple Fv regions can be connected to confer binding ability to more than a target, or chimeric heavy and light chain combinations can be adopted. Any appropriate method can be used to clone antibody variable regions and produce recombinant antibodies.

[0297] In some embodiments, a suitable nucleic acid encoding the variable region of the heavy chain and / or light chain is obtained and inserted into an expression vector, which can be transfected into a standard recombinant host cell. A variety of such host cells can be used. In some embodiments, mammalian host cells may be advantageous for efficient processing and production. Typical mammalian cell lines that can be used for this purpose include CHO cells, 293 cells, or NSO cells. The 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 coding sequence expression. They can be recovered by separating antibodies or antigen-binding fragments from the culture. The expression system can be designed to include a signal peptide so that the resulting antibody is secreted into the culture medium; however, intracellular production is also possible.

[0298] 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 of the variable regions of the antibodies produced by any of the above hybridomas.

[0299] The polynucleotide encoding the antibody or antigen binding fragment can be, for example, DNA, cDNA, RNA, or synthetically produced DNA or RNA or recombinantly produced chimeric nucleic acid molecules, which contain any of those polynucleotides alone or in combination. In some embodiments, the polynucleotide is part of a vector. Such a vector can contain other genes, such as marker genes, which allow the beta to become a vector in a suitable host cell and under suitable conditions.

[0300] In some embodiments, the polynucleotide is operably connected to an expression control sequence to allow expression in a prokaryotic or eukaryotic cell. The expression of the polynucleotide includes the polynucleotide being transcribed into a translatable mRNA. It is well known to those skilled in the art to ensure that the regulatory elements expressed in eukaryotic cells, preferably mammalian cells, are well known. They can include regulatory sequences that promote transcription initiation and optionally promote poly-A signals that terminate transcription and transcript stability. Other regulatory elements can include transcription and translation 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 Escherichia coli, and examples of regulatory elements that allow expression in eukaryotic host cells are CMV-promoters, SV40 promoters, RSV promoters (Rous sarcoma virus), CMV enhancers, SV40 enhancers or globin introns in AOX1 or GAL1 promoters in yeast or mammals and other animal cells.

[0301] In addition to the elements responsible for transcription initiation, such regulatory elements can also include transcription termination signals downstream of the polynucleotide, such as SV40-poly-A sites or tk-poly-A sites. In addition, depending on the expression system adopted, the leader sequence that can guide the polypeptide to the cell compartment or secrete it into the culture medium can be added to the coding sequence of the polynucleotide and has been described previously. The leader sequence is assembled in the appropriate phase with translation, initiation and termination sequences, and preferably, the protein of the translation or a part thereof can be directed to be secreted into, for example, the leader sequence in the extracellular culture medium. Optionally, a heterologous polynucleotide sequence encoding a fusion protein can be used, the fusion protein comprising a C- or N-terminal recognition peptide, which imparts desired characteristics, for example, the stable or simplified purification of the recombinant product expressed.

[0302] In some embodiments, the polynucleotide encoding at least the variable domain of the light chain and / or the heavy chain can encode the variable domain of two immunoglobulin chains or only one. Similarly, the polynucleotides can be under the control of the same promoter, or can be controlled separately for expression. In addition, some aspects relate to vectors, particularly plasmids, cosmids, viruses and phages routinely used in genetic engineering, which contain polynucleotides encoding the variable domains of the immunoglobulin chains of antibodies or antigen-binding fragments; optionally in combination with polynucleotides encoding the variable domains of another immunoglobulin chain of antibodies.

[0303] In some embodiments, expression control sequences are provided with eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, but control sequences of 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) can be used to deliver polynucleotides or vectors to target cell populations (e.g., engineered cells to express antibodies or antigen binding fragments). A variety of suitable methods can be used to construct recombinant viral vectors. In some embodiments, polynucleotides and vectors can be reconstructed into liposomes to be delivered to target cells. A vector containing polynucleotides (e.g., heavy and / or light variable domain encoding sequences of immunoglobulin chains and expression control sequences) can be transferred to a host cell by a suitable method, and the method described varies according to the type of the cell host.

[0304] Therefore, in one aspect of the present invention, a method for preparing a pharmaceutical composition comprising a selective inhibitor of RGMc (e.g., a neutralizing antibody) is provided, the method comprising the following steps: i) identifying the ability of an antibody or antigen-binding fragment to selectively bind to RGMc relative to RGMa and RGMb; ii) identifying the ability of the antibody or antigen-binding fragment to inhibit / neutralize RGMc activity in vivo based on step (i); iii) selecting an inhibitory / neutralizing antibody based on steps (i) and (ii) to be formulated into a pharmaceutical composition. In one embodiment, step (i) comprises positive selection and optionally also comprises 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 determining an iron parameter selected from the following: 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 hepatic hepcidin level and / or serum hepcidin level. In some embodiments, step (ii) comprises identifying neutralizing antibodies or antigen-binding fragments capable of increasing serum iron levels and / or inhibiting hepcidin expression in vivo. In some embodiments, step (i) comprises confirming selectivity for RGMc relative to RGMa and RGMb.

[0305] In another embodiment, the antibody or antigen binding fragment produced by the methods disclosed herein binds to an epitope that excludes a BMP6 binding site. In some embodiments, the epitope excludes a regeneration protein binding site. In some embodiments, the epitope excludes a BMP6 binding site and a regeneration protein binding site. In some embodiments, the antibody or antigen binding fragment preferentially binds to membrane-bound RGMc compared to soluble RGMc. In some embodiments, the antibody induces internalization of the antibody-antigen complex.

[0306] In another embodiment, the pharmaceutical compositions produced by the methods disclosed herein are formulated for intravenous or subcutaneous administration.

[0307] Modification

[0308] Antibodies or antigen-binding fragments thereof of the present invention may be modified with detectable labels or detectable groups, 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 detecting and separating RGMc. Suitable techniques may be used to directly couple or conjugate a detectable substance or group to a polypeptide of the present invention or indirectly couple or conjugate a polypeptide of the present invention through an intermediate such as, for example, a linker (e.g., a cleavable linker). 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, dichlorotriazinamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent materials include luminol; non-limiting examples of bioluminescent materials include luciferase, luciferin and aequorin; and examples of suitable radioactive materials include radioactive metal ions, for example, α-emitters or other radioisotopes, for example, 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), 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).

[0309] The detectable substance can be directly coupled or conjugated to the antibodies of the present disclosure that specifically bind to RGMc or indirectly coupled or conjugated through an intermediate (such as, for example, a linker) using appropriate techniques. Any antibody provided herein conjugated to a detectable substance can be used in any suitable diagnostic assay, such as those described herein.

[0310] In addition, the antibodies or antigen-binding fragments thereof of the present disclosure may also be modified with drugs. The drug may be coupled or conjugated directly to the polypeptide of the present disclosure or indirectly via an intermediate such as, for example, a linker (eg, a cleavable linker) using appropriate techniques.

[0311] Pharmaceutical compositions and preparations

[0312] 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 pharmaceutically acceptable carrier (excipient) (including, for example, a buffer) to form a pharmaceutical composition. Such formulations can be used to treat diseases or disorders involving RGMc signaling. In particularly preferred embodiments, the formulations can be used to treat anemia as described herein.

[0313] The pharmaceutical composition of the present invention can be administered to a patient to alleviate the indication associated with RGMc. For example, the indication associated with RGMc can be iron-restricted anemia. 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), cancer-induced anemia, and / or chemotherapy-induced anemia.

[0314] "Acceptable" means that the carrier is compatible with the active ingredient of the composition (and preferably capable of stabilizing the active ingredient) and is not deleterious to the subject to be treated. Examples of pharmaceutically acceptable excipients (carriers) (including buffers) will be apparent to those skilled in the art and have been described previously. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover.

[0315] The pharmaceutical composition used in the method of the present invention may comprise a pharmaceutically acceptable carrier, excipient or stabilizer in the form of a lyophilized formulation or an aqueous solution (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover).

[0316] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. 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) can be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0317] The pharmaceutical compounds described herein may include one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound and do not impart any undesirable toxicological effects (see, e.g., 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, e.g., hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, etc., 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, etc. Base addition salts include those derived from alkaline earth metals (eg, sodium, potassium, magnesium, calcium, etc.), as well as those derived from nontoxic organic amines (eg, N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.).

[0318] The pharmaceutical compositions described herein may also include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, 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 chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0319] 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 (e.g., olive oil) and injectable organic esters (e.g., ethyl oleate). Suitable fluidity can be maintained, for example, by the use of coating materials (e.g., lecithin), by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0320] These compositions may also include adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. The presence of microorganisms can be ensured by the above-mentioned sterilization procedures and by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, etc. It may also be necessary to include isotonic agents, such as sugars, sodium chloride, etc. in the composition. In addition, the absorption of injectable drug forms can be extended by including agents that delay absorption (e.g., aluminum monostearate and gelatin).

[0321] Other acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol 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 1 0 residues) polypeptides; proteins, such as serum albumin, gelatin 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., zinc protein complexes); and / or nonionic surfactants, such as TWEEN TM 、PLURONICS TM Or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

[0322] In some embodiments, the pharmaceutical compositions described herein comprise liposomes containing antibodies that specifically bind to RGMc, which can be prepared by any suitable method, such as 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. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with extended circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be produced by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter having a defined pore size to produce liposomes having a desired diameter.

[0323] The antibodies described herein can also be embedded in microcapsules (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions, for example, prepared by coacervation techniques or by interfacial polymerization. Exemplary techniques have been described previously, see, e.g., Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[0324] In other examples, the pharmaceutical compositions described herein can be formulated into sustained release forms. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers comprising antibodies, which are in the form of molded 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), L-glutamic acid and 7-ethyl-L-glutamate copolymers, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyrate.

[0325] Pharmaceutical compositions for in vivo administration must be sterile. This is easily accomplished, for example, by filtration through a sterile filter membrane. The therapeutic antibody composition is typically placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle.

[0326] 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.

[0327] In order to prepare solid compositions such as tablets, the main active ingredient can be mixed with a pharmaceutical carrier, such as conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talcum powder, stearic acid, magnesium stearate, dicalcium phosphate or gum and other pharmaceutical diluents (such as water) to form a solid preformulated composition comprising a uniform mixture of the disclosed compounds or their non-toxic pharmaceutically acceptable salts. When these preformulated compositions are referred to as uniform, it means that the active ingredient is evenly dispersed throughout the composition, so that the composition can be easily subdivided into equivalent unit dosage forms, such as tablets, pills and capsules. The solid preformulated composition is then subdivided into unit dosage forms of the above type comprising 0.1 mg to about 500 mg of the active ingredient of the present invention. The tablets or pills of the new composition can be coated or otherwise compounded to provide a dosage form with the advantage of prolonged action. For example, a tablet or pill may include an inner dose and an outer dose component, the latter being the form of an encapsulation on the former. The two components may be separated by an enteric layer, which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to delay its release. A variety of materials can be used for such enteric layers or coatings, including a variety of polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0328] Suitable surface-active agents include, in particular, non-ionic agents such as polyoxyethylene sorbitan (e.g., Tween TM 20, 40, 60, 80 or 85) and other sorbitans (e.g. Span TM 20, 40, 60, 80 or 85). Compositions with surface active agents conveniently contain between 0.05 and 5% of surface active agent, and may be between 0.1 and 2.5%. It will be appreciated that other ingredients may be added if desired, such as mannitol or other pharmaceutically acceptable vehicles.

[0329] Commercially available fat emulsions such as Intralipid TM , Liposyn TM Infonutrol TM 、Lipofundin TM Lipiphysan TM To prepare a suitable emulsion. The active ingredient can be dissolved in a premixed emulsion composition, or it can be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and formed into an emulsion when mixed with a phospholipid (e.g., lecithin, soybean lecithin, or soybean lecithin) and water. It should be understood that other ingredients, such as glycerol or glucose, can be added to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, for example between 5% and 20%.

[0330] The emulsion composition can be prepared by combining the antibody of the present invention with Intralipid TM Or those prepared by mixing its components (soybean oil, lecithin, glycerin and water).

[0331] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the composition is administered via the oral or nasal respiratory route for local or systemic effect.

[0332] Compositions in preferably sterile pharmaceutically acceptable solvents may be nebulized by use of gases. Nebulized solutions may be breathed directly from the nebulizing device, or the nebulizing device may be connected to a mask, tent, or intermittent positive pressure breathing machine. Solutions, suspensions, or powder compositions may be administered preferably orally or nasally from a device that delivers the formulation in an appropriate manner.

[0333] Therapeutic uses, treatment methods, patient populations

[0334] Abnormality of iron homeostasis is associated with many diseases that may be difficult to diagnose and treat. Such disorders can be roughly divided 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 divided into two main forms: absolute iron deficiency (AID) and functional iron deficiency (FID). The definition of AID is the reduction of iron reserves in the body, and FID is a disorder in which the iron supply (e.g., iron mobilization) to the bone marrow is dysregulated or insufficient while the systemic iron reserves are normal or increased.

[0335] 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 therapies are associated with undesirable side effects. For example, it has been shown that patients with chronic anemia who receive frequent iron supplements (e.g., IV iron) may experience iron overload. Too much iron in the body may be highly toxic, which may affect many organs, leading to various serious conditions, such as liver disease, heart disease, diabetes, hormone abnormalities, and immune system dysfunction. Similarly, patients who receive blood transfusions also have toxic risks associated with iron overload. For example, in patients who receive multiple blood transfusions, especially patients with major thalassemia, sickle cell disease, myelodysplastic syndrome, aplastic anemia, hemolytic anemia, and refractory sideroblastic anemia (who may become dependent on blood transfusions), excess iron from transfused red blood cells (about 250 mg per transfusion) gradually accumulates in various tissues, leading to morbidity and death. Therefore, excessive iron in the body caused by treatment can lead to serious adverse reactions, including toxicity to cardiovascular, gastrointestinal, immune, bone / cartilage, reproductive, and renal systems.

[0336] As an additional or alternative treatment option to IV iron, ESA therapy (e.g., EPO) has been widely administered to a wide range of patient populations, including those with cancer-related and chemotherapy-induced anemia. However, paradoxically, recent preclinical and clinical studies have shown that ESAs may potentially accelerate tumor growth and compromise the survival of cancer patients.

[0337] HIF stabilizers, instead of exogenously added erythropoietin or its equivalent, are intended to stimulate the body's ability to produce endogenous erythropoietin in order to increase red blood cells via the HIF signaling cascade. It is hypothesized that this approach could potentially reduce risk factors associated with ESA therapy, such as increased cancer progression and serious adverse cardiac events (e.g., stroke). However, to date, improved safety has not been clearly established.

[0338] Recently, a monoclonal antibody that specifically binds to RGMa / RGMc has been shown to increase serum iron in rats and cynomolgus monkeys by downregulating hepcidin ( et al. (2015) AAPS J. 20157;17(4):930–93). In this case, the researchers observed no overt toxicity in healthy animals that received the RGMa / RGMc antibody, and the recovery period after the dosing period ( After 12 weeks, normalization of blood parameters was seen. However, potential risks associated with the simultaneous inhibition of RGMa, such as neurological and immunomodulatory effects, could not be discerned from published work.

[0339] Therefore, there is a need for improved therapies for achieving iron homeostasis that can be effectively and safely used to treat patients suffering from diseases and disorders involving an imbalance in iron homeostasis, such as anemia.

[0340] Therefore, in one aspect, the present invention provides an improved method for treating and / or preventing a disorder related to iron metabolism, particularly a disorder related to iron deficiency (e.g., anemia). RGMc-selective inhibitors (e.g., monoclonal antibodies and antigen-binding fragments thereof that selectively bind and inhibit RGMc rather than RGMa / b) can be used to treat a disorder related to functional iron deficiency in a subject. Therapeutic use comprises administering one or more RGMc-selective inhibitors according to the present invention in an amount effective to treat the subject. Treatment may include alleviating, alleviating, normalizing or maintaining one or more symptoms / parameters of anemia, such as serum iron levels, transferrin saturation (TAST), reticulocyte hemoglobin content (CHr), reticulocyte count, red blood cell count, hemoglobin and hematocrit. RGMc-selective inhibitors can provide clinical benefits in terms of the degree of therapeutic effect and / or the kinetics or timing of such effect (e.g., faster relief, longer effects, etc.). RGMc-selective inhibitors can provide clinical benefits in terms of reducing the toxicity or adverse events associated with the therapy intended to treat anemia or the underlying disease causing anemia.

[0341] Conditions that can be treated and / or prevented using the methods of the present invention include any disease, disorder or syndrome associated with a disorder in iron metabolism. A disorder in iron metabolism may be associated with a disturbance in one or more of iron uptake, iron absorption, iron transport, iron storage, iron processing, iron mobilization and iron utilization. Typically, a disorder in iron metabolism can result in iron overload or iron deficiency.

[0342] The disease or disorder of iron metabolism can be any disease or disorder in which the iron homeostasis in the subject is disturbed. This homeostasis depends on the correct regulation of appropriate plasma iron levels. The iron bound to transferrin circulates in the plasma, and transferrin is the carrier of iron delivery into the cell. Plasma transferrin is usually saturated with iron about 30%. Therefore, transferrin saturation must be maintained at an appropriate physiological level in response to a variety of signals from pathways with iron consumption.

[0343] 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. This disruption of iron homeostasis may also lead to anemia of chronic disease, in which subjects with the disease exhibit high blood hepcidin levels. The term "anemia of chronic disease" (ACD) refers to any anemia that occurs due to, for example, prolonged infection, inflammation, neoplastic disorders, etc. The anemia that occurs is usually characterized by shortened erythrocyte lifespan and sequestration of iron in macrophages, which results in a reduction in the amount of iron available to make new erythrocytes. 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 disorders.

[0344] In addition, a subject with 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 arrhythmia, liver disease, diabetes, infertility, impotence, depression, mood or psychiatric disorders, poor cognitive ability or neurodegenerative disease, iron-refractory iron deficiency anemia, anemia of chronic kidney disease, resistance to erythropoiesis stimulating agents, aplastic anemia, cancer, aplastic anemia, paroxysmal nocturnal hemoglobinuria, von Willebrand disease, and hereditary hemorrhagic telangiectasia in hemophilia.

[0345] Further conditions include diseases and disorders associated with infection, inflammation, and tumors, including, for example, inflammatory infections (e.g., lung abscess, tuberculosis, etc.), non-infectious inflammatory 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 may also result from chemotherapy treatment or conditions such as blood loss from pregnancy, menstruation, infancy and childhood, injury.

[0346] Conditions associated with iron overload include primary and secondary iron overload diseases, syndromes or disorders including, but not limited to, hereditary hemochromatosis, porphyria cutanea tarda, hereditary globulocytosis, hypochromic anemias, hysererythropoietic anemia (CDAI), dyserythropoietic anemia congenital (CDAII), faciogenital dysplasia (FGDY), Aarskog syndrome, atransferrinemia, sideroblastic anemia (SA), pyridoxine-responsive sideroblastic anemia, erythrocyte enzyme diseases such as glucose-6-phosphate dehydrogenase (G6PD) or pyruvate kinase deficiency (PKD), and hemoglobinopathies such as thalassemia and sickle cell. Some studies have shown that there is an association between disorders of iron metabolism (e.g., thalassemia and hemochromatosis) and many disease states (e.g., 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).

[0347] Studies have also shown that iron metabolism plays a role in many other disease states, including cardiovascular disease (see, for example, 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).

[0348] Recently, a role for hepcidin and iron in neurological disorders has been proposed. For example, elevated levels of hepcidin have been shown in various neurological diseases, including, for example, 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).

[0349] In addition, iron metabolism may also play a key 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). In fact, elevated levels of prohepcidin have been shown in brain tissue of patients with restless legs syndrome, while decreased levels of prohepcidin have been observed in cerebrospinal fluid of early-onset disease (Clardy et al., J Neurol Sci 2006; 247: 173-9). Low iron reserves 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). Even in the presence of HH, low iron levels in the brain of RLS patients persist (Haba-Rubio J. et al., J Neurol Neurosurg Psychiatry 2005; 76: 1009-10). In addition, RLS patients have lower expression of transferrin receptors in the brain microvasculature, indicating lower transport of iron across the BBB (Connor JR et al., Brain 2011; 134: 959-68). Interestingly, restless legs syndrome is considered a comorbidity of rheumatoid arthritis - both diseases are associated with iron dysregulation (John A. Gjevre and Regina M. Taylor Gjevre, Autoimmune Diseases 2013; Article ID 352782).

[0350] i. Iron-refractory iron deficiency anemia (IRIDA)

[0351] Iron-refractory iron deficiency anemia is an autosomal recessive genetic disease caused by mutations in Tmprss6. TMPRSS6 is a negative regulator of hepcidin, which produces soluble RGMc (s-RGMc) by cutting membrane-bound RGMc, which serves as a decoy receptor for BMP6. Reduced BMP6 signaling leads to a reduction in hepcidin expression. Therefore, mutations that inhibit TMPRSS6 function promote the accumulation of membrane-bound RGMc, which leads to an increase in hepcidin expression and iron deficiency anemia. There is too little iron in the blood of patients with IRIDA, which leads to small red blood cell size (microerythrocytes) and pale color (too little hemoglobin). Other phenotypes of the disease may include low transferrin saturation, low serum ferritin, and low serum iron levels. Unfortunately, the high hepcidin levels in these patients make it refractory to oral administration of iron.

[0352] Given that the clear mechanism of IRIDA results in an increase in membrane-bound RGMc (and increased hepcidin levels), agents that specifically bind to RGMc and reduce hepcidin levels would also be beneficial while limiting potential side effects of targeting related RGM proteins (e.g., RGMa and RGMb).

[0353] Therefore, RGMc-selective inhibitors (e.g., monoclonal antibodies disclosed herein, derivatives thereof, monoclonal antibodies competing therewith, and engineered molecules comprising antigen-binding fragments thereof) can be used to treat IRIDA in subjects (e.g., human patients). Therefore, in one aspect, a method for treating, preventing, or ameliorating symptoms associated with IRIDA is disclosed. In one embodiment, the method comprises administering an RGMc antagonist to a patient suffering from IRIDA. In another embodiment, the method comprises administering an antibody that specifically binds to RGMc and inhibits its function to a patient suffering from IRIDA. In another embodiment, the RGMc-specific antibody does not bind to (or has reduced binding 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-blocks) antigen binding with one or more RGMc antibodies disclosed herein.

[0354] ii. Anemia of chronic disease (ACD)

[0355] Anemia of chronic disease (eg, anemia of chronic inflammation) is a form of anemia resulting from chronic infection, chronic immune activation (eg, inflammation), and / or malignancy, and is the most common form of anemia in hospitalized patients.

[0356] Typically, ACD begins with an inflammatory response, followed by the release of inflammatory cytokines that mediate disease progression. Cytokines reduce the production of erythrocytes, promote their lysis, and stimulate macrophages to store and retain iron in the form of ferritin, ultimately leading to insufficient iron availability. In addition, there is a large increase in interleukin 6 (IL-6), which stimulates the expression of hepcidin, which in turn causes the degradation of ferroportin, thereby preventing the release of iron from macrophages and enterocytes into the circulation. In addition to the inflammatory response, other mechanisms may also play a role in ACD, for example, reduced erythropoiesis by reducing the ability of the bone marrow to respond to erythropoietin.

[0357] As mentioned 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), inflammatory diseases of the gastrointestinal tract (such as inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis), diabetes, and heart failure. Although inflammatory anemia usually develops slowly, anemia of critical illness can develop rapidly in patients hospitalized for severe acute infection, trauma, or other conditions that may cause inflammation. In addition, certain drug treatments, such as chemotherapy, can rapidly induce inflammation and anemia.

[0358] Other inflammatory diseases associated with anemia may include, for example, Castleman syndrome, chronic kidney disease, diabetes, heart failure / heart disease, idiopathic autoimmune hemolytic anemia, idiopathic pulmonary hypertension, inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus, and systemic juvenile idiopathic arthritis. Infectious diseases that cause anemia may include, for example, endocarditis, Helicobacter pylori infection, hepatitis, human immunodeficiency virus, and malaria. Nervous system diseases associated with anemia may include, for example, acute ischemic stroke, amyotrophic lateral sclerosis, focal cerebral ischemia / reperfusion, intracranial cerebral hemorrhage, and subarachnoid hemorrhage.

[0359] Therefore, RGMc selective inhibitors (e.g., monoclonal antibodies disclosed herein, derivatives thereof, monoclonal antibodies competing therewith, and engineered molecules comprising antigen-binding fragments thereof) can be used to treat ACD in subjects (e.g., human patients) (e.g., one or more ACD indications contained herein). Therefore, in one aspect, a method for treating anemia of chronic disease (ACD) (e.g., preventing or ameliorating symptoms associated therewith) in human patients is disclosed. In some embodiments, the method comprises administering an RGMc antagonist to a patient suffering from ACD. In some embodiments, the method comprises administering an antibody that specifically binds to RGMc and inhibits its function to a patient suffering from ACD. In some embodiments, the RGMc-specific (RGMc-selective) antibody does not bind to (or has reduced binding 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, the RGMc-specific (RGMc-selective) antibody competes (e.g., cross-competes or cross-blocks) antigen binding with one or more RGMc antibodies disclosed herein.

[0360] iii. Cancer-related anemia

[0361] Approximately 30-90% of cancer patients are affected by anemia (Knight et al., Am J Med 2004; 116 Suppl 7A: 11S-26S). For example, approximately 32% of patients with non-Hodgkin's lymphoma and 49% of patients with gynecological cancers suffer from anemia at the time of diagnosis (Moullet et al., Ann Oncol 1998; 8: 1109-1115 and Ludwig et al., Eur J Cancer 2004; 40: 2293-2306). Anemia may be caused by factors related to the cancer itself (cancer-induced anemia) or as a result of chemotherapy (chemotherapy-induced anemia). In addition, radiation therapy to the bones 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) decreased production of functional red blood cells (RBCs); 2) increased destruction of RBCs; and 3) blood loss. However, the causes of anemia may be multifactorial, which may complicate evaluation and treatment. Anemia may be caused by bleeding, hemolysis, nutritional deficiencies, genetic disorders, renal insufficiency, hormonal dysfunction, and / or combinations thereof. In addition, cancer cells themselves may cause and / or exacerbate anemia by infiltrating the bone marrow, producing cytokines that cause iron chelation (resulting in reduced RBC production), shortening RBC survival time, and / or altering coagulation.

[0362] In addition, cancer patients may experience chronic blood loss at the tumor site due to vascular or organ damage. Other factors that may lead to anemia in cancer patients include loss of appetite, immune-mediated antibody-induced hemolysis, or changes in coagulation ability.

[0363] Finally, anemia has also been identified as a poor prognostic factor for cancer patients. For example, it is predicted that the risk of death increases by 65% ​​and the average annual medical costs per patient increase by nearly four times. Therefore, the goal of treating anemia in cancer patients is to improve the patient's quality of life (QoL) and reduce dependence on blood transfusions. Blood transfusions are associated with potential risks of infectious disease transmission, transfusion reactions, lung injury, and alloimmunization. Blood transfusions may also increase the risk of death and morbidity (including stroke, myocardial infarction, acute renal failure, and cancer recurrence) (Annals of Oncology 21 (Suppl. 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).

[0364] As used herein, the term "cancer" refers to a physiological condition in multicellular eukaryotic organisms that is generally characterized by unregulated cell proliferation and malignancy. The term broadly encompasses solid and liquid malignancies, including tumors, blood cancers (e.g., leukemias, lymphomas, and myelofibrosis), and myelofibrosis. Cancers that cause anemia may include, for example, acute leukemias, diffuse large B-cell lymphomas, Hodgkin's lymphomas, lymphocytic leukemias, multiple myeloma, myelodysplastic syndromes, myeloproliferative diseases (e.g., myelofibrosis), non-Hodgkin's lymphomas, 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, liver cancer, 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).

[0365] Therefore, RGMc selective inhibitors (e.g., monoclonal antibodies disclosed herein, derivatives thereof, monoclonal antibodies competing therewith, and engineered molecules comprising antigen-binding fragments thereof) can be used to treat cancer-induced or cancer-related anemia in subjects (e.g., human patients). Therefore, in one aspect, a method for treating anemia caused by cancer (e.g., preventing or ameliorating symptoms associated therewith). In some embodiments, the method comprises administering an RGMc antagonist to a patient suffering from anemia caused by cancer. 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, 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 to (or has reduced binding 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, the RGMc-specific (RGMc-selective) antibody competes (e.g., cross-competes or cross-blocks) antigen binding with one or more RGMc antibodies disclosed herein.

[0366] iv. Treatment-induced anemia

[0367] Various therapies administered to patients are associated with undesirable side effects, including anemia in the treated patients. In some cases, patients may already be suffering from anemia, but the therapy they receive may further aggravate the condition.

[0368] A significant proportion of patients receiving cancer treatments, such as chemotherapy and radiation therapy, experience treatment-induced anemia. Typically, these therapies are designed to kill cancer cells in the body, but also affect healthy cells, including blood cells (e.g., hematopoietic cells and hematopoietic stem cells), which leads to anemia.

[0369] It has been shown that chemotherapeutic agents can induce anemia by directly damaging the hematopoiesis of the bone marrow. In addition, the effect of cytotoxicity on renal function can cause anemia by reducing the production of erythropoietin. In particular, it is known that platinum-based therapies commonly used in lung cancer, ovarian cancer, and head and neck cancer can induce anemia by bone marrow and kidney toxicity (Groopman et al., J Natl Cancer Inst 1999; 91: 1616-1634). In addition, it has also been shown that repeated cycles of chemotherapy can increase the severity of anemia in these patients (Ludwig et al., Eur J Cancer 2004; 40: 2293-2306).

[0370] 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 following the use of nivolumab have also been reported, including cases of anemia in patients receiving ipilimumab (anti-CTLA-4 antibody) and alloimmunisation (anti-PD-1 antibody). Therefore, the term "chemotherapy-induced anemia" covers anemia that is caused or increases the chance / risk of occurrence by the administration of anticancer agents / drugs (including immunotherapy).

[0371] Finally, as mentioned above, the presence of anemia has been suggested to be a prognostic factor for cancer survival (Caro JJ et al., Cancer 2001 15; 91(12): 2214-21). Therefore, there is a strong rationale for treating anemia in cancer patients or patients undergoing chemotherapy, as it may lead to improved prognosis. In addition, given the well-known association between cancer, chemotherapy, and anemia, the National Comprehensive Cancer Network (NCCN) regularly publishes guidelines for the diagnosis, evaluation, and treatment of cancer and chemotherapy-induced anemia (e.g., see Rodgers et al., Cancer-and Chemotherapy Induced Anemia, JNCCN 2012; 10: 628-53).

[0372] Therefore, RGMc selective inhibitors (e.g., monoclonal antibodies disclosed herein, derivatives thereof, monoclonal antibodies competing therewith, and engineered molecules comprising antigen-binding fragments thereof) can be used to treat treatment-induced anemia (e.g., chemotherapy-induced anemia) in a subject (e.g., a human patient).

[0373] Thus, in one aspect, a method for treating, preventing or ameliorating symptoms associated with chemotherapy-induced anemia is provided. In one embodiment, the method comprises administering an RGMc antagonist to a patient suffering from chemotherapy-induced anemia.

[0374] 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 to (or has reduced binding 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-blocks) antigen binding with one or more RGMc antibodies disclosed herein.

[0375] v.Chronic kidney disease (CKD)

[0376] Anemia is a common complication associated with chronic kidney disease (CKD). In fact, between 2007 and 2010, approximately 14% of U.S. adults had CKD, and the prevalence of anemia was nearly twice as high in CKD patients, at 15.4% compared to 8.4% in the general population (Stauffer and Fan, PLoS One 2014;9:e84943).

[0377] While it is well known that the kidneys act as filters for the blood, removing waste products and controlling the balance of fluids and electrolytes, a lesser known role of the kidneys is the production of erythropoietin, which stimulates the production of red blood cells. Therefore, disturbances in kidney function have the potential to cause anemia. In addition, the underlying inflammation in patients with CKD may also cause and / or worsen anemia in these patients.

[0378] CKD is characterized by a gradual loss of kidney function over a period of time (months or years). Causes of CKD include, for example, diabetes, hypertension, glomerulonephritis, and polycystic kidney disease. However, there are also idiopathic cases (i.e., the cause is unknown), which are usually associated with small kidneys.

[0379] Treatment to slow or stop the progression of CKD usually requires treating the original disease, but controlling blood pressure with angiotensin-converting enzyme inhibitors or angiotensin II receptor antagonists is a common treatment because they have been found to slow the progression of the disease. However, in severe cases (e.g., end-stage renal disease), the patient will have to undergo dialysis or even a kidney transplant. The patient may be dialysis-dependent (e.g., a long-term hemodialysis patient); the patient may be non-dialysis-dependent.

[0380] Anemic CKD patients can be treated with oral or intravenous iron. In some cases, treatment may include injection of genetically engineered forms of erythropoietin (EPO) and / or blood transfusions. However, iron replacement is not always effective; for example, some patients have a low response to ESA treatment or develop a low response. In addition, ESA (e.g., EPO treatment) is associated with an increased likelihood of cardiovascular events (e.g., heart attack and stroke). Therefore, there is a need for improved therapies that can regulate iron levels and have reduced side effects.

[0381] Therefore, RGMc selective inhibitors (e.g., monoclonal antibodies disclosed herein, derivatives thereof, monoclonal antibodies competing therewith, and engineered molecules comprising antigen-binding fragments thereof) can be used to treat cancer-induced or cancer-related anemia in subjects (e.g., human patients). Therefore, in one aspect, a method for treating, preventing, or ameliorating symptoms associated with chronic kidney disease (e.g., anemia) is provided. In one embodiment, the method comprises administering an RGMc antagonist to a patient suffering from chronic kidney disease. 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 to (or has reduced binding 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-blocks) antigen binding with one or more RGMc antibodies disclosed herein.

[0382] In any of the above-mentioned embodiments for treating anemia or a disease characterized by anemia or associated with anemia, the RGMc inhibitor therapy described herein can partially or completely replace one or more current or standard therapies applied to the patient, such as ESA, HIF stabilizers, iron supplements (e.g., IV iron) and blood transfusions. In some embodiments, adding RGMc inhibitor therapy to a treatment regimen according to the present invention allows the patient to receive this treatment with a reduced dosage or dosing frequency. This can alleviate or avoid harmful side effects or other risks, such as iron overload in the patient. In some embodiments, RGMc inhibitor therapy can completely replace other therapies to treat the anemia of the patient.

[0383] In any of the above embodiments for treating anemia or a disease characterized by or associated with anemia, the RGMc inhibitor therapy described herein can be used in combination with another therapy as an additional therapy or adjuvant therapy. In certain embodiments, the patient can receive or continue to receive other therapies to treat anemia, but at a lower dose or with a lower frequency. In some embodiments, when the RGMc inhibitor is used in combination with the therapy, patients who are resistant or poorly responsive to the therapy can be made more responsive (e.g., sensitized) to the therapy.

[0384] Selection of subjects for treatment with RGMc inhibitors

[0385] The treatment methods described herein may also include measuring one or more factors to determine the presence and / or severity of a disease (e.g., anemia) in a subject. Typically, anemia is defined as female Hb<12g / dl, and male Hb<13g / dl. Although measuring hemoglobin is recognized as a measure of anemia, determining the type of anemia (AID or FID) and the cause of anemia may involve many different tests. For example, there are several well-known tests that can help determine the type and severity of anemia in a subject, including, but not limited to, hemoglobin (Hb), serum ferritin (SF), transferrin saturation (TSAT), soluble transferrin receptor (sTfR), ferritin index (FI=sTfR / log SF), hypochromic reticulocytes (CHR), and C-reactive protein (CRP) measurements. Mean corpuscular volume (MCV) or mean corpuscular hemoglobin concentration (MCHC) are also measurements that may be associated with anemia types.

[0386] The exact criteria for defining AID and FID are the subject of debate and vary according to study, disease, gender, and jurisdiction. For example, without being bound by a particular theory, FID can be defined as 1) serum transferrin saturation (TSAT) <20% and ferritin ≥100 ng / mL (Ludwig et al., Ann Oncol 2013;24(7):1886-1892); 2) TSAT <16% and ferritin >100 ng / ml (mild) or serum ferritin 30-100 ng / ml (moderate) (Bach et al., Clinical Interventions InAging 2014;9:1187-1196); 3) TSAT 20-50% and ferritin 30-800 ng / ml (Gilreath et al., Am J Hematol 2014;89(2):203-212); 4) TSAT <20% and ferritin >30 ng / ml and >100 ng / ml in cancer patients (Ludwig et al., Wien Klin Wochenschr. Am J Hematol 2014;89(2):203-212); 2015; 127: 907-919) or 5) TSAT < 20% and ferritin > 30 ng / ml (female) or > 40 ng / ml (male) (Hedenus et al., Med Oncol 2014; 31(12): 302). In other cases, the percentage of hypochromic erythrocytes can be used to detect FID (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 other cases, FID is defined as a patient with Hb < 110 g / l, iron-restricted erythropoiesis (% hypohemoglobin > 5), systemic inflammation (CRP> 10), and optionally a ferritin value of 30-800 ng / ml (Neoh et al., Support Care Cancer 2017; 25: 1209-1214).

[0387] On the other hand, without being bound by a particular theory, AID can be defined as, for example, 1) TSAT < 20% and ferritin < 30 ng / ml (Ludwig et al., Ann Oncol 2013; 24(7): 1886-1892); 2) TSAT < 20% and ferritin < 100 ng / ml in cancer patients (Ludwig et al., Wien Klin Wochenschr 2015; 127: 907-919); or 3) TSAT < 20% and ferritin levels < 40 ug / L (Hashemi et al., Int J Hematol Oncol Stem Cell Res 2017; 11(3): 192-198).

[0388] Therapeutic methods may also include measurement of other markers as discussed herein and known in the art, such as hepcidin (see, e.g., US20150202224A1 and Kroot et al., Clinical Chemistry 2011; 57(12): 1650-1669), neoplastic protein, growth differentiation factor 15 (GDF-15), neutrophil gelatinase-associated lipocalin (NGAL), interleukin 6 (IL-6), and / or BMP6.

[0389] In addition, identification of other risk factors and / or diseases can help determine the presence and type of anemia, for example, the patient may have an infection, cancer, or chronic kidney disease. Genetic testing can also help determine specific iron-related diseases, for example, iron-refractory iron deficiency anemia (IRIDA), thalassemia, hereditary spherocytosis, hereditary elliptocytosis, abetalipoproteinemia, sickle cell anemia, congenital dyserythropoietic anemia, hereditary hemochromatosis (HH), and juvenile hemochromatosis.

[0390] The subject in any method described herein can be a subject suffering from, diagnosed with, suspected of having iron deficiency anemia or a disease, illness or condition related to iron deficiency anemia or a subject at risk of its occurrence. The subject can be a mammal, which can be human or non-human. In a specific embodiment, the subject is a human subject. In another embodiment, the subject can be a human male subject. In another embodiment, the subject can be a human female subject. In another embodiment, the subject can be an intensive care patient, receiving chemotherapy, recovering from surgery, or may be at risk of infection, cancer, autoimmune disease or disorder, chronic organ disease and / or inflammation or suffer from infection, cancer, autoimmune disease or disorder, chronic organ disease and / or inflammation, and / or chronic rejection of organs after solid organ transplantation. Infection can be acute or chronic. Infection may be viral, bacterial, parasitic or fungal. Cancer can be any cancer, for example, hematology or solid tumors. 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 may be chronic kidney disease, wherein the subject may be on dialysis or not. The viral infection may be hepatitis B or C infection, or human immunodeficiency virus infection. Any disease or disorder may be the underlying cause of ACD. The surgery may be perioperative or postoperative. The surgery may be tumor surgery.

[0391] It is usually necessary to assess the subject in need of treatment by a medical history and physical examination consistent with the disease or condition in question. The diagnosis of various conditions that can be treated by the methods described herein is within the technical scope of the art. The RGMc antagonists described herein can be applied to subjects with clinical needs for delivery of iron or subjects suffering from functional iron deficiency, such as subjects receiving erythropoietin treatment. It is within the capabilities of those skilled in the art to determine whether parenteral or intravenous iron treatment is needed. For example, needs can be assessed by monitoring the iron status of the subject. The diagnosis of iron deficiency can be based on appropriate laboratory tests as described above, for example, according to the Hg levels of Table 2 or below baseline levels>2g / dl, and / or MCV below 80 femtoliters (fL) of red blood cells. Other measurements for diagnosing iron deficiency can be based on measurement of serum ferritin (SF), transferrin saturation (TSAT), soluble transferrin receptor (sTfR), ferritin index (FI = sTfR / logSF), hypochromic reticulocytes (CHR), C-reactive protein (CRP), mean corpuscular volume (MCV) and / or mean corpuscular hemoglobin concentration (MCHC).

[0392] Table 2. WHO hemoglobin thresholds as of 2011

[0393] Age or gender group Hb threshold (g / dl) Children (0.5-5.0 years old) 11.0 Children (5-12 years old) 11.5 Teenagers (12-15 years old) 12.0 Female, non-pregnant (>15 years old) 12.0 Female, pregnant 11.0 Male (>15 years old) 13.0

[0394] The subject in need of treatment can also be determined by diagnosing a subject with a disease, disorder or condition related to iron deficiency anemia. For example, many chronic renal failure patients receiving erythropoietin will need iron to maintain target iron levels. As another example, most hemodialysis patients will need to be administered iron due to dialysis-related blood loss and resulting negative iron balance.

[0395] The monitoring frequency may depend on the disease, disorder or condition that the subject suffers from or is at risk for. For example, in subjects starting erythropoietin therapy, iron index is monitored monthly. As another example, in subjects who have reached a target range Hb or are receiving intravenous iron therapy, TSAT and ferritin levels may be monitored every 3 months.

[0396] The iron status of a subject can indicate absolute iron deficiency (AID) or functional iron deficiency (FID), both of which can be treated with the compositions and treatment methods described herein. Absolute iron deficiency occurs when the amount of iron is insufficient to meet the body's needs. This deficiency may be due to insufficient iron intake, reduced bioavailability of dietary iron, increased utilization of iron, or chronic blood loss. Long-term iron deficiency can lead to iron deficiency anemia, a microcytic hypochromic anemia in which iron storage is insufficient. AID is usually indicated when TSAT <20% and ferritin <100ng / mL.

[0397] TSAT is the ratio of serum iron to total iron binding capacity multiplied by 100. Of the transferrin available for binding iron, the TSAT value tells the clinician how much serum iron is actually bound. For example, a value of 15% means that 15% of the iron binding sites of transferrin are occupied by iron.

[0398] Although the total body iron reserves are sufficient, FID may still occur when iron is not released fast enough to keep up with the demand of bone marrow for erythropoiesis. In these cases, ferritin levels may be normal or high, but the iron supply to the erythroid system is limited, as indicated by low transferrin saturation and increased number of microcytic hypochromic erythrocytes. FID can be characterized by the following features: insufficient response of hemoglobin 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 80fL) or mean corpuscular hemoglobin concentration (MCHC; e.g., less than 34 ± 2g / dl) in severe cases. Functional iron deficiency (i.e., iron storage is considered sufficient but cannot be used for iron delivery) is usually indicated in the case of TSAT <20% and ferritin>100ng / mL.

[0399] For example, the need for iron therapy can be assessed according to 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. DOQI provides the best clinical practice for the treatment of anemia in chronic renal failure. The DOQI guidelines detail intravenous iron therapy for kidney disease based on hemoglobin, transferrin saturation (TSAT) and ferritin levels.

[0400] Other guidelines for iron management and determination of AID and FID in patients with cancer and / or chemotherapy are also recommended, for example, see Steinmetz et al., Ther Adv Hematol 2012; 3(3):177-91, Mikhael et al., Curr Oncol 2007; 14:209-217, 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), American Society of Hematology (ASH) and American Society of Clinical Oncology (ASCO) (see, e.g., Rizzo et al., Blood 2010; 100:2303-2320), National Comprehensive Cancer Network (NCN) and the National Cancer Institute (NCI) 2010; 101:197-2011). The National Cancer Network (NCCN) guidelines 3.2018 version of Cancer-and Chemotherapy-induced Anemia, the European Society for Medical Oncology (ESMO) (see, e.g., Aepro et al., Annals of Oncology 2018; 0: 1-15), and the National Institute for Health and Care Excellence (NICE) (see, e.g., Technology appraisal guidance issued on November 26, 2014; nice.org.uk / guidance / ta323).

[0401] The subject in need of treatment can be determined by the subject's target iron level. For example, the subject's target hemoglobin level can be selected to be 11.0 g / dL to 12.0 g / dL (hematocrit is approximately 33% to 36%). In order to achieve the target hemoglobin with the optimal erythropoietin dose, sufficient iron can be provided to maintain TSAT ≥ 20% and ferritin 100 ng / mL. Achieving the target hemoglobin level with the optimal erythropoietin dose is related to providing enough iron to maintain TSAT above 20%.

[0402] Iron therapy can be given to maintain target hemoglobin while preventing iron deficiency and also preventing iron overload. The dosage of iron is adjusted to maintain target levels of hemoglobin, hematocrit, and laboratory parameters of iron storage are within the ordinary skill of the art. For example, in the case of anemia or iron deficiency in a subject, iron can be administered when the patient's ferritin is <800 ng / mL, TSAT <50% and / or hemoglobin is <12 g / dL. Iron overload can be avoided by stopping the administration of iron when TSAT>50% and / or ferritin>800 ng / mL.

[0403] If the subject is not anemic or iron deficient, but still requires an RGMc antagonist, for example, a subject with restless legs syndrome, then hemoglobin and TSAT levels are not necessarily relevant, and ferritin > 800 can still provide a general cutoff point for administration.

[0404] Application

[0405] In order to implement the method disclosed herein, the above-mentioned pharmaceutical composition of effective amount can be applied to the subject (such as human) in need of treatment by suitable route, wherein the suitable route is such as intravenous administration, such as push injection or continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, inhalation or local route. Commercially available sprayers for liquid preparations, including jet sprayers and ultrasonic sprayers, can be used for administration. Liquid preparations can be directly atomized and lyophilized powders can be reconstituted and atomized. Alternatively, fluorocarbon preparations and metered dose inhalers can be used to atomize antibodies or their antigen-binding fragments that specifically bind to RGMc, or inhaled in the form of lyophilized and ground powders.

[0406] 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, sports animals, pets, primates, horses, dogs, cats, mice and rats. The human subject in need of treatment can be a human patient suffering from, at risk of, or suspected of having an indication associated with RGMc (such as those mentioned above, for example, anemia). The subject suffering from the indication associated with RGMc can be determined by routine medical examinations, for example, laboratory tests, organ function tests, CT scans or ultrasound. Subjects suspected of having any such indication may show one or more symptoms of the indication. A subject at risk of an indication can be a subject having one or more risk factors for the indication.

[0407] As used herein, the terms "effective amount" and "effective dose" refer to any amount or dosage of a compound or composition sufficient to achieve the intended purpose, i.e., a desired biological or medical (clinical) response in a tissue or subject at 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, achieving clinically significant results associated with RGMc inhibition. For example, clinically significant effects may include increased serum iron levels, recovery or normalization of hemoglobin or red blood cell counts, increased transferrin saturation, and the like. In certain embodiments, clinically significant effects may include reducing toxicity or risk of harm, such as cardiovascular risk (e.g., stroke or heart failure) and cancer. In some embodiments, clinically significant effects may include reducing the need for other therapies such as iron supplements. In some embodiments, clinically significant effects may include improving the quality of life or the overall health of the patient's sense of well-being. In some embodiments, clinically significant effects may include accelerating or accelerating the alleviation of one or more parameters indicating anemia. This alleviation may be achieved by an additional therapy or combination therapy comprising an RGMc inhibitor and another therapy (e.g., ESA, HIF stabilizer, etc.). For example, typically, when starting EPO therapy (e.g., monotherapy), treatment requires 1-2 months (e.g., 6-8 weeks) to show an effect. Therefore, patients with severe anemia may also require IV iron or blood transfusions during this period. However, in combination with RGMc selective inhibitors such as those disclosed herein, the onset of therapeutic action may be significantly faster (e.g., 1-4 weeks), which can reduce the need for additional IV iron or blood transfusions.

[0408] As is known to 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, physical condition, size, sex and weight, the duration of treatment, the nature of co-treatment (if any), the specific route of administration and similar factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be solved only by routine experimentation. It is generally preferred to use the maximum dose of a single component or a combination thereof, i.e., the highest safe dose according to reasonable medical judgment. However, it will be understood by those of ordinary skill in the art that a patient may insist on a lower dose or a tolerable dose for medical reasons, psychological reasons or indeed any other reason.

[0409] Empirical factors, such as half-life, will generally help determine the dosage. For example, antibodies compatible with the human immune system, such as humanized antibodies or fully human antibodies, can be used to extend the half-life of the antibody and prevent the antibody from being attacked by the host's immune system. The frequency of administration can be determined and adjusted during the course of treatment, and is generally but not necessarily based on the treatment and / or inhibition and / or improvement and / or delay of RGMc-related indications. Alternatively, a sustained continuous release formulation of an antibody that specifically binds to RGMc may be suitable. Various formulations and devices for achieving sustained release are apparent to those skilled in the art and are within the scope of the present disclosure.

[0410] In one embodiment, the dosage of an antibody that specifically binds to RGMc as described herein can be determined empirically in an individual who has been given one or more administrations of the antibody. Increasing doses of an RGMc antagonist are given to the individual. To assess efficacy, an index of RGMc-related indications can be followed. For example, methods for measuring anemia are well known in the art and are further described herein.

[0411] The present invention encompasses the recognition that agents that are able to specifically modulate RGMc signaling relative to RGMa and / or RGMb may provide improved safety when used as drugs. Accordingly, the present invention encompasses antibodies and antigen-binding fragments thereof that specifically bind to and inhibit activation of RGMc but not RGMa and / or RGMb, thereby conferring specific inhibition of RGMc signaling in vivo while minimizing undesirable side effects caused by affecting RGMa and / or RGMb signaling.

[0412] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are non-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 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 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.

[0413] Typically, for administration of any of the antibodies described herein, the initial candidate dosage may be about 0.1-20 mg / kg, such as 1-20 mg / kg per administration, for example, weekly, every 2 weeks, every 3 weeks, monthly, every three months, etc. For example, a patient may receive an injection of about 1-10 mg / kg every 1 week, every 2 weeks, every 3 weeks, or every 4 weeks, etc., in an amount effective for treating a disease (e.g., cancer), wherein the amount is well tolerated (within an acceptable range of toxicity or adverse events).

[0414] For purposes of this disclosure, typical dosages (per administration, e.g., injection and infusion) can be in any range of about 0.1 mg / kg to 1 mg / kg to 2 mg / kg to 3 mg / kg to 5 mg / kg to 10 mg / kg to 20 mg / kg to 30 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, treatment is continued until the desired symptom suppression occurs or until a therapeutic level sufficient to alleviate the RGMc-related indication or its symptoms is reached.

[0415] Exemplary dosing regimens include administering an initial dose, followed by administering one or more maintenance doses, wherein the latter is generally lower than the former. For example, an initial dose may be between about 2 and 30 mg / kg, such as once a week or twice a week. Thereafter, a maintenance dose may follow, for example, between about 0.1 and 20 mg / kg (e.g., 1, 2, 3, 5, 10, 15 mg / kg), such as once a week, once every other week, once a month, etc. However, other dosage regimens may be useful, depending on the pharmacokinetic decay pattern that the implementer wishes to achieve. Pharmacodynamic (PD) experiments show that there is a continuous PD effect (e.g., serum iron level increases) at least 3 weeks after administering an antibody disclosed herein (e.g., Ab8 to a preclinical animal model (e.g., a rat model); 20 mg / kg). It is not desirable to be bound by any particular theory, and this continuous post-administration effect may be advantageous, because the frequency of administration can be reduced in a subject (e.g., a human subject) to which the antibody is administered while maintaining a clinically effective serum concentration. In some embodiments, the dosing frequency is once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks or once every 10 weeks; or once a month, once every 2 months or once every 3 months or longer. The progress of the therapy is easily monitored by conventional techniques and analysis. The dosing regimen (including the antibody used) can vary over time.

[0416] In some embodiments, for normal weight adult patients, a dosage ranging from about 0.3 to 5.00 mg / kg may be administered. The specific dosage regimen, e.g., dosage, time, and repeated dosages will depend on the specific individual and the individual's medical history, as well as the characteristics of the individual drug (e.g., the half-life of the drug and other relevant considerations).

[0417] For purposes of this disclosure, the appropriate dosage of an antibody that specifically binds to RGMc will depend on the specific antibody (or combination thereof) used, the type and severity of the indication, whether the antibody is being administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to antagonists, and the judgment of the attending physician. In some embodiments, the clinician will administer 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 can be continuous or intermittent, depending on, for example, the physiological condition of the recipient, whether the purpose of administration is therapeutic or preventive, and other factors known to those skilled in the art. Administration of an antibody that specifically binds to RGMc can be substantially continuous over a predetermined period of time, or can be administered in a series of spaced doses, for example before, during, or after the onset of an indication associated with RGMc (e.g., anemia).

[0418] As used herein, the term "treat" refers to the application or administration of a composition comprising one or more active agents to a subject having an RGMc-related indication, a symptom of the indication, or a susceptibility to the indication, with the purpose of curing, restoring, alleviating, alleviating, altering, remedying, improving, enhancing, or influencing the indication, a symptom of the indication, or a susceptibility to the indication.

[0419] Alleviating RGMc-related indications with antibodies that specifically bind to RGMc includes delaying the development or progression of the indication, or reducing the severity of the indication. Alleviating an indication does not necessarily require a cure. As used herein, "delaying" the development of an indication associated with an RGMc-related indication means delaying, hindering, slowing down, delaying, stabilizing and / or postponing the progression of the indication. Depending on the indication being treated and / or the individual's medical history, the delay can be a different length of time. A method of "delaying" or alleviating the development of an indication or delaying the onset of an indication is a method that reduces the likelihood of one or more symptoms of an indication occurring within a given time frame and / or reduces the degree of symptoms within a given time frame compared to a situation where the method is not used. Such comparisons are typically based on clinical studies using multiple subjects sufficient to give statistically significant results.

[0420] Combination therapy

[0421] The present disclosure also includes pharmaceutical compositions and related methods for use as combination therapies for treating subjects who can benefit from RGMc inhibition in vivo. In any of these embodiments, such subjects may receive a combination therapy comprising a first composition comprising at least one RGMc antagonist described herein, e.g., an antibody or an antigen-binding fragment thereof, used in combination with a second composition comprising at least one other therapeutic agent intended to treat the same or overlapping disease or clinical condition. The first and second compositions may act on the same cell target or a discrete cell target. In some embodiments, the first and second compositions may treat or alleviate a group of symptoms or aspects of the same or overlapping disease or clinical condition. In some embodiments, the first and second compositions may treat or alleviate a group of separate symptoms or aspects of a disease or clinical condition. To give just one example, a first composition may treat a disease or condition (e.g., CKD or cancer), while a second composition may treat inflammation, decreased erythropoiesis, or anemia associated with the same disease, etc. Such combination therapies may be administered in conjunction with one another. In the context of a combination therapy, the phrase "in conjunction with..." means that in a subject receiving the combination therapy, the therapeutic effect of the first therapy overlaps temporally and / or spatially with the therapeutic effect of the second therapy. Thus, the combination therapies may be formulated as a single formulation for simultaneous administration, or as separate formulations for sequential administration of the therapies.

[0422] In a preferred embodiment, the combination therapy produces a synergistic effect in the treatment of the disease.The term "synergistic" refers to an additive effect (eg, greater efficacy) that is greater than the sum of each monotherapy.

[0423] In some embodiments, combination therapy can achieve a more rapid therapeutic effect than single therapies.

[0424] In some embodiments, the efficacy of the combination therapy comprising the pharmaceutical composition described herein is generally equivalent to the efficacy of another therapy (e.g., a monotherapy of a second medicament), but the combination therapy is associated with fewer adverse side effects or less toxicity associated with the second medicament than the monotherapy of the second medicament. In some embodiments, such a combination therapy allows a lower dose of the second medicament but maintains overall efficacy. Such a combination therapy may be particularly suitable for patient populations requiring long-term treatment and / or involving pediatric patients.

[0425] Therefore, in one aspect, the present invention provides pharmaceutical compositions and methods in combination therapies for reducing RGMc activity and treating or preventing diseases or conditions associated with RGMc signaling as described herein. Therefore, the method or pharmaceutical composition also comprises a second therapy. In some embodiments, the second therapy can be used to treat or prevent diseases or conditions associated with RGMc signaling. The second therapy can reduce or treat at least one symptom associated with the target disease. The first and second therapies can exert their biological effects through similar or unrelated mechanisms of action; or one or both of the first and second therapies can exert their biological effects through multiple mechanisms of action. For example, in the case of anemia, a RGMc inhibitor can be combined (e.g., sequentially or simultaneously) with a second therapy that increases erythropoiesis (e.g., EPO therapy, blood transfusion, etc.). Alternatively, in the case of anemia of chronic disease, a RGMc inhibitor can be combined (e.g., sequentially or simultaneously) with a second therapy (e.g., chemotherapy, radiotherapy, immunotherapy) for treating a basic disease (e.g., cancer and immune disease).

[0426] It should be understood that for each of the embodiments described, the pharmaceutical compositions described herein can have the first and second therapies in the same pharmaceutically acceptable carrier or in different pharmaceutically acceptable carriers. It should also be understood that in the embodiments described, the first and second therapies can be administered simultaneously or sequentially.

[0427] One or more RGMc inhibitors (e.g., antibodies or antigen-binding fragments thereof) of the invention can be administered to a patient who has received or is receiving one or more additional therapeutic agents. Examples of other therapeutic agents that can be used with RGMc antibodies or the invention include, but are not limited to, erythropoietin stimulators (ESAs), hypoxia-inducible factor (HIF) stabilizers, hepcidin antagonists, iron supplements (e.g., oral or intravenous), anti-inflammatory drugs, and / or anticancer drugs.

[0428] ESAs are drugs that stimulate the bone marrow to produce red blood cells and are used to treat various types of anemia in patients. However, even if there is increased red blood cell production, anemia will persist if there is not enough iron. This is because red blood cells require hemoglobin to transport oxygen, and iron is essential in the synthesis of hemoglobin. Therefore, if there is not enough iron, the newly produced red blood cells will lack hemoglobin and will be hypochromic (lacking the red hemoglobin pigment) and microcytic (smaller than normal).

[0429] Thus, in one embodiment, the RGMc antagonists described herein (e.g., RGMc antibodies) can be administered to patients who have received or are currently receiving ESAs for the treatment of iron-related diseases. Examples of ESAs include, but are not limited to, Epoetin alpha, Epoetin beta, Epoetin delta (e.g., ), Epoetin ω (e.g., ), Darbepoetin alfa (Lupin pharma), (Nanogen Pharmaceutical Biotech), (Intas pharma), (Amgen), (Chugai Pharma), (manufactured by Janssen-Cilag), (Sandoz), (Pooyeshdarou Biopharmaceutical Co., Ltd.), (Probiomed), (produced by Hoffmann–La Roche), (Produced by Stada / Hospira), (manufactured by Panacea Biotechnology Co., Ltd.), Erypro Safe TM (Biocon Co., Ltd.), (Produced by Serum Institute of India Ltd.), (Emcure Pharmaceuticals), Erykine (Intas Biopharmaceuticals), (Wockhardt Biotech), Espogen TM (produced by LG Life Sciences), ReliPoietin TM (Reliance Life Sciences), Shanpoietin TM (Shantha Biotech Ltd.), (Produced by Cadila Healthcare Co., Ltd.), (rHuEPO) (produced by Shenyang Sunshine Pharmaceutical Co., Ltd.) and China (Produced by CinnaGen Biopharmaceuticals).

[0430] ESAs are generally effective, but there is evidence that even at high doses, a subset of patients do not respond to treatment (Solak Y et al., Blood Purification. 2016; 42(2): 160-7). In addition, multiple studies have shown that the use of ESAs may lead to higher rates of cerebrovascular and cardiovascular (CV) events and mortality (Biggar P et al., Kidney Research and Clinical Practice 2017 Sep; 36(3): 209-223). ESAs may also be associated with an increased risk of cancer in some patients, and in some cases, with a poorer response to anticancer therapy. Therefore, without being bound by a particular theory, the use of RGMc inhibitors in patients who have received or are currently receiving ESAs may improve the response rate and reduce the dose of ESAs required to produce an effect, thereby reducing the occurrence of adverse reactions.

[0431] Hypoxia inducible factor (HIF) prolyl hydroxylase (PH) inhibitors (also known as HIF-PHIs or HIF stabilizers) are a class of agents used to treat anemia. These agents work by stabilizing the HIF complex and stimulating endogenous erythropoietin production. Therefore, HIF-PH inhibitors that increase erythropoietin production can be supplemented by drugs that increase serum iron levels (e.g., RGMc inhibitors). Therefore, in some embodiments, the RGMc inhibitors described herein (e.g., RGMc antibodies) can be administered to patients who have received or are receiving HIF stabilizers or prolyl hydroxylase (PH) inhibitors to treat iron-related diseases. Examples of HIF stabilizers or HIF-PH inhibitors include, but are not limited to, roxadustat (FG-4592), valdustat (AKB-6548), daptostat (GSK-1278863), molistat (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-quinolyl]carbonyl]glycine), JNJ-42041935 (1-[6-chloro-5-(trifluoromethoxy)-1H-benzimidazol-2-yl]-1H-pyrazole-4-carboxylic acid), N-oxalylglycine (2-(carboxymethylamino)-2-oxoacetic acid), 1,4-dihydrophenanthroline-4-one-3-carboxylic acid, and Adaptaquin (7-[(4-chlorophenyl)[(3-hydroxy-2-pyridyl)amino]methyl]-8-quinoline).

[0432] In some embodiments, the RGMc-specific inhibitors described herein can be administered to patients who have received or are receiving iron supplements (e.g., oral or intravenous (iv) iron). Examples of intravenous iron preparations include, but are not limited to, iron dextran, iron hydroxide-dextran complexes, iron hydroxide-sucrose iron complexes, iron gluconate, sodium iron gluconate, iron sucrose, saccharate iron, nano-iron oxide (ferumoxytol), carboxymaltose iron, and isomaltose iron. Examples of oral iron supplements may include, but are not limited to, ferric pyrophosphate, ferrous gluconate, ferrous sulfate, ferrous fumarate, ferrous carbonate, and carbonyl iron.

[0433] Intravenous and oral iron supplements are generally effective, but studies have shown a variety of toxicities or adverse events associated with iron. Examples of adverse events include cytotoxicity, tubular necrosis, 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, the use of RGMc-specific inhibitors together with intravenous or oral iron can increase the response rate and reduce the dose of iron required for therapeutic effect, thereby reducing the occurrence and / or extent of adverse events / toxicity.

[0434] It is recognized that certain drugs intended to treat various diseases often induce or exacerbate anemia in the treated patient (e.g., treatment or drug-induced anemia, such as chemotherapy-induced anemia and radiation-induced anemia). In some embodiments, the patient is treated with a myelosuppressive drug, which may cause side effects including anemia. Such patients may benefit from the RGMc inhibitors of the present disclosure used in combination with myelosuppressive therapy. Examples of myelosuppressive therapy include, but are not limited to, pegylated interferon α-2a, interferon α-n3, pegylated interferon α-2b, aldesleukin, gemtuzumab tuzumab, interferon alfacon-1, rituximab, ibritumomab tiuxetan, tositumomab, alemtuzumab, bevacizumab, L-phenylalanine, bortezomib, cladribine, carmustine, amsacrine, chlorambucil, raltitrexed, mitomycin, bexarotene, long-acting Chundesin, floxuridine, thioguanine, vinorelbine, dexrazoxane, sorafenib, streptozotocin, gemcitabine, teniposide, epirubicin, chloramphenicol, lenalidomide, hexamethylmelamine, zidovudine, cisplatin, oxaliplatin, cyclophosphamide, fluorouracil, propylthiouracil, pentostatin, methotrexate, carbamazepine, vinblastine, linezolid, imatinib, clofarabine, pemetrexed, daunorubicin, irinotecan, methimazole, etoposide glycosides, dacarbazine, temozolomide, tacrolimus, sirolimus, nitrogen mustard, azacitidine, carboplatin, actinomycin D, 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, temsirolimus, bosutinib, bendamustine, cabazitaxel, eribulin, ruxolitinib, carfilzomib, tofacitinib, ponatinib, pomalidomide, atezolizumab (obinutuzumab), tedizolid phosphate, blinatumomab, ibrutinib, palbociclib, olaparib, dinutuximab, and colchicine.

[0435] In some embodiments, the RGMc specific inhibitor described herein can be administered to patients who have received or are receiving cancer treatment. Such cancer therapy can include, but is not limited to, chemotherapy, radiotherapy / radiotherapy, small molecule drugs or therapeutic antibodies, such as cancer vaccines; engineered immune cell therapy; chemotherapy; radiotherapy; 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 (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, etc. Other examples of other therapeutic agents that can be used with RGMc specific inhibitors include, but are not limited to, indoleamine 2,3-dioxygenase (IDO) inhibitors, tyrosine kinase inhibitors, Ser / Thr kinase inhibitors, dual specificity kinase inhibitors. In some embodiments, such agents can be PI3K inhibitors, PKC inhibitors, or JAK inhibitors. In some embodiments, the JAK inhibitor can be a JAK1 inhibitor and / or a JAK2 inhibitor, which can be used to treat myeloproliferative diseases, such as primary myelofibrosis.

[0436] It is also understood that other cancer treatments, such as radiation therapy, can lead to anemia in cancer patients. Thus, in one embodiment, the RGMc-specific inhibitors described herein can be used in combination with radiation therapy.

[0437] In some embodiments, the additional agent to be administered as a combination therapy or used in combination with an RGMc inhibitor is or comprises an inhibitor of a member of the TGFβ superfamily of growth factors or a regulator thereof. In some embodiments, such an inhibitor is or comprises 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 a subtype selective inhibitor of TGFβ1, such as a TGFβ1 activation inhibitor. In some embodiments, the TGFβ inhibitor is a subtype selective inhibitor of TGFβ3, such as a TGFβ3 activation inhibitor.

[0438] In some embodiments, the combination therapy for treating cancer in a subject comprises a cancer immunotherapy (e.g., a checkpoint inhibitor), a TGFβ1-selective inhibitor, and a RGMc-selective inhibitor disclosed herein, wherein optionally, the checkpoint inhibitor is a PD-(L)1 antibody, and further optionally, the TGFβ1-selective inhibitor is a TGFβ1-selective activation inhibitor.

[0439] In some embodiments, the RGMc-specific inhibitors described herein can be used in combination with other compounds, drugs, and / or agents used to treat inflammation-related diseases or chronic kidney disease. Such compounds, drugs and / or agents may 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 sympathetic blockers, bicarbonate supplements, insulin, vitamin D supplements, urate reducers (e.g., allopurinol), HMG-CaA reductase inhibitors (statins), pirfenidone, TGFβ pathway inhibitors, anti-inflammatory drugs (including nonsteroidal anti-inflammatory drugs), antioxidants inflammation modulators (e.g., bardoxolone methyl), endothelin 1 antagonists (e.g., avosentan and atrasentan), advanced glycan end-product inhibitors, aspirin, metformin, aminoguanidine, calcitriol, erythropoietin, androgens, phosphate binders, vitamin B12 and vitamin B6 and their derivatives (e.g., pyridoxamine).

[0440] In some embodiments, the RGMc-specific inhibitors described herein can be combined with granulocyte colony stimulating factor (G-CSF) or its derivatives, such as or Used in combination. G-CSF is a glycoprotein used to stimulate the production of blood cells (such as white blood cells or hematopoietic stem cells) and enhance their ability to function. Therefore, G-CSF can be used to reduce the risk of infection, anemia and bleeding problems in patients after receiving bone marrow suppression therapy (such as chemotherapy). However, the use of G-CSF has side effects, including: thrombocytopenia, nausea, fever, bone pain, elevated lactate dehydrogenase, elevated alkaline phosphatase, ecchymosis, back pain, epistaxis (nose bleeding), cough and / or dyspnea (shortness of breath). Therefore, it is expected that the RGMc-specific inhibitor described herein can supplement the pharmacological effects of G-CSF (for example, reducing anemia and promoting red blood cell production) and / or reduce the effective dose of G-CSF, thereby reducing side effects and improving tolerance. Therefore, in one embodiment, the RGMc-specific inhibitor described herein can be applied to patients who have received or are receiving G-CSF therapy. In another embodiment, the RGMc-specific inhibitor described herein reduces the effective dose of G-CSF required to achieve favorable pharmacological effects such as promoting blood cell production and / or reducing anemia. In another embodiment, the RGMc-specific inhibitors described herein complement the effects of G-CSF, for example, further increasing red blood cell production and / or reducing anemia.

[0441] The combination therapies contemplated herein can advantageously utilize lower doses of the administered therapeutic agent, thereby avoiding possible toxicities or complications associated with various monotherapies. In some embodiments, the use of the RGMc-specific inhibitors described herein can make patients who respond poorly or do not respond to treatment (e.g., standard of care) more responsive. In some embodiments, the use of the RGMc-specific inhibitors described herein can allow for reduced treatment (e.g., standard of care) doses that still produce equivalent clinical efficacy in patients, but produce fewer or lower levels of drug-related toxicity or adverse events.

[0442] Inhibition of RGMc activity

[0443] The methods disclosed herein include methods for inhibiting RGMc activity in one or more biological systems. Such methods may include contacting one or more biological systems with antibodies and / or compositions disclosed herein. Inhibitors (e.g., antibodies) and / or compositions according to such methods may include, but are not limited to, biomolecules, including, but not limited to, recombinant proteins, protein complexes, and / or antibodies or antigenic fragments thereof, as described herein.

[0444] The inhibitors that selectively bind to RGMc described herein, e.g., antibodies and antigen-binding fragments thereof, can be used in a wide range of applications where it is desired to modulate RGMc activity. In one embodiment, the invention provides a method of inhibiting RGMc activation by exposing a RGMc peptide or protein to an inhibitor that selectively binds to RGMc (e.g., an antibody or an antigen-binding fragment thereof). The foregoing method can be performed in vitro, e.g., to inhibit RGMc activity in cultured cells. The foregoing 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 effects of RGMc inhibition are to be evaluated.

[0445] Any inhibitor described herein, eg, an antibody or antigen-binding fragment thereof, that selectively binds to RGMc, as well as any pharmaceutical composition comprising such an inhibitor or antibody, is suitable for use in the methods of the invention.

[0446] Therefore, in one aspect of the invention, a method of inhibiting RGMc activity is provided by exposing RGMc to an inhibitor, such as an antibody or an antigen binding fragment thereof, that selectively binds to RGMc. In some embodiments, the inhibitor (e.g., an antibody or an antigen binding fragment thereof) competes with a BMP for binding to RGMc. In some embodiments, the BMP is BMP2 and / or BMP6. In some embodiments, the BMP is BMP6.

[0447] In another embodiment, the inhibitor (e.g., an antibody or an antigen-binding fragment thereof) inhibits the binding of regeneration protein to RGMc. In some embodiments, the inhibitor (e.g., an antibody or an antigen-binding fragment thereof) competes with regeneration protein for binding to RGMc.

[0448] In some embodiments, the inhibitor (e.g., an antibody or an antigen-binding fragment thereof) does not inhibit the binding of regeneration protein to RGMc. In some embodiments, the inhibitor (e.g., an antibody or an antigen-binding fragment thereof) does not compete with regeneration protein for binding to RGMc.

[0449] In another embodiment, an inhibitor (e.g., an antibody or antigen-binding fragment thereof) inhibits the binding of RGMc to other binding partners or proteins complexed with RGMc. For example, other binding partners complexed with RGMc, BMP and / or regenerin include HFE, TFR2, ALK2, ALK3, BMPR2, ACVR2A and TMPRSS6.

[0450] In another embodiment, the antibody or antigen binding fragment thereof that inhibits RGMc activity can also regulate other biological processes (e.g., downstream effects). For example, the antibody or antigen binding fragment thereof that inhibits RGMc activity reduces the expression of hepcidin in the subject. In some embodiments, the antibody or antigen binding fragment thereof that inhibits RGMc activity increases the expression of iron transporter in the subject. In some embodiments, the antibody or antigen binding fragment thereof that inhibits RGMc activity increases the transferrin saturation of the subject. In some embodiments, the antibody or antigen binding fragment thereof that inhibits RGMc activity increases the erythropoiesis of the subject. In some embodiments, the antibody or antigen binding fragment thereof that inhibits RGMc activity reduces the unsaturated iron binding capacity (UIBC) of the subject. In some embodiments, the antibody or antigen binding fragment thereof that inhibits RGMc activity reduces the total iron binding capacity (TIBC) of the subject. In some embodiments, the antibody or antigen binding fragment thereof that inhibits RGMc activity increases the serum ferritin level of the subject.

[0451] 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, sports animals, pets, primates, horses, dogs, cats, mice, and rats. The human subject in need of treatment can be a human patient who suffers from an indication associated with RGMc (e.g., those described above, such as anemia), is at risk of becoming ill, or is suspected of suffering from an indication associated with RGMc (e.g., those described above, such as anemia).

[0452] Thus, in one embodiment, a method for increasing serum iron levels in a subject is provided, the method comprising administering to the subject a pharmaceutical composition of the present invention (e.g., a 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 present invention (e.g., a RGMc-specific inhibitor disclosed herein) in an amount effective to downregulate hepcidin in the subject. In some embodiments, the pharmaceutical composition reduces hepatic hepcidin mRNA levels. In some embodiments, the pharmaceutical composition reduces hepatic hepcidin protein levels, serum (circulating) hepcidin protein levels, or both.

[0453] In one embodiment, a method for increasing serum iron levels in a subject is provided, the method comprising administering a pharmaceutical composition of the present invention (e.g., a RGMc specific inhibitor disclosed herein) to a subject in an amount that effectively increases the expression of iron transporter in the subject compared to a baseline. In some embodiments, a method for increasing the expression of iron transporter in a subject is provided, the method comprising administering a pharmaceutical composition of the present invention (e.g., a RGMc specific inhibitor disclosed herein) to a subject in an amount that effectively increases the expression of iron transporter in the subject. In some embodiments, the pharmaceutical composition increases intestinal endothelial cell (intestinal) iron transporter protein levels. In some embodiments, the pharmaceutical composition increases hepatocyte (liver) iron transporter protein levels. In some embodiments, the pharmaceutical composition increases macrophage iron transporter protein levels. In some embodiments, the pharmaceutical composition increases adipocyte iron transporter protein levels.

[0454] In some embodiments, a method for increasing transferrin saturation in a subject is provided, the method comprising administering a pharmaceutical composition of the invention (e.g., a RGMc-specific inhibitor disclosed herein) to a subject in an amount effective to increase the transferrin saturation (TSAT) of 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.

[0455] In some embodiments, the method increases the transferrin saturation in a subject to a level of ≥15%, ≥16%, ≥17%, ≥18%, ≥19%, ≥20%, ≥21%, ≥22%, ≥23%, ≥24%, or ≥25%. In some embodiments, the method increases the transferrin saturation in a subject to a level of ≥15%. In some embodiments, the method increases the transferrin saturation in a subject to a level of ≥16%. In some embodiments, the method increases the transferrin saturation in a subject to a level of ≥17%. In some embodiments, the method increases the transferrin saturation in a subject to a level of ≥18%. In some embodiments, the method increases the transferrin saturation in a subject to a level of ≥19%. In some embodiments, the method increases the transferrin saturation in a subject to a level of ≥20%. In some embodiments, the method increases the transferrin saturation in a subject to a level of ≥21%. In some embodiments, the method increases the transferrin saturation in a subject to a level of ≥22%. In some embodiments, the method increases the transferrin saturation in a subject to a level of ≥23%. In some embodiments, the method increases the transferrin saturation in a subject to a level of ≥24%. In some embodiments, the method increases the transferrin saturation in a subject to a level of ≥25%.

[0456] In some embodiments, a method for increasing erythropoiesis in a subject is provided, the method comprising administering to the subject a pharmaceutical composition of the invention (eg, an RGMc-specific inhibitor disclosed herein) in an amount effective to increase erythropoiesis in the subject.

[0457] 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., a 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 method increases Hb to a level of ≥13 g / dl.

[0458] 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 invention (e.g., a RGMc-specific inhibitor disclosed herein) in an amount effective to reduce the unsaturated iron binding capacity in the subject. The typical range of UIBC in a subject is 120–470 ug / dL (21–84 umol / L), although levels may vary from population to population. Thus, in some embodiments, the subject's UIBC is ≥400, ≥450, ≥500, ≥550, ≥600, ≥650, ≥700, ≥750, or ≥800 ug / dL. In other embodiments, an effective amount of a RGMc-specific inhibitor reduces the subject's UIBC by ≥50, ≥100, ≥150, ≥200, ≥250, ≥300, ≥350, or ≥450 ug / dL.

[0459] 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., a RGMc-specific inhibitor disclosed herein) in an amount effective to reduce total iron binding capacity in the subject. The typical range of TIBC is 255–450 ug / dL, although levels vary from population to population. 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 the subject's TIBC by ≥50, ≥100, ≥150, ≥200, ≥250, ≥300, ≥350, or ≥450 ug / dL.

[0460] 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 invention (e.g., a RGMc-specific inhibitor disclosed herein) in an amount effective to increase the serum ferritin level of the subject. In some embodiments, the method increases the serum ferritin level of 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 the serum ferritin level of the subject by ≥5 ng / ml compared to baseline. In some embodiments, the method increases the serum ferritin level of the subject by ≥10 ng / ml compared to baseline. In some embodiments, the method increases the serum ferritin level of the subject by ≥15 ng / ml compared to baseline. In some embodiments, the method increases the serum ferritin level of the subject by ≥20 ng / ml compared to baseline. In some embodiments, the method increases the serum ferritin level of the subject by ≥30 ng / ml compared to baseline. In some embodiments, the method increases the serum ferritin level of the subject by ≥40 ng / ml, as compared to baseline. In some embodiments, the method increases the serum ferritin level of the subject by ≥50 ng / ml, as compared to baseline. In some embodiments, the method increases the serum ferritin level of the subject by ≥60 ng / ml, as compared to baseline. In some embodiments, the method increases the serum ferritin level of the subject by ≥70 ng / ml, as compared to baseline. In some embodiments, the method increases the serum ferritin level of the subject by ≥80 ng / ml, as compared to baseline. In some embodiments, the method increases the serum ferritin level of the subject by ≥90 ng / ml, as compared to baseline. In some embodiments, the method increases the serum ferritin level of the subject by ≥100 ng / ml, as compared to baseline.

[0461] In some embodiments, the method increases serum ferritin in a subject to a level of ≥30 ng / ml. In some embodiments, the method increases serum ferritin in a subject to a level of ≥40 ng / ml. In some embodiments, the method increases serum ferritin in a subject to a level of ≥50 ng / ml. In some embodiments, the method increases serum ferritin in a subject to a level of ≥60 ng / ml. In some embodiments, the method increases serum ferritin in a subject to a level of ≥70 ng / ml. In some embodiments, the method increases serum ferritin in a subject to a level of ≥80 ng / ml. In some embodiments, the method increases serum ferritin in a subject to a level of ≥90 ng / ml. In some embodiments, the method increases serum ferritin in a subject to a level of ≥100 ng / ml.

[0462] 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 invention (e.g., a 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, ACD is anemia associated with cancer. In another embodiment, ACD is anemia associated with chronic kidney disease (CKD). In some embodiments, the anemia is chemotherapy-induced anemia.

[0463] Kits for alleviating diseases / disorders associated with RGMc-related indications

[0464] The present disclosure also provides kits for alleviating diseases / disorders associated with RGMc-related indications (e.g., anemia).Such kits may include one or more containers comprising an inhibitor that selectively binds to RGMc, e.g., an antibody or antigen-binding fragment thereof.

[0465] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include a description of administering an inhibitor (e.g., an antibody or an antigen-binding fragment thereof) that selectively binds to RGMc to treat, delay the onset of, or alleviate a target disease as described herein. The kit may further include a description of selecting an individual suitable for treatment based on identifying whether the individual suffers from the target disease. In other embodiments, the instructions include a description of administering the antibody or an antigen-binding fragment thereof to an individual at risk for the target disease.

[0466] Instructions for use of an inhibitor that selectively binds to RGMc (e.g., an antibody or an antigen-binding fragment thereof) typically include information about the dosage, dosing regimen, and route of administration for the intended treatment. The container may be a unit dose, bulk (e.g., a multi-dose package), or a subunit dose. The instructions provided in the kit of the present disclosure are typically written instructions on a label or package insert (e.g., a sheet of paper included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. The label or package insert may indicate that the composition is used to treat a disease or disorder associated with a TGFβ-related indication, delay its onset, and / or alleviate the disease or disorder. Instructions for practicing any of the methods described herein may be provided.

[0467] The kit of the present disclosure can be provided in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc. Packaging for use in combination with specific devices such as inhalers, nasal administration devices (e.g., nebulizers), or infusion devices (e.g., miniature pumps) is also contemplated. The kit can have a sterile access port (e.g., the container can be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). The container can also have a sterile access port (e.g., the container can be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is an inhibitor that selectively binds to RGMc as described herein, e.g., an antibody or an antigen-binding fragment thereof.

[0468] The kit may optionally provide other components such as a buffer and interpretative information. Typically, the kit comprises a container and a label or package insert on or a...

Claims

1. An isolated antibody that specifically binds to human repulsive guidance molecule C (RGMc), 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); c) the antibody inhibits or reduces the interaction between RGMc and BMP6; and d) the antibody comprises H-CDR1, H-CDR2 and H-CDR3 sequences as shown in SEQ ID NOs: 30, 31 and 32, respectively, and L-CDR1, L-CDR2 and L-CDR3 sequences as shown in SEQ ID NOs: 33, 34 and 35, respectively.

2. The antibody of claim 1, wherein the antibody comprises: a) a heavy chain variable region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 36; and / or b) a variable light chain variable region having an amino acid sequence that is at least 90% identical to SEQ ID NO:

37.

3. The antibody of claim 1, wherein the antibody comprises: c) the heavy chain variable region sequence shown in SEQ ID NO: 36; and d) The light chain variable region sequence shown in SEQ ID NO:

37.

4. The antibody of any one of claims 1-3, wherein the antibody is a fully human antibody or a humanized antibody.

5. The antibody of any one of claims 1-3, wherein the antibody is a human IgG1, IgG2 or IgG4 antibody. The antibody of claim 5 , wherein the antibody is a human IgG4 antibody.

7. The antibody of claim 6, wherein the human IgG4 antibody comprises a Ser to Pro backbone substitution that results in an IgG1-like hinge.

8. The antibody of any one of claims 1 to 3, wherein the antibody has a concentration of less than 0.1×10 -9 (0.1nM) K D Value combined with RGMc.

9. The antibody of any one of claims 1-3, wherein the antibody induces internalization of the antibody-antigen complex.

10. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

11. Use of the composition of claim 10 in the preparation of a medicament for treating anemia in a human subject, wherein the treatment comprises administering the medicament to the subject in an amount effective to treat the anemia.

12. The use according to claim 11, wherein the anemia is iron-refractory iron deficiency anemia (IRIDA) or anemia of chronic disease (ACD).

13. The use according to claim 11, wherein the anemia is cancer-related anemia or chemotherapy-induced anemia.

14. The use according to claim 12, wherein the ACD is anemia of chronic kidney disease (CKD).

15. The use according to claim 14, wherein the subject is dialysis-dependent or non-dialysis-dependent.

16. The use according to any one of claims 11 to 15, wherein the medicament is administered in combination with an additional therapeutic agent.

17. The use according to claim 16, wherein the additional therapeutic agent is an erythropoietin stimulating agent (ESA), a HIF stabilizer, an iron supplement or a blood transfusion.

18. The use of claim 16, wherein the medicament reduces toxicity associated with ESA, HIF stabilizers, iron supplements or blood transfusions.

19. Use according to claim 18, wherein the toxicity comprises risk of iron overload or cancer.

20. The use according to any one of claims 11-15, wherein the subject: a) receiving iron therapy but benefiting from a reduction in the dose of iron therapy; b) had received iron therapy but discontinued it due to toxicity; c) already receiving iron therapy and benefiting from a dose reduction of iron therapy; d) are at risk of developing cancer; and / or e) has been diagnosed with cancer.

21. The use according to any one of claims 11-15, wherein the subject has received erythropoietin (EPO / ESA) therapy, HIF stabilizer therapy, IV iron supplementation, or a combination thereof.

22. The use of claim 21, wherein the subject has exhibited or is at risk for an adverse event associated with EPO therapy, HIF stabilizer therapy, or IV iron supplementation.

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