antagonists

Abstract

To provide pharmaceuticals for treating or preventing anemia, such as moderate to severe anemia.SOLUTION: The invention relates to bone morphogenetic protein 6 (BMP6) antagonists, such as antibodies and fragments, as well as methods, uses and combinations.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to bone morphogenetic protein 6 (BMP6) antagonists, such as antibodies and fragments, as well as methods, uses and combinations. [Background technology]

[0002] Bone morphogenetic protein 6 (BMP6) is a key regulator of hepcidin, a small peptide secreted by the liver that is a major regulator of iron metabolism in mammals. Anti-BMP6 antagonists, such as antibodies, have been developed for use in methods for treating or preventing anemia (see, for example, WO2016 / 098079, US2016 / 0176956A1). See also WO2017 / 191437, which discloses the combination of anti-BMP6 antagonists with erythropoietin-stimulating agents (ESAs).

[0003] Anemia is a major disease affecting 25% of the world's population, or over 1.7 billion people, particularly pregnant women, newborns, and children. Over 40% of anemias reflect a dysfunction in the homeostatic control of iron uptake, storage, and recycling. This dysregulation is the result of a variety of chronic diseases, including infection (e.g., HIV, hepatitis), inflammation (e.g., rheumatoid arthritis), cancer, and kidney disease. The devastating impact of diseases that cause dysregulation of iron homeostasis can be seen in the United States, where anemia affects 10% of the 40 million adults over the age of 65, and one-third of these are caused by chronic disorders.

[0004] Standard treatment focuses on blood transfusions and treatment with EPO or ESAs such as Aranesp® (Amgen, Inc.).

[0005] The HAMP gene encodes hepcidin, a 25-amino acid peptide hormone produced by the liver. Hepcidin primarily functions by regulating the flux of iron from cells by regulating the amount of the iron transporter ferroportin present on the cell surface of cells involved in iron transport. Hepcidin interacts with ferroportin, which is primarily expressed in macrophages and duodenal enterocytes, causing ferroportin internalization and degradation (Ganz and Nemeth, 2011; Ramey et al., 2010). BMP6 induces hepcidin expression through a mechanism involving several receptors and cofactors. These include BMP class I and II receptors, matriptase-2, neogenin, HFE, and transferrin receptors 1 and 2, which are essential for inducing HAMP expression and are members of the repulsive guidance molecule family RGMc (hemojuvelin, HJV) and RGMb (DRAGON). The SMAD pathway is thought to be triggered by the initial interaction of BMP6 with the IBMP class receptor, likely involving HJV. This leads to the association of BMP6 with autophosphorylated BMPRII and the activation of BMPRI, inducing the SMAD cascade. Phosphorylated SMADs 1, 5, and 8, and ultimately SMAD4, translocate to the nucleus, interact with BMP response elements in the HAMP gene regulatory region, and induce hepcidin expression. In mice, liver-specific disruption of the SMAD4 signaling molecule or the type I receptors Alk2 and Alk3 reduces hepcidin expression, similar to BMP6 knockout, confirming that these factors are also part of the associated pathway (Steinbicker et al., 2011; Wang et al., 2005). ·Ganz, T., Nemeth, E., 2011. The Hepcidin-Ferroportin System as a Therapeutic Target in Anemias and Iron Overload Disorders. Hematology 2011, 538-542. ·Ramey, G., Deschemin, JC, Durel, B., Canonne-Hergaux, F., Nicolas, G., Vaulont, S., 2010. Hepcidin targets ferroportin for degradation in hepatocytes. Haematologica 95,501-504. ·Steinbicker,AU,Bartnikas,TB,Lohmeyer,LK,Leyton,P.,Mayeur,C.,Kao,SM,Pappas,AE,Peterson,RT,Bloch,DB,Yu,PB,Fleming,MD,Bloch,KD,2011.Perturbation of hepcidin expression by BMP type I receptor deletion induces iron overload in mice.Blood 118,4224-4230. ·Wang, R.-H., Li, C., Xu, Summary of the Invention [Means for solving the problem]

[0006] The present invention provides the following: An antibody or fragment comprising a binding site that specifically binds to bone morphogenetic protein 6 (BMP6), wherein the binding site comprises a VH domain, and the VH domain comprises a CDRH3 sequence of the VH domain comprising sequence number 114.

[0007] An antibody or fragment comprising a binding site that specifically binds to BMP6, wherein the binding site comprises a VH domain comprising sequence number 114, or an amino acid sequence at least 70% identical thereto.

[0008] An antibody or fragment comprising a binding site that specifically binds to BMP6, wherein the binding site comprises a VL domain, and the VL domain comprises a CDRL3 sequence of the VL domain comprising sequence number 123.

[0009] An antibody or fragment comprising a binding site that specifically binds to BMP6, wherein the binding site comprises a VL domain comprising sequence number 123, or an amino acid sequence at least 70% identical thereto.

[0010] An antibody or fragment that specifically binds to bone morphogenetic protein 6 (BMP6) and comprises (i) a heavy chain amino acid sequence comprising SEQ ID NO: 116, or an amino acid sequence that is at least 70% identical thereto, and / or (ii) a light chain sequence comprising SEQ ID NO: 125, or an amino acid sequence that is at least 70% identical thereto.

[0011] An antibody or fragment that (i) specifically binds to a human BMP6 epitope that is identical to the epitope bound by an antibody of the present invention, and / or (ii) competes with an antibody of the present invention for binding to human BMP6.

[0012] The present invention also provides the following configuration. In a first configuration, the present invention provides a method for manufacturing a semiconductor device comprising: Provided is an antibody or fragment comprising a binding site that specifically binds to bone morphogenetic protein 6 (BMP6), wherein the binding site comprises a VH domain encoded by a nucleotide sequence derived from the recombination of a human VH gene segment, a D gene segment, and a JH gene segment, wherein the VH gene segment is selected from IGHV3-11 and IGHV1-3.

[0013] In a second configuration, the present invention provides a method for manufacturing a semiconductor device comprising: Antibodies or fragments that specifically bind to BMP6 and comprise the CDRH3 sequence of an anti-BMP6 antibody according to the present invention, or said CDRH3 sequence containing 3, 2 or 1 amino acid substitution(s) are provided.

[0014] In a third configuration, the present invention provides a method for manufacturing a semiconductor device comprising: Provided is an antibody or fragment comprising a VH domain that specifically binds to BMP6 and comprises the CDRH3 sequence of an antibody selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, and CL-58713, or said sequence comprising three, two, or one amino acid substitution(s).

[0015] In a fourth configuration, the present invention provides a method for manufacturing a semiconductor device comprising: Antibodies or fragments comprising a binding site that specifically binds to BMP6 are provided, wherein the binding site comprises a VH domain comprising the amino acid sequence of the VH domain of an antibody selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, and CL-58713, or amino acids at least 70% identical thereto.

[0016] In a fifth configuration, the present invention provides a method for manufacturing a semiconductor device comprising: An antibody or fragment comprising a binding site that specifically binds to BMP6 is provided, wherein the binding site comprises a VL domain encoded by a nucleotide sequence derived from the recombination of human VL gene segments and JL gene segments, and the VL gene segment is selected from IGKV3-20, IGKV1-5, and IGKV3-15.

[0017] In a sixth configuration, the present invention provides a method for manufacturing a semiconductor device comprising: Antibodies or fragments that specifically bind to BMP6 and comprise the CDRL3 sequence of an anti-BMP6 antibody of the present invention, the CDRL3 sequence comprising three, two, or one amino acid substitution(s) are provided.

[0018] In a seventh configuration, the present invention provides a method for manufacturing a semiconductor device comprising: Provided is an antibody or fragment comprising a VL domain that specifically binds to BMP6 and comprises the CDRL3 (and optionally the CDRH3) sequence of an antibody selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, and CL-58713, or each of said sequence(s) comprising 3, 2, or 1 amino acid substitution(s).

[0019] In an eighth configuration, the present invention provides a method for manufacturing a semiconductor device comprising: Antibodies or fragments comprising a binding site that specifically binds to BMP6 are provided, wherein the binding site comprises a VL domain comprising the amino acid sequence of the VL domain of an antibody selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, and CL-58713, or amino acids at least 70% identical thereto.

[0020] In a ninth configuration, the present invention provides a method for manufacturing a semiconductor device comprising: The present invention provides an antibody or fragment thereof that specifically binds to BMP6 and comprises the heavy chain amino acid sequence of an antibody selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, and CL-58713, or an antibody or fragment thereof that is at least 70% identical to the heavy chain amino acid sequence.

[0021] In a tenth configuration, the present invention provides a method for manufacturing a semiconductor device comprising: The present invention provides an antibody or fragment thereof that specifically binds to BMP6 and comprises the light chain amino acid sequence of an antibody selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, and CL-58713, or an antibody or fragment thereof that is at least 70% identical to the light chain amino acid sequence.

[0022] In an eleventh configuration, the present invention provides a method for manufacturing a semiconductor device comprising: Antibodies or fragments are provided that specifically bind to a human BMP6 epitope that is identical to the epitope bound by an antibody of the present invention (eg, CL-58838).

[0023] In a twelfth configuration, the present invention provides a method for manufacturing a semiconductor device comprising: Antibodies or fragments that compete with the antibodies of the invention for binding to human BMP6 are provided.

[0024] In a thirteenth configuration, the present invention provides a method for manufacturing a semiconductor device comprising: The anti-BMP6 antibody or fragment of the present invention is provided for treating or preventing a BMP6-mediated disease or condition (optionally anemia) in a subject.

[0025] In a fourteenth configuration, the present invention provides a method for manufacturing a semiconductor device comprising: A combination of an amount of an anti-BMP6 antibody or fragment and an amount of ESA (optionally comprising multiple doses of the antibody and / or ESA) is provided, wherein the antibody or fragment is according to the invention.

[0026] In a fifteenth configuration, the present invention provides a method for manufacturing a semiconductor device comprising: There is provided the use of an antibody, fragment or combination of the invention in the manufacture of a medicament for administration to a subject to treat or prevent a BMP6-mediated disease or condition, optionally anemia.

[0027] In a sixteenth configuration, the present invention provides a method for manufacturing a semiconductor device, comprising: A method for treating or preventing a BMP6-mediated disease or condition (optionally anemia) in a subject is provided, the method comprising administering to the subject a therapeutically effective amount of an antibody, fragment or combination of the invention, whereby the BMP6-mediated disease or condition is treated or prevented.

[0028] In a seventeenth configuration, the present invention provides a method for manufacturing a semiconductor device, comprising: Pharmaceutical compositions are provided comprising an antibody, fragment, or combination of the invention and a pharmaceutically acceptable excipient, diluent, or carrier.

[0029] In an eighteenth configuration, the present invention provides a method for manufacturing a semiconductor device, comprising: Nucleic acids encoding the VH and / or VL domains of the antibodies or fragments of the invention are provided.

[0030] In a nineteenth configuration, the present invention provides a method for manufacturing a semiconductor device, comprising: Nucleic acids are provided that encode a VH domain comprising the amino acid sequence of the VH domain of an antibody selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, and CL-58713, or that are at least 70% identical thereto.

[0031] In a twentieth configuration, the present invention provides: (a) a nucleotide sequence that is at least 70% identical to the sequence of SEQ ID NO: 115, 520, or 521, and / or (b) a nucleic acid comprising a nucleotide sequence that is at least 70% identical to the sequence of SEQ ID NO: 124, 522 or 523 (e.g., in a host cell, such as a CHO or HEK293 or Cos cell).

[0032] (a) a nucleotide sequence that is at least 70% identical to the sequence of SEQ ID NO: 115, and / or (b) a nucleic acid comprising a nucleotide sequence that is at least 70% identical to the sequence of SEQ ID NO: 124 (e.g., in a host cell, such as a CHO or HEK293 or Cos cell).

[0033] (a) a nucleotide sequence that is at least 70% identical to the sequence of SEQ ID NO: 520, and / or (b) a nucleic acid comprising a nucleotide sequence that is at least 70% identical to the sequence of SEQ ID NO: 522 (e.g., in a host cell, such as a CHO or HEK293 or Cos cell).

[0034] (a) a nucleotide sequence that is at least 70% identical to the sequence of SEQ ID NO: 521, and / or (b) a nucleic acid comprising a nucleotide sequence that is at least 70% identical to the sequence of SEQ ID NO: 523 (e.g., in a host cell, such as a CHO or HEK293 or Cos cell).

[0035] In a twenty-first configuration, the present invention provides: (a) a nucleotide sequence that is at least 70% identical to the sequence of SEQ ID NO: 115, 520, or 521, and / or (b) a combination (e.g., in a host cell, e.g., a CHO or HEK293 or Cos cell) of a first and second nucleic acid, each comprising a nucleotide sequence that is at least 70% identical to the sequence of SEQ ID NO: 124, 522 or 523.

[0036] (a) a nucleotide sequence that is at least 70% identical to the sequence of SEQ ID NO: 115, and / or (b) a combination (e.g., in a host cell, e.g., a CHO or HEK293 or Cos cell) of a first and a second nucleic acid, each comprising a nucleotide sequence that is at least 70% identical to the sequence of SEQ ID NO: 124.

[0037] (a) a nucleotide sequence that is at least 70% identical to the sequence of SEQ ID NO: 520, and / or (b) a combination (e.g., in a host cell, e.g., a CHO or HEK293 or Cos cell) of a first and a second nucleic acid, each comprising a nucleotide sequence that is at least 70% identical to the sequence of SEQ ID NO: 522.

[0038] (a) a nucleotide sequence that is at least 70% identical to the sequence of SEQ ID NO: 521, and / or (b) a combination (e.g., in a host cell, e.g., a CHO or HEK293 or Cos cell) of a first and a second nucleic acid, each comprising a nucleotide sequence that is at least 70% identical to the sequence of SEQ ID NO: 523.

[0039] In a twenty-second configuration, the present invention provides a method for manufacturing a semiconductor device, comprising: Nucleic acids encoding the heavy and / or light chains of the antibodies or fragments of the invention are provided.

[0040] In a twenty-third configuration, the present invention provides a method for manufacturing a semiconductor device, comprising: A nucleic acid encoding a heavy chain comprising an amino acid sequence that is at least 70% identical to SEQ ID NO:116 is provided.

[0041] In a twenty-fourth configuration, the present invention provides a method for manufacturing a semiconductor device, comprising: A nucleic acid encoding a light chain comprising an amino acid sequence that is at least 70% identical to SEQ ID NO:125 is provided.

[0042] In a twenty-fifth configuration, the present invention provides: (a) a nucleotide sequence that is at least 70% identical to a selected heavy chain sequence of an antibody selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, and CL-58713; and / or (b) a nucleic acid (e.g., in a host cell, e.g., a CHO or HEK293 or Cos cell) comprising a nucleotide sequence that is at least 70% identical to a selected sequence of an antibody selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, and CL-58713.

[0043] (a) a nucleotide sequence that is at least 70% identical to a sequence selected from SEQ ID NOs: 512, 514, 516, 518, and 519, and / or (b) a nucleic acid (e.g., in a host cell, e.g., a CHO or HEK293 or Cos cell) comprising a nucleotide sequence that is at least 70% identical to a sequence selected from SEQ ID NOs: 513, 515, and 517.

[0044] (a) a nucleotide sequence at least 70% identical to SEQ ID NO: 512, and / or (b) a nucleic acid comprising a nucleotide sequence that is at least 70% identical to SEQ ID NO: 513 (e.g., in a host cell, such as a CHO or HEK293 or Cos cell).

[0045] (a) a nucleotide sequence at least 70% identical to SEQ ID NO: 516, and / or (b) a nucleic acid comprising a nucleotide sequence that is at least 70% identical to SEQ ID NO: 517 (e.g., in a host cell, such as a CHO or HEK293 or Cos cell).

[0046] (a) a nucleotide sequence at least 70% identical to SEQ ID NO: 518, and / or (b) a nucleic acid comprising a nucleotide sequence that is at least 70% identical to SEQ ID NO: 513 (e.g., in a host cell, such as a CHO or HEK293 or Cos cell).

[0047] (a) a nucleotide sequence at least 70% identical to SEQ ID NO: 519, and / or (b) a nucleic acid comprising a nucleotide sequence that is at least 70% identical to SEQ ID NO: 513 (e.g., in a host cell, such as a CHO or HEK293 or Cos cell).

[0048] (a) a nucleotide sequence at least 70% identical to SEQ ID NO: 518, and / or (b) a nucleic acid comprising a nucleotide sequence that is at least 70% identical to SEQ ID NO: 517 (e.g., in a host cell, such as a CHO or HEK293 or Cos cell).

[0049] (a) a nucleotide sequence at least 70% identical to SEQ ID NO: 519, and / or (b) a nucleic acid comprising a nucleotide sequence that is at least 70% identical to SEQ ID NO: 517 (e.g., in a host cell, such as a CHO or HEK293 or Cos cell).

[0050] (a) a nucleotide sequence at least 70% identical to SEQ ID NO: 514, and / or (b) a nucleic acid comprising a nucleotide sequence that is at least 70% identical to SEQ ID NO: 515 (e.g., in a host cell, such as a CHO or HEK293 or Cos cell).

[0051] (a) a nucleotide sequence at least 70% identical to SEQ ID NO: 516, and / or (b) a nucleic acid comprising a nucleotide sequence that is at least 70% identical to SEQ ID NO: 517 (e.g., in a host cell, such as a CHO or HEK293 or Cos cell).

[0052] (a) a nucleotide sequence at least 70% identical to SEQ ID NO: 518, and / or (b) a nucleic acid comprising a nucleotide sequence that is at least 70% identical to SEQ ID NO: 513, 515, or 517 (e.g., in a host cell, such as a CHO or HEK293 or Cos cell).

[0053] (a) a nucleotide sequence at least 70% identical to SEQ ID NO: 519, and / or (b) a nucleic acid comprising a nucleotide sequence that is at least 70% identical to SEQ ID NO: 513, 515, or 517 (e.g., in a host cell, such as a CHO or HEK293 or Cos cell).

[0054] In a twenty-sixth configuration, the present invention provides a method for manufacturing a semiconductor device, comprising: A vector comprising the nucleic acid(s) is provided, and optionally the vector is a CHO or HEK293 vector.

[0055] In a twenty-seventh configuration, the present invention provides a method for manufacturing a semiconductor device, comprising: A host cell containing the nucleic acid(s) or vector is provided.

[0056] In a twenty-eighth configuration, the present invention provides a method for manufacturing a semiconductor device, comprising: The antibodies, fragments, combinations, vectors, host cells, uses or methods described herein are provided.

[0057] In a twenty-ninth configuration, the present invention provides: An antibody or fragment that specifically binds to a bone morphogenetic protein (BMP) for use in a method for treating or preventing a disease or condition in a human or animal subject caused by a hemojuvelin (HJV)-deficient BMP-BMP receptor (BMPR) complex, the method comprising administering the antibody or fragment to the subject to inhibit the formation of the complex and / or inhibit the induction of intracellular signaling by such a complex in the subject, thereby treating or preventing an HJV-independent BMP-BMPR-mediated disease or condition.

[0058] An antibody or fragment that specifically binds to a bone morphogenetic protein (BMP) for use in a method for treating or preventing hemojuvelin-independent anemia or osteoporosis in a human or animal subject, the method comprising administering the antibody or fragment to the subject to inhibit the formation of a hemojuvelin (HJV)-deficient BMP-BMP receptor (BMPR) complex and / or inhibit the induction of intracellular signaling by such a complex in the subject, thereby treating or preventing HJV-independent anemia or osteoporosis.

[0059] An antibody or fragment that specifically binds to a bone morphogenetic protein (BMP) for use in a method for treating or preventing hemojuvelin (HJV)-independent anemia or osteoporosis in a human or animal subject, the method comprising administering the antibody to the subject, thereby treating or preventing HJV-independent anemia or osteoporosis.

[0060] The present invention also provides such methods for treating diseases or conditions such as anemia or osteoporosis. [Brief explanation of the drawings]

[0061] [Figure 1] Establishment of the assay window using the HepG2 hepcidin reporter gene cell line and various human BMP ligands as promoters. The function of the Red Firefly luciferase gene under the control of the human hepcidin promoter regulatory element in HepG2 cells was tested by stimulating the cells with increasing concentrations of various human BMP proteins (R&D Systems or Peprotech). The assay performance was tested in two different media settings: MEM containing 1% FBS (A) and hybridoma medium containing 25% MEM (B; see Example 1 for details). [Figure 2] The HepG2 Han Luciferase reporter gene cell assay window was evaluated using various human or mouse BMP6 ligands (R&D Systems or Peprotech) added at increasing concentrations as enhancers. BMP6 was diluted in MEM containing 1% FBS. The total assay volume was 60 μl using 25% HMM (see Example 1 for further details and reagents used). [Figure 3] The HepG2 Hank's luciferase reporter gene cell assay window was evaluated using a fixed concentration of human (Peprotech) or mouse BMP6 (R&D Systems) at 1 nM in MEM containing 1% FBS and commercially available mouse anti-BMP6 monoclonal antibodies MAB507 (A) and MAB2365 (B) from R&D Systems, diluted in hybridoma maintenance medium (HMM) (see Example 1 for further details and reagents). [Figure 4]Serum titration of immunization regime KM089 was performed using five bmp6- / - Kymice mice by a reverse DELFIA® assay (Perkin Elmer). IgG antibodies were captured via the Fc domain with anti-mouse IgG (goat anti-mouse IgG, Southern Biotech 1030-01) in serum at various serial dilutions. Then, the antibodies were incubated with biotinylated BMP6 and detected using DELFIA Eu-N1 europium-labeled streptavidin (Perkin Elmer). The cutoff (signals below this value were considered negative) was defined as the negative control mean of all replicates + 3x standard deviation. The KMBM code in the legend refers to Kymouse™ bmp6- / - animals. [Figure 5] Two micrograms per lane of purified human BMP6 (Peprotech) was separated by SDS-PAGE and stained with Coomassie Blue (A) under reducing (R) or nonreducing (NR) conditions. Western blots from these gels were then blotted onto membranes and probed with purified human anti-BMP6 antibodies as a label. Bound antibody was detected with anti-human kappa light chain-horseradish peroxidase (HRP) and peroxidase by enhanced chemiluminescence (CL-58838 and antibody A) or anti-human Fc-alkaline phosphatase (AP) and phosphatase 5-bromo-4-chloro-3-indolylphosphate (BCIP) and nitroblue tetrazolium (NBT) colorimetric substrate conversion (antibody B). [Figure 6-1]Receptor dimerization assay based on U2OS cells transfected with either BMPRIA (ALK3, CD292) and BMPRII (T-ALK, Figure A) or BMPRIA (ALK6) and BMPRII (T-ALK, Figure B). Ligand-driven dimerization with BMP6 (Peprotech 120-06, SEQ ID NO: 2) generated a chemiluminescent signal. Human anti-BMP6 IgG4 antibody CL-58838 and anti-BMP6 antibodies A and B (A), human anti-BMP6 IgG4 antibodies 1-8 (B), and mouse monoclonal antibody MAB507 (R&D Systems, A and B) were added at increasing concentrations (11-point dilutions). Data are representative of two experiments in each case. Appropriate isotype control IgG was used in both experiments. [Figure 6-2] See Figure 6-1. [Figure 7-1] Cross-reactivity profile of purified CL-58838 IgG4 (SEQ ID NO: 116 and SEQ ID NO: 125) with human BMP2 (A), BMP4 (B), BMP5 (C), BMP7 (D), and BMP9 (E, details described in Example 7). The effect of the anti-BMP6 antibody CL-58838 on BMP-driven activation of a Hank's luciferase reporter gene in HepG2 cells was investigated by adding increasing amounts of CL-58838. As a positive control, relevant BMP-specific antibodies (all R&D Systems) were added to inhibit activation of the relevant BMP. [Figure 7-2] See Figure 7-1. [Figure 7-3] See Figure 7-1. [Figure 8-1] Plotted transferrin saturation % (TSAT) in normal rats from Table 12 (for Figures 8A-D), Table 13 (for Figures 8E-G), and Table 14 (for Figures 8H and I) after a single intravenous injection of anti-BMP-6 or isotype IgG control antibody. [Figure 8-2] See Figure 8-1. [Figure 8-3] See Figure 8-1. [Figure 8-4] See Figure 8-1. [Figure 8-5] See Figure 8-1. [Figure 9] Results of % transferrin saturation (TSAT) in normal rats after a single intravenous injection of various doses of CL-58838 (A) or 1 mg / kg of CL-58838 and antibody A (B, Table 15). [Figure 10] Results of % transferrin saturation (TSAT) in normal rats after a single subcutaneous injection of various doses of CL-58838 (A) or 1 mg / kg of CL-58838 and antibodies A and B (B, Table 16). [Figure 11-1] Pharmacokinetic (PK) profiles of human anti-BMP6 IgG4 antibody CL-58838 after a single intravenous injection at various doses (A) or compared to antibody A at a 1 mg / kg dose (B). PK profiles after subcutaneous (sc) injection of CL-58838 at various doses (C) or antibodies A and B at a 1 mg / kg dose (D). Results plotted as IgG [ng / ml] over time [h]. [Figure 11-2] See Figure 11-1. [Figure 12-1] Results of the rat PG-PS model of ACD. Transferrin saturation (TSAT) (A), hemoglobin (g / dL) (B), MCH (pg) (C), and serum hepcidin (ng / mL) levels at weeks 0 (D), 1 (E), and 2 (F) after treatment initiation (*ρ<0.05, **p<0.01, ***p<0.001). [Figure 12-2] See Figure 12-1. [Figure 12-3] See Figure 12-1. [Figure 12-4] See Figure 12-1. [Figure 13-1] Transferrin saturation % (TSAT) after a single intravenous dose of CL-58838 with antibodies A and B at 3 mg / kg (A), and CL-58838 at various doses (B) in cynomolgus monkeys, and plasma hepcidin levels after a single intravenous dose of CL-58838 with antibodies A and B at 3 mg / kg (C), and CL-58838 at various doses (D) in cynomolgus monkeys. [Figure 13-2] See Figure 13-1. [Figure 14]PK profile of CL-58838 after a single intravenous injection in cynomolgus monkeys at various doses (A), and a comparison of the PK profiles of CL-58838 at 3 mg / kg with antibodies A and B (B). [Figure 15] Amino acid sequence alignment of antibody Vh and Vk sequences highly related to CL-58838 based on V region usage and CDRH3 (Table 6). The top row shows the germline sequences of the IMGT V region genes IGHV3-11 and IGKV3-20 encoded in the bmp6- / - Kymouse used in the present invention. Amino acid positions that differ from the germline V region sequences of selected antibody sequences below are boxed. Antibodies selected for in vivo evaluation are shown in bold (Table 6). [Figure 16] Homogeneous time-resolved FRET (HTRF) assays demonstrate the ability of antibodies highly related to CL-58838, based on CDRH antibodies identified by V-region usage and NGS sequencing (Table 6), to compete with labeled CL-58838 (10 nM) for binding to human BMP6 present at 32 nM. Competing antibodies were added as a concentration range over an 11-point dilution range starting at a final concentration of 3 μM. [Figure 17-1] Experimental setup and results from Example 17. Graphical representation of the experimental time course (A), decision diagram for rat enrollment and EPO application (B), hemoglobin [g / dL] (C), MCV [fL] (D), MCH [pg] (E), and applied EPO dose [%] (F). ANCOVA analysis of change from baseline at week 4 using Fisher's exact test (F) was used with baseline as a covariate (C-E). Data are presented as mean ± SEM (*ρ<0.05, **p<0.01, ***p<0.001). [Figure 17-2] See Figure 17-1. [Figure 17-3] See Figure 17-1. [Figure 17-4] See Figure 17-1. [Figure 18-1]Experimental setup and results of Example 18. Graphical representation of the experimental time course (A), hemoglobin [g / dL] (B), MCV [fL] (C), MCH [pg] (D), liver HAMP mRNA levels [fold expression], plasma iron levels [μg / dL] (F), and TSAT values [%]. Analysis of variance with Dunnett's correction for multiple comparisons versus EPO was applied. Results are shown for EPO versus EPO + CL-58838. Data are presented as mean ± SEM (*ρ<0.05, **p<0.01, ***p<0.001). [Figure 18-2] See Figure 18-1. [Figure 18-3] See Figure 18-1. [Figure 18-4] See Figure 18-1. [Figure 19-1] Results from Example 19. Hemoglobin values [g / dL] comparing treatment groups after administration of 1 μg / kg EPO in combination with different CL-58838 concentrations (A). Hemoglobin values [g / dL] after administration of 10 mg / kg CL-58838 in combination with different EPO doses (B). MCV values [fL] and TSAT [%] (D) of mice after administration of 1 μg / kg EPO in combination with different CL-58838 concentrations (C). MCV values [fL] of mice after administration of 1 mg / kg CL-58838 in combination with different EPO doses (E). Hepatic HAMP mRNA levels [fold expression] (F) among all treatment groups. Analysis of variance with Dunnett's correction for multiple comparisons versus group II was applied. Data are presented as mean ± SEM (*ρ<0.05, **p<0.01, ***p<0.001). [Figure 19-2] See Figure 19-1. [Figure 19-3] See Figure 19-1. DETAILED DESCRIPTION OF THE INVENTION

[0062] definition Unless otherwise defined herein, scientific and technical terms shall have the meaning commonly understood by those skilled in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin "exempli gratia" and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example." In the specification and claims, the term "about" is used to modify, for example, the amounts, concentrations, volumes, processing temperatures, processing times, yields, flow rates, pressures, and similar values and ranges of components in compositions used in describing embodiments of the present disclosure. The term "about" refers to variations in quantity that may occur, for example, through typical measuring and handling procedures for making a compound, composition, concentrate, or formulation for use, through accidental errors in these procedures, differences in purity in the manufacture, source, or starting materials or components used to carry out the method, and similar straightforward considerations. The term "about" also encompasses amounts that vary with aging of a formulation having a particular initial concentration or mixture, and amounts that vary by mixing or processing a formulation having a particular initial concentration or mixture. When modified by the term "about," the appended claims include equivalents of these quantities.

[0063] As used herein, "administering" or "administration" refers to the act of injecting or otherwise physically delivering an exogenous substance (e.g., an anti-hBMP6 antibody provided herein) to a patient by mucosal delivery, intradermal delivery, intravenous delivery, intramuscular delivery, and / or any other method described herein or known in the art. When a disease or symptom thereof is treated, administration of the substance typically occurs after the onset of the disease or symptom thereof. When a disease or symptom thereof is prevented, administration of the substance typically occurs before the onset of the disease or symptom thereof.

[0064] The terms "antibody," "immunoglobulin," or "Ig" may be used interchangeably herein and refer to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing, at at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) variants, multispecific antibodies such as bispecific antibodies (including biconjugated antibodies), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antigenic portion of an antibody, and any other modified immunoglobulin molecule containing an antigen recognition site, so long as it exhibits the desired biological activity. The term "antibody" can also refer to a Y-shaped glycoprotein with a molecular weight of approximately 150 kDa, consisting of four polypeptide chains (two light (L) chains and two heavy (H) chains). There are five mammalian Ig heavy chain isotypes, designated by the Greek letters alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ). The type of heavy chain defines the class of antibody, i.e., IgA, IgD, IgE, IgG, and IgM, respectively. The gamma and alpha classes are further divided into subclasses, e.g., IgG1, hIgG2, mIgG2A, mIgG2B, IgG3, IgG4, IgA1, and IgA2, based on differences in constant domain sequence and function. In mammals, there are two types of immunoglobulin light chains: lambda and kappa. The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the antibody heavy or light chain. The variable domains of the heavy and light chains are called "V" and "V" respectively. H " and "V L ". These domains are generally the most variable parts of an antibody (compared to other antibodies of the same class) and contain the antigen-binding sites. An example of an antibody is a heavy-chain-only (i.e., H2) antibody that contains a heavy chain dimer (5'-VH-(optional hinge)-CH2-CH3-3') and lacks light chains.

[0065] The antibodies described herein may be oligoclonal, polyclonal, monoclonal (including full-length monoclonal), camelized, chimeric, CDR-grafted, multispecific, bispecific (including biconjugated), catalytic, chimeric, humanized, fully human, or anti-idiotypic antibodies, including antibodies that may be labeled in soluble or conjugated form, as well as fragments, variants, or derivatives thereof, either alone or in combination with other amino acid sequences provided by known techniques. Antibodies may be from any species. The antibodies described herein may be naked or conjugated to other molecules, such as toxins, radioisotopes, etc.

[0066] The terms "antigen-binding site," "antigen-binding domain," "antigen-binding region," "antigen-binding fragment," and similar terms refer to the portion of an antibody (e.g., the complementarity-determining region (CDR)) that contains the amino acid residues that interact with an antigen and confer to the binder its specificity and affinity for the antigen. The antigen-binding region can be derived from any animal species, including rodents (e.g., rabbits, rats, or hamsters) and humans. Preferably, the antigen-binding region is of human origin.

[0067] Antigen-binding fragments described herein may include single-chain Fvs (scFvs), single-chain antibodies, single-chain domain antibodies, domain antibodies, Fv fragments, Fab fragments, F(ab') fragments, F(ab') fragments, antibody fragments exhibiting desired biological activity, disulfide-stabilized variable regions (dsFvs), dimeric variable regions (diabodies), anti-idiotypic (anti-Id) antibodies (e.g., anti-Id antibodies against antibodies), intrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from any of the above antibody fragments and epitope-binding fragments. In particular, antibodies and antibody fragments described herein may include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing an antigen-binding site. Digestion of antibodies with the enzyme papain produces two identical antigen-binding fragments, also known as "Fab" fragments and "Fc" fragments, which lack antigen-binding activity but have the ability to crystallize. As used herein, "Fab" refers to an antibody fragment containing one constant domain and one variable domain from each of the heavy and light chains. The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain and includes native-sequence Fc regions and variant Fc regions. "Fc fragment" refers to the carboxy-terminal portions of both H chains held together by disulfides. The effector function of an antibody is determined by the sequence in the Fc region, and this region is also recognized by Fc receptors (FcRs) found on certain types of cells. Digestion of an antibody with the enzyme pepsin results in an F(ab')2 fragment in which the two arms of the antibody molecule remain linked and contain two antigen-binding sites. The F(ab')2 fragment has the ability to cross-link antigens.

[0068] The term "derived from the recombination of" in reference to gene segments is readily apparent to one of skill in the art, who understands that B cells recombine their variable region gene segments to produce a variable domain coding sequence. For example, the term "derived from the recombination of a human VH gene segment, a D gene segment, and a JH gene segment" refers to the recombination of one human VH gene segment, where one D gene segment and one JH gene segment together form a rearranged VDJ sequence encoding a heavy chain antibody variable domain. Junctional and somatic hypermutation may also be characteristic of the process, whereby the resulting recombined VDJ sequence contains one or more nucleotide additions, substitutions, or deletions (e.g., p-additions and / or n-additions) not constituted by the germline V, D, and J sequences. Equivalents would be those of the Vκ and Jκ gene segments for kappa light chain variable domains, and Vλ and Jλ for lambda light chain variable domains. It is intended that any post-translational modifications may also be incorporated into the variable domain.

[0069] "Fv" as used herein refers to the minimum antibody fragment that retains both the antigen recognition and binding sites. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain associated by tight non-covalent or covalent bonds. In this configuration, the three CDRs of each variable domain interact to form a V H -V L The six CDRs define an antigen-binding site on the surface of the dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0070] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible natural mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic determinant or epitope. In contrast, polyclonal antibody preparations typically contain different antibodies directed against different antigenic determinants (or epitopes). As used herein, the term "monoclonal antibody" encompasses both intact and full-length monoclonal antibodies, as well as antibody fragments (Fab, Fab', F(ab')2, Fv, etc.), single-chain (scFv) variants, fusion proteins containing antibody portions, and any other modified immunoglobulin molecule containing an antigen recognition site. Furthermore, "monoclonal antibody" refers to such antibodies produced by any number of methods, including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals.

[0071] Monoclonal antibodies herein may include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies from a particular species belonging to a particular antibody class or subclass, while the remaining chain(s) are identical to or homologous to corresponding sequences in antibodies from another species belonging to a different antibody class or subclass, as well as fragments of such antibodies that exhibit the desired biological activity.

[0072] The term "humanized antibody" refers to a subset of chimeric antibodies in which a "hypervariable region" from a non-human immunoglobulin (donor antibody) replaces residues from a hypervariable region in a human immunoglobulin (recipient antibody). Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin sequence and all or substantially all of the framework regions being those of a human immunoglobulin sequence, except that the framework regions may contain one or more substitutions which improve antibody performance, e.g., binding affinity, isomerization, immunogenicity, etc.

[0073] The term "bispecific antibody" refers to an antibody that contains specificity for two target molecules, and includes DVD-Ig (see DiGiammarino et al., "Design and generation of DVD-Ig™ molecules for dual-specific targeting," Meth. Mo. Biol., 2012, 889, 145-156), mAb 2 (See WO2008 / 003103, mAb 2type, the description of which is incorporated herein by reference), FIT-Ig (WO2015 / 103072, the description of the FIT-Ig backbone is incorporated herein by reference), mAb-dAb, Dock-Lock, Fab-arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, κλ-body, orthogonal Fab, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 Formats include, but are not limited to, KIH, scFv-CH-CL-scFv, F(ab')2-scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCab, ImmTAC, knobs-in-holes, knobs-in-hole with a common light chain, knobs-in-hole with a common light chain and charge pair, charge pair, charge pair with a common light chain, DT-IgG, DutaMab, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, and zybody. For a review of bispecific formats, see Spiess, C., et al., Mol. Immunol. (2015).In another embodiment, the bispecific molecule comprises another non-Ig format, such as a T-cell receptor binding domain, an immunoglobulin superfamily domain, a jawless variable lymphocyte receptor, a fibronectin domain (e.g., Adnectin™), an antibody constant domain (e.g., a CH3 domain, e.g., CH2 and / or CH3 of an Fcab™) (wherein the constant domain is not a functional CH1 domain), an scFv, (scFv)2, sc-diabody, scFab, centyrin and epitope binding domains derived from a scaffold selected from CTLA-4 (Evibody™), a lipocalin domain, a protein A domain, a fibronectin domain (e.g., Adnectin™), ... These include antibodies fused to protein A such as Z domains (e.g., Affibody™ or SpA), A domains (e.g., Avimer™ or Maxibody™), heat shock proteins (such as epitope binding domains derived from GroEI and GroES), transferrin domains (e.g., Transbodies), ankyrin repeat proteins (e.g., DARPins™), peptide aptamers, C-type lectin domains (e.g., Tetranectin™), human gamma-crystallin or human ubiquitin (affilin), PDZ domains, scorpion toxins, and Kunitz-type domains of human protease inhibitors.

[0074] In one embodiment, the bispecific antibody is a mAb 2 mAb 2 is a V from an intact antibody fused to a modified constant region H and V L domains, which have been engineered to form antigen-binding sites known as "Fcabs." Fcab / mAb 2 The technology behind the format is described in more detail in WO2008 / 003103 and in mAb 2 The format description is incorporated herein by reference.

[0075] In another embodiment, the bispecific antibody is a "dual binding antibody." As used herein, a "dual binding antibody" refers to an antibody in which both antigen-binding domains are VH / V L and bispecific antibodies formed by a pair, including FIT-Ig (see WO2015 / 103072, incorporated herein by reference), mAb-dAb, Dock-Lock, Fab-arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, κλ-body, orthogonal Fab, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, diabody-CH3, triple body, miniantibody, and the like. These include, but are not limited to, minibodies, scFv-CH3KIH, scFv-CH-CL-scFv, F(ab')2-scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCab, ImmTAC, knob-in-hole, knob-in-hole with a common light chain, knob-in-hole with a common light chain and charge pair, charge pair, charge pair with a common light chain, DT-IgG, DutaMab, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, and scFv4-Ig.

[0076] The terms "hypervariable region," "CDR region," or "CDR" refer to the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops. Generally, the antigen-binding site of an antibody is composed of six hypervariable regions (V H Three in (CDRH1, CDRH2, CDRH3) and V L These regions of the heavy and light chains of an antibody confer antigen-binding specificity to the antibody. CDRs can be defined according to the Kabat system (Kabat, EA et al., 1991, "Sequences of Proteins of Immunological Interest", 5th (See, edit., NIH Publication no. 91-3242, USDapartment of Health and Human Services). Other systems can be used to define CDRs, such as the system devised by Chothia et al. (See, Chothia, C. & Lesk, AM, 1987, "Canonical structures for the hypervariable regions of immunoglobulins", J. Mol. Biol., 196, 901-917) and the IMGT system (See, Lefranc, MP, 1997, "Unique database numbering system for immunogenetic analysis", Immunol. Today, 18, 50). Antibodies typically contain three heavy chain CDRs and three light chain CDRs. The term CDR(s) is used herein to refer to one or several of these regions. Those skilled in the art can easily compare different systems of nomenclature and determine whether a particular sequence can be defined as a CDR.

[0077] A "human antibody" is an antibody that possesses an amino acid sequence corresponding to that of an antibody produced by a human and / or is produced using any of the techniques for producing human antibodies, excluding humanized antibodies that contain non-human antigen-binding residues. The term "specifically binds" refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of the target in the presence of a homogeneous population of molecules, including biological molecules. For example, an antibody that specifically binds to a target (which may be an epitope) is an antibody that binds to this target with greater affinity, avidity, more readily, and / or longer than it binds to other targets. In one embodiment, the extent of binding of the antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA).

[0078] An antibody or fragment thereof that specifically binds to a hBMP6 antigen may be cross-reactive with related antigens. Preferably, an antibody or fragment thereof that specifically binds to a hBMP6 antigen does not cross-react with other antigens (although it may optionally cross-react with BMP6 from different species, such as rhesus monkeys or mice). An antibody or fragment thereof that specifically binds to a hBMP6 antigen can be identified, for example, by immunoassay, BIAcore™, or other techniques known to those skilled in the art. An antibody or fragment thereof specifically binds to a BMP6 antigen if it binds to the hBMP6 antigen with higher affinity than to any cross-reactive antigens, as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, a specific or selective reaction is at least 2 times the background signal or noise, more typically more than 10 times (e.g., more than 15 times, more than 20 times, more than 50 times, or more than 100 times) the background. For a discussion of antibody specificity, see, e.g., pages 332-336 of Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York.

[0079] The term "aliphatic amino acid" means that the amino acid R group is nonpolar and hydrophobic. Hydrophobicity increases with the number of C atoms in the hydrocarbon chain. Glycine, alanine, valine, leucine, and isoleucine are aliphatic amino acids.

[0080] The term "aromatic amino acid" means that the amino acid R group contains an aromatic ring system. Phenylalanine, tyrosine, and tryptophan are aromatic amino acids. The term "hydroxyl-containing amino acid" means that the amino acid R group contains a hydroxyl group and is hydrophilic. Serine, cysteine, threonine, and methionine are hydroxyl-containing amino acids.

[0081] The term "basic amino acid" means that the amino acid R group contains nitrogen and is basic at neutral pH. Histidine, lysine, and arginine are basic amino acids.

[0082] The term "cyclic amino acid" means that the amino acid R group has an aliphatic ring structure. Proline is the only cyclic aliphatic amino acid.

[0083] The term "acidic amino acid" means that the amino acid R group is polar and negatively charged at physiological pH. Aspartic acid and glutamic acid are acidic amino acids.

[0084] The term "amide amino acid" means that the amino acid R group contains an amide group. Asparagine and glutamine are amide amino acids.

[0085] As used herein, "approval number" and "marketing authorization number" refer to a number issued by a regulatory agency when that agency determines that a particular medical product and / or composition may be sold and / or released in the agency's area of jurisdiction. As used herein, "regulatory agency" refers to, for example, one of the agencies responsible for evaluating the safety and effectiveness of medical products and / or compositions and controlling the release / sale of such products and / or compositions in a given area. The Food and Drug Administration (FDA) in the United States and the European Medicines Agency (EPA) in Europe are just two examples of such regulatory agencies. Other non-limiting examples include the SDA, MPA, MHPRA, IMA, ANMAT, the Hong Kong Department of Health-Drug Office, CDSCO, Medsafe, and KFDA.

[0086] As used herein, "buffer" refers to a chemical agent that can absorb a certain amount of acid or base without undergoing a strong fluctuation in pH.

[0087] As used herein, the term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which a therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions.

[0088] The terms "chemotherapeutic agent" or "chemotherapy" typically refer to therapeutic agents whose primary purpose is to destroy cancer cells by interfering with their ability to grow or proliferate. Many different types of chemotherapy drugs exist, with over 50 approved chemotherapy drugs available. Chemotherapy drugs can be classified based on how they work. Alkylating agents kill cancer cells by directly attacking DNA, the genetic material of genes. Cyclophosphamide is an alkylating agent. Antimetabolites interfere with the production of DNA, suppressing cell growth and proliferation. An example of an antimetabolite is 5-fluorouracil (5-FU). Antitumor antibiotics are produced from natural substances, such as fungi in the soil. They interfere with important cellular functions, including the production of DNA and cellular proteins. Doxorubicin and bleomycin belong to this group of chemotherapy drugs. Plant alkaloids prevent cells from dividing normally. Vinblastine and vincristine are plant alkaloids obtained from the periwinkle plant. Steroid hormones slow the growth of some hormone-dependent cancers. For example, tamoxifen is used to treat breast cancer, which depends on the hormone estrogen for growth. DNA damage response (DDR) inhibitors, such as PARP inhibitors, block DNA repair mechanisms after single- or double-strand breaks.

[0089] Examples of chemotherapeutic agents include adriamycin, doxorubicin, 5-fluorouracil, cytosine arabinoside (Ara-C), cyclophosphamide, thiotepa, taxotere (docetaxel), busulfan, cytoxin, taxol, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone, vincleistine, vinorelbine carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycin, esperamicin (see U.S. Pat. No. 4,675,187), melphalan, and other related nitrogen mustards. Suitable toxins and chemotherapeutic agents are described in Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co. 1995) and Goodman and Gilman's The Pharmacological Basis of Therapeutics, 7 th Ed. (MacMillan Publishing Co. 1985). Another example of a chemotherapeutic agent is the class of toxins conjugated to antibodies, including, but not limited to, pyrrolobenzodiazepines, maytansanoids, calicheamicin, and the like. Other suitable toxins and / or chemotherapeutic agents will be known to those skilled in the art.

[0090] As used herein, the term "composition" is intended to encompass a product containing the specified ingredients (e.g., an antibody of the invention), optionally in the specified amounts, and any product that results directly or indirectly from the combination of the specified ingredients, optionally in the specified amounts.

[0091] As used herein, the terms "comprising" or "comprises" are used in reference to antibodies, fragments, uses, compositions, methods, and their respective component(s) that are essential to the method or composition, but are open to including unspecified elements, whether essential or not.

[0092] The term "consisting of" refers to the antibodies, fragments, uses, compositions, methods, and their respective components described herein, and does not include any elements not listed in the description of that embodiment.

[0093] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.

[0094] As used herein, in the context of a polypeptide, the term "derivative" includes a polypeptide comprising the amino acid sequence of an hBMP6 polypeptide, a fragment of an hBMP6 polypeptide, or an antibody or fragment that specifically binds to an hBMP6 polypeptide that has been modified by the introduction of amino acid substitutions, deletions, or additions. As used herein, the term "derivative" also includes an hBMP6 polypeptide, a fragment of an hBMP6 polypeptide, or an antibody that specifically binds to an hBMP6 polypeptide that has been chemically modified, for example, by the covalent attachment of any type of molecule to the polypeptide. For example, and without limitation, an hBMP6 polypeptide, a fragment of an hBMP6 polypeptide, or an hBMP6 antibody may be chemically modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A derivative is modified in a manner that differs from the native or starting peptide or polypeptide in either the type or location of the attached molecule. A derivative further includes the deletion of one or more chemical groups naturally present on the peptide or polypeptide. Derivatives of hBMP6 polypeptides, hBMP6 polypeptide fragments, or hBMP6 antibodies may be chemically modified using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Furthermore, derivatives of hBMP6 polypeptides, hBMP6 polypeptide fragments, or hBMP6 antibodies may contain one or more non-classical amino acids. Polypeptide derivatives possess similar or identical functions as the hBMP6 polypeptides, hBMP6 polypeptide fragments, or hBMP6 antibodies described herein.

[0095] As used herein, the term "effector function" (or "effector-enabled") refers to one or more of antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC)-mediated responses, Fc-mediated phagocytosis or antibody-dependent cellular phagocytosis (ADCP), and antibody recycling via the FcRn receptor.

[0096] An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired effect, including a therapeutic or prophylactic result. A "therapeutically effective amount" refers to the minimum concentration necessary to achieve a measurable improvement or prevention of a particular disorder. The therapeutically effective amount herein may vary depending on factors such as the patient's disease state, age, sex, and weight, and the ability of the antibody to elicit a desired response in an individual. A therapeutically effective amount is also one in which any toxic or adverse effects of the antibody are outweighed by the beneficial effects of treatment. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic effect. In some embodiments, an effective amount of an antibody of the present invention is from about 0.1 mg / kg (mg of antibody per kg of subject body weight) to about 100 mg / kg. In certain embodiments, an effective amount of an antibody provided herein is about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, 3 mg / kg, 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg (or a range therein). In some embodiments, "effective amount," as used herein, also refers to the amount of an antibody of the invention to achieve a specified result (e.g., inhibition of cellular hBMP6 biological activity).

[0097] As used herein, the term "epitope" refers to a localized region on the surface of an antigen, such as an hBMP6 polypeptide or hBMP6 polypeptide fragment, that can bind to one or more antigen-binding regions of an antibody, has antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably a human, and can elicit an immune response. An epitope with immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal. An epitope with antigenic activity is a portion of a polypeptide to which an antibody specifically binds, as determined by any method known in the art, such as the immunoassays described herein. An antigenic epitope need not necessarily be immunogenic. Epitopes usually consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics and specific charge characteristics. The region of a polypeptide that contributes to an epitope may be contiguous amino acids of the polypeptide, or an epitope may be assembled from two or more non-contiguous regions of the polypeptide. An epitope may or may not be a three-dimensional surface feature of an antigen. In certain embodiments, the hBMP6 epitope is a three-dimensional surface feature of the hBMP6 polypeptide (e.g., in the trimeric form of the hBMP6 polypeptide). In other embodiments, the hBMP6 epitope is a linear feature of the hBMP6 polypeptide (e.g., in the trimeric or monomeric form of the hBMP6 polypeptide). The antibodies provided herein may specifically bind to an epitope of the monomeric (denatured) form of hBMP6, an epitope of the trimeric (native) form of hBMP6, or an epitope of both the monomeric (denatured) and trimeric (native) forms of hBMP6. In certain embodiments, the antibodies provided herein specifically bind to an epitope of the trimeric form of hBMP6, but do not specifically bind to the monomeric form of hBMP6.

[0098] As used herein, the term "excipient" generally refers to an inert substance used as a diluent, vehicle, preservative, binder, or stabilizer for a drug, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkylsulfonates, caprylates, etc.), surfactants (e.g., SDS, polysorbates, nonionic surfactants, etc.), sugars (e.g., sucrose, maltose, trehalose, etc.), and polyols (e.g., mannitol, sorbitol, etc.). See also Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa., the entire contents of which are incorporated herein by reference.

[0099] As used herein, in the context of a peptide or polypeptide, the term "fragment" refers to a peptide or polypeptide that comprises less than the full-length amino acid sequence. Such fragments can result, for example, from truncation at the amino terminus, truncation at the carboxy terminus, and / or internal deletion of a residue(s) from the amino acid sequence. Fragments can result, for example, from alternative RNA splicing or from in vivo protease activity. In certain embodiments, the BMP6 fragment comprises a polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of an hBMP6 polypeptide or an antibody that specifically binds to an hBMP6 polypeptide. In certain embodiments, a fragment of an hBMP6 polypeptide or an antibody that specifically binds to an hBMP6 antigen retains at least one, at least two, or at least three functions of the polypeptide or antibody.

[0100] The term "free" can refer to a polypeptide, such as BMP6, or a fragment or variant thereof, combined with a buffer, where the polypeptide is not bound to a cell surface or cell membrane. Thus, the term "free" can refer to a polypeptide capable of surface expression (i.e., containing one or more transmembrane or membrane-binding domains), but which is not currently expressed on the surface of a cell or bound to a protein expressed on the surface of a cell. A free polypeptide can also refer to a free recombinant or natural or unbound polypeptide. In the context of phage display, the free antigen can be selected in solution (referred to herein as "soluble selection") or adsorbed to a surface, for example, the surface of a 96-well plate (referred to herein as "biopanning selection").

[0101] As used herein, the term "fusion protein" refers to a polypeptide comprising the amino acid sequence of an antibody and the amino acid sequence of a heterologous polypeptide or protein (i.e., a polypeptide or protein that is not normally part of an antibody (e.g., a non-anti-BMP6 antigen antibody)). The term "fusion," when used in connection with BMP6 or an anti-BMP6 antibody, refers to the conjugation of a peptide or polypeptide, or a fragment, variant, and / or derivative thereof, with a heterologous peptide or polypeptide. Preferably, the fusion protein retains the biological activity of BMP6 or an anti-BMP6 antibody. In certain embodiments, the fusion protein comprises the VH domain, VL domain, VH CDRs (one, two, or three VH CDRs), and / or VL CDRs (one, two, or three VL CDRs) of a BMP6 antibody, and the fusion protein specifically binds to a BMP6 epitope.

[0102] The term "heavy chain," when used in reference to antibodies, refers to five different types, designated alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the heavy chain constant domain. These different types of heavy chains are well known and give rise to five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, respectively, including four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4. Preferably, the heavy chain is a human heavy chain. In the human population, multiple heavy chain constant region alleles exist for each immunoglobulin or immunoglobulin subclass. The nucleotide and amino acid sequences of these allelic variants are accessible on publicly available databases, such as IMGT, ENSEMBL Swiss-Prot, and Uniprot. Allelic variants can also be identified in various genome sequencing projects. In one embodiment, the antibodies and antibody fragments disclosed herein are derived from human IGHG1. * 01 (SEQ ID NOs: 340, 341, and 537), IGHG1 * 02 (SEQ ID NOs: 340, 341, and 537), IGHG1 * 03 (SEQ ID NOs: 523 and 524), IGHG1 * 04 (SEQ ID NOs: 525 and 526), and IGHG1 * In one embodiment, the antibodies and antibody fragments disclosed herein comprise a heavy chain encoded by an IgG1 constant region allele, including, but not limited to, human IGHG2. * 01 (SEQ ID NOs: 527 and 528), IGHG2 * 02 (SEQ ID NOs: 529 and 530), IGHG2 * 03 (SEQ ID NOs: 527 and 528), IGHG2 * 04 (SEQ ID NOs: 531 and 532), IGHG2 * 05 (SEQ ID NOs: 527 and 528), and IGHG2 *In one embodiment, the antibodies and antibody fragments disclosed herein comprise proteins encoded by IgG2 constant region alleles, including, but not limited to, human IGHG3. * 01, IGHG3 * 02, IGHG3 * 03, IGHG3 * 04, IGHG3 * 05, IGHG3 * 06, IGHG3 * 07, IGHG3 * 08, IGHG3 * 09, IGHG3 * 10.IGHG3 * 11.IGHG3 * 12, IGHG3 * 13, IGHG3 * 14, IGHG3 * 15, IGHG3 * 16, IGHG3 * 17, IGHG3 * 18, and IGHG3 * In one embodiment, the antibody or antibody fragment disclosed herein is an antibody or antibody fragment encoding a protein encoded by an IgG3 constant region allele, including, but not limited to, human IGHG4. * 01 (see, e.g., the sequence listing herein), IGHG4 * 02 (see, e.g., the sequence listing herein), IGHG4 * 03 (see, e.g., the sequence listing herein), and IGHG4 *In another example, the heavy chain is a truncated IgG isotype, e.g., truncated IgG4. In certain embodiments, the antibodies of the present invention comprise a human gamma 4 constant region. In another embodiment, the heavy chain constant region does not bind to an Fc-γ receptor and comprises, for example, a Leu235Glu mutation. In another embodiment, the heavy chain constant region comprises a Ser228Pro mutation to increase stability. In another embodiment, the heavy chain constant region is IgG4-PE (see, e.g., the sequence listing herein). In another embodiment, the antibodies and antibody fragments disclosed herein are encoded by murine IGHG1. * 01 or IGHG1 * In one embodiment, the antibodies and antibody fragments disclosed herein comprise a heavy chain constant region encoded by a mouse IgG1 constant region allele, including but not limited to mouse IGHG2A. * 01, IGHG2A * 02, IGHG2B * 01, IGHG2B * 02, IGHG2C * 01, IGHG2C * 02, or IGHG2C * In one embodiment, the antibody or antibody fragment disclosed herein comprises a heavy chain constant region encoded by a mouse IgG2 constant region allele, including, but not limited to, mouse IGHG3. * The present invention also includes proteins encoded by murine IgG3 constant region alleles, including but not limited to 01.

[0103] As used herein, the term "host" refers to an animal, preferably a mammal, and most preferably a human.

[0104] As used herein, the term "host cell" refers to a particular subject cell transfected with a nucleic acid molecule, and to the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in subsequent generations, or due to integration of the nucleic acid molecule into the host cell genome. The term " in combination with " in the context of administering other therapies refers to the use of two or more therapies.The use of the term " in combination with " does not limit the order that therapies are administered to the subject with disease.The first therapy can be administered before (for example, 1 minute, 45 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks), simultaneously with, or after (for example, 1 minute, 45 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks) the second therapy is administered to the subject who has had, has, or is susceptible to BMP6-mediated disease. Any additional therapy may be administered in any order with other additional therapies. In certain embodiments, the antibodies of the present invention may be administered in combination with one or more therapies (e.g., therapies other than the antibodies of the present invention that are currently being administered to prevent, treat, manage, and / or ameliorate a BMP6-mediated disease). Non-limiting examples of therapies that may be administered in combination with the antibodies of the present invention include analgesics, anesthetics, antibiotics, or immunomodulators, or any other drugs listed in the United States Pharmacopeia and / or Physician's Package Inserts.

[0105] As used herein, "injection device" refers to a device designed to perform injections, which includes temporarily fluidly connecting the injection device to human tissue, typically subcutaneous tissue. Injection further includes administering a quantity of liquid medication into the tissue and disconnecting or removing the injection device from the tissue. In some embodiments, the injection device may be an intravenous or IV device, a type of injection device used when the target tissue is blood in the circulatory system, e.g., a venous blood vessel. A common, but non-limiting, example of an injection device is a needle and syringe.

[0106] As used herein, "instructions" refers to written, printed, or graphic material on the immediate container of an item, e.g., written material displayed on a vial containing a pharmaceutically active agent, or a display detailing the composition and use of a product of interest included in a kit containing the composition of interest. The instructions describe the method of treatment intended to be administered or performed.

[0107] An "isolated" or "purified" antibody or protein is one that has been identified, separated, and / or recovered from components of its production environment (e.g., natural or recombinant). For example, the antibody or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody is derived, or, if chemically synthesized, substantially free of chemical precursors or other chemicals. The term "substantially free of cellular material" includes preparations of antibodies in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes preparations of antibodies having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins"). If the antibody is recombinantly produced, the antibody is also preferably substantially free of culture medium, i.e., culture medium accounts for less than about 20%, 10%, or 5% of the volume of the protein preparation. If the antibody is produced by chemical synthesis, the antibody is preferably substantially free of chemical precursors or other chemicals, i.e., the antibody is separated from chemical precursors or other chemicals involved in the synthesis of the protein. Accordingly, such preparations of antibodies have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In preferred embodiments, the antibodies of the invention are isolated or purified.

[0108] The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues that are more variable than other amino acid residues (i.e., hypervariable) in the heavy chain variable region of an antibody, or antigen-binding portion thereof (Kabat et al., (1971) Ann. NY Acad. Sci., 190:382-391 and Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable regions typically range from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3.

[0109] As used herein, "label" or "labeled" refers to the addition of a detectable moiety to a polypeptide, such as a radiolabel, a fluorescent label, an enzyme label, a chemiluminescent label, or a biotinyl group or gold. Radioisotopes or radionuclides include: 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 115 In, 125 I, 131Fluorescent labels may include rhodamine, lanthanide fluorophores, or FITC; enzyme labels may include horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase. Additional labels include, by way of example and not limitation, enzymes such as glucose-6-phosphate dehydratase ("G6PDH"), alpha-D-galactosidase, glucose oxidase, glucose amylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, and peroxidase; dyes (e.g., cyanine dyes, e.g., Cy5™, Cy5.5™, or Cy7™); additional fluorescent labels or agents such as fluorescein and its derivatives, fluorescent dyes, GFP (GFP for "green fluorescent protein"), other fluorescent proteins (e.g., mCherry, mTomato), dansyl, umbelliferone, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine; fluorophores such as lanthanide cryptates and chelates, e.g., europium (Perkin Elmer and Cisbio assays); chemiluminescent labels or chemifluorescent agents such as isoluminol, luminol, and dioxetanes; sensitizers; coenzymes; enzyme substrates; particles such as latex or carbon particles; metal sols; microcrystals; liposomes; cells, etc., which may be further labeled with dyes, catalysts, or other detectable groups; molecules such as biotin, digoxigenin, or 5-bromodexuridine; toxin moieties such as a toxin moiety selected from the group consisting of Pseudomonas exotoxin (PE or a cytotoxic fragment or variant thereof), diphtheria toxin or a cytotoxic fragment or variant thereof, botulinum toxin A, B, C, D, E, or F, ricin or a cytotoxic fragment thereof, e.g., ricin A, abrin or a cytotoxic fragment thereof, saporin or a cytotoxic fragment thereof, pokeweed antiviral toxin or a cytotoxic fragment thereof, and bryodin 1 or a cytotoxic fragment thereof.

[0110] The term "light chain," when used in reference to an antibody, refers to an immunoglobulin light chain, of which there are two types in mammals: lambda (λ) and kappa (κ). Preferably, the light chain is a human light chain. Preferably, the light chain constant region is a human constant region. Multiple light chain constant region alleles exist in the human population. The nucleotide and amino acid sequences of these allelic variants are accessible on publicly available databases such as IMGT, ENSEMBL Swiss-Prot, and Uniprot. In one embodiment, the antibody or antibody fragment disclosed herein is an antibody or antibody fragment encoding IGKC. * 01 (see, e.g., the sequence listing herein), IGKC * 02 (see, e.g., the sequence listing herein), IGKC * 03 (see, e.g., the sequence listing herein), IGKC * 04 (see, e.g., the sequence listing herein), and IGKC * 05 (see, e.g., the sequence listing herein). In one embodiment, the antibody or antibody fragment disclosed herein encodes a protein encoded by a human kappa constant region allele, including, but not limited to, IGLC1. * 01 (see, e.g., the sequence listing herein), IGLC1 * 02 (see, e.g., the sequence listing herein), IGLC2 * 01 (see, e.g., the sequence listing herein), IGLC2 * 02 (see, e.g., the sequence listing herein), IGLC2 * 03 (see, e.g., the sequence listing herein), IGLC3 * 01 (see, e.g., the sequence listing herein), IGLC3 * 02 (see, e.g., the sequence listing herein), IGLC3 * 03 (see, e.g., the sequence listing herein), IGLC3 * 04 (see, e.g., the sequence listing herein), IGLC6 * 01 (see, e.g., the sequence listing herein), IGLC7 * 01 (see, e.g., the sequence listing herein), IGLC7 * 02 (see, e.g., the sequence listing herein), IGLC7* In another embodiment, the antibodies and antibody fragments disclosed herein comprise proteins encoded by human lambda constant region alleles, including, but not limited to, IGKC. * 01.IGKC * 03, or IGKC * In another embodiment, the antibodies and antibody fragments disclosed herein comprise a light chain constant region encoded by a mouse kappa constant region allele, including, but not limited to, IGLC1. * 01, IGLC2 * 01, or IGLC3 * and light chain constant regions encoded by mouse lambda constant region alleles, including, but not limited to, 01.

[0111] "Percent (%) amino acid sequence identity" and "homology" in the context of peptide, polypeptide, or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to a particular peptide or polypeptide sequence after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEG ALIGN™ (DNASTAR) software. In one embodiment, the percent homology is about 70%. In one embodiment, the percent homology is about 75%. In one embodiment, the percent homology is about 80%. In one embodiment, the percent homology is about 85%. In one embodiment, the percent homology is about 90%. In one embodiment, the percent homology is about 92%. In one embodiment, the percent homology is about 95%. In one embodiment, the % homology is about 97%. In one embodiment, the % homology is about 98%. In one embodiment, the % homology is about 99%. In one embodiment, the % homology is 100%.

[0112] The terms "native" or "natural" when used in reference to biological materials, e.g., nucleic acid molecules, polypeptides, host cells, refer to those found in nature and that have not been manipulated by human beings.

[0113] As used herein, "packaging" refers to the manner in which components are grouped together and / or contained in a unit suitable for distribution and / or use. Packaging can include, for example, boxes, bags, syringes, ampoules, vials, tubes, clamshell packaging, barriers, and / or containers for maintaining sterility, labeling, etc.

[0114] As used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias, for use in animals, and more particularly in humans.

[0115] As used herein, the terms "polynucleotide," "nucleotide," "nucleic acid," "nucleic acid molecule," and other similar terms are used interchangeably and include DNA, RNA, mRNA, and the like.

[0116] As used herein, the terms "prevent," "preventing," and "prevention" refer to the complete or partial inhibition of the onset, recurrence, development, or metastasis of an hBMP6-mediated disease and / or symptoms associated therewith brought about by the administration of a therapy or combination of therapies provided herein (e.g., a combination of prophylactic or therapeutic agents, such as an antibody of the invention).

[0117] The term "soluble" refers to a polypeptide, such as BMP6 and its variants or fragments, that lacks one or more transmembrane or cytoplasmic domains found in its native or membrane-bound form. In one embodiment, a "soluble" form of BMP6 lacks both the transmembrane and cytoplasmic domains.

[0118] The term "subject" or "patient" refers to any animal, including, but not limited to, mammals. As used herein, the term "mammal" refers to any vertebrate that either nurses its young and gives birth to live young (eutherian or placental mammals) or lays eggs (metatherian or non-placental mammals). Examples of mammalian species include, but are not limited to, humans and other primates (including non-human primates such as chimpanzees and other apes and monkeys); livestock animals such as cows, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals, including rodents such as mice, rats (including cotton rats), and guinea pigs; and birds, including poultry, wild birds, and game birds, such as chickens, turkeys, and other pheasants, ducks, and geese.

[0119] As used herein, "substantially all" refers to at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.

[0120] As used herein, the term "therapeutic agent" refers to any agent that can be used in the treatment, management, or amelioration of a BMP6-mediated disease and / or symptoms associated therewith. In certain embodiments, the term "therapeutic agent" refers to an antibody of the present invention. In certain other embodiments, the term "therapeutic agent" refers to an agent other than an antibody of the present invention. Preferably, the therapeutic agent is an agent that is known to be useful for, has been used for, or is currently being used to treat, manage, or ameliorate a BMP6-mediated disease or one or more symptoms associated therewith. In certain embodiments, the therapeutic agent is a fully human anti-BMP6 antibody, such as a fully human anti-BMP6 monoclonal antibody.

[0121] As used herein, the term "therapy" refers to any protocol, method, and / or agent that can be used to prevent, manage, treat, and / or ameliorate a BMP6-mediated disease (e.g., cancer). In certain embodiments, the term "therapy(s)" refers to biologic, supportive, and / or other therapies known to those skilled in the art, such as medical professionals, that are useful in preventing, managing, treating, and / or ameliorating a BMP6-mediated disease. The terms "treat," "treatment," and "treating" refer to the reduction or amelioration of the progression, severity, and / or duration of an hBMP6-mediated disease (e.g., cancer) brought about by the administration of one or more therapeutic agents (including, but not limited to, the administration of one or more prophylactic or therapeutic agents, such as an antibody of the invention). In certain embodiments, such terms refer to the reduction or inhibition of binding of hBMP6 to a BMP receptor or HJV, and / or the inhibition or reduction of one or more symptoms associated with a BMP6-mediated disease, such as anemia.

[0122] The term "variable region" or "variable domain" refers to the portions of the light and heavy chains, typically the amino-terminal 120-130 amino acids in the heavy chain and the 100-110 amino acids in the light chain, which vary widely in sequence among antibodies and are used in the binding and specificity of each particular antibody to its particular antigen. The sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while the more highly conserved regions in the variable domains are called framework regions (FRs). The CDRs of BMP6 and the heavy chain are primarily responsible for the interaction of the antibody with the antigen. The numbering of amino acid positions used herein is based on Kabat et al. (1991) Sequences of proteins of immunological interest. (USDepartment of Health and Human Services, Washington, DC) 5 th (Kabat et al.), ed. In a preferred embodiment, the variable region is a human variable region.

[0123] Definitions of common terms in cell and molecular biology are given in "The Merck Manual of Diagnosis and Therapy," 19 th Edition (published by Merck Research Laboratories), 2006 (ISBN 0-911910-19-0), Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology (published by Blackwell Science Ltd.), 1994 (ISBN 0-632-02182-9), Benjamin Lewin, Genes X (published by Jones & Bartlett Publishing), 2009 (ISBN-10:0763766321), Kendrew et al. (Eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference (published by VCH Publishers, Inc.), 1995 (ISBN 1-56081-569-8), and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.

[0124] Unless otherwise stated, the present invention is not limited to or incorporates the teachings of any of the following publications: Sambrook et al., Molecular Cloning: A Laboratory Manual (4th ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1995); Methods in Enzymology: Guide to Molecular Cloning Techniques Vol. 152, S.L. Berger and A.R. Kimmel Eds., Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (John E. Coligan, et al., ed., John Wiley and Sons, Inc.); Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et al. ed., John Wiley and Sons, Inc.); and Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss, 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998), all of which are incorporated herein by reference in their entirety. Other terms are defined herein in the description of various aspects of the invention.

[0125] BMP6 and iron Iron is essential for all forms of life and must be supplied from the environment, so its availability and use in the body are tightly regulated. A key regulator of iron homeostasis is a 25-amino acid peptide hormone called hepcidin. Hepcidin is produced by the liver and regulates the expression of the iron transporter molecule ferroportin, thereby retaining iron in duodenal enterocytes and macrophages, the primary iron uptake and storage compartments. Hepcidin itself is regulated by iron levels through homeostatic control mechanisms following immune system activation and erythropoiesis during infection and / or inflammation. Importantly, hepcidin levels are elevated in chronic inflammatory conditions, infections, and certain cancers. Elevated hepcidin levels sequester iron in enterocytes, macrophages, and hepatocytes, thereby suppressing hemoglobin synthesis and erythropoiesis. This results in anemia despite normal iron store levels. Hepcidin gene expression is regulated by a soluble factor called BMP6 (bone morphogenetic protein 6). BMP6 is considered a master regulator because cytokines alone (or other BMPs) cannot overcome the lack of BMP6 signaling in its absence. Therefore, we have identified BMP6 as an important drug target for controlling abnormal dysregulation of iron homeostasis in anemia, such as anemia of chronic disease (ACD).

[0126] BMP6 is a highly conserved soluble protein factor that is considered the "master" regulator of hepcidin production in mice and humans. Thus, administration of BMP6 to mice increases hepcidin levels and reduces blood and serum iron, while BMP6 inhibitors do the opposite. Additionally, knockout of the mouse BMP6 gene or human mutations within the BMP6 pathway support the central role of BMP6 in regulating hepcidin and blood and serum iron levels. Furthermore, preclinical and clinical evaluations targeting BMP6 have been conducted, respectively, by administering anti-BMP6 antibodies to rodents or cynomolgus monkeys, or by neutralizing BMP6 using HJV-Fc (FMX-8, Ferrumax Inc.) in Phase I trials to increase available iron levels. Andriopoulos Jr.B, Corradini E, Xia Y, Faasse SA, Chen S, Grgurevic L, Knutson MD, Pietrangelo A, Vukicevic S, Lin HY and Babitt.2009.BMP-6 is a key endogenous regulator of hepcidin expression and iron Reference is made to metabolism.Nat.Genet.41(4), 482-487, WO2016 / 098079 and US8980582.

[0127] Anti-BMP6 antibodies and fragments The present invention provides various anti-BMP6 antibodies and fragments (such as Fab or scFv fragments), uses, methods and combinations (e.g., with ESAs). Examples are described in the numbered clauses below.

[0128] 1. An antibody or fragment comprising a binding site that specifically binds to bone morphogenetic protein 6 (BMP6), wherein the binding site comprises a VH domain encoded by a nucleotide sequence derived from the recombination of human VH, DH and JH gene segments, and the VH gene segment is selected from IGHV3-11 and IGHV1-3.

[0129] For example, the VH gene segment is IGHV3-11, and the D and JH gene segments are human gene segments. For example, the VH gene segment is IGHV1-3, and the D and JH gene segments are human gene segments. Optionally, the VH segment is human IGHV3-11. * 01 gene segment. Alternatively, optionally, the VH segment is a human IGHV1-3 * 01 gene segment.

[0130] In one example, specific binding can be measured using KD, K off and / or K on In one example, specific binding is with a KD of 1 pM to 5 nM.

[0131] Those skilled in the art are familiar with databases and other sources of information about antibody gene segments from humans and other species. For example, the IMGT database (www.IMGT.org), version as of September 1, 2018, is a suitable source.

[0132] See Tables 7 and 8, which show antibodies based on IGHV1-3. Surprisingly, this human VH gene segment produces anti-BMP6 antibodies with desirable anti-BMP6 properties, such as those described in the Examples.

[0133] See Table 9, which lists antibodies based on IGHV3-11. Surprisingly, this human VH gene segment produces anti-BMP6 antibodies with desirable anti-BMP6 properties, such as those described in the Examples. Thus, for example, the antibody or fragment comprises a CDRH3 sequence selected from SEQ ID NOs: 110, 113, 290, 293, 308, 311, 272, and 275. For example, the antibody or fragment comprises a CDRL3 sequence selected from SEQ ID NOs: 119, 122, 299, 302, 317, 320, 281, and 284. For example, the antibody or fragment comprises a CDRH3 sequence selected from SEQ ID NOs: 110, 113, 290, 293, 308, 311, 272, and 275, and a CDRL3 sequence selected from SEQ ID NOs: 119, 122, 299, 302, 317, 320, 281, and 284, respectively.

[0134] For example, the antibody or fragment comprises a CDRH3 sequence selected from SEQ ID NOs: 110 and 113, and a CDRL3 sequence selected from SEQ ID NOs: 119 and 122. For example, the antibody or fragment comprises an anti-BMP6 binding site, wherein the binding site comprises a VH domain comprising SEQ ID NO: 110 paired with a VL domain comprising SEQ ID NO: 119. For example, the antibody or fragment comprises an anti-BMP6 binding site, wherein the binding site comprises a VH domain comprising SEQ ID NO: 113 paired with a VL domain comprising SEQ ID NO: 122.

[0135] For example, the antibody or fragment comprises the CDRH3 sequence of an antibody selected from CL-58838, CL-58835, CL-58756, and CL-58722, and optionally the CDRL3 of such a selected antibody. For example, the antibody or fragment comprises the CDRH1 and CDRH3 sequences of an antibody selected from CL-58838, CL-58835, CL-58756, and CL-58722, and optionally the CDRL2 of such a selected antibody. For example, the antibody or fragment comprises the CDRH1 and CDRH2 sequences of an antibody selected from CL-58838, CL-58835, CL-58756, and CL-58722. For example, the antibody or fragment comprises the CDRH2 and CDRH3 sequences of an antibody selected from CL-58838, CL-58835, CL-58756, and CL-58722. For example, the antibody or fragment comprises an anti-BMP6 binding site, wherein the binding site comprises a VH domain comprising the CDRH3 sequence of CL-58838 paired with the VL domain of CL-58838.

[0136] For example, the antibody or fragment comprises an anti-BMP6 binding site, wherein the binding site comprises a VH domain comprising SEQ ID NO: 114, 294, 312, or 276, optionally paired with a VL domain comprising SEQ ID NO: 123, 303, 321, or 285, respectively. For example, the antibody or fragment comprises an anti-BMP6 binding site, wherein the binding site comprises a VH domain comprising SEQ ID NO: 114, optionally paired with a VL domain comprising SEQ ID NO: 123.

[0137] For example, the antibody or fragment comprises an anti-BMP6 binding site, wherein the binding site comprises the VH domain of an antibody selected from CL-58838, CL-58835, CL-58756, and CL-58722, optionally paired with the VL domain of such selected antibody. For example, the antibody or fragment comprises an anti-BMP6 binding site, wherein the binding site comprises the VH domain of CL-58838 paired with the VL domain of CL-58838.

[0138] 2. The antibody or fragment of clause 1, wherein the DH gene segment is a human gene segment selected from IGHD3-10, IGHD6-19, IGHD7-27, IGHD4-23, IGHD5-18, IGHD3-22 and IGHD3-16.

[0139] Optionally, the DH gene segment is IGHD3-10 * 01, IGHD6-19 * 01, IGHD7-27 * 02, IGHD4-23 * 01, IGHD5-18 * 01, IGHD3-22 * 01 and IGHD3-16 * 02 is selected.

[0140] 3. The antibody or fragment of clause 1 or 2, wherein the JH gene segment is a human gene segment selected from IGHJ3, IGHJ4 and IGHJ5.

[0141] Optionally, the JH gene segment is IGHJ3 * 02, IGHJ4 * 02 and IGHJ5 * 02 is selected.

[0142] 4. An antibody or fragment that specifically binds to bone morphogenetic protein 6 (BMP6) and comprises the CDRH3 sequence of an anti-BMP6 antibody according to any one of clauses 1 to 3.

[0143] 5. A VH domain that specifically binds bone morphogenetic protein 6 (BMP6) and comprises the CDRH3 sequence of any anti-BMP6 antibody disclosed herein (e.g., any antibody selected from the antibodies or fragments listed in any of Tables 4-11 herein), or an antibody or fragment comprising said CDRH3 comprising 3, 2, or 1 amino acid substitution(s).

[0144] 6. An antibody or fragment (optionally according to any of clauses 1-5) that specifically binds bone morphogenetic protein 6 (BMP6), and comprising a VH domain comprising the CDRH3 sequence of an antibody selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713, or said sequence comprising three, two or one amino acid substitution(s).

[0145] Optionally, the VH domain comprises a CDRH3 sequence selected from SEQ ID NO: 110 or 113, or said selected sequence comprises 3, 2 or 1 amino acid substitution(s).

[0146] 7.VH domains are (i) CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183 , CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL 7. The antibody or fragment of clause 6, comprising: (i) a CDRH3 sequence of an antibody selected from CL-58650, CL-58679, CL-58680 and CL-58713, or said CDRH3 sequence comprising three, two or one amino acid substitution(s); and (ii) a CDRH1 sequence of said selected antibody, or said CDRH1 sequence comprising three, two or one amino acid substitution(s).

[0147] Optionally, the VH domain of the antibody or fragment comprises (a) a CDRH3 sequence of SEQ ID NO: 110 or 113, or said CDRH3 sequence comprising 3, 2 or 1 amino acid substitution(s), and (b) a CDRH1 sequence of SEQ ID NO: 108 or 111, or said CDRH1 sequence comprising 3, 2 or 1 amino acid substitution(s).

[0148] 8.VH domain is (iii) CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-7518 3, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL 8. The antibody or fragment of clause 6 or 7, comprising: (i) a CDRH3 sequence of an antibody selected from CL-58650, CL-58679, CL-58680 and CL-58713, or said CDRH3 sequence comprising three, two or one amino acid substitution(s); and (ii) a CDRH2 sequence of said selected antibody, or said CDRH2 sequence comprising three, two or one amino acid substitution(s).

[0149] Optionally, the VH domain of the antibody or fragment comprises (c) a CDRH3 sequence of SEQ ID NO: 110 or 113, or said CDRH3 sequence comprising 3, 2 or 1 amino acid substitution(s), and (d) a CDRH2 sequence of SEQ ID NO: 109 or 112, or said CDRH2 sequence comprising 3, 2 or 1 amino acid substitution(s).

[0150] Optionally, the VH domain of the antibody or fragment comprises: (e) a CDRH3 sequence of SEQ ID NO: 110 or 113, or said CDRH3 sequence comprising three, two or one amino acid substitution(s); (f) a CDRH1 sequence of SEQ ID NO: 108 or 111, or said CDRH1 sequence comprising three, two or one amino acid substitution(s); and (g) a CDRH2 sequence of SEQ ID NO: 109 or 112, or said CDRH2 sequence comprising three, two or one amino acid substitution(s).

[0151] 9. An antibody or fragment comprising a binding site that specifically binds to bone morphogenetic protein 6 (BMP6), wherein the binding site is selected from the group consisting of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, C An antibody or fragment (optionally according to any of clauses 1 to 8) comprising a VH domain comprising the amino acid sequence of the VH domain of an antibody selected from CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713, or an antibody or fragment which is at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical thereto.

[0152] For example, the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0153] Optionally, the VH domain of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 114, or a heavy chain variable domain amino acid sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 114. For example, the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0154] 10. The antibody or fragment of any of clauses 1-9, comprising a first and a second copy of said VH domain.

[0155] In one example, the antibody or fragment comprises a binding site comprising a VH domain of the invention paired with a VL domain of the invention, wherein the binding site is capable of specifically binding to BMP6 (e.g., mature BMP6, e.g., human and / or cynomolgus BMP6). For example, the antibody or fragment comprises two of such binding sites.

[0156] 11. An antibody or fragment (optionally according to any of clauses 1 to 10) comprising a binding site that specifically binds bone morphogenetic protein 6 (BMP6), wherein the binding site comprises a VL domain encoded by a nucleotide sequence derived from the recombination of human VL and JL gene segments, and the VL gene segment is selected from IGKV3-20, IGKV1-5 and IGKV3-15.

[0157] For example, the VL gene segment can be IGKV3-20, e.g., IGKV3-20 * 01. For example, the VL gene segment is IGKV1-5, e.g., IGKV1-5 * For example, the VL gene segment is IGKV3-15, e.g., IGKV3-15 * The number is 01.

[0158] 12. The antibody or fragment of clause 11, wherein the VL is a Vκ and the JL gene segment is a human gene segment selected from IGKJ1 and IGKJ3.

[0159] Optionally, the JL gene segment is IGKJ1 * 01 and IGKJ3 * Selected from 01.

[0160] 13. An antibody or fragment that specifically binds to bone morphogenetic protein 6 (BMP6) and comprises the CDRL3 sequence of the anti-BMP6 antibody of clause 11 or 12.

[0161] 14. An antibody or fragment (optionally according to any of clauses 1-13) that specifically binds to bone morphogenetic protein 6 (BMP6) and comprises a VL domain comprising the CDRL3 sequence of any anti-BMP6 antibody disclosed herein (e.g., any antibody selected from the antibodies or fragments listed in any of Tables 4-11 herein), or said selected CDRL3 comprising 3, 2 or 1 amino acid substitution(s).

[0162] 15. The antibody or fragment of clause 14, comprising a VH domain comprising the CDRH3 sequence of said selected antibody.

[0163] 16. An antibody or fragment (optionally according to any of clauses 1-15) that specifically binds to bone morphogenetic protein 6 (BMP6), and comprising a VL domain comprising a CDRL3 sequence selected from SEQ ID NO: 119 or 122, or said selected CDRL3 sequence comprising 3, 2 or 1 amino acid substitution(s).

[0164] 17. An antibody or fragment (optionally according to any of clauses 1-16) that specifically binds bone morphogenetic protein 6 (BMP6), and comprising a VL domain comprising the CDRL3 (and optionally the CDRH3) sequence of an antibody selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713, or each such sequence comprising 3, 2 or 1 amino acid substitution(s).

[0165] Optionally, the VL domain comprises a CDRL3 sequence selected from SEQ ID NO: 119 or 122, or said selected sequence comprising 3, 2 or 1 amino acid substitution(s), and / or optionally, the VH domain comprises a CDRH3 sequence selected from SEQ ID NO: 119 or 122, or said selected sequence comprising 3, 2 or 1 amino acid substitution(s).

[0166] 18.VL domains are (i) CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500 , CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL- 58680 and CL-58713, or each said CDR3 sequence(s) comprising 3, 2 or 1 amino acid substitution(s); and (ii) the CDRL1 (and optionally CDRH1) sequence of said selected antibody, or each said CDR1 sequence(s) comprising 3, 2 or 1 amino acid substitution(s).

[0167] Optionally, the VL domain of the antibody or fragment comprises (a) a CDRL3 sequence of SEQ ID NO: 119 or 122, or said CDRL3 sequence comprising 3, 2 or 1 amino acid substitution(s), and (b) a CDRL1 sequence of SEQ ID NO: 117 or 120, or said CDRL1 sequence comprising 3, 2 or 1 amino acid substitution(s).

[0168] 19.VL domains are (iii) CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500 , CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58 680 and CL-58713, or each said CDR3 sequence(s) comprising 3, 2 or 1 amino acid substitution(s); and (iv) the CDRL2 (and optionally CDRH2) sequence of said selected antibody, or each said CDR2 sequence(s) comprising 3, 2 or 1 amino acid substitution(s).

[0169] Optionally, the VL domain of the antibody or fragment comprises (c) a CDRL3 sequence of SEQ ID NO: 119 or 122, or said CDRL3 sequence comprising 3, 2 or 1 amino acid substitution(s), and (d) a CDRL2 sequence of SEQ ID NO: 118 or 121, or said CDRL2 sequence comprising 3, 2 or 1 amino acid substitution(s).

[0170] Optionally, the VL domain of the antibody or fragment comprises (e) a CDRL3 sequence of SEQ ID NO: 119 or 122, or said CDRL3 sequence containing 3, 2 or 1 amino acid substitution(s), (f) a CDRL1 sequence of SEQ ID NO: 117 or 120, or said CDRL1 sequence containing 3, 2 or 1 amino acid substitution(s), and (g) a CDRL2 sequence of SEQ ID NO: 118 or 121, or said CDRL2 sequence containing 3, 2 or 1 amino acid substitution(s).

[0171] 20. An antibody or fragment comprising a binding site that specifically binds to bone morphogenetic protein 6 (BMP6), wherein the binding site is selected from the group consisting of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, C 20. An antibody or fragment (optionally according to any of clauses 1 to 19) comprising a VL domain comprising the amino acid sequence of the VL domain of an antibody selected from CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713, or an antibody or fragment which is at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical thereto.

[0172] For example, the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0173] Optionally, the VL domain of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 123, or a heavy chain variable domain amino acid sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 123. For example, the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0174] 21. The antibody or fragment of any of clauses 1 to 20, comprising a first and a second copy of said VL domain.

[0175] In one example, the antibody or fragment comprises a binding site comprising a VL domain of the invention paired with a VH domain, wherein the binding site is capable of specifically binding to BMP6 (e.g., mature BMP6, e.g., human and / or cynomolgus monkey BMP6). For example, the antibody or fragment comprises two of such binding sites.

[0176] 22. An antibody or fragment (optionally according to any of clauses 1-21) that specifically binds bone morphogenetic protein 6 (BMP6) and comprises the heavy chain amino acid sequence of an antibody selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713, or amino acids at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical thereto.

[0177] For example, the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0178] 23. An antibody or fragment (optionally according to any of clauses 1-22) that specifically binds bone morphogenetic protein 6 (BMP6) and comprises the light chain amino acid sequence of an antibody selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713, or amino acids at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical thereto.

[0179] For example, the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0180] 24. The antibody or fragment of clause 23, comprising the light chain amino acid sequence of said selected antibody, or amino acids at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical thereto.

[0181] For example, the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0182] 25. An antibody or fragment (optionally according to any of clauses 1 to 24) which specifically binds to a human BMP6 epitope which is identical to the epitope bound by an antibody according to any of clauses 1 to 24.

[0183] 26. The antibody or fragment of clause 25, wherein the epitope is identified by an independent amino acid scan or by X-ray crystallography.

[0184] Contact amino acid residues involved in the interaction between an antibody and an antigen can be determined by various methods known to those skilled in the art.

[0185] In one embodiment, serial substitution of amino acids in the antigen sequence (using standard molecular biology techniques to mutate the DNA of the antigen's coding sequence), in this case, substitution of BMP6 with alanine (also known as alanine scanning) or another unrelated amino acid, can provide residues that reduce or eliminate the ability of the antibody to recognize the antigen of interest. Binding can be assessed using standard techniques, including, but not limited to, SPR, HTRF, and ELISA (described elsewhere herein). Other substitutions can be made to enhance disruption of binding, such as altering the side chain charge of antigen sequence amino acids (e.g., changing lysine to glutamic acid) or exchanging polar and nonpolar residues (e.g., changing serine to leucine). Alanine scanning or other amino acid substitution methods can be performed either with recombinant soluble antigen or, if the target is a cell membrane target, directly on cells using transient or stable expression of the mutated version.

[0186] In one embodiment, protein crystallography can be used to determine contact residues between an antibody and an antigen (i.e., to determine the epitope to which the antibody binds), and crystallography allows direct visualization of contact residues involved in antibody-antigen interactions. In addition to standard X-ray crystallography, cryo-electron microscopy has been used to determine contact residues between antibodies and HIV capsid proteins (see Lee, Jeong Hyun, et al., "Antibodies to a conformational epitope on gp41 neutralize HIV-1 by destabilizing the Env spike," Nature Communications, 6, (2015)).

[0187] In one embodiment, if an antibody recognizes a linear epitope, short peptides can be generated based on the antigen sequence, and antibody binding to these peptides can be assessed using standard techniques, including, but not limited to, SPR, HTRF, and ELISA (described elsewhere herein). Further investigation of the epitope can be provided by performing an alanine scan on any peptides that show binding. Instead of linear peptides, structural scans can be performed using Pepscan technology (http: / / www.pepscan.com / ), which uses chemical attachment of peptides to a backbone, and has been used to determine non-continuous epitopes on CD20-targeting antibodies (Niederfellner, Gerhard, et al., "Epitope characterization and crystal structure of GA101 provide insights into the molecular basis for type I / II distinction of CD20 antibodies." Blood, 118.2, (2011), 358-367).

[0188] In one embodiment, limited protein digestion and mass spectrophotometric analysis can be used to identify binding epitopes. The antibody-antigen complex is digested with a protease, such as, but not limited to, trypsin. The digested complex peptides are compared with digestion mass spectrophotometric analysis of the antibody alone and the antigen alone to determine whether a specific epitope is protected by complex formation. Competitive binding, a further step involving amino acid substitution, can be used to narrow down the individual amino acid residues involved in the interaction (see, for example, Suckau, Detlev, et al., "Molecular epitope identification by limited proteolysis of an immobilized antigen-antibody complex and mass spectrometric peptide mapping," Proceedings of the National Academy of Sciences, 87, 24, (1990), 9848-9852).

[0189] Thus, in one embodiment, contact residues of the epitope are identified using an unrelated amino acid scan (e.g., an alanine scan). In another embodiment, the unrelated amino acid scan (e.g., an alanine scan) is performed using a technique selected from SPR, HTRF, ELISA, X-ray crystallography, cryo-electron microscopy, and limited protein digestion in combination with mass spectrometry. In one embodiment, the unrelated amino acid scan (e.g., an alanine scan) is performed using HTRF. In one embodiment, the unrelated amino acid scan (e.g., an alanine scan) is performed using ELISA. When an alanine scan is performed using either ELISA or HTRF, an amino acid residue is identified as contributing to the epitope if there is at least a 25% reduction in signal. In one embodiment, the signal reduction is at least 30%. In one embodiment, the signal reduction is at least 35%. In one embodiment, the signal reduction is at least 40%. In one embodiment, the signal reduction is at least 45%. In one embodiment, the signal reduction is at least 50%. In one embodiment, the signal reduction is at least 55%. In one embodiment, the signal reduction is at least 60%. In one embodiment, the signal reduction is at least 70%. In one embodiment, the signal reduction is at least 75%. In one embodiment, the signal reduction is at least 80%. In one embodiment, the signal reduction is at least 85%. In one embodiment, the signal reduction is at least 90%. When an alanine scan is performed using SPR, an amino acid residue is identified as contributing to the epitope if there is at least a 10-fold reduction in affinity. In one embodiment, the affinity reduction is at least 15-fold. In one embodiment, the affinity reduction is at least 20-fold. In one embodiment, the affinity reduction is at least 30-fold. In one embodiment, the affinity reduction is at least 40-fold. In one embodiment, the affinity reduction is at least 50-fold. In one embodiment, the affinity reduction is at least 100-fold.

[0190] In one embodiment, contact residues of the epitope are identified by X-ray crystallography. In one embodiment, contact residues of the epitope are identified by cryo-electron microscopy. In one embodiment, contact residues of the epitope are identified by a combination of limited protein digestion and mass spectrometry.

[0191] 27. The antibody or fragment of clause 26, wherein the contact residues of the epitope are defined by at least a 10-fold reduction in affinity in an unrelated amino acid scan, e.g., an alanine scan, as determined by SPR.

[0192] In one embodiment, the reduction in affinity is at least 15-fold. In one embodiment, the reduction in affinity is at least 20-fold. In one embodiment, the reduction in affinity is at least 30-fold. In one embodiment, the reduction in affinity is at least 40-fold. In one embodiment, the reduction in affinity is at least 50-fold. In one embodiment, the reduction in affinity is at least 100-fold. SPR can be performed as described herein.

[0193] 28. An antibody or fragment (optionally according to any of clauses 1 to 27) that competes with an antibody of any of clauses 1 to 27 for binding to human BMP6.

[0194] Optionally, competition is determined by surface plasmon resonance (SPR) or ELISA. Those skilled in the art will be familiar with, for example, these techniques and standard conditions.

[0195] In one embodiment, the antibody or fragment competes with hBMP6 (or a fusion protein thereof) for binding to cell surface-expressed hBMP6 (e.g., in a dose-dependent manner). In one embodiment, the antibody or fragment competes with hBMP6 (or a fusion protein thereof) for binding to soluble hBMP6 (e.g., in a dose-dependent manner).

[0196] Optionally, the competition for binding to hBMP6 is carried out using SPR, which can be performed as described herein.

[0197] 29. The antibody or fragment of any of clauses 1-28, which specifically binds to human BMP6 comprising SEQ ID NO: 562, and / or cynomolgus monkey BMP6 comprising SEQ ID NO: 564, and / or rat BMP6 comprising SEQ ID NO: 563.

[0198] Optionally, an antibody or fragment of the invention specifically binds to the amino acid sequence of SEQ ID NO: 562. Optionally, an antibody or fragment of the invention specifically binds to the amino acid sequence of SEQ ID NO: 563. Optionally, an antibody or fragment of the invention specifically binds to the amino acid sequence of SEQ ID NO: 564.

[0199] In one example, the BMP6 herein is human, mouse, or cynomolgus monkey BMP6.

[0200] In one embodiment, the antibody or fragment binds to cynomolgus BMP6 with an affinity of less than 1 nM (e.g., 1 nM to 0.01 pM, or 1 nM to 0.1 pM, or 1 nM to 1 pM). In one embodiment, the antibody or fragment binds to cynomolgus BMP6 with an affinity of less than 10 nM (e.g., 10 nM to 0.01 pM, or 10 nM to 0.1 pM, or 10 nM to 1 pM). In one embodiment, the antibody or fragment binds to cynomolgus BMP6 with an affinity of less than 0.1 nM (e.g., 0.1 nM to 0.01 pM, or 0.1 nM to 0.1 pM, or 0.1 nM to 1 pM). In one embodiment, the antibody or fragment binds to cynomolgus BMP6 with an affinity of less than 0.01 nM (eg, 0.011 nM to 0.01 pM or 0.01 nM to 0.1 pM).

[0201] In one embodiment, the antibody or fragment binds to cynomolgus BMP6 with an affinity within 2-fold of its affinity to hBMP6. In one embodiment, the antibody or fragment binds to cynomolgus BMP6 with an affinity within 4-fold of its affinity to hBMP6. In one embodiment, the antibody or fragment binds to cynomolgus BMP6 with an affinity within 5-fold of its affinity to hBMP6. In one embodiment, the antibody or fragment binds to cynomolgus BMP6 with an affinity within 6-fold of its affinity to hBMP6. In one embodiment, the antibody or fragment binds to cynomolgus BMP6 with an affinity within 8-fold of its affinity to hBMP6. In one embodiment, the antibody or fragment binds to cynomolgus BMP6 with an affinity within 10-fold of its affinity to hBMP6. "hBMP6" herein refers to human BMP6, e.g., human BMP6 disclosed herein, e.g., including SEQ ID NO:562.

[0202] In one embodiment, the antibody or fragment does not detectably bind to cynomolgus monkey BMP6. In one embodiment, the antibody or fragment does not detectably bind to murine (e.g., mouse and / or rat) BMP6.

[0203] In one embodiment, the antibody or fragment binds to mouse (e.g., mouse and / or rat) BMP6 with an affinity of less than 1 nM (e.g., 1 nM to 0.01 pM, or 1 nM to 0.1 pM, or 1 nM to 1 pM). In one embodiment, the antibody or fragment binds to mouse BMP6 with an affinity of less than 10 nM (e.g., 10 nM to 0.01 pM, or 10 nM to 0.1 pM, or 10 nM to 1 pM). In one embodiment, the antibody or fragment binds to mouse BMP6 with an affinity of less than 0.1 nM (e.g., 0.1 nM to 0.01 pM, or 0.1 nM to 0.1 pM, or 0.1 nM to 1 pM). In one embodiment, the antibody or fragment binds to mouse BMP6 with an affinity of less than 0.01 nM (e.g., 0.011 nM to 0.01 pM or 0.01 nM to 0.1 pM).

[0204] Optionally, the antibody or fragment comprises an effector-enabled or effector-neutral constant region, such as a human constant region, e.g., an effector-null human constant region, e.g., an IgG4 constant region or an IgG1 constant region, and optionally the constant region is IgG4-PE or disabled IgG1. Optionally, the antibody or fragment comprises a murine (e.g., mouse and / or rat) constant region. Optionally, the antibody or fragment comprises any of the heavy chain constant region sequences described herein.

[0205] Optionally, the constant region has CDC and / or ADCC activity.

[0206] 30. The antibody or fragment of any of clauses 1-29, wherein the antibody or fragment comprises a human constant region, e.g., an IgG4 constant region or an IgG1 constant region.

[0207] For example, the constant region comprises a heavy chain constant region, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 429, 431, 433, 435, 437, 439, 440, 442, 444, 446, 448, 450, 454, or 456. Optionally, the heavy chain C region is an IGHG1 C region comprising the amino acid sequence of SEQ ID NO: 429, 431, 433, 435, or 437. Optionally, the heavy chain C region is an IGHG2 C region comprising the amino acid sequence of SEQ ID NO: 439, 440, 442, or 444. Optionally, the heavy chain C region is an IGHG4 C region comprising the amino acid sequence of SEQ ID NO: 446, 448, 450, 454, or 456, preferably SEQ ID NO: 454, preferably SEQ ID NO: 456. In one example, the heavy chain C region is encoded by a nucleic acid comprising SEQ ID NO: 451, 452, or 453.

[0208] In one example (optionally in addition to a heavy chain region as in the immediately preceding paragraph), the constant region comprises a light chain constant region, which comprises the amino acid sequence of SEQ ID NO: 458, 460, 462, 464, 466, 468, 470, 473, 476, 478, 480, 482, 484, 486, 488 or 490. Optionally, the light chain C region is an IGKC C region comprising the amino acid sequence of SEQ ID NO: 458, 460, 462, 464 or 466, preferably SEQ ID NO: 458. Optionally, the light chain C region is an IGLC C region comprising the amino acid sequence of SEQ ID NO: 468, 470, 473, 476, 478, 480, 482, 484, 486, 488 or 490.

[0209] 31. The antibody or fragment of clause 30, wherein the constant region is an IgG4-PE constant region.

[0210] Optionally, the antibody or fragment comprises a heavy chain constant region, wherein the constant region comprises the amino acid sequence of SEQ ID NO:454.

[0211] The anti-BMP6 antibody or fragment according to the present invention comprises a constant region, such as a human constant region, for example an effector-null human constant region, for example an IgG4 constant region or an IgG1 constant region, optionally wherein the constant region is IgG4-PE or a deleted IgG1 as defined in the sequence listing of the present invention.

[0212] In other embodiments, the antibody or fragment has any of the isotypes or constant regions described herein. In one embodiment, the constant region is wild-type human IgG1. For example, the constant region is an effector-enabled IgG1 constant region, optionally having ADCC and / or CDC activity. In one embodiment, the constant region has been engineered to enhance ADCC and / or CDC and / or ADCP. In another embodiment, the constant region has been engineered to enhance effector function.

[0213] The IgG4 constant region may be any of the IgG4 constant region amino acid sequences or may be encoded by any of the nucleic acid sequences in the sequence listing herein. The heavy chain constant region may be an IgG4 containing both the Leu235Glu mutation and the Ser228Pro mutation. This "IgG4-PE" heavy chain constant region (see the sequence listing for an example) is effector null.

[0214] An alternative effector-null human constant region is an IgG1 mAb containing the L235A and / or G237A mutations (e.g., LAGA, see Sequence Listing). * In one embodiment, the antibody or antibody fragment disclosed herein comprises an IgG1 heavy chain constant region, the sequence of which contains an alanine at positions 235 and / or 237 (EU index numbering).

[0215] The potency of Fc-mediated effects can be enhanced by engineering the Fc domain by any of the techniques that will be apparent to those skilled in the art. In another embodiment, the antibodies and fragments disclosed herein can contain a triple mutation (M252Y / S254T / T256E) that enhances binding to FcRn.

[0216] 32. The antibody or fragment (e.g., a bispecific antibody) of any of clauses 1-31, further comprising antigen-binding sites that specifically bind to another target antigen (optionally human hemojuvelin, a transferrin receptor (e.g., TFR2) or a BMP receptor (e.g., BMPRI or BMPRII), or that bind to BMP6 and another BMP (e.g., BMP2, 4, 7 or 9).

[0217] For example, a bispecific antibody specifically binds to BMP6 and BMP2. For example, a bispecific antibody specifically binds to BMP6 and HJV. For example, a bispecific antibody specifically binds to BMP6 and BMPRI (i.e., BMPR1, e.g., BMPR1A or BMPR1B). For example, a bispecific antibody specifically binds to BMP6 and BMPRII. For example, a bispecific antibody specifically binds to BMP6 and 2. For example, a bispecific antibody specifically binds to BMP6 and 4. For example, a bispecific antibody specifically binds to BMP6 and 9. For example, a bispecific antibody specifically binds to BMP6 and 7. For example, a bispecific antibody specifically binds to BMP6 and TFR2.

[0218] In one example, the further binding site is an agonist binding site of said further antigen. In one example, the further binding site is an antagonist binding site of said further antigen.

[0219] In one example, the further binding site is an antibody binding site comprising a VH and a VL, a constant domain of an antibody (Fcab binding site), or a non-immunoglobulin binding site (e.g., a fibronectin domain). Optionally, the antigen binding site is any antigen binding site disclosed herein.

[0220] For example, the antibody or fragment is a bispecific antibody or fragment, for example, the antibody or fragment is a bispecific antibody or fragment or a fusion protein comprising an antibody or fragment thereof according to clauses 1 to 31. Bispecific antibody has the meaning given above.

[0221] In one example, the antibody, fragment or fusion protein of clause 24 or 24a is a DVD-Ig, mAb 2 , FIT-Ig, mAb-dAb, Dock-Lock, SEEDbody, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, minibody, knob-in-hole, knob-in-hole with common light chain, knob-in-hole with common light chain and charge pair, charge pair, charge pair with common light chain, especially mAb 2 mAb, e.g., knobs-in-hole, common light chain, and charge pair and FIT-Ig with knobs-in-hole, common light chain, and common light chain 2 and bispecific formats selected from knobs-in-holes with FIT-Ig.

[0222] In one embodiment, the bispecific format is DVD-Ig, mAb 2, FIT-Ig, mAb-dAb, Dock-Lock, Fab-Arm Exchange, SEEDbody, Triomab, LUZ-Y, Fcab, κλ-body, Orthogonal Fab, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, Intrabody, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, Triple Body, Miniantibody, Minibody, TriBi Minibody, scFv-CH3KIH, scFv- The antibody is selected from CH-CL-scFv, F(ab')2-scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCab, ImmTAC, knobs-in-hole, knobs-in-hole with a common light chain, knobs-in-hole with a common light chain and charge pair, charge pair, charge pair with a common light chain, DT-IgG, DutaMab, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, and zybody.

[0223] In one embodiment, the bispecific format is DVD-Ig, FIT-Ig, mAb-dAb, Dock-Lock, Fab-arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, κλ-body, Orthogonal Fab, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, triplebody, miniantibody, minibody, TriBi minibody, scFv -CH3KIH, scFv-CH-CL-scFv, F(ab')2-scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCab, ImmTAC, knob-in-hole, knob-in-hole with common light chain, knob-in-hole with common light chain and charge pair, charge pair, charge pair with common light chain, DT-IgG, DutaMab, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, and zybodies, such as DVD-Ig, FIT-Ig, mAb-dAb, Dock-Lock, SEEDbody, scDiabody-Fc, diabody-Fc, tandem-scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, minibody, knobs-in-hole, knobs-in-hole with a common light chain, knobs-in-hole with a common light chain and charge pair, charge pair, charge pair with a common light chain, especially knobs-in-hole, knobs-in-hole with a common light chain, knobs-in-hole with a common light chain and charge pair, and FIT-Ig, such as FIT-Ig.

[0224] In one embodiment, the bispecific format is DVD-Ig, mAb 2, mAb-dAb, Dock-Lock, Fab-Arm Exchange, SEEDbody, Triomab, LUZ-Y, Fcab, κλ-body, Orthogonal Fab, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, Intrabody, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, Triple Body, Miniantibody, Minibody, TriBi Minibody, scFv-CH3KIH, scFv-CH-C L-scFv, F(ab')2-scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCab, ImmTAC, knob-in-hole, knob-in-hole with a common light chain, knob-in-hole with a common light chain and charge pair, charge pair, charge pair with a common light chain, DT-IgG, DutaMab, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, and zybodies, e.g., DVD-Ig, mAbs 2 , mAb-dAb, Dock-Lock, SEEDbody, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, minibody, knob-in-hole, knob-in-hole with common light chain, knob-in-hole with common light chain and charge pair, charge pair, charge pair with common light chain, mAb, among others 2 , knobs-in-hole, knobs-in-hole with a common light chain, knobs-in-hole with a common light chain and charge pair, and knobs-in-hole with a common light chain, e.g., mAb 2 is selected from.

[0225] In one embodiment, the bispecific format is DVD-Ig, mAb-dAb, Dock-Lock, Fab-arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, κλ-body, Orthogonal Fab, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, triplebody, miniantibody, minibody, TriBi minibody, scFv-CH 3KIH, scFv-CH-CL-scFv, F(ab')2-scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCab, ImmTAC, knob-in-hole, knob-in-hole with common light chain, knob-in-hole with common light chain and charge pair, charge pair, charge pair with common light chain, DT-IgG, DutaMab, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, and zybodies such as DVD-Ig, mAb-dAb, Dock-Lock, SEEDbody, scDiabody-Fc, diabody-Fc, tandem-scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, minibody, knobs-in-hole, knobs-in-hole with a common light chain, knobs-in-hole with a common light chain and charge pair, charge pair, charge pair with a common light chain, especially selected from knobs-in-hole, knobs-in-hole with a common light chain, knobs-in-hole with a common light chain and charge pair, and knobs-in-hole with a common light chain.

[0226] 33. The anti-BMP6 antibody or fragment according to any of clauses 1 to 32 for treating or preventing a BMP6-mediated disease or condition (e.g., anemia) in a subject.

[0227] In one example, the subject is a human. Alternatively, the subject is a non-human animal. In one example, the subject is an adult. In one example, the subject is an infant. In one example, the subject is a human CKD patient undergoing dialysis treatment. In one example, the subject is a human with end-stage renal disease.

[0228] In one example, the antibody or fragment of the invention is for treating or preventing a disease or condition in a subject (e.g., a human) selected from anemia, pulmonary arterial hypertension (PAH) (e.g., primary PAH or secondary PAH), cerebral cavernous hemangioma (CCM) (e.g., familial CCM or sporadic CCM), restless legs syndrome (RLS), cancer (e.g., breast cancer, pancreatic cancer, colorectal cancer, salivary gland cancer, esophageal cancer, or melanoma), cancer metastasis, systemic sclerosis, Sjogren's syndrome, endothelial-mesenchymal transition (EndoMT), cardiovascular disease, atherosclerosis, systemic sclerosis-associated pulmonary fibrosis, and cardiac fibrosis.

[0229] Examples of diseases or conditions mediated by EndoMT are cardiovascular disease, atherosclerosis, systemic sclerosis-associated pulmonary fibrosis, cardiac fibrosis, PAH, tumorigenesis, tumor invasion, tumor metastasis, fibrotic diseases, and cancer-associated fibroblast generation (e.g., pancreatic cancer).

[0230] In one example, the disease or condition is in a human. In one example, the disease or condition is in an animal.

[0231] In one example, the antibody or fragment of the present invention is for treating or preventing a TIGIT-mediated disease or condition in a human, e.g., selected from neoplastic or non-neoplastic diseases, chronic viral infections, and malignant tumors such as melanoma, Merkel cell carcinoma, non-small cell lung cancer (squamous and non-squamous), renal cell carcinoma, bladder cancer, head and neck squamous cell carcinoma, mesothelioma, virus-induced cancers (e.g., cervical cancer and nasopharyngeal carcinoma), soft tissue sarcoma, hematological tumors such as Hodgkin's and non-Hodgkin's disease, and diffuse large B-cell lymphoma (e.g., melanoma, Merkel cell carcinoma, non-small cell lung cancer (squamous and non-squamous), renal cell carcinoma, bladder cancer, head and neck squamous cell carcinoma, and mesothelioma, or e.g., virus-induced cancers (e.g., cervical cancer and nasopharyngeal carcinoma) and soft tissue sarcoma).

[0232] In one example, the BMP6-mediated disease or condition is a neurodegenerative disease, disorder, or condition selected from, for example, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, primary progressive multiple sclerosis, secondary progressive multiple sclerosis, corticobasal degeneration, Rett's syndrome, a retinal degenerative disorder selected from age-related macular degeneration and retinitis pigmentosa, anterior ischemic optic neuropathy, glaucoma, uveitis, depression, trauma-related stress or post-traumatic stress disorder, frontotemporal dementia, dementia with Lewy bodies, mild cognitive impairment, posterior cortical atrophy, primary progressive aphasia, and progressive supranuclear palsy or age-related dementia; in particular, the neurodegenerative disease, disorder, or condition is selected from Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, and Huntington's disease, e.g., Alzheimer's disease.

[0233] In one example, the antibodies, fragments, and combinations of the invention are administered or are intended for intravenous administration to a subject. In one example, the antibodies, fragments, and combinations of the invention are administered or are intended for subcutaneous administration to a subject.

[0234] 34. The antibody or fragment of clause 33, wherein the antibody or fragment is administered to the subject simultaneously or sequentially with an erythropoietin-stimulating agent (ESA).

[0235] 35. A combination of an amount of an anti-BMP6 antibody or fragment with an amount of an ESA (e.g., comprising multiple doses of the antibody and / or ESA), wherein the antibody or fragment is as described in any one of clauses 1 to 34.

[0236] Also provided is a medical kit comprising the combination, a first sterile container containing the amount of the antibody or fragment, and a second sterile container containing the amount of the ESA, optionally with instructions for use of the combination to treat anemia in a subject.

[0237] In one example, the combination is for treating or preventing anemia in a subject, and for four consecutive weeks, a total dose of antibody and a total dose of ESA are administered to the subject in a ratio of X:Y, where X is between 10 and 2x10. 6 and Y=4, e.g., X is 10 to 2x10 6 micrograms and Y=4 micrograms.

[0238] In one example, treatment increases (or is intended to increase) one, more, or all of Hb concentration, mean corpuscular hemoglobin (MCH) and transferrin saturation in the subject.Those skilled in the art will be familiar with these parameters and the method of determining them, for example, using one or more serum samples from the subject.For example, transferrin saturation measured as percentage is the value of serum iron divided by total iron binding capacity.

[0239] In one example, at the start of treatment, the subject suffers from anemia of chronic disease (ACD), and optionally the anemia is associated with chronic inflammation (e.g., the subject suffers from arthritis) or bacterial infection (e.g., a streptococcal infection), or the subject is a chronic kidney disease (CKD) patient.

[0240] 36. (a) a method for preventing a decrease in a subject's blood hemoglobin level to below 10 g / dL, the method comprising administering to the subject an antibody or fragment and an erythropoiesis-stimulating agent (ESA); (b) a method for increasing blood hemoglobin to a level of at least 10 g / dL in a subject suffering from anemia, comprising administering to the subject an antibody or fragment and an erythropoiesis-stimulating agent (ESA), wherein the anemia is treated; (c) a method for treating or preventing anemia in a subject suffering from an inflammatory disease or condition, comprising administering to the subject an antibody or fragment and an erythropoiesis-stimulating agent (ESA), wherein the anemia is treated or prevented; (d) a method for eliminating or reducing the need for iron administration or blood transfusions in a subject suffering from anemia, comprising administering to the subject an antibody or fragment and an erythropoiesis-stimulating agent (ESA), wherein the need is eliminated or reduced; (e) a method for treating or preventing anemia in a subject suffering from a microbial infection, the method comprising administering to the subject an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA); (f) a method of reducing the administration of an erythropoiesis-stimulating agent (ESA) for treating anemia to a subject suffering from anemia, the method comprising administering an antibody or fragment and an ESA, wherein anemia is treated in the subject; or (g) The antibody, fragment or combination of any one of clauses 1-34 for use in a method of treating or reducing the risk of anemia in a subject suffering from or at risk of anemia, the method comprising administering to the subject the antibody or fragment and a low dose of an erythropoiesis-stimulating agent (ESA), wherein the anemia is treated or the risk of anemia is reduced in the subject.

[0241] 37. The ESA epoetin alfa administered at a weekly dose of less than 1000, 1500, 2500, 5000, 11000, 18000, 34000, or 90000 units, and optionally the subject has previously received epoetin alfa therapy at <1500, 1500-2499, 2500-4999, 5000-10999, 11000-17999, 18000-33999, 34000-89999, or ≥ 90000 units per week, respectively; b. Darbepoetin alfa or Aranesp®, administered at a weekly dose of less than 6.25, 10, 12.5, 20, 25, 40, 60, 100, or 200 mcg, and optionally, the subject has previously received weekly darbepoetin alfa or Aranesp® treatment at 6.25, 10, 12.5, 20, 25, 40, 60, 100, or 200 mcg, respectively; or c. The antibody, fragment or combination of any one of clauses 1-34 and 36, wherein the antibody, fragment or combination is darbepoetin alfa or Aranesp® and is administered at a weekly dose of less than 6.25, 10, 20, 40, 60, 100 or 200 mcg, and optionally the subject has previously received 1500-2499, 2500-4999, 5000-10999, 11000-17999, 18000-33999, 34000-89999 or ≧90000 Units of epoetin alfa therapy per week, respectively.

[0242] 38. The antibody, fragment or combination of any one of clauses 1-34, 36 and 37, for maintaining or increasing blood hemoglobin levels to at least 10 g / dL in a subject at least 13 or 14 days after the subject receives the anti-BMP6 antibody or fragment and an ESA.

[0243] 39. Use of an antibody, fragment or combination according to any of clauses 1 to 38 in the manufacture of a medicament for administration to a subject for treating or preventing a BMP6-mediated disease or condition, such as anemia.

[0244] 40. A method for treating or preventing a BMP6-mediated disease or condition (e.g., anemia) in a subject, comprising administering to said subject a therapeutically effective amount of an antibody, fragment or combination described in any one of clauses 1 to 38, wherein the BMP6-mediated disease or condition is thereby treated or prevented.

[0245] The disease or condition can be any of those disclosed herein.

[0246] 41. The use according to clause 39 or the method according to clause 40, wherein the BMP6-mediated disease or condition is anemia.

[0247] 42. The method further comprises administering to the subject an additional therapy, e.g., an additional therapeutic agent, optionally wherein the additional therapeutic agent is: (a) Intravenous iron, (b) ESAs (e.g., EPOs), (c) ActRIIa inhibitors, (d) ActRIIb inhibitors, (e) IL-6 or IL-6 receptor inhibitors (e.g., anti-IL-6 or IL-6 receptor antibodies); (f) TNF alpha or TNF alpha receptor inhibitors (e.g., anti-TNF alpha or TNF alpha receptor antibodies), (g) HJV inhibitors (e.g., anti-HJV antibodies), (h) a BMP inhibitor (e.g., an additional anti-BMP antibody or fragment) (e.g., the BMP is BMP2, 4, 5, 6, 7, or 9); (i) matriptase-2 (MTP2) agonists (e.g., matriptase-2 (MTP2) agonist antibodies), (j) HIF-PH inhibitors, (k) transferrin receptor 2 (TFR2) inhibitors, (l) HFE inhibitors, (m) NRf2 inhibitors, (n) transforming growth factor beta superfamily type 1 activin receptor-like kinase (ALK) receptor inhibitors; (o) activin receptor inhibitors (e.g., activin receptor Fc fusions); (p) a GDF11 inhibitor, and (q) a myostatin inhibitor.

[0248] Optionally, the additional agent is Luspatercept™ or Sotatercept™. Optionally, the additional agent is a TGF-β superfamily inhibitor. In one example, the additional agent is a transforming growth factor beta superfamily type 1 activin receptor-like kinase (ALK) receptor inhibitor, an ALK2 inhibitor, an ALK3 inhibitor, an ALK4 inhibitor, an ALK5 inhibitor, or an ALK7 inhibitor.

[0249] In one example, the additional agent is an IL-6 or IL-6R inhibitor, such as sarilumab, bovalilizumab, or tocilizumab (e.g., Kevzara® or Actemra®).

[0250] In one example, the additional agent is a TNF-alpha or TNF-alpha receptor inhibitor, for example, adalimumab, Humira®, Remicade® or Enbrel®, Simponi®. In one embodiment, the NRf2 inhibitor improves the efficacy of the anti-BMP6 antibody or fragment by disrupting the more iron feedback loop that induces more BMP6 expression in the treated subject.

[0251] The present disclosure alternatively includes generic versions of branded drugs, the disclosure of which is incorporated herein by reference for possible use in the present invention, for example, as part of a combination.

[0252] In one example, the combination comprises an inhibitor of BMP6 and HJV, or BMP6 and HFE, or BMP6 and TFR2, or BMP6 and BMP2, or BMP6 and BMP4, or BMP6 and ALK2, wherein the BMP6 inhibitor comprises an antibody or fragment of the invention.

[0253] 43. A pharmaceutical composition comprising an antibody, fragment or combination according to any one of clauses 1 to 38 and 42 and a pharmaceutically acceptable excipient, diluent or carrier, optionally in combination with a further therapeutic agent selected from those described above (e.g. clause 42).

[0254] 44. A pharmaceutical composition according to clause 43 for treating and / or preventing a BMP6-mediated condition or disease, such as anemia.

[0255] Suitable diseases and conditions include anemia, pulmonary arterial hypertension (PAH) (e.g., primary PAH or secondary PAH), cerebral cavernous hemangioma (CCM) (e.g., familial CCM or sporadic CCM), restless legs syndrome (RLS), cancer (e.g., breast cancer, pancreatic cancer, colorectal cancer, salivary gland cancer, esophageal cancer, or melanoma), cancer metastasis, systemic sclerosis, Sjogren's syndrome, endothelial-mesenchymal transition (EndoMT), cardiovascular disease, atherosclerosis, systemic sclerosis-associated pulmonary fibrosis, and cardiac fibrosis.

[0256] Examples of diseases or conditions mediated by EndoMT are cardiovascular disease, atherosclerosis, systemic sclerosis-associated pulmonary fibrosis, cardiac fibrosis, PAH, tumorigenesis, tumor invasion, tumor metastasis, fibrotic diseases, and cancer-associated fibroblast generation (e.g., pancreatic cancer).

[0257] 45. A pharmaceutical composition according to clause 43 or 44 in combination with a label or instructions for use in treating and / or preventing said disease or condition in a human, wherein optionally the label or instructions includes a marketing authorization number (e.g., an FDA or EMA approval number), and optionally the kit includes an IV or injection device containing the antibody or fragment.

[0258] 46. A nucleic acid encoding the VH domain and / or the VL domain of the antibody or fragment of any one of clauses 1 to 32.

[0259] 47. A nucleic acid encoding a VH domain comprising the amino acid sequence of the VH domain of an antibody selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, and CL-58713, or an amino acid sequence at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical thereto.

[0260] For example, the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0261] Optionally, a nucleic acid encoding a VH domain comprising the amino acid sequence of SEQ ID NO: 114, or an amino acid sequence at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical thereto, is provided. For example, the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0262] 48. A nucleic acid encoding a VL domain comprising the amino acid sequence of the VL domain of an antibody selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, and CL-58713, or an amino acid sequence at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical thereto.

[0263] Optionally, the nucleic acid also encodes a VH domain comprising the amino acid sequence of the VH domain of the selected antibody, or an amino acid sequence at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical thereto. For example, the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0264] Optionally, a nucleic acid encoding a VL domain comprising the amino acid sequence of SEQ ID NO: 123, or an amino acid sequence at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical thereto, is provided. For example, the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0265] 49. (a) a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical to the sequence of SEQ ID NO: 115, 520 or 521, and / or (b) a nucleic acid (e.g., in a host cell, e.g., a CHO or HEK293 or Cos cell) comprising a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical to the sequence of SEQ ID NO: 124, 522 or 523.

[0266] Alternatively, the following is provided: (a) a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the sequence of SEQ ID NO: 115, and / or (b) a nucleic acid (e.g., in a host cell, e.g., a CHO or HEK293 or Cos cell) comprising a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the sequence of SEQ ID NO: 124.

[0267] (a) a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the sequence of SEQ ID NO: 520, and / or (b) a nucleic acid (e.g., in a host cell, e.g., a CHO or HEK293 or Cos cell) comprising a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the sequence of SEQ ID NO: 522.

[0268] (a) a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the sequence of SEQ ID NO: 521, and / or (b) a nucleic acid (e.g., in a host cell, e.g., a CHO or HEK293 or Cos cell) comprising a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the sequence of SEQ ID NO: 523.

[0269] (a) a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical to the sequence of SEQ ID NO: 115, 520 or 521, and / or (b) a combination of a first and a second nucleic acid (e.g., in a host cell, e.g., a CHO or HEK293 or Cos cell) comprising a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical to the sequence of SEQ ID NO: 124, 522 or 523, respectively.

[0270] (a) a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the sequence of SEQ ID NO: 115, and / or (b) a combination of a first and a second nucleic acid (e.g., in a host cell, e.g., a CHO or HEK293 or Cos cell), each comprising a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the sequence of SEQ ID NO: 124.

[0271] (a) a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the sequence of SEQ ID NO: 520, and / or (b) a combination of a first and a second nucleic acid (e.g., in a host cell, e.g., a CHO or HEK293 or Cos cell), each comprising a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the sequence of SEQ ID NO: 522.

[0272] (a) a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the sequence of SEQ ID NO: 521, and / or (b) a combination of a first and a second nucleic acid (e.g., in a host cell, e.g., a CHO or HEK293 or Cos cell), each comprising a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the sequence of SEQ ID NO: 523.

[0273] For example, with respect to (a), the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0274] For example, with respect to (b), the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0275] In any instance where percent identity is referred to herein, in one instance there is 100% identity.

[0276] 50. A nucleic acid encoding the heavy and / or light chain of the antibody or fragment according to any one of clauses 1 to 32.

[0277] 51. A nucleic acid encoding a heavy chain comprising an amino acid sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the sequence of SEQ ID NO:116.

[0278] For example, the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0279] 52. A nucleic acid encoding a light chain comprising an amino acid sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the sequence of SEQ ID NO: 125.

[0280] For example, the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0281] 53. (a) a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to a selected heavy chain sequence of an antibody selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, and CL-58713; and / or (b) a nucleic acid (e.g., in a host cell, e.g., a CHO or HEK293 or Cos cell) comprising a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical to a selected sequence of an antibody selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, and CL-58713.

[0282] Preferably, the antibodies selected in (a) and (b) are the same antibody, e.g., CL-58838. Alternatively, for example, in a host cell, e.g., a CHO or HEK293 or Cos cell, the first nucleic acid comprises (a) and the second nucleic acid comprises (b).

[0283] All of the nucleic acids of the invention herein can be expressed in host cells, such as CHO or HEK293 or Cos cells, to express the variable domains or chains of the antibodies or fragments of the invention.

[0284] For example, the following is provided: (a) a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to a sequence selected from SEQ ID NOs: 512, 514, 516, 518, and 519; and / or (b) a nucleic acid (e.g., in a host cell, such as a CHO or HEK293 or Cos cell) comprising a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical to a sequence selected from SEQ ID NOs: 513, 515 and 517.

[0285] (a) a nucleotide sequence at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 512, and / or (b) a nucleic acid comprising a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 513 (e.g., in a host cell such as a CHO or HEK293 or Cos cell).

[0286] (a) a nucleotide sequence at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 514, and / or (b) a nucleic acid comprising a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 515 (e.g., in a host cell such as a CHO or HEK293 or Cos cell).

[0287] (a) a nucleotide sequence at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 516, and / or (b) a nucleic acid comprising a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 517 (e.g., in a host cell, such as a CHO or HEK293 or Cos cell).

[0288] (a) a nucleotide sequence at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 518, and / or (b) a nucleic acid comprising a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 513, 515 or 517 (e.g., in a host cell such as a CHO or HEK293 or Cos cell).

[0289] (a) a nucleotide sequence at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 519, and / or (b) a nucleic acid comprising a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 513, 515 or 517 (e.g., in a host cell such as a CHO or HEK293 or Cos cell).

[0290] For example, with respect to (a), the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0291] For example, with respect to (b), the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0292] 54. A vector comprising a nucleic acid(s) (e.g., a nucleic acid(s) according to any one of clauses 46-53), optionally wherein the vector is a CHO or HEK293 vector.

[0293] 55. A host cell comprising a nucleic acid(s) (eg a nucleic acid(s) according to any one of clauses 46 to 53) or a vector of clause 54.

[0294] Optionally, the VH gene segment is IGHV1-3 * 01 and IGHV3-11 * 01. Optionally, the VL gene segment is selected from IGKV1-5*03, IGKV3-20 * 01 and IGKV3-15 * Selected from 01.

[0295] In one example, the VH, DH, and JH are selected from IGHV1-3, IGHD3-10, and IGHJ4 (e.g., IGHV1-3 * 01, IGHD3-10 * 01 and IGHJ4 * 02).

[0296] In one example, the VH, DH and JH are IGHV1-3, IGHD3-10 and IGHJ3, e.g., IGHV1-3 * 01, IGHD3-10 * 01 and IGHJ3 * The number is 02.

[0297] In one example, the VH, DH and JH are IGHV3-11, IGHD6-19 and IGHJ4, e.g., IGHV3-11 * 01, IGHD6-19 * 01 and IGHJ4 * The number is 02.

[0298] In one example, the VH, DH and JH are IGHV1-3, IGHD7-27 and IGHJ4, e.g., IGHV1-3 * 01, IGHD7-27 * 02 and IGHJ4 * The number is 02.

[0299] In one example, the VH, DH and JH are IGHV1-3, IGHD4-23 and IGHJ4, e.g., IGHV1-3 * 01, IGHD4-23 * 01 and IGHJ4 * The number is 02.

[0300] In one example, the VH, DH and JH are IGHV1-3, IGHD5-18 and IGHJ4, e.g., IGHV1-3 * 01, IGHD5-18 * 01 and IGHJ4 * The number is 02.

[0301] In one example, the VL and JL are IGKV1-5 and IGKJ1, e.g., IGKV1-5 * 03 and IGKJ1 * The number is 01.

[0302] In one example, the VL and JL are IGKV3-20 and IGKJ1, e.g., IGKV3-20 * 01 and IGKJ1 * The number is 01.

[0303] In one example, the VL and JL are IGKV3-15 and IGKJ3, e.g., IGKV3-15 * 01 and IGKJ3 * The number is 01.

[0304] In one example, the VL and JL are IGKV3-20 and IGKJ3, e.g., IGKV3-20 * 01 and IGKJ3 * The number is 01.

[0305] In one example, the antibody or fragment comprises an HCDR3 length of 9, 10, 11, or 12 residues, such as 10 residues, for example 11 residues. In one example, the antibody or fragment comprises an LCDR3 length of 7, 8, or 9 residues, such as 8 residues, for example 9 residues. In one example, each VH domain of the antibody or fragment comprises 1 to 11 non-germline residues, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 non-germline residues. In one example, each VL domain of the antibody or fragment comprises 3 to 8 non-germline residues, for example 3, 4, 5, 6, 7, or 8 non-germline residues.

[0306] In one embodiment, the CDR sequences herein are determined according to Kabat. Alternatively, the CDR sequences are determined according to IMGT.

[0307] In one example, the selected antibody is CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, or CL-58713. In one example, the selected antibody is CL-58838.

[0308] In one example, the selected antibody comprises a heavy chain of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, or CL-58713. In one example, the selected antibody comprises a heavy chain of CL-58838.

[0309] In one example, the heavy chain of an antibody or fragment of the invention is a human gamma-1, gamma-2, gamma-3, gamma-4, mu, delta, epsilon, or alpha isotype, preferably a gamma isotype (e.g., an IgG4 isotype). In one example, the light chain of an antibody or fragment of the invention comprises a human kappa constant region. Alternatively, in one example, the light chain of an antibody or fragment of the invention comprises a human lambda constant region.

[0310] Optionally, the antibody is a four-chain antibody comprising a heavy chain dimer combined with a light chain dimer. In one example, the heavy chain comprises one or a combination of heavy chain CDRs or CDRs disclosed herein, and / or the light chain comprises one or a combination of heavy chain CDRs or CDRs disclosed herein, such as from the same selected antibody. In one example, the heavy chain comprises a VH domain disclosed herein, and / or the light chain comprises a VL domain disclosed herein, such as from the same selected antibody. In one example, the heavy and light chains are derived from the same selected antibody, such as any antibody disclosed in the sequence listing herein or in the tables of the examples herein.

[0311] In one example, the selected antibody comprises the light chain of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, or CL-58713. In one example, the selected antibody comprises the light chain of CL-58838.

[0312] In one example, the selected antibody comprises the variable domain of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, or CL-58713. In one example, the selected antibody comprises the variable domain of CL-58838.

[0313] In one example, the selected antibody comprises the VH domain of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, or CL-58713. In one example, the selected antibody comprises the VH domain of CL-58838.

[0314] In one example, the selected antibody comprises the VH and VL domains of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, or CL-58713. In one example, the selected antibody comprises the VH and VL domains of CL-58838.

[0315] Optionally, the VH segment is a human IGHV3-11 gene segment, e.g., the VH is encoded by a nucleotide sequence derived from the recombination of human IGHV3-11 and IGHJ4 (e.g., human gene segment IGHV3-11 * 01 and IGHJ4 * 02, IGHV3-11, IGHD6-19, IGHJ4, or IGHV3-11 * 01, IGHD6-19 * 01 and IGHJ4 * 02). Optionally, JH is IGHJ4 * 02. Optionally, the VL is encoded by a nucleotide sequence derived from the recombination of a human VL gene segment and a JL gene segment, wherein the VL gene segment is selected from IGKV1-5, IGKV3-20, and IGKV3-15. Optionally, the VL is human IGKV3-20 (e.g., IGKV3-20 * 01). Optionally, JL is IGKJ1 (e.g., IGKJ1 * 01). For example, the VL is human IGKV3-20 (e.g., IGKV3-20 * 01) and human IGKJ1 (e.g., IGKJ1 * 01) and is encoded by a nucleotide sequence derived from the recombination of

[0316] In one example, the binding site comprises a VH / VL pair that specifically binds to human BMP6 (e.g., human BMP6 comprising or consisting of the bolded sequence of SEQ ID NO: 1 in the sequence listing herein). In one example, the antibody or fragment comprises two (e.g., two and no more than two) copies of the binding site.

[0317] In one example, the antibody or fragment comprises an HCDR3 length of 9 to 12 residues, and / or the antibody or fragment comprises an LCDR3 length of 7 to 9 residues. In one example, the antibody or fragment comprises an HCDR3 length of 9, 10, 11, or 12 residues, such as 10 residues, for example 11 residues. In one example, the antibody or fragment comprises an LCDR3 length of 7, 8, or 9 residues, such as 8 residues, for example 9 residues. In one example, each VH domain of the antibody or fragment comprises 1 to 11 non-germline residues, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 non-germline residues. In one example, each VL domain of the antibody or fragment comprises 3 to 8 non-germline residues, for example 3, 4, 5, 6, 7, or 8 non-germline residues.

[0318] Optionally, the antibody or fragment competes with CL-58838 (e.g., CL-58838 in IgG format, e.g., IgG-PE) for binding to BMP6 (e.g., human BMP6, e.g., mature human BMP6, e.g., BMP6 comprising or consisting of the sequence of mature BMP6 disclosed in the sequence listing herein, i.e., the bolded sequence of SEQ ID NO: 1), as determined by SPR.

[0319] Optionally, the amino acid substitutions are conservative amino acid substitutions, and optionally each conservative substitution is selected from the group (1) to (6): 1) Alanine (A), serine (S), threonine (T), 2) Aspartic acid (D), glutamic acid (E), 3) Asparagine (N), Glutamine (Q), 4) Arginine (R), Lysine (K), 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V), and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0320] Any SPR herein is, for example, surface plasmon resonance (SPR) at 37° C. and pH 7.6.

[0321] Optionally, any BMP6 herein is human BMP6 (e.g., in in vitro testing), e.g., hBMP6 (Peprotech 120-06).

[0322] In one example, the antibody or fragment of the invention may be administered in a volume of, for example, 5x10 6 M -1 ×s -1 , or about 5x10 6 M -1 ×s -1 In one example, the antibody or fragment of the invention binds to human BMP6 with a Ka of, for example, 4 or 5s -1 , or about 4 or 5 seconds -1 In one example, the antibody or fragment of the present invention binds to human BMP6 with a KD of, for example, 0.07 or 0.14 nM or about 0.07 or 0.14 nM. In one embodiment, the fragment is a Fab fragment. In one embodiment, the fragment is an scFv.

[0323] In one example, the antibody comprises heavy chains, each heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:116, and each light chain comprising or consisting of the amino acid sequence of SEQ ID NO:125.

[0324] In one example, the antibody or fragment comprises heavy chain VH domains, each VH comprising or consisting of the amino acid sequence of SEQ ID NO: 418, and each light chain VL domain comprising or consisting of the amino acid sequence of SEQ ID NO: 426.

[0325] Surrogate antibodies or fragments: The present invention, in any of its configurations, may relate to antibodies or fragments (surrogate antibodies or fragments) as follows:

[0326] Optionally (Option 1), the antibody or fragment is a. each heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 403 or 566, and each light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 411, or b. Each heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:419, and each light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:427.

[0327] Optionally (option 2), the antibody or fragment is a. each heavy chain comprising the VH domain amino acid sequence of SEQ ID NO: 402 or 565, and each light chain comprising the VL domain amino acid sequence of SEQ ID NO: 410, or b. each heavy chain comprising a VH domain amino acid sequence of SEQ ID NO: 418, and each light chain comprising a VH domain amino acid sequence of SEQ ID NO: 426; and c. Optionally, the heavy chain is human gamma-1 (e.g., IGHG1 * 01), gamma-4 (e.g., IGHG4 * 01 or IGHG4 * 01-PE) constant region, or the amino acid sequence of SEQ ID NO: 429, 437, 446, 454, or 456.

[0328] Optionally, the antibody or fragment competes with a reference antibody for binding BMP6, where the reference antibody is mAb507 (R&D Systems) or a surrogate antibody (e.g., an Option 1 or Option 2 antibody as defined herein). Competition can be, for example, by SPR or ELISA, or in a functional assay such as an assay described herein (e.g., in the Examples). The BMP6 can be human BMP6 (e.g., a mature BMP6 comprising the sequence of SEQ ID NO: 562), rat BMP6 (e.g., a mature BMP6 comprising the sequence of SEQ ID NO: 56), or cynomolgus BMP6 (e.g., a mature BMP6 comprising the sequence of SEQ ID NO: 564).

[0329] Human IgG heavy chain genes naturally encode a C-terminal lysine. This residue is rarely found in antibodies isolated from serum and is present at low, but variable, levels on therapeutic antibodies expressed in mammalian cell culture systems. Because C-terminal lysine clipping occurs naturally in serum and is not known to affect overall antibody function, it can be removed from the heavy chain coding sequence to provide a uniform "lysine-clipped" heavy chain and, therefore, a uniform preparation. Thus, IgG antibodies, constant regions, or heavy chains shown herein to terminate at the C-terminus with a G may alternatively be provided in a form terminating in a GK (i.e., a lysine attached C-terminal to the indicated G).

[0330] Examples of options 1 and 2 are as follows: Option 1a: In one example, the antibody comprises heavy chains, each heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 403, and each light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 411.

[0331] Option 1b: In one example, the antibody comprises heavy chains, each heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 566, and each light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 411.

[0332] Option 1c: In one example, the antibody comprises heavy chains, each heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 419, and each light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 427.

[0333] Option 2a: In one example, the antibody or fragment comprises heavy chain VH domains, each VH comprising or consisting of the amino acid sequence of SEQ ID NO: 402, and each light chain VL domain comprising or consisting of the amino acid sequence of SEQ ID NO: 410.

[0334] Option 2b: In one example, the antibody or fragment comprises heavy chain VH domains, each VH comprising or consisting of the amino acid sequence of SEQ ID NO: 565, and each light chain VL domain comprising or consisting of the amino acid sequence of SEQ ID NO: 410.

[0335] In one example, the antibody or fragment comprises heavy chain VH domains, each VH comprising or consisting of the amino acid sequence of SEQ ID NO: 114, and each light chain VL domain comprising or consisting of the amino acid sequence of SEQ ID NO: 123.

[0336] Optionally, the antibody or fragment competes with the reference antibody for binding to the amino acid sequence of SEQ ID NO: 1. Additionally or alternatively, optionally, the antibody or fragment competes with the reference antibody for binding to the amino acid sequence of SEQ ID NO: 492. Additionally or alternatively, optionally, the antibody or fragment competes with the reference antibody for binding to the amino acid sequence of SEQ ID NO: 491. Additionally or alternatively, optionally, the antibody or fragment competes with the reference antibody for binding to the amino acid sequence of SEQ ID NO: 4. Additionally or alternatively, the antibody or fragment competes with the reference antibody for binding to a mature version of any one or more of these.

[0337] Optionally, the antibody or fragment competitively inhibits binding of soluble hemojuvelin (HJV) to BMP6. Optionally, the HJV herein is human HJV.

[0338] Optionally, the antibody or fragment does not competitively inhibit binding of soluble hemojuvelin (HJV) to BMP6.

[0339] As used herein, "inhibit," "inhibition," "inhibiting," and the like refer to the ability of an antagonist (e.g., an antibody or fragment thereof) to bind to an epitope (e.g., of hBMP6) that partially or completely prevents the binding of another antigen. If the epitope bound by the antagonist completely blocks the binding site of the ligand, ligand binding is completely prevented (this can be physical blocking in the case of overlapping epitopes, or steric blocking if the antagonist is large enough to prevent ligand binding to its unique epitope), and the ligand is not removed from circulation. Thus, the concentration of circulating ligand may appear to increase. If the epitope bound by the antagonist partially blocks the binding site of the ligand, the ligand may be able to bind, but only weakly (in the case of partial inhibition) or bind in an orientation different from the natural binding interaction. In this case, some of the ligand may be removed from circulation, but not as much as if the ligand binding site were completely free and available for binding. Thus, inhibition refers to the physical interaction between the ligand and the receptor. Inhibition can be measured by HTRF, which is described in more detail elsewhere herein and in Mathis (1995) Clinical Chemistry 41(9), 1391-1397. Inhibition can also be measured by flow cytometry, where the receptor is expressed on cells, or by ELISA, where the receptor is adsorbed to a plate.

[0340] Optionally, the antibody comprises a VH domain encoded by a VDJ region sequence, wherein the VDJ is derived from the recombination of a VH, D, and JH gene segment, and wherein the VH is a human germline (i) VH1-3, (ii) VH2-5, or (iii) VH3-15 gene segment. Additionally or alternatively, optionally, the antibody comprises a VL domain encoded by a VJ region sequence, wherein the VJ is derived from the recombination of a VL and a JL gene segment, and wherein the VL is a human germline (iv) Vκ3-20, (v) Vλ3-1, (vi) Vκ1-17, or (vii) Vλ1-40.

[0341] Optionally, the antibody or fragment binds to BMP6 with greater affinity (lower KD as determined by SPR) than it binds to BMP7, and / or optionally binds to BMP6 with greater affinity than BMP5.

[0342] for example, (a) the antibody or fragment binds to BMP6 with greater affinity (lower KD as determined by SPR) than it binds to BMP7, and optionally binds to BMP6 with greater affinity than BMP5; (b) The antibody or fragment competes with a reference antibody for binding BMP6, the reference antibody being mAb507 (R&D Systems) or a surrogate antibody (e.g., Option 1 or Option 2 antibody).

[0343] Optionally, the antibody of the present invention has an affinity (KD) for binding BMP6 of 1 pM to 5 nM, and optionally the binding is determined by SPR using a Fab of the antibody at 37°C, pH 7.6.

[0344] Optionally, antibodies are 1x10 -5 ~1×10 -3 S -1 Off-rate (K) for binding BMP6 off ), and optionally binding is determined by SPR using the Fab of the antibody at 37°C, pH 7.6.

[0345] Optionally, antibodies are 1x10 5 ~1×10 7 M -1 S -1 On-rate (K) for binding BMP6 on ), and optionally binding is determined by SPR using the Fab of the antibody at 37°C, pH 7.6.

[0346] In one example, the antibody (e.g., as a Fab) or fragment may be (a) 2, 3, 4, 5, or 10 pM to 3, 4, or 5 nM; (b) 1-10 pM-5 nM, (c) 10 pM to 3, 4, or 5 nM; (d) 50 or 80 pM to 200 nM; (e) 50 or 80 pM to 150 nM, or (f) has an affinity (KD) for binding BMP6 (e.g., human BMP6) of 50 or 80 pM to 100 nM.

[0347] In one example, the KD is (or is about) 5-15 pM (e.g., 10 pM). In one example, the KD is (or is about) 2-5 nM (e.g., 3 nM). In one example, the KD is (or is about) 100-400 pM (e.g., 140 or 390 pM).

[0348] In one example, the antibody (e.g., as a Fab) or fragment may be (a) 1x10 -5 ~5×10 -4 S -1 , (b) 1x10 -5 ~6×10 -4 S -1 , (c)1x10 -5 ~7×10 -4 S -1 , (d) 1x10 -5 ~8×10 -4 S -1 , (e) 2x10 -5 ~1×10 -3 S -1 , (f)2x10 -5 ~5×10 -4 S -1 , (g)2x10 -5 ~6×10 -4 S -1 , (h)2x10 -5 ~7×10 -4 S -1 ,or (i) 2x10 -5 ~8×10 -4 S-1 The off-rate (K) for binding BMP6 (e.g., human BMP6) off )

[0349] In one example, K off is 5x10 -4 S -1 (or about) (e.g., when the KD is 2 nM to 400 pM (or about), when the KD is 2 to 5 nM (e.g., 3 nM) (or about), or when the KD is 100 to 400 pM (e.g., 140 or 390 pM) (or about). off is 3x10 -5 S -1 is (or is about) 5 to 15 pM (e.g., 10 pM) (for example, when the KD is (or is about) 5 to 15 pM (e.g., 10 pM)).

[0350] In one example, the antibody (e.g., as a Fab) or fragment may be (a) 1x10 5 ~1x10 6 M -1 S -1 , (b) 1x10 5 ~2x10 6 M -1 S -1 , (c)1x10 5 ~3x10 6 M -1 S -1 , (d) 1x10 5 ~4x10 6 M -1 S -1 , (e) 1x10 5 ~5x10 6 M -1 S -1 , (f)2x10 5 ~5x10 6 M -1 S -1 , (g)3x10 5 ~5x10 6 M-1 S -1 , (h)4x10 5 ~5x10 6 M -1 S -1 , (i) 5x10 5 ~5x10 6 M -1 S -1 ,or (j)6x10 5 ~5x10 6 M -1 S -1 On-rate (K) for binding BMP6 (e.g., human BMP6) on )

[0351] In one example, K on is 1 or 2x10 -5 M -1 S -1 is (or is about) (e.g., when the KD is 2 to 5 nM (e.g., 3 nM)). on 1-4, 1, 2, 3 or 4x10 -6 M -1 S -1 (or about the same) (e.g., when the KD is 5 to 400 pM (e.g., 140 or 390 pM) or 5 to 15 pM (e.g., 10 pM)).

[0352] As provided herein or in other embodiments, the anti-BMP6 antibody or fragment may have a K of less than 50 nM, less than 40 nM, or less than 30 nM as determined by surface plasmon resonance. D In another embodiment, the anti-BMP6 antibody or fragment may bind to a BMP6, e.g., a human BMP6, having a K of less than 20 nM, less than 15 nM, or less than 10 nM as determined by surface plasmon resonance. D The anti-BMP6 antibody or fragment may bind to BMP6, e.g., human BMP6, having a K of less than 8 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM as determined by surface plasmon resonance. DIt can bind to BMP6 having the formula K, for example, human BMP6. D can be 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, or 0.1 nM or less.

[0353] In another embodiment, K D is in the range of 0.01 to 1 nM, or in the range of 0.05 to 2 nM, or in the range of 0.05 to 1 nM. D The may be with respect to hBMP6, cynomolugus monkey (i.e., "cyno") BMP6 and / or mouse BMP6.

[0354] In another embodiment, the anti-BMP6 antibodies described herein have a K of about 0.5-10 μM, e.g., about 1-8 μM or about 1-7 μM. ON In another embodiment, the K ON The rate is about 1-5 μM, e.g., about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, or about 3 μM. ON The rate is about 3.5 μM, about 4 μM, about 4.5 μM, about 5 μM or about 5.5 μM.

[0355] In another embodiment, the anti-BMP6 antibodies described herein have a K of about 0.01 to 100 mM, e.g., about 0.1 to 50 mM or about 0.5 to 50 mM. OFF In another embodiment, the K OFF In another embodiment, the K OFF The rate is about 0.6 mM, about 0.7 mM, about 0.8 mM or about 0.9 mM.

[0356] The invention also provides the following methods (or antibodies or fragments of the invention for use in such methods). 1. A method of treating anemia in a subject, the method comprising: (a) on day 1 (D0), administering an anti-BMP6 antibody or fragment to a subject; (b) administering, for at least three consecutive weeks, multiple doses of an erythropoietin-stimulating agent (ESA) for said period beginning on D0, wherein said subject's blood hemoglobin (Hb) concentration is increased from the baseline concentration on D0 throughout said period; (c) throughout the entire period; (i) the Hb concentration is equal to or greater than 100% of the baseline Hb concentration, during which the Hb concentration reaches at least 120% of baseline; and / or (ii) the Hb concentration increases by at least 1 g / dl above baseline.

[0357] Optionally, the antibody, fragment, or combination inhibits iron release by human hepatocytes, for example, in an in vitro assay or in humans. Those skilled in the art will be aware of standard assays such as those mentioned in the Examples herein.

[0358] Optionally, the antibody, fragment, or combination is for treating or preventing a BMP6-mediated disease or condition in a human, as disclosed herein, by inhibiting iron release by human hepatocytes in the human. Optionally, the antibody, fragment, or combination is for treating or preventing anemia, PAH, or fibrosis in a human, by inhibiting iron release by human hepatocytes in the human.

[0359] Optionally, the antibody, fragment, or combination is for treating or preventing a BMP6-mediated disease or condition in a human, as disclosed herein, by inhibiting HAMP gene expression in human hepatocytes in the human. Optionally, the antibody, fragment, or combination is for treating or preventing anemia, PAH, or fibrosis in a human, by inhibiting HAMP gene expression in human hepatocytes in the human.

[0360] Optionally, the antibody, fragment, or combination is for treating or preventing a BMP6-mediated disease or condition in a human, as disclosed herein, by inhibiting hepcidin or its expression in human hepatocytes in the human. Optionally, the antibody, fragment, or combination is for treating or preventing anemia, PAH, or fibrosis in a human, by inhibiting hepcidin or its expression in human hepatocytes in the human.

[0361] Optionally, the antibody, fragment, or combination is for treating or preventing a BMP6-mediated disease or condition in a human, such as those disclosed herein, by inhibiting BMP6 activation of HAMP gene expression in the human (e.g., in liver cells thereof). Optionally, the antibody, fragment, or combination is for treating or preventing anemia, PAH, or fibrosis in a human, by inhibiting BMP6 activation of HAMP gene expression in the human (e.g., in liver cells thereof).

[0362] Optionally, the antibody, fragment, or combination is for treating or preventing a BMP6-mediated disease or condition as disclosed herein in a human by inhibiting HJV-mediated activation of HAMP gene expression in a human (e.g., in its liver cells). Optionally, the antibody, fragment, or combination is for treating or preventing anemia, PAH, or fibrosis in a human by inhibiting HJV-mediated activation of HAMP gene expression in a human (e.g., in its liver cells). In one example, the antibody or fragment competitively inhibits the binding of HJV to BMP6 in vitro and / or in a human. In vitro competition can be determined, for example, by standard SPR or ELISA.

[0363] In one example, the antibody or fragment inhibits human BMP6-induced luciferase expression in HepG2 cells under the control of the HAMP regulatory region in vitro.

[0364] Optionally, the antibody, fragment, or combination is for treating or preventing a BMP6-mediated disease or condition in a human, such as those disclosed herein, by inhibiting BMP binding in the human (e.g., in hepatocytes thereof). Optionally, the antibody, fragment, or combination is for treating or preventing anemia, PAH, or fibrosis in a human, by inhibiting BMP binding in the human (e.g., in hepatocytes thereof). In one example, the antibody or fragment binds to an epitope where HJV contacts BMP6, forming an HJV-BMP6 complex that can activate HMP gene expression in human hepatocytes.

[0365] In one example, the human cell is a HepG2 cell in vitro. Further details are provided in the Examples herein. In one example, the inhibition is determined in an in vitro HepG2 cell assay, for example, by the inhibition of a reporter gene under the control of one or more human HMP regulatory elements in vitro. For example, the regulatory elements include response elements to pSMAD(BMP) and pSTAT(IL6). In one example, the assay is performed using human BMP6, cynomolgus monkey BMP6, rat BMP6, or mouse BMP6, and / or the assay is performed using human HJV, cynomolgus monkey HJV, rat HJV, or mouse HJV. In one example, the reporter is a luciferase gene. In one example, the antibody or fragment neutralizes BMP activation of reporter gene expression in the assay, for example, the neutralization is at least 20, 30, 40, 50, 60, 70, 80, 90, or 95%, or is complete neutralization.

[0366] In one example, the antibody or fragment competes with a reference antibody for binding to BMP6, for example, as determined in vitro using a labeled reference antibody, for example, by SPR, ELISA, or in a HepG2 assay (for example, the HepG2 assay described herein).In one example, the competition reduces the binding by at least 20, 30, 40, 50, 60, 70, 80, 90, or 95%, or completely inhibits binding.For example, the reference antibody is MAb507 or MAb2365, and for example, the reference antibody is a surrogate antibody (for example, option 1 or option 2 antibody).

[0367] In one example, the BMP6 of the present invention is human BMP6 (Peprotech #120-06) (SEQ ID NO: 2). In one example, the BMP6 of the present invention is any other human BMP6 disclosed herein.

[0368] In one example, the antibody or fragment may be present in a concentration of 1x10 as determined by surface plasmon resonance (SPR), e.g., at room temperature or rtp. -4 S -1 Binds to human BMP6 with the following off-rates: See Examples.

[0369] In one example, the antibody is selected from the group consisting of CL-66833, CL-57890, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102, CL-57859, CL-58832, CL-57945, CL-75714, CL-75605, CL-75565, CL-75539, CL-75520, CL-75519, CL-75511, CL-75506, CL-75500, CL-75496, CL-75194, CL-75183, CL-58722, CL-58713, CL-58 680, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650 and CL-58756, e.g., selected from CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102 and CL-57945, e.g., selected from CL-58722, CL-58713, CL-58680, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650 and CL-58756.

[0370] Alternatively, the reference antibody herein is selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, and CL-58713. Optionally, the reference antibody is CL-58838.

[0371] In one example, the antibody competes with a reference antibody for binding to human BMP6, and the reference antibody comprises a VH amino acid sequence selected from SEQ ID NOs: 24, 42, 114, 132, 96, 78, 60, 258, 240, 222, 204, 186, 168, 150, 276, 384, 366, 348, 294, 330, and 312C, e.g., selected from SEQ ID NOs: 24, 42, 114, 132, 96, 78, and 60, e.g., selected from SEQ ID NOs: 276, 384, 366, 348, 294, 330, and 312; and / or the reference antibody comprises a VH amino acid sequence selected from SEQ ID NOs: 33, 51, 123, 141, 105, 87, 69, 267, 249, 231, 213, The antibody of the present invention comprises (e.g., comprises, respectively) a VL amino acid sequence selected from SEQ ID NOs: 195, 177, 159, 285, 393, 375, 357, 303, 339, and 321, for example, selected from SEQ ID NOs: 33, 51, 123, 141, 105, 87, and 69, for example, selected from SEQ ID NOs: 285, 393, 375, 357, 303, 339, and 321. In one example, the reference antibody is an IgG4 (e.g., IgG4-PE) antibody. In one example, the reference antibody is an IgG1 antibody. In one example, the antibody of the present invention is an IgG4 (e.g., IgG4-PE) antibody. In one example, the antibody of the present invention is an IgG1 antibody. In one example, the antibody of the present invention preferentially binds to human BMP6 over human BMP5 and / or human BMP7. Binding or competition can be determined by, for example, SPR or ELISA, as known to those skilled in the art.

[0372] In one example, the antibody competes with a reference antibody for binding to human BMP6, and the reference antibody is selected from the sequence listing herein, or selected from the table in the Examples, or is selected from the following: CL-66833, CL-57890, 42 CL-57931, 114 CL-58838, 132 CL-58851, 96 CL-58252, 78 CL-58102, CL-57859, CL-58832, 60 CL-57945, 258 CL-75714, CL-75605, 240 CL-75565, 222 CL-75539, 204 CL-75520, CL-75519, CL-75511, 186 CL-75506, 168 The reference antibody comprises the VH amino acid sequence of the VH of an antibody selected from CL-75500, CL-75496, CL-75194, 150 CL-75183, 276 CL-58722, 384 CL-58713, 366 CL-58680, 348 CL-58679, CL-58921, CL-58676, 294 CL-58835, 330 CL-58650, and 312 CL-58756, and / or the reference antibody comprises the VL amino acid sequence of the VH of the selected antibody. In one example, the reference antibody is an IgG4 (e.g., IgG4-PE) antibody. In one example, the reference antibody is an IgG1 antibody. In one example, the antibody of the present invention is an IgG4 (e.g., IgG4-PE) antibody. In one example, the antibody of the present invention is an IgG1 antibody. In one example, an antibody of the invention preferentially binds to human BMP6 over human BMP5 and / or human BMP7. Binding or competition can be determined, for example, by SPR or ELISA, as known to those skilled in the art.

[0373] In one example, the antibody or fragment of the invention is CL-66833, CL-57890, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102, CL-57859, CL-58832, CL-57945, CL-75714, CL-75605, CL-75565, CL-75539, CL-75520, CL-75715, CL-75721, CL-75731, CL-75741, CL-75742, CL-75743, CL-75744, CL-75745, CL-75746, CL-75747, CL-75748, CL-75749, CL-75751, CL-75752, CL-75753, CL-75754, CL-75755, CL-75756, CL-75757, CL-75758, CL-75759, CL-75760, CL-75761, CL-75762, CL-75763, CL-75764, CL-75765, CL-75766, CL-75767, CL-75768, CL-75769, CL-75770, CL-75771, CL-75772, CL-75773, CL-75774, CL-75775, CL-75776, CL-75777, CL-75778, CL-7 and CL-58722, CL-58713, CL-58680, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650, or CL-58756.

[0374] In one example, an antibody or fragment of the present invention comprises the VH and VL domains of CL-66833. In one example, an antibody or fragment of the present invention comprises the VH and VL domains of CL-57931. In one example, an antibody or fragment of the present invention comprises the VH and VL domains of CL-58838. In one example, an antibody or fragment of the present invention comprises the VH and VL domains of CL-58851. In one example, an antibody or fragment of the present invention comprises the VH and VL domains of CL-58252. In one example, an antibody or fragment of the present invention comprises the VH and VL domains of CL-58102. In one example, an antibody or fragment of the present invention comprises the VH and VL domains of CL-57945.

[0375] In one example, the selected antibody is CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, or CL-58102. In one example, the selected antibody is CL-66833. In one example, the selected antibody is CL-58838.

[0376] In one example, the selected antibody comprises the variable domain of CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, or CL-58102. In one example, the selected antibody comprises the variable domain of CL-66833. In one example, the selected antibody comprises the variable domain of CL-58838.

[0377] In one example, the selected antibody comprises the VH domain of CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, or CL-58102. In one example, the selected antibody comprises the VH domain of CL-66833. In one example, the selected antibody comprises the VH domain of CL-58838.

[0378] In one example, the selected antibody comprises the VH and VL domains of CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, or CL-58102. In one example, the selected antibody comprises the VH and VL domains of CL-66833. In one example, the selected antibody comprises the VH and VL domains of CL-58838.

[0379] In one example, the antibody or fragment of the invention is directed to the human gene segments IGHV3-11 and IGHJ4 (e.g., human gene segment IGHV3-11 * 01 and IGHJ4 * 02, IGHV3-11, IGHD6-19, IGHJ4, or IGHV3-11 * 01, IGHD6-19 * 01 and IGHJ4 * Additionally or alternatively, optionally, the antibody or fragment of the invention comprises a VH domain encoded by a nucleotide sequence that is a combination of the human gene segments IGKV3-20 and IGKJ1 (e.g., IGKV3-20 * 01 and IGKJ1 * 01). Additionally or alternatively, optionally, the antibody or fragment of the invention comprises an HCDR3 of CL-58835.

[0380] Optionally, the antibody or fragment of the invention is selected from the group consisting of CL-66833, CL-57890, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102, CL-57859, CL-58832, CL-57945, CL-75714, CL-75605, CL-75565, CL-75539, CL-75520, CL-75519, CL-75511, CL-75506, CL-75500, CL-7549 6, CL-75194, CL-75183, CL-58722, CL-58713, CL-58680, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650 and CL-58756, e.g., selected from CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102 and CL-57945. Optionally, the antibody or fragment of the present invention comprises the HCDR1 and / or HCDR2 of the selected antibody.

[0381] Optionally, the antibody or fragment of the invention is selected from the group consisting of CL-66833, CL-57890, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102, CL-57859, CL-58832, CL-57945, CL-75714, CL-75605, CL-75565, CL-75539, CL-75520, CL-75519, CL-75511, CL-75506, CL-75500, CL-7549 6, CL-75194, CL-75183, CL-58722, CL-58713, CL-58680, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650, and CL-58756, e.g., selected from CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102, and CL-57945. Optionally, the antibody or fragment of the present invention comprises HCDR2 and / or HCDR3 of the selected antibody.

[0382] Optionally, the antibody or fragment of the invention is selected from the group consisting of CL-66833, CL-57890, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102, CL-57859, CL-58832, CL-57945, CL-75714, CL-75605, CL-75565, CL-75539, CL-75520, CL-75519, CL-75511, CL-75506, CL-75500, CL-7549 6, CL-75194, CL-75183, CL-58722, CL-58713, CL-58680, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650 and CL-58756, e.g., selected from CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102 and CL-57945. Optionally, the antibody or fragment of the present invention comprises the HCDR1 and / or HCDR3 of the selected antibody.

[0383] Optionally, the antibody or fragment of the invention is selected from the group consisting of CL-66833, CL-57890, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102, CL-57859, CL-58832, CL-57945, CL-75714, CL-75605, CL-75565, CL-75539, CL-75520, CL-75519, CL-75511, CL-75506, CL-75500, CL-75496, CL-75194, CL-75183, CL-58722, CL-58713, CL-5868 0, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650, and CL-58756, e.g., selected from CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102, and CL-57945, e.g., selected from CL-58722, CL-58713, CL-58680, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650, and CL-58756. Optionally, the antibody or fragment of the present invention comprises the VH of the selected antibody.

[0384] Optionally, the antibody or fragment of the invention is selected from the group consisting of CL-66833, CL-57890, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102, CL-57859, CL-58832, CL-57945, CL-75714, CL-75605, CL-75565, CL-75539, CL-75520, CL-75519, CL-75511, CL-75506, CL-75500, CL-75496, CL-75194, CL-75183, CL-58722, CL-58713, CL-5868 0, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650, and CL-58756, e.g., selected from CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102, and CL-57945, e.g., selected from CL-58722, CL-58713, CL-58680, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650, and CL-58756. Optionally, the antibody or fragment of the present invention comprises the VH of the selected antibody.

[0385] Optionally, the antibody or fragment of the invention is selected from the group consisting of CL-66833, CL-57890, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102, CL-57859, CL-58832, CL-57945, CL-75714, CL-75605, CL-75565, CL-75539, CL-75520, CL-75519, CL-75511, CL-75506, CL-75500, CL-75496, CL-75194, CL-75183, CL-58722, CL-58713, CL-5868 0, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650, and CL-58756, e.g., selected from CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102, and CL-57945, e.g., selected from CL-58722, CL-58713, CL-58680, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650, and CL-58756. Optionally, the antibody or fragment of the present invention comprises the light chain of the selected antibody.

[0386] Optionally, the antibody or fragment of the invention is selected from the group consisting of CL-66833, CL-57890, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102, CL-57859, CL-58832, CL-57945, CL-75714, CL-75605, CL-75565, CL-75539, CL-75520, CL-75519, CL-75511, CL-75506, CL-75500, CL-75496, CL-75194, CL-75183, CL-58722, CL-58713, CL-5868 0, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650, and CL-58756, e.g., selected from CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102, and CL-57945, e.g., selected from CL-58722, CL-58713, CL-58680, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650, and CL-58756. Optionally, the antibody or fragment of the present invention comprises the heavy chain of the selected antibody.

[0387] In one example, the antibody selected is CL-58835.

[0388] Optionally, the antibodies of the invention comprise a human IgG4 constant region.

[0389] Preferably, an antibody or fragment thereof that specifically binds to hBMP6 does not cross-react with other antigens (but may optionally cross-react with BMP6 from different species, such as rhesus monkeys, cynomolgus monkeys, or mice, and / or optionally cross-react with different BMPs, such as BMP2, 4, or 9).An antibody or fragment thereof that specifically binds to a BMP6 antigen can be identified, for example, by immunoassay, BIAcore™, or other techniques known to those skilled in the art.An antibody or fragment thereof specifically binds to a hBMP6 antigen when it binds to the hBMP6 antigen with higher affinity than to any cross-reactive antigens, as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA).Typically, a specific or selective reaction is at least 2 times the background signal or noise, more typically more than 10 times the background. For a discussion of antibody specificity, see, e.g., pages 332-336 of Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York.

[0390] Contact amino acid residues involved in the interaction between an antibody and an antigen, such as BMP6, can be determined by a variety of methods known to those skilled in the art.

[0391] In one embodiment, if the antibody recognizes a linear epitope, short peptides can be generated based on the antigen sequence and binding of the antibody to these peptides can be assessed using standard techniques.

[0392] In one embodiment, limited protein digestion and mass spectrophotometric measurements can be used to identify binding epitopes.

[0393] In one embodiment, contact residues of the epitope are identified by X-ray crystallography. In one embodiment, contact residues of the epitope are identified by cryo-electron microscopy. In one embodiment, contact residues of the epitope are identified by a combination of limited protein digestion and mass spectrometry.

[0394] In another embodiment, the anti-BMP6 antibodies (and fragments) described herein provide improved transient expression levels compared to other anti-BMP6 antibodies and fragments. Thus, in one embodiment, the anti-BMP6 antibody (or fragment) is expressed in HEK293 cells, e.g., HEK293T cells, at an expression level of about 100 μg / mL, or in the range of about 100-350 μg / mL. In another embodiment, the expression level is greater than about 350 μg / mL.

[0395] In another embodiment, the anti-BMP6 antibody (or fragment) is expressed in CHO cells, e.g., Expi-CHO cells, at an expression level of about 100 μg / mL, or in the range of about 100-350 μg / mL, hi another embodiment, the expression level is greater than about 350 μg / mL.

[0396] In another embodiment, the anti-BMP6 antibody (or fragment) is expressed in CHO cells, e.g., Expi-CHO cells or CHO-E7 EBNA cells, at an expression level of about 100 μg / mL, or in the range of about 100-350 μg / mL. In another embodiment, the expression level is greater than about 350 μg / mL. For example, the antibody comprises the VH and VL domains of any one of CL-58838, formatted as human IgG1 or human IgG4 (e.g., IgG4-PE).

[0397] In any of these expression systems, expression is carried out in a volume of about 0.5 mL to 3 mL, for example, about 0.5 mL to 2 mL. In any of these expression systems, the anti-BMP6 antibody (or fragment) can be expressed from a pTT5 vector. In any of these expression systems, the anti-BMP6 antibody (or fragment) can be expressed in combination with a lipid transfection reagent and, optionally, in CHO cells, such as Expi-CHO cells. In any of these expression systems, the anti-BMP6 antibody (or fragment) can be expressed in combination with a PEI transfection reagent and, optionally, in CHO cells, such as CHO-E7 EBNA cells. In any of these expression systems, the anti-BMP6 antibody (or fragment) can be expressed in combination with a helper plasmid (e.g., an AKT helper plasmid) and, optionally, in CHO cells, such as CHO-E7 EBNA cells.

[0398] In any of these expression systems, the expression level is about 100 μg / mL to about 1500 μg / mL, for example, about 100 μg / mL to about 1000 μg / mL, or about 200 μg / mL to about 1000 μg / mL, or about 350 μg / mL to about 1000 μg / mL. In any of these expression systems, the lower limit of expression can be about 100 μg / mL, about 200 μg / mL, about 300 μg / mL, or about 400 μg / mL. In another embodiment, the lower limit of expression can be about 500 μg / mL, about 600 μg / mL, about 700 μg / mL, or about 800 μg / mL. In any of these expression systems, the upper limit of expression can be about 2000 μg / mL, about 1800 μg / mL, about 1600 μg / mL, or about 1500 μg / mL. In another embodiment, the upper limit of expression can be about 1250 μg / mL, about 1000 μg / mL, about 900 μg / mL, or about 800 μg / mL.

[0399] In another embodiment, the expression system is a Lonza expression system, such as the Lonza X-Ceed® system. In the Lonza expression system, expression can be carried out in volumes of about 30 mL to 2 L, e.g., 50 mL to 1 L, or 1 L to 2 L. In the Lonza expression system, the anti-BMP6 antibody (or fragment) can be expressed in combination with electroporation, optionally without any helper plasmid. In the Lonza expression system, the anti-BMP6 antibody (or fragment) can be expressed at levels of about 1 g / L, about 900 mg / L, about 800 mg / L, or about 700 mg / L. In another embodiment, in the Lonza expression system, the anti-BMP6 antibody (or fragment) can be expressed at levels of about 600 mg / L, about 500 mg / L, or about 400 mg / L. In the Lonza expression system, the anti-BMP6 antibody (or fragment) can be expressed at a level of about 400 mg / L to about 2 g / L, e.g., about 500 mg / L to about 1.5 g / L, or about 500 mg / L to about 1 g / L. In another embodiment, the expression level is greater than 1 g / L. In another embodiment, the anti-BMP6 antibody provides an improved half-life over other anti-BMP6 antibodies.

[0400] In one embodiment, the antibody or fragment is a human antibody or fragment. In one embodiment, the antibody or fragment is a fully human antibody or fragment. In one embodiment, the antibody or fragment is a fully human monoclonal antibody or fragment.

[0401] In one embodiment, the antibody or fragment is a humanized antibody or fragment. In one embodiment, the antibody or fragment is a humanized monoclonal antibody or fragment.

[0402] Contact amino acid residues involved in the interaction of an antibody with an antigen can be determined by various methods known to those skilled in the art, such as alanine scanning, protein crystallography, mass spectrometry, or any other technique that will be apparent to those skilled in the art.

[0403] In one embodiment, the listed CDRs contain one amino acid substitution, which may be a conservative amino acid substitution. In one embodiment, the listed CDRs contain two amino acid substitutions, which may be conservative amino acid substitutions. In one embodiment, the listed CDRs contain three amino acid substitutions, which may be conservative amino acid substitutions. In one embodiment, the listed CDRs contain four amino acid substitutions, which may be conservative amino acid substitutions. In one embodiment, the listed CDRs contain five amino acid substitutions, which may be conservative amino acid substitutions. In one embodiment, the listed CDRs contain six amino acid substitutions, which may be conservative amino acid substitutions.

[0404] Amino acid substitutions include modifications in which an amino acid is replaced with a different naturally occurring amino acid residue. Such substitutions can be classified as "conservative," in which an amino acid residue contained in a polypeptide is replaced with another naturally occurring amino acid having similar characteristics in terms of polarity, side chain functionality, or size. Such conservative substitutions are well known in the art. Substitutions encompassed by the present invention can also be "non-conservative," in which an amino acid residue present in a peptide is replaced with an amino acid having different properties, such as a naturally occurring amino acid from a different group (e.g., replacing a charged or hydrophobic amino acid with alanine), or a naturally occurring amino acid is replaced with a non-conventional amino acid.

[0405] In one embodiment, conservative amino acid substitutions are as described herein. For example, substitutions may be: Y for F, T for S or K, P for A, E for D or Q, N for D or G, R for K, G for N or A, T for S or K, D for N or E, I for L or V, F for Y, S for T or A, R for K, G for N or A, K for R, A for S, K, or P. In another embodiment, conservative amino acid substitutions may be Y replaced by F, T replaced by A or S, I replaced by L or V, W replaced by Y, M replaced by L, N replaced by D, G replaced by A, T replaced by A or S, D replaced by N, I replaced by L or V, F replaced by Y or L, S replaced by A or T, and A replaced by S, G, T, or V.

[0406] combination The antibodies or fragments of the present invention can be used in combination with ESAs to treat or prevent anemia, particularly moderate to severe anemia (i.e., blood hemoglobin levels below 9.5 g / dL). Such combinations can be effective in treating anemias such as those caused by anemia of chronic disease (ACD), inflammation, or infection, and can result in maintenance or elevation of blood hemoglobin levels that are statistically significant compared to the use of an anti-BMP6 antibody alone. Furthermore, such effects can be durable for several weeks (even after a single dose of an anti-BMP6 antibody is administered). Furthermore, the combination therapy of the present invention is useful for ESA-sparing anemia therapy, i.e., allowing treatment with lower ESA doses than standard ESAs. This is useful in light of the potentially harmful side effects of ESAs. The present invention can also be useful for treating anemia in subjects who are refractory to ESAs or who have a poor response to standard ESA therapy. The present invention can effectively maintain blood hemoglobin outside the range of moderate to severe anemia and / or prevent a decrease in blood hemoglobin to such range, and is therefore useful in reducing the need for iron or blood transfusion therapy.

[0407] The present invention is useful for the therapy of anemia in the setting of inflammatory disease and microbial infection.

[0408] For this purpose, the present invention provides the following configurations 1 to 13. 1. A method for maintaining a blood hemoglobin level of at least 10 g / dL in a subject, comprising administering to the subject an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA).

[0409] 2. A method for preventing a decrease in a subject's blood hemoglobin level to below 10 g / dL, comprising administering to the subject an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA).

[0410] 3. A method for increasing blood hemoglobin to a level of at least 10 g / dL in a subject suffering from anemia, comprising administering to the subject an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA), wherein the anemia is treated.

[0411] 4. A method for treating or preventing moderate or severe anemia in a subject, comprising administering to the subject an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA), wherein the anemia is treated or prevented.

[0412] 5. A method for treating or preventing anemia in a subject suffering from an inflammatory disease or condition, comprising administering to the subject an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA), wherein the anemia is treated or prevented.

[0413] 6. A method for eliminating or reducing the need for iron administration or blood transfusion in a subject suffering from anemia, comprising administering to the subject an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA), thereby eliminating or reducing the need.

[0414] In one example, one or more or all of labile plasma iron (LPI), enhanced LPI (eLPI), and non-transferrin-bound iron (NTBI) are reduced in a subject. In one example, one or more or all of labile plasma iron (LPI), enhanced LPI (eLPI), and non-transferrin-bound iron (NTBI) are reduced in a subject.

[0415] 7. A method for treating or preventing anemia in a subject suffering from a microbial infection, comprising administering to the subject an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA).

[0416] 8. A method for reducing the administration of an erythropoiesis-stimulating agent (ESA) for treating anemia to a subject suffering from anemia, the method comprising administering an anti-BMP6 antagonist and the ESA, wherein anemia is treated in the subject.

[0417] 9. A method for treating anemia or reducing the risk of anemia in a subject suffering from or at risk of anemia, comprising administering to the subject an anti-BMP6 antagonist and a low dose of an erythropoiesis-stimulating agent (ESA), wherein the anemia is treated or the risk of anemia is reduced in the subject.

[0418] 10. A therapeutic regimen for treating or preventing anemia in a subject suffering from or at risk of anemia, comprising administering to the subject an anti-BMP6 antagonist and an ESA simultaneously or sequentially; a. On day 0, the subject is administered an antagonist, and by day 7 (e.g., on day 1), the subject is administered an ESA; or b. On day 0, an ESA is administered to the subject, and by day 7 (e.g., on day 1), an antagonist is administered to the subject; or c. On day 0, the antagonist and ESA are administered simultaneously to the subject; or d. On day 0, the subject is already receiving an ESA, and on day 0, the subject is administered an antagonist; or e. On day 0, the subject has already received an antagonist, and on day 0, the subject is administered an ESA; A treatment regimen whereby on or after day 14 the subject has a blood hemoglobin level of at least 10 g / dL and said anemia is treated or prevented.

[0419] 11. A combination therapy for use in any of the methods or regimens of claims 1-10 for treating or preventing anemia in a subject, wherein the combination a. anti-BMP6 antagonists; b.ESA, and c. Combination therapy, optionally including instructions for use in the method or regimen.

[0420] 12. An anti-BMP6 antagonist for use in any of the methods or regimens of compositions 1 to 11 for treating or preventing anemia in a subject.

[0421] 13. An anti-bone morphogenetic protein 6 (BMP6) antagonist for use in a method for treating or preventing anemia in a subject, the method comprising administering the anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA) to the subject, wherein the anemia is treated or prevented.

[0422] When an "anti-BMP6 antagonist" is referred to herein, the antagonist may be any anti-BMP6 antibody or fragment disclosed herein, such as a surrogate antibody or fragment (described elsewhere herein), or an antibody or fragment as claimed or mentioned in the Summary of the Invention or in the Examples, such as Tables 4-11.

[0423] In one embodiment, the antagonist comprises or consists of an anti-BMP6 antibody or fragment, and the method comprises: (a) on day 1 (D0), administering an anti-BMP6 antibody or fragment to a subject; (b) administering multiple doses of an ESA for at least three consecutive weeks, the period beginning on D0, wherein the subject's blood Hb concentration is increased from the baseline concentration at D0 throughout the period; As a result, (i) the Hb concentration is equal to or greater than 100% of the baseline Hb concentration for the entire period, and during the period the Hb concentration reaches at least 120% of the baseline; and / or (ii) Hb concentration increases by at least 1 g / dl over baseline for the entire period.

[0424] Aspects of the present invention are as follows, and these aspects (and all unnumbered paragraphs) can be combined with any other configurations, embodiments, features, aspects, or provisions of the present invention described herein, and the antagonists (e.g., anti-BMP6 antibodies or fragments) or ESAs of the present invention can be provided for use in (or can be used in) the methods in the following aspects. 1. A method for maintaining a blood hemoglobin level of at least 10, 10.5, 11, 11.5, 12, 12.5, or 13 g / dL in a subject, comprising administering to the subject an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA).

[0425] In one example, the Hb level in the subject is below 11, 11.5 or 12 g / dl.

[0426] 2. A method for preventing a subject's blood hemoglobin level from decreasing to less than 10, 10.5, 11, 11.5, 12, 12.5, or 13 / dL, comprising administering to the subject an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA).

[0427] 3. A method for increasing blood hemoglobin to a level of at least 10, 10.5, 11, 11.5, 12, 12.5, or 13 g / dL in a subject suffering from anemia, comprising administering to the subject an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA), wherein the anemia is treated.

[0428] In one example of any embodiment, the subject suffers from moderate or severe anemia before administering the BMP6 antagonist.In one embodiment, the result of the method is that the subject does not have anemia or has mild (and not moderate or severe) anemia.

[0429] 4. A method for treating or preventing moderate or severe anemia in a subject, comprising administering to the subject an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA), wherein the anemia is treated or prevented.

[0430] 5. A method for treating or preventing anemia in a subject suffering from an inflammatory disease or condition, comprising administering to the subject an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA), wherein the anemia is treated or prevented.

[0431] In one example, the inflammatory disease or condition is selected from the group consisting of microbial infection (e.g., bacterial infection) or rheumatoid arthritis inflammation. In one example, the anemia is anemia of inflammation (also known as anemia of chronic disease, ACD).

[0432] 6. A method for eliminating or reducing the need for iron administration or blood transfusion in a subject suffering from anemia, comprising administering to the subject an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA), thereby eliminating or reducing the need.

[0433] In one embodiment, the method reduces the iron dose (eg, weekly, biweekly, or monthly dose) or dosing frequency.

[0434] 7. A method for treating or preventing anemia in a subject suffering from a microbial (e.g., bacterial) infection, comprising administering to the subject an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA).

[0435] 8. A method for reducing the administration of an erythropoiesis-stimulating agent (ESA) for treating anemia to a subject suffering from anemia, the method comprising administering an anti-BMP6 antagonist and the ESA, wherein anemia is treated in the subject.

[0436] 9. A method for treating anemia or reducing the risk of anemia in a subject suffering from or at risk of anemia, comprising administering to the subject an anti-BMP6 antagonist and a low dose of an erythropoiesis-stimulating agent (ESA), wherein the anemia is treated or the risk of anemia is reduced in the subject.

[0437] The dosage is lower than the standard dosage that is typically used for treating or reducing anemia in subjects, for example, humans or adults, such as men or women.The typical dosage for treatment or prevention will be easily clear to those skilled in the art.See, for example, embodiment 10.

[0438] Epogen is typically formulated in vials in multiple formulations. Single-dose vials formulated in an isotonic sodium chloride / sodium citrate buffer are supplied in multiple strengths. Each 1 mL vial contains 2000, 3000, 4000, or 10,000 units of epoetin alfa, albumin (human) (2.5 mg), citric acid (0.06 mg), sodium chloride (5.9 mg), and sodium citrate (5.8 mg) in water for injection, USP (pH 6.9 ± 0.3). Formulated in isotonic sodium chloride / sodium phosphate buffer, single-dose 1 mL vials contain 40,000 units of epoetin alfa albumin (human) (2.5 mg), citric acid (0.0068 mg), sodium chloride (5.8 mg), sodium citrate (0.7 mg), sodium phosphate dibasic (1.8 mg), and phosphate monobasic monohydrate (1.2 mg) in water for injection, USP (pH 6.9 ± 0.3). Multi-dose 2 mL vials contain 10,000 units of epoetin alfa, albumin (human) (2.5 mg), benzyl alcohol (1%), sodium chloride (8.2 mg), and sodium citrate (1.3 mg) per mL of water for injection, USP (pH 6.1 ± 0.3). Each multi-dose 1 mL vial contains 20,000 units of epoetin alfa, albumin (human) (2.5 mg), benzyl alcohol (1%), sodium chloride (8.2 mg), citric acid (0.11 mg), and sodium citrate (1.3 mg) in 1 mL of water for injection, USP (pH 6.1±0.3). In one example of the invention, the ESA is administered in one of these formulations.

[0439] 10.ESA epoetin alfa administered at a weekly dose of less than 1000, 1500, 2500, 5000, 11000, 18000, 34000, or 90000 units, and optionally the subject has previously received epoetin alfa therapy at <1500, 1500-2499, 2500-4999, 5000-10999, 11000-17999, 18000-33999, 34000-89999, or ≥ 90000 units per week, respectively; b. Darbepoetin alfa or Aranesp®, administered at a weekly dose of less than 6.25, 10, 12.5, 20, 25, 40, 60, 100, or 200 mcg, and optionally, the subject has previously received weekly darbepoetin alfa or Aranesp® treatment at 6.25, 10, 12.5, 20, 25, 40, 60, 100, or 200 mcg, respectively; or c. The method of any of embodiments 1-9, wherein the darbepoetin alfa or Aranesp® is administered at a weekly dose of less than 6.25, 10, 20, 40, 60, 100, or 200 mcg, and optionally the subject has previously received between 1,500 and 2,499, 2,500 and 4,999, 5,000 and 10,999, 11,000 and 17,999, 18,000 and 33,999, 34,000 and 89,999, or greater than or equal to 90,000 units of epoetin alfa therapy per week, respectively.

[0440] 11. The method of any of embodiments 1-10, wherein the anti-BMP6 antagonist is an antibody and is administered every 1, 2, or 3 weeks, or monthly, every 2 months, or every 3 months, etc., at a total dose of 30 mg / kg or less (e.g., 0.1-30 mg / kg). Administration can be, for example, intravenous or subcutaneous, and the subject is a human, such as an adult.

[0441] 12. A therapeutic regimen for treating or preventing anemia in a subject suffering from or at risk of anemia, comprising administering to the subject an anti-BMP6 antagonist and an ESA simultaneously or sequentially; a. On day 0, an antagonist is administered to the subject, and on days 56, 28, 14, or 7 (e.g., days 1, 6, or 7), an ESA is administered to the subject; or b. On day 0, an ESA is administered to the subject, and on days 56, 28, 14, or 7 (e.g., days 1, 6, or 7), an antagonist is administered to the subject; or c. On day 0, the antagonist and ESA are administered simultaneously to the subject; or d. On day 0, the subject is already receiving an ESA, and on day 0, the subject is administered an antagonist; or e. On day 0, the subject has already received an antagonist, and on day 0, the subject is administered an ESA; A treatment regimen whereby on day 14 or later (e.g., day 28, 56, or 70), the blood hemoglobin level is at least 10, 10.5, 11, 11.5, 12, 12.5, or 13 g / dL in the subject, and said anemia is treated or prevented.

[0442] Optionally, the antagonist is administered a second time by day 7 (eg, the antagonist is administered on day 6).

[0443] 13. The method or regimen of any of embodiments 1-12, wherein the anti-BMP6 antagonist and the ESA are administered to the subject no more than 7 days apart.

[0444] 14. The method or regimen of any one of aspects 1-13, wherein the method or regimen maintains a blood hemoglobin level in the subject above 10 g / dL in the subject.

[0445] 15. The method or regimen of any of embodiments 1-14, wherein the method or regimen maintains or increases blood hemoglobin levels to at least 10 g / dL in the subject at least 13 or 14 days after the subject receives the anti-BMP6 antagonist and an ESA.

[0446] 16. The method or regimen of embodiment 14 or 15, wherein the anti-BMP6 antagonist and the ESA are administered to the subject no more than one day apart.

[0447] 17. The method or regimen of any of embodiments 1-16, wherein the anti-BMP6 antagonist and the ESA are administered to the subject simultaneously.

[0448] 18. The method or regimen of any of aspects 1-17, wherein the subject's blood hemoglobin level is prevented from falling to below 10, 10.5, 11, 11.5, 12, 12.5, or 13 g / dL (e.g., on day 14).

[0449] 19. The method or regimen of any of aspects 1-18, wherein the subject's blood hemoglobin is increased to a level of at least 10, 10.5, 11, 11.5, 12, 12.5, or 13 g / dL (e.g., on day 14).

[0450] 20. The method or regimen of any of embodiments 1-19, wherein moderate or severe anemia is prevented in the subject (e.g., on day 14).

[0451] 21. The subject is a. suffers from an inflammatory disease or condition, or b. Have an infectious disease? c. Have kidney disease, d. Have HIV or are undergoing treatment for HIV; or e. have cancer, The method or regimen of any of embodiments 1-20, wherein moderate or severe anemia is treated or prevented in the subject.

[0452] In one example, the subject is suffering from HIV infection, or is suffering from HIV, hepatitis, rheumatoid arthritis, chronic kidney disease, or end-stage renal disease. For example, the infection is a gram-negative bacterial infection. For example, the infection is a gram-positive bacterial infection.

[0453] HIV-infected people treated with anti-HIV therapy may develop anemia. Therefore, the present invention can be useful for treating or preventing anemia in such patients. In one example, the method or regimen treats or prevents anemia in HIV-infected people treated with anti-HIV therapy, for example, zidovudine is administered at a dose of less than 4200 mg / week.

[0454] Cancer patients treated with anti-cancer chemotherapy (e.g., immunotherapy, such as by administering an immune checkpoint inhibitor to a subject, such as an anti-CTLA4, anti-PD-L1, anti-TIGIT, anti-ICOS, or anti-PD1 antibody) may develop anemia. Therefore, the present invention may be useful for treating or preventing anemia in such patients. In one example, a method or regimen treats or prevents anemia in a human suffering from cancer. In the art, an ESA, such as erythropoietin, is typically administered to such patients intravenously or subcutaneously initially at a dose of 150 units / kg three times a week, and alternatively at 40,000 units subcutaneously once a week until the course of chemotherapy is completed. In one example, the present invention treats or prevents anemia in a human cancer patient by administering an ESA to a human intravenously or subcutaneously three times a week at a total weekly dose of less than 150 units / kg, or less than 450 units / kg for one week, or at less than 40,000 units subcutaneously weekly.

[0455] ESA therapy has been used in the art to reduce the need for red blood cell (RBC) transfusion in patients, for example, patients undergoing surgery.Therefore, ESA therapy is used in human patients with preoperative hemoglobin levels above 10 g / dL but below 13 g / dL, who are at high risk of preoperative and postoperative blood loss due to surgery, such as elective surgery, non-cardiac surgery, non-vascular surgery, etc. ESA is administered subcutaneously at 300 units / kg once a day for 15 consecutive days (10 days before surgery, the day of surgery, and 4 days after surgery), or alternatively, 600 units / kg is administered subcutaneously four times, 21, 14, and 7 days before surgery and on the day of surgery. In one example, the invention treats or prevents anemia in human surgical patients, where an ESA is administered to a human at less than 300 units / kg once daily for 15 consecutive days (10 days before surgery, the day of surgery, and 4 days after surgery), or for a total 15-day dose of less than 4500 units / kg, or less than 600 units / kg in 3-5 or 4 doses, for example, 21 days, 14 days, 7 days before surgery and on the day of surgery.

[0456] 22. The method of embodiment 21, wherein moderate or severe anemia is treated or prevented in the subject.

[0457] 23. The method or regimen of any of embodiments 1-22, wherein the subject is a mammal.

[0458] 24. A combination therapy for use in any of the methods or regimens of aspects 1-23 for treating or preventing anemia in a subject, wherein the combination a. anti-BMP6 antagonists; b. ESA, and c. Combination therapy, optionally including instructions for use in the method or regimen.

[0459] 25. An anti-BMP6 antagonist for use in any of the methods or regimens of embodiments 1 to 24 for treating or preventing anemia in a subject.

[0460] 26. A combination of embodiment 24 or an antagonist of embodiment 25 for treating or preventing moderate or severe anemia.

[0461] 27. A combination of the antagonist of any one of aspects 24-26 in combination with an anti-inflammatory agent.

[0462] 28. The method, regimen, combination, or antagonist of any of embodiments 1-27, wherein the antagonist comprises an anti-BMP6 antibody binding site, e.g., the antagonist is an antibody or an anti-BMP6 trap.

[0463] In one example, the trap comprises a human BMP6 receptor domain fused to a human antibody Fc region. In one embodiment, the Fc comprises a human gamma-1 or -4 heavy chain constant region.

[0464] 29. The method, regimen, combination, or antagonist of any of embodiments 1-28, wherein the ESA is erythropoietin.

[0465] 30. The method or regimen of any one of aspects 1-23, 28, and 29, wherein an anti-inflammatory agent is administered to the subject.

[0466] In one example, the present invention uses an anti-BMP6 monoclonal antibody (mAb) to mobilize endogenous iron stores, increase hemoglobin synthesis, and optionally increase erythropoiesis. In one aspect, the present invention can reduce the need for simultaneous and common use of intravenous iron or blood transfusions in ACD patients. Additionally or alternatively, the present invention can reduce the dose underlying standard treatment with ESAs (e.g., EPO), or make patients who are unresponsive (or hyporesponsive) to ESAs (e.g., EPO) responsive to co-administration of ESAs with anti-BMP6 antagonists. Additionally or alternatively, the present invention can treat or prevent anemia in patients whose anemia is refractory or unresponsive to standard ESA treatment. ESAs are popular in patients with uncontrolled hypertension or with pure red cell aplasia (PRCA, a type of anemia) caused by receiving an ESA (e.g., darbepoetin alfa, such as Arenesp®, or epoetin alfa, such as Epogen® or Procrit®).

[0467] Thus, in one embodiment of the present invention, a subject (e.g., a human) i. Refractory or non-responsive to ESAs (e.g., darbepoetin alfa or epoetin alfa), ii. have or have had high blood pressure (e.g., uncontrolled high blood pressure); or iii. Have or have had pure red cell aplasia (e.g. caused by administration of ESAs such as darbepoetin alfa or epoetin alfa).

[0468] "Refractory" in the context of drug therapy, such as ESA therapy, is readily apparent to one of skill in the art and means, for example, that a subject is ESA-resistant or has a poor response (i.e., less than average response) to an ESA, and the anemia is not being effectively treated by standard therapy with an ESA.

[0469] ESAs are typically used to maintain hemoglobin at a minimum level, which both minimizes transfusions and best meets the patient's needs. As explained above, the present invention, in its various configurations, aspects, examples, and embodiments, is useful for ESA-sparing anemia therapy, i.e., allows treatment with a lower than standard ESA dose. This is useful in view of the potentially harmful side effects of ESAs. Tables A-D provide relevant information in this regard. [Table 1]

[0470] In one example, the subject is a chronic kidney disease (CKD) patient not undergoing dialysis. In one example, the subject is a chronic kidney disease (CKD) patient undergoing dialysis. In one example, the subject is a chemotherapy patient (e.g., undergoing or undergoing cancer chemotherapy). [Table B continues] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0471] In one embodiment, the treatment or prevention of the present invention reduces in a subject the occurrence or risk of one or more side effects listed in Table B, e.g., one or more of the "common," "more common," or "very common" side effects.

[0472] In one aspect, the present invention provides a method of ESA therapy with reduced side effects in a subject suffering from or at risk of anemia, comprising administering an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA) to the subject, wherein the anemia is treated or prevented. Optionally, the occurrence or risk of one or more ESA side effects listed in Table B (e.g., one or more of the "common," "more common," or "very common" side effects) is reduced. In one example, the therapy is anemia treatment. In one example, the therapy is anemia prevention. In one example, the anemia is moderate or severe anemia. [Table 3]

[0473] In one embodiment, the treatment or prevention of the present invention reduces the occurrence or risk of one or more side effects listed in Table C in a subject, e.g., shortened overall survival and / or increased risk of tumor progression or recurrence, and the subject is a breast cancer, non-small cell lung cancer, head and neck cancer, lymphoid cancer, and cervical cancer patient, or the risk of having a cardiovascular or thromboembolic reaction, such as stroke.

[0474] In one aspect, the present invention provides a method of ESA therapy with reduced side effects in a subject suffering from or at risk of anemia, the method comprising administering an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA) to the subject, wherein the anemia is treated or prevented. Optionally, the occurrence or risk of one or more ESA side effects listed in Table C (e.g., shortened overall survival and / or increased risk of tumor progression or recurrence, where the subject is a breast cancer, non-small cell lung cancer, head and neck cancer, lymphoid cancer, or cervical cancer patient, or the risk of having a cardiovascular or thromboembolic reaction, such as stroke) is reduced. In one example, the therapy is anemia treatment. In one example, the therapy is anemia prevention. In one example, the anemia is moderate anemia. In one example, the anemia is moderate to severe anemia. In one example, the anemia of the present invention is anemia caused by myelosuppressive chemotherapy. [Table 4]

[0475] Aspects of the present invention provide (i) and (ii). (i) A method for reducing the administration of an erythropoiesis-stimulating agent (ESA) for treating anemia to a subject suffering from anemia, the method comprising administering an anti-BMP6 antagonist and the ESA, wherein anemia is treated in the subject.

[0476] (ii) A method for treating anemia or reducing the risk of anemia in a subject suffering from or at risk of anemia, comprising administering to the subject an anti-BMP6 antagonist and a low dose of an erythropoiesis-stimulating agent (ESA), wherein anemia is treated or the risk of anemia is reduced in the subject.

[0477] In these example embodiments, the ESA epoetin alfa administered at a weekly dose of less than 1000, 1500, 2500, 5000, 11000, 18000, 34000, or 90000 units, and optionally the subject has previously received epoetin alfa therapy at <1500, 1500-2499, 2500-4999, 5000-10999, 11000-17999, 18000-33999, 34000-89999, or ≥ 90000 units per week, respectively; b. Darbepoetin alfa or Aranesp®, administered at a weekly dose of less than 6.25, 10, 12.5, 20, 25, 40, 60, 100, or 200 mcg, and optionally, the subject has previously received weekly darbepoetin alfa or Aranesp® treatment at 6.25, 10, 12.5, 20, 25, 40, 60, 100, or 200 mcg, respectively; or c. Darbepoetin alfa or Aranesp®, administered at a weekly dose of less than 6.25, 10, 20, 40, 60, 100, or 200 mcg, and optionally, the subject has previously received 1500-2499, 2500-4999, 5000-10999, 11000-17999, 18000-33999, 34000-89999, or ≧90000 units of epoetin alfa per week, respectively.

[0478] In one example, the ESA (i) epoetin alfa, administered at a weekly dose ranging from 3,000 to 30,000 units; or (ii) darbepoetin alfa or Aranesp®, administered in a weekly dose ranging from 15 to 100 mcg; and The subject is a human, for example an adult.

[0479] In one example, blood hemoglobin is elevated to or maintained at a level greater than 10 g / dL.

[0480] In one example, the subject is an adult. In one example, the subject is an infant. In one example, the subject is a human CKD patient undergoing dialysis treatment. In one example, the subject is a human with end-stage renal disease.

[0481] In the method of the present invention, a therapeutically effective amount or a prophylactically effective amount of antagonist and ESA is administered to a subject.In one example, the anti-BMP6 antagonist and ESA are administered to a subject at intervals of 10, 14, 21 or 28 days or less.For example, the anti-BMP6 antagonist and ESA are administered to a subject at intervals of 1 month or 2 months or less.

[0482] Examples of erythropoiesis-stimulating agents (ESAs) are epoetin alfa, Epogen®, Dynepo®, Eprex®, erythropoietin, darbepoetin alfa, Aranesp®, epoetin beta, NeoRecormon®, methoxypolyethylene glycol-epoetin beta, Mircera®, and Procrit®. In one embodiment, the ESA of the invention is any one of these or a combination of two or more of these.

[0483] In one example, the ESA comprises or consists of a recombinant erythropoietin, for example, selected from the table below: Erythropoietin has various glycosylation patterns, resulting in alpha, beta, delta, and omega forms. [Table 5]

[0484] In one example, the ESA of the present invention is selected from the group consisting of alpha, beta, delta, zeta and omega forms.

[0485] In one example, the ESA is a hypoxia-inducible factor prolyl hydroxylase (HIF-PH) inhibitor, such as roxadustat or FG-4592. HIF is the primary regulator of red blood cell (RBC) production in the body and represents a potentially novel mechanism for treating anemia. This novel mechanism of action is called a hypoxia-inducible factor prolyl hydroxylase (HIF-PH) inhibitor. HIF-PH inhibitors act by mimicking the body's natural response to anemia, allowing for controlled, adaptive stimulation of the body's erythropoietic system. This system-wide activation results in both increased red blood cell (RBC) production and improved stability of the bone marrow iron supply, ensuring the proper incorporation of iron required for such RBC production into hemoglobin. This adaptive simulation closely resembles the natural response induced when humans ascend to high altitudes. At higher altitudes, low levels of oxygen circulating in the bloodstream result in reduced HIF-PH activity in relevant cells in the kidneys and liver. Reduced HIF-PH activity stabilizes and increases intracellular levels of the proteins HIF1α and HIF2α (collectively referred to as HIFα). For most cells, the stabilization of HIF2α is greater than that of HIF1α, ultimately leading to increased erythropoietin (EPO) secretion and subsequent increased RBC production.

[0486] HIF-PH inhibitors work by blocking the action of prolyl hydroxylase enzymes, which promote the degradation of HIFα proteins. When degradation is inhibited, levels of these HIFα proteins increase in cells. These HIFs are the key protein mediators that allow the body and all of its individual cells to adapt to changes in oxygen levels. Both HIFα proteins are constantly produced, and their levels in cells are regulated by the activity of HIF-PH enzymes, which target HIFα proteins for degradation. HIF1α helps cells survive very low oxygen conditions, while HIF2α helps cells and the body adapt to moderate changes in oxygen levels, such as those that occur with altitude changes from sea level to 7,500 feet. When HIFα is stabilized, it binds to the protein HIFβ and translocates to the cell's nucleus. Together, they induce gene signals for the production of EPO and several other proteins. HIF-PH inhibitors increase HIFα levels by inhibiting HIF-PH enzymes in the body, in the same way that reduced oxygen increases HIFα levels. Sustained stabilization of HIFα (either by staying at higher altitudes or by daily administration of HIF-PH inhibitors) increases hemoglobin and RBC levels to increase the amount of oxygen circulating in the blood.

[0487] An example of an anti-BMP6 antibody is MAB507 (monoclonal mouse IgG2B, clone #74219), commercially available from R&D Systems. Other suitable antibodies are disclosed in US8980582, WO2016 / 098079, and US2016 / 0176956A1, the disclosures of which (and the sequences of antibodies, variable regions, and CDRs therein) are expressly incorporated by reference herein for potential use in the present invention.

[0488] In one embodiment, the antagonist comprises or consists of an antibody or antigen-binding fragment thereof that binds to human BMP-6 (SEQ ID NO: 1), comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises complementarity-determining regions (CDRs) LCDR1, LCDR2, and LCDR3, and the HCVR comprises CDRs HCDR1, HCDR2, and HCDR3, wherein LCDR1 is the polypeptide of SEQ ID NO: 2, LCDR2 is the polypeptide of SEQ ID NO: 3, LCDR3 is the polypeptide of SEQ ID NO: 4, HCDR1 is the polypeptide of SEQ ID NO: 5, HCDR2 is the polypeptide of SEQ ID NO: 6 or SEQ ID NO: 7, and HCDR3 is the polypeptide of SEQ ID NO: 8. The SEQ ID NOs in this paragraph are those disclosed in US8980582, and these sequences are expressly incorporated herein by reference for their possible use in the present invention and for possible inclusion in one or more claims herein.

[0489] In one embodiment, the antagonist comprises or consists of an antibody or antigen-binding fragment thereof that binds to human BMP-6 (SEQ ID NO: 1), comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises complementarity-determining regions (CDRs) LCDR1, LCDR2, and LCDR3, and the HCVR comprises CDRs HCDR1, HCDR2, and HCDR3, wherein LCDR1 is the polypeptide of SEQ ID NO: 2, LCDR2 is the polypeptide of SEQ ID NO: 3, LCDR3 is the polypeptide of SEQ ID NO: 4, HCDR1 is the polypeptide of SEQ ID NO: 5, HCDR2 is the polypeptide of SEQ ID NO: 6, and HCDR3 is the polypeptide of SEQ ID NO: 8. The SEQ ID NOs in this paragraph are those disclosed in US8980582, and these sequences are expressly incorporated herein by reference for their possible use in the present invention and for possible inclusion in one or more claims herein.

[0490] In one embodiment, the antagonist comprises or consists of an antibody or antigen-binding fragment thereof that binds to human BMP-6 (SEQ ID NO: 1), comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, and the HCVR comprises CDRs HCDR1, HCDR2, and HCDR3, wherein LCDR1 is the polypeptide of SEQ ID NO: 2, LCDR2 is the polypeptide of SEQ ID NO: 3, LCDR3 is the polypeptide of SEQ ID NO: 4, HCDR1 is the polypeptide of SEQ ID NO: 5, HCDR2 is the polypeptide of SEQ ID NO: 7, and HCDR3 is the polypeptide of SEQ ID NO: 8. The SEQ ID NOs in this paragraph are those disclosed in US8980582, and these sequences are expressly incorporated herein by reference for their possible use in the present invention and for possible inclusion in one or more claims herein.

[0491] In one embodiment, the antagonist comprises or consists of an antibody or antigen-binding fragment thereof that binds to human BMP-6 (SEQ ID NO: 1), comprising a LCVR and a HCVR, wherein the LCVR is the polypeptide of SEQ ID NO: 9, and the HCVR is the polypeptide of SEQ ID NO: 10 or SEQ ID NO: 11. In a further embodiment, the antagonist comprises or consists of an antibody or antigen-binding fragment thereof that binds to human BMP-6 (SEQ ID NO: 1), comprising a LCVR and a HCVR, wherein the LCVR is the polypeptide of SEQ ID NO: 9, and the HCVR is the polypeptide of SEQ ID NO: 10. In another embodiment, the antagonist comprises or consists of an antibody or antigen-binding fragment thereof that binds to human BMP-6 (SEQ ID NO: 1), comprising a LCVR and a HCVR, wherein the LCVR is the polypeptide of SEQ ID NO: 9, and the HCVR is the polypeptide of SEQ ID NO: 11. The SEQ ID NOs in this paragraph are those disclosed in US8980582, and these sequences are expressly incorporated herein by reference for their possible use in the present invention and for possible inclusion in one or more claims herein.

[0492] In one embodiment, the antagonist comprises or consists of an antibody that binds to human BMP-6 (SEQ ID NO: 1), comprising an LCVR and an HCVR, wherein the LCVR is the polypeptide of SEQ ID NO: 9, and the HCVR is the polypeptide of SEQ ID NO: 10 or SEQ ID NO: 11. In a further embodiment, the present invention provides an antibody that binds to human BMP-6 (SEQ ID NO: 1), comprising an LCVR and an HCVR, wherein the LCVR is the polypeptide of SEQ ID NO: 9, and the HCVR is the polypeptide of SEQ ID NO: 10. In another embodiment, the antagonist comprises or consists of an antibody that binds to human BMP-6 (SEQ ID NO: 1), comprising an LCVR and an HCVR, wherein the LCVR is the polypeptide of SEQ ID NO: 9, and the HCVR is the polypeptide of SEQ ID NO: 11. The SEQ ID NOs in this paragraph are those disclosed in US8980582, and these sequences are expressly incorporated herein by reference for their possible use in the present invention and for possible inclusion in one or more claims herein.

[0493] In one embodiment, the antagonist comprises or consists of an antibody that binds to human BMP-6 (SEQ ID NO: 1), comprising a light chain (LC) and a heavy chain (HC), wherein the LC is the polypeptide of SEQ ID NO: 12, and the HC is the polypeptide of SEQ ID NO: 13 or SEQ ID NO: 14. In a further embodiment, the antagonist comprises or consists of an antibody that binds to human BMP-6 (SEQ ID NO: 1), comprising a LC and a HC, wherein the LC is the polypeptide of SEQ ID NO: 12, and the HC is the polypeptide of SEQ ID NO: 13. In another embodiment, the antagonist comprises or consists of an antibody that binds to human BMP-6 (SEQ ID NO: 1), comprising a LC and a HC, wherein the LC is the polypeptide of SEQ ID NO: 12, and the HC is the polypeptide of SEQ ID NO: 14. The SEQ ID NOs in this paragraph are those disclosed in US8980582, and these sequences are expressly incorporated herein by reference for their possible use in the present invention and for possible inclusion in one or more claims herein.

[0494] In one embodiment, the antagonist comprises or consists of an antibody that binds to human BMP-6 (SEQ ID NO: 1), comprising two light chains and two heavy chains, each light chain being a polypeptide of SEQ ID NO: 12 and each heavy chain being a polypeptide of SEQ ID NO: 13. In one embodiment, the antagonist comprises or consists of an antibody that binds to human BMP-6 (SEQ ID NO: 1), comprising two light chains and two heavy chains, each light chain being a polypeptide of SEQ ID NO: 12 and each heavy chain being a polypeptide of SEQ ID NO: 14. The SEQ ID NOs in this paragraph are those disclosed in US8980582, and these sequences are expressly incorporated herein by reference for their possible use in the present invention and for possible inclusion in one or more claims herein.

[0495] In one embodiment, the present invention provides a pharmaceutical composition comprising an anti-BMP6 antagonist of the present invention (e.g., an antibody, or a BMP6-binding fragment thereof) and an acceptable carrier, diluent, or excipient. More specifically, the composition of the present invention further comprises one or more additional therapeutic agents, such as an ESA and / or an anti-inflammatory agent. Suitable anti-inflammatory agents may be antibodies or antibody fragments, such as anti-TNF-alpha antibodies (e.g., adalimumab, Humira®, infliximab, Remicade®, golimumab, Simponi®), or traps (e.g., etanercept or Enbrel®), or anti-TNFR antibodies or antibody fragments, or anti-IL6R antibodies (e.g., sarilumab, tocilizumab, or Actemra®).

[0496] In one example, the anti-BMP6 antagonist, antibody, or fragment is at a concentration of 1×10 as determined by common methods known in the art, e.g., using a surface plasmon resonance (SPR) biosensor at 37° C. -8 Less than 1 x 10 -9 Binds to BMP6 with a KD less than M.

[0497] "Effective amount" refers to the amount of an antagonist (e.g., an antibody) or ESA of the invention, or a pharmaceutical composition of the invention, that elicits the biological or clinical response or desired therapeutic effect in a subject, mammal, or human, that is being sought by a researcher, physician, or other clinician. An effective amount may vary depending on factors such as the individual's disease state, age, sex, and weight, and the ability of the antibody and / or ESA to elicit the desired response in the individual. An effective amount is also one in which any toxic or adverse effects are outweighed by the therapeutically beneficial effects.

[0498] Terms such as "treatment," "treat," and "treating" are intended to include slowing or reversing the progression of disorders such as anemia, moderate anemia, severe anemia, or decreased blood hemoglobin. These terms also include improving, alleviating, alleviating, eliminating, or reducing one or more symptoms of a disorder or condition (such as anemia, moderate anemia, severe anemia, or decreased blood hemoglobin), even if the disorder or condition is not actually completely eliminated. A subject or patient refers to a mammal that will benefit from the inhibition of BMP-6 activity, preferably a human with a disease, disorder, or condition (e.g., anemia or at risk of anemia). The term "preventing" refers to, for example, reducing the risk of a disease or condition, such as anemia.

[0499] Pharmaceutical compositions comprising the ESA, anti-BMP6 antagonist antibody, or antigen-binding fragment thereof, combination, or the like of the present invention can be administered parenterally (e.g., subcutaneously, intravenously, intraperitoneally, intramuscularly, or transdermally). Administration can be to a subject alone or in combination with a pharmaceutically acceptable carrier and / or diluent in single or multiple doses. The pharmaceutical compositions, combinations, or antagonists of the present invention can be administered by methods well known in the art (e.g., see Remington: The Science and Practice of Pharmacy, 1999). thed. (1995), A. Gennaro et al., Mack Publishing Co.), and may contain or be combined with one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0500] In one example, the subject suffers from moderate or severe anemia before administration of the BMP6 antagonist, and the moderate or severe anemia is treated.In one embodiment, the subject suffers from moderate anemia before administration, and after treatment, the subject has mild anemia or no anemia.In one embodiment, the subject suffers from severe anemia before administration, and after treatment, the subject has mild, moderate, or no anemia.In one embodiment, after treatment, the subject has mild anemia or no anemia, and is not moderate or severe anemia.In another embodiment, after treatment, the subject does not have anemia.In one embodiment, the subject has a blood hemoglobin level of less than 9.5 g / dL before administration, and after treatment, the subject has a blood hemoglobin level of at least 10, 11, 12, 13, or 14 g / dL.

[0501] Anemia is generally considered when the hemoglobin concentration falls below 11 g / dL in pregnant women, 12 g / dL in non-pregnant women, and 13 g / dL in men.

[0502] The severity of anemia is classified according to the following hemoglobin concentration ranges: A hemoglobin level of 9.5-13.0 g / dL is considered mild anemia. A hemoglobin level of 8.0-9.5g / dL is considered moderate anemia. A hemoglobin concentration of less than 8.0 g / dL is considered severe anemia.

[0503] In one example, the hemoglobin level is measured at sea level or equivalent.

[0504] In one embodiment, the subject is a human male, for example, an adult or an infant. In one embodiment, the subject is a human female, for example, an adult or an infant, for example, a non-pregnant woman or a pregnant woman. In one example, the human is a dialysis patient. The infant can be a human over one month old.

[0505] In one example, the method is a method for eliminating or reducing the need for iron administration or transfusions in a subject suffering from anemia, e.g., reducing the dose of iron or the frequency of iron dosing in a subject.

[0506] The present invention can include administering anti-BMP6 antagonist and ESA simultaneously or sequentially.In one example, the antagonist and ESA are administered at intervals of 1 month, 4 weeks, 3 weeks, 2 weeks, 1 week, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day or less.As exemplified herein, the administration of the antagonist and ESA can be effective when the interval is 7 days or less (for example, 1 day or less).In one example, the anti-BMP6 antagonist and ESA are administered to the subject at intervals of 10, 14, 21 or 28 days or less.

[0507] In one example, the ESA is administered 2, 3, or 4 times per week. In one example, the ESA is administered 1, 2, 3, or 4 times per month, or within an 8-week period. In one example, the ESA (e.g., epoetin alfa) is administered at a total dose of <3000, 2900, 2800, 2700, 2600, <2500, 2500, 2400, 2300, 2200, 2100, <2000, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, or 1000 units / kg per week. In another example, the ESA is administered 1, 2, 3, or 4 times per month, or within an 8-week period. In one example, the ESA (e.g., darbepoetin alfa) is administered at a total dose of <15, <30, 12, 11, 10, 9, 8, 7, 6, or 5 mcg / kg per week.

[0508] In one example, the ESA and / or antagonist is administered to the subject intravenously or subcutaneously.

[0509] In one example, the anemia is in a subject undergoing or having undergone zidovudine treatment.

[0510] Optionally, any configuration of the present invention also comprises: (a) increasing or maintaining elevated blood iron, for example, to treat or reduce the risk of anemia; (b) treating iron deficiency; (c) treating or reducing the risk of anemia of chronic inflammation (ACI); (d) treating or reducing the risk of anemia of chronic disease (ACD); (e) treating or reducing the risk of anemia associated with cancer, renal conditions, or GvHD; (f) increasing blood or serum iron concentrations; (g) increasing reticulocyte count; (h) increasing red blood cell count; (i) increasing hemoglobin; and (j) increasing the hematocrit of a subject (e.g., a human).

[0511] In one embodiment, the present invention is for modulating (eg, increasing) erythropoiesis in a subject.

[0512] In one embodiment, the subject is a human comprising the BMP6 gene SNP rs111588693, which may be correlated with an increased propensity for anemia.

[0513] In one example, the anemia is anemia of chronic disease (ACD), such as anemia of cancer or anemia of chronic kidney disease (CKD). Certain chronic diseases, such as cancer, kidney disease, and autoimmune disorders, can lead to ACD when excessive inflammatory cytokines cause dysregulation of iron homeostasis, reduced erythropoiesis, and reduced red blood cell lifespan. Hepcidin has been identified as an important hormone involved in iron homeostasis, and high levels of hepcidin are associated with iron-limited erythropoiesis seen in ACD. BMP-6 has been shown to increase hepcidin expression. In one example, the present invention is for reducing or maintaining reduced hepcidin levels in a subject.

[0514] Anemia of CKD is an early and common complication in patients with CKD. Anemia of cancer is anemia caused by hematological malignancies and some solid tumors, while chemotherapy-induced (e.g., immunotherapy-induced) anemia is anemia caused by treatment of cancer patients with chemotherapy agents. Anemia of CKD exacerbates diabetic neuropathy, cardiovascular disease, and retinopathy, among other conditions. Cancer-related anemia is associated with an increased relative risk of mortality. Current treatment options for cancer-related anemia are limited to blood transfusions because erythropoiesis-stimulating agents are only indicated for chemotherapy-induced anemia.

[0515] In one example, the subject suffers from a chronic disease such as cancer (e.g., hematological malignancy or solid tumor), kidney disease, autoimmune disorder or chemotherapy-induced anemia. In one example, the subject (e.g., human) suffers from CKD and one or more of diabetic neuropathy, cardiovascular disease and retinopathy.

[0516] In one example, anemia is hepcidin-related iron-restricted anemia.In one example, anemia is iron-refractory iron deficiency anemia (IRIDA).In one embodiment, IRIDA is caused by the defect of the target TMPRSS6 gene, for example, IRIDA is caused by TMPRSS6 gene mutation (for example, SNP such as rs855791, rs2543519, rs2235324 or rs1421312).

[0517] In one example, the method is a method of treating or preventing Sjogren's syndrome in addition to, or instead of, treating or preventing anemia.

[0518] In one example, the present invention is for increasing blood iron levels, serum iron levels, reticulocyte count, red blood cell count, hemoglobin, and / or hematocrit in a subject (eg, a human).

[0519] In one embodiment, the present invention provides the use of an anti-BMP6 antagonist and an ESA for the manufacture of a medicament. In a further embodiment, the present invention provides the use of an anti-BMP6 antagonist and an ESA for the manufacture of a medicament for the treatment or prevention of anemia, e.g., moderate to severe anemia. In another embodiment, the present invention provides the use of an anti-BMP6 antagonist and an ESA for the manufacture of a medicament for the treatment of anemia of chronic disease. In another embodiment, the present invention provides the use of an anti-BMP6 antagonist and an ESA for the manufacture of a medicament for the treatment of anemia of chronic kidney disease. In another embodiment, the present invention provides the use of an anti-BMP6 antagonist and an ESA for the manufacture of a medicament for the treatment of anemia of cancer. In one embodiment, the present invention provides the use of an anti-BMP6 antagonist and an ESA for the manufacture of a medicament for the treatment of IRIDA. In a further embodiment, the present invention provides the use of an anti-BMP6 antagonist and an ESA for the manufacture of a medicament for the treatment of IRIDA, wherein IRIDA is caused by a TMPRSS6 gene mutation (e.g., an SNP such as rs855791, rs2543519, rs2235324, or rs1421312). In one embodiment, the present invention provides the use of an anti-BMP6 antagonist and an ESA for the manufacture of a medicament for the treatment of Sjögren's syndrome.

[0520] Embodiment Embodiments of the present invention are as follows, and these embodiments (and all unnumbered paragraphs) can be combined with any other configuration, clause, paragraph, example, feature, or aspect of the present invention described herein, and the antagonists (e.g., anti-BMP6 antibodies or fragments) or ESAs of the present invention can be provided for use in (or can be used in) the methods in the following embodiments.

[0521] 1. An anti-bone morphogenetic protein 6 (BMP6) antagonist for use in a method for treating or preventing anemia in a subject, the method comprising administering the anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA) to the subject, wherein the anemia is treated or prevented.

[0522] In one embodiment, the method is for treating anemia in a subject, and the anemia is treated.

[0523] In one example, the antagonist comprises or consists of an anti-BMP6 antibody or fragment, such as a human, humanized, or chimeric antibody. As an alternative to an antibody or fragment, a different BMP6 antagonist, such as anti-BMP6 trap or HJV-Fc, is contemplated by the present invention.

[0524] 2. The antagonist comprises or consists of an anti-BMP6 antibody or fragment, and the method comprises: (a) on day 1 (D0), administering an anti-BMP6 antibody or fragment to a subject; (b) administering multiple doses of an ESA for at least three consecutive weeks, the period beginning on D0, wherein the subject's blood Hb concentration is increased from the baseline concentration at D0 throughout the period; As a result, (i) the Hb concentration is equal to or greater than 100% of the baseline Hb concentration for the entire period, and during the period the Hb concentration reaches at least 120% of the baseline; and / or (ii) the Hb concentration is increased by at least 1 g / dl over the entire period of time.

[0525] In any embodiment herein, in one example, the Hb concentration increases by at least 1 g / dl, e.g., at least 1.5, 2, or 2.5 g / dl, over the entire period of time above baseline. In one example, the Hb concentration is 11, 11.5, or 12 g / dl or less in a subject, e.g., an adult male or female human.

[0526] Hb concentration and MCH (see below) can be determined using one or more blood samples obtained from the subject, for example, as determined using blood samples taken at the end of each week of the period (and at baseline determined using a sample taken at D0).

[0527] 3. A combination of an amount of an anti-BMP6 antibody or fragment with an amount of an ESA (e.g., comprising multiple doses of the ESA) for use in a method for treating anemia, wherein the antibody, fragment and method are as described in embodiment 2.

[0528] 4. The combination of embodiment 3, wherein the method comprises obtaining a single dose from the amount of antibody or fragment, wherein the single dose is administered to the subject on DO, and obtaining multiple doses of the ESA, wherein at least one dose is administered weekly from DO.

[0529] In any of the embodiments herein, in one example, the first dose of ESA is administered on D0.

[0530] 5. The combination of embodiment 3 or 4, wherein the antibody or fragment is comprised by a pharmaceutical composition, wherein the antibody or fragment is mixed with a dose of ESA for administration to said subject on DO.

[0531] 6. A medical kit comprising the combination of any one of embodiments 3 to 5, a first sterile container containing said amount of antibody or fragment, and a second sterile container containing said amount of ESA, and optionally instructions for carrying out the method.

[0532] 7. The antagonist comprises or consists of an erythropoietin-stimulating agent (ESA) (e.g., constituted by multiple doses of the ESA), and the method comprises: (a) on day 1 (D0), administering an anti-BMP6 antibody or fragment to a subject; (b) administering multiple doses of the ESA for at least three consecutive weeks, the period beginning on D0, wherein the subject's blood hemoglobin (Hb) concentration is increased from the baseline concentration at D0 throughout the period; Such, (i) the Hb concentration is equal to or greater than 100% of the baseline Hb concentration for the entire period, and during the period the Hb concentration reaches at least 120% of the baseline; and / or (ii) the Hb concentration is increased by at least 1 g / dl over the entire period of time.

[0533] 8. (iii) The antagonist, combination or kit of any one of embodiments 2-7, wherein the Hb concentration on the last day of said consecutive weekly period is at least 120% of the Hb concentration on the 7th day immediately preceding said last day.

[0534] 9. The antagonist, combination or kit of any one of embodiments 2-8, wherein the ESA is administered to the subject within 24 hours of administration of the anti-BMP6 antibody or fragment.

[0535] 10. The antagonist, combination or kit of any one of embodiments 2-9, wherein said consecutive weekly periods consist of 3 or 4 consecutive weekly periods.

[0536] 11. The antagonist, combination, or kit of any one of embodiments 2-10 (e.g., embodiment 10), wherein during said period, Hb concentration reaches an increase over baseline in the range of 1 to 8 g / dl.

[0537] In any embodiment herein, in one example, during the period, the Hb concentration reaches an increase over baseline in the range of 1 to 3, 2.5, 2, 1.5, or 1.25 g / dl, e.g., the Hb concentration reaches an increase of 1 to 2 g / dl.

[0538] 12. The antagonist, combination or kit of any one of embodiments 2-11, wherein the period consists of 3 or 4 consecutive weeks, at the end of which Hb levels reach at least 150% of baseline.

[0539] 13. The period consists of 3 or 4 consecutive weeks; (a) the Hb concentration is equal to or greater than 110% of the baseline Hb concentration for the entire period, and during the period the Hb concentration reaches at least 150% of the baseline; and / or (b) the Hb concentration increases by at least 1 g / dl over the entire period, and during the period, the Hb concentration reaches an increase over baseline in the range of 1 to 8 g / dl, e.g., the Hb concentration reaches an increase of 1 to 2 g / dl.

[0540] 14. The antagonist, combination or kit of any one of embodiments 2 to 13, wherein said period the antibody or fragment is administered at DO.

[0541] 15. The antagonist, combination or kit of embodiment 14, wherein the antibody or fragment is administered to the subject as a single dose on DO.

[0542] In any of the embodiments herein, in one example, the antibody or fragment is administered to the subject as a single dose on DO, where the single dose is administered to the subject in one or more aliquots.

[0543] 16. The antagonist, combination or kit of any one of embodiments 2-15, wherein the first ESA dose is administered on DO or no more than 2 days thereafter.

[0544] 17. The antagonist, combination or kit of any one of embodiments 2-16, wherein the ESA dose is administered on day 4 to 9 (e.g. day 7) immediately after DO.

[0545] 18. The antagonist, combination or kit of any one of embodiments 2-17, wherein the ESA dose is administered immediately after DO, on days 12 to 16 (e.g., day 14).

[0546] 19. The antagonist, combination or kit of any one of embodiments 2-18, wherein the ESA dose is administered immediately after DO, on days 19 to 23 (e.g., day 21).

[0547] In any embodiment herein, in one example, the ESA dose is administered immediately after D0 on (i) days 4-9 (e.g., day 7), (ii) days 12-16 (e.g., day 14), and (iii) days 19-23 (e.g., day 21).

[0548] 20. The antagonist, combination or kit of any one of embodiments 2-19, wherein the equivalent of 4 ESA doses is administered during said period.

[0549] As used herein, the term "equivalent amount" contemplates that multiple aliquots of an ESA can be administered (e.g., on the same day or sequentially), with the aliquots representing a total dose of the ESA. In one example, the ESA is darbepoetin alfa or Aranesp®, and the dose ranges from 15 to 100 mcg (micrograms), or from 30 to 100 mcg. In one example, the ESA is epoetin alfa, and the dose ranges from 3,000 to 30,000 units (i.e., units refer to international units, also known in various languages as IU, UI, IE, ME, and NE).

[0550] Generally, as used herein, a dose (e.g., of an antibody, fragment, or ESA) can be administered in one aliquot or multiple aliquots (e.g., on the same day, simultaneously, within a 30, 1, or 24 hour time frame).

[0551] 21. The antagonist, combination or kit of any one of embodiments 2-20, wherein the ESA is administered to the subject during each of the first and second weeks after the initial ESA dose.

[0552] 22. The antagonist, combination or kit of any one of embodiments 2-21, wherein the ESA is administered to the subject during each of the first, second and third weeks after the initial ESA dose.

[0553] 23. The antagonist, combination or kit of embodiment 21 or 22, wherein the ESA is administered as a single dose at the end of each said week, optionally said period consisting of 3 or 4 weeks starting from DO.

[0554] In one example of any of the embodiments, the period consists of 4 weeks starting on D0, and anemia is treated in the fourth week.

[0555] 24. The antagonist, combination or kit of any one of embodiments 2-23, wherein during said period no more than 4 doses of an ESA, and optionally a single dose of said antibody or fragment, are administered to the subject.

[0556] 25. Over the period (which is 4 consecutive weeks), the subject is administered a total dose of antibody and a total dose of ESA in a ratio of X:Y, where X is between 10 and 2x10. 6 25. The antagonist, combination or kit of any one of embodiments 2-24, wherein and Y=4.

[0557] In one example, the total weekly dose of the ESA (e.g., when the subject is a human) is 10, 15, 16, 18, 200, or 300 mcg (micrograms). For example, the total weekly dose is 10 to 80, 15 to 80, or 30 to 80 mcg. For example, the ESA comprises or consists of darbepoetin alfa, epoetin alfa, or any other ESA disclosed herein. In one example, each dose (or weekly dose) of the ESA is administered to the subject in the range of 1.5 to 2 mcg / kg of the ESA.

[0558] In certain configurations, the method relates to reducing or sparing the administration of an ESA, where, for example, the total weekly dose of the ESA (e.g., when the subject is human) is 1 to 20 mcg, e.g., 1 to 15 mcg. In one example where ESA sparing or reduction is present, each dose (or weekly dose) of ESA is administered to the subject in the range of 0.01, or 0.1 to 0.3, or 1 mcg / kg of ESA, e.g., 0.01 to 0.3, or 0.1 to 0.3, or 0.01 to 1, or 0.1 to 1 mcg / kg.

[0559] 26. The antagonist, combination or kit of any one of embodiments 2-25, wherein the Hb concentration at the end of said period (e.g., 3 or 4 consecutive weeks) is at least 130% of the Hb concentration in control anemic patients of the same species who received an anti-BMP6 antibody or fragment in the same dosing regimen as the subject, except for patients who did not receive an ESA during said period.

[0560] 27. The antagonist, combination or kit of embodiment 26, wherein the Hb concentration at the end of said period is significantly higher than said control at the end of said period, as determined by a p-value of p<0.0001.

[0561] 28. The antagonist, combination or kit of any one of embodiments 2-27, wherein the mean corpuscular hemoglobin (MCH) at the end of said period is at least 109% of the MCH in control anemic patients of the same species who received an anti-BMP6 antibody or fragment in the same dosage regimen as the subject, excluding patients who did not receive an ESA during said period.

[0562] Mean corpuscular hemoglobin (MCH) is the average mass of hemoglobin per red blood cell in a blood sample.

[0563] 29. The antagonist, combination, or kit of any one of embodiments 2-28, wherein the Hb concentration at the end of the period (e.g., 3 or 4 consecutive weeks) is at least 120% of the Hb concentration in control anemic patients of the same species who received the ESA in the same dosage regimen as the subject, except for patients who did not receive an anti-BMP6 antibody or fragment during the period. Optionally, the control patients received a control IgG4 antibody that does not specifically bind to BMP6 (e.g., the BMP6 antibody and the control antibody are administered to the subject and control patients at the same doses, respectively). Optionally, X is between 10 and 2x10 5 , 2x10 4 or 2x10 3 is.

[0564] 30. The antagonist, combination or kit of embodiment 29, wherein the Hb concentration at the end of said period is significantly higher than said control at the end of said period, as determined by a p-value of p<0.0001.

[0565] 31. The antagonist, combination or kit of any one of embodiments 2-30, wherein the mean corpuscular hemoglobin (MCH) at the end of said period (e.g., 3 or 4 consecutive weeks) is at least 119% of the MCH in control anemic patients of the same species who received said ESA on the same dosing regimen as the subject, excluding patients who did not receive an anti-BMP6 antibody or fragment during said period.

[0566] 32. The antagonist, combination or kit of embodiment 31, wherein the MCH at the end of said period is significantly higher than the control at the end of said period, as determined by a p-value of p<0.0001.

[0567] 33. The antagonist, combination or kit of any one of embodiments 2-32, wherein the subject on DO suffers from anemia of chronic disease (ACD), and optionally the anemia is associated with chronic inflammation (e.g., the subject suffers from arthritis) or bacterial infection (e.g., a streptococcal infection), and the subject is a chronic kidney disease (CKD) patient.

[0568] 34. The antagonist, combination or kit of any one of embodiments 2-33, wherein anemia in said subject at the end of said period is less severe than at D0.

[0569] 35. The antagonist of embodiment 1, wherein the antagonist comprises or consists of an anti-BMP6 antibody or fragment.

[0570] 36. The antibody or fragment competes with a reference antibody for binding BMP6, the reference antibody being mAb507 (R&D Systems) or a. each heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or 2, and each light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 3, or b. The antagonist, combination or kit of any one of embodiments 2 to 34, which is an antibody comprising each heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 4, and each light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 5.

[0571] Competition herein can be determined, for example, by SPR (e.g., at 37°C, pH 7.6, optionally as Fab), by ELISA, by fluorescence-activated cell sorting (FACS), or in a homogeneous time-resolved fluorescence (HTRF) assay. SPR can be performed using Biacore™, Proteon™, or another standard SPR technology. In one embodiment, competition is determined by ForteBio Octet® Bio-Layer Interferometry (BLI), a technique readily apparent to those skilled in the art.

[0572] Alternatively, the reference antibody is any of the anti-BMP6 antibodies disclosed in WO2016 / 098079 (the sequences and disclosure relating to such antibodies incorporated herein for potential use in the present invention).

[0573] 37. The antagonist, combination, or kit of any one of embodiments 2 to 36, wherein the antibody or fragment competes with said reference antibody for binding to SEQ ID NO: 6. SEQ ID NO: 6 can be used as the peptide itself, as part of a larger peptide, or as part of a BMP6 protein (e.g., wild-type human BMP6 or recombinantly produced BMP6).

[0574] Additionally or alternatively, the antibody or fragment competes with the reference antibody for binding to an additional sequence selected from the group consisting of SEQ ID NOs: 7-19. The additional sequence can be a peptide itself, a portion of a larger peptide (e.g., comprising SEQ ID NO: 6), or a portion of a BMP6 protein (e.g., wild-type human BMP6 or recombinantly produced BMP6, e.g., comprising SEQ ID NO: 6). For example, the antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 7. For example, the antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 8. For example, the antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 9. For example, the antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 10. For example, the antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 11. For example, the antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 12. For example, the antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 13. For example, the antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 14. For example, an antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 15. For example, an antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 16. For example, an antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 17. For example, an antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 18. For example, an antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 19. The sequence ID numbers in this paragraph and in the embodiments herein are those disclosed in WO2017 / 191437, the disclosure of which is expressly incorporated herein by reference.

[0575] 38. The antagonist, combination, or kit of any one of embodiments 2-37, wherein the antibody or fragment competitively inhibits binding of soluble hemojuvelin (HJV) to BMP6.

[0576] 39. The antagonist, combination, or kit of any one of embodiments 1-38, wherein the antibody or fragment does not competitively inhibit the binding of soluble hemojuvelin (HJV) to BMP6 (e.g., as determined by SPR, HTRF, or ELISA).

[0577] 40. The antagonist, combination, or kit of any one of embodiments 2-39, wherein the antibody comprises a VH domain encoded by a VDJ region sequence, wherein the VDJ is derived from the recombination of a VH gene segment, a D gene segment, and a JH gene segment, and wherein the VH is a human germline (i) VH1-3, (ii) VH2-5, or (iii) VH3-15 gene segment.

[0578] 41. The antagonist, combination, or kit of any one of embodiments 2-40, wherein the antibody comprises a VL domain encoded by a VJ region sequence, wherein the VJ is derived from the recombination of a VL gene segment and a JL gene segment, and wherein the VL is human germline (iv) Vκ3-20, (v) Vλ3-1, (vi) Vκ1-17, or (vii) Vλ1-40.

[0579] 42. The antagonist, combination, or kit of any one of embodiments 2 to 41, wherein the antibody or fragment binds to BMP6 with a stronger affinity (lower KD as determined by SPR) than it binds to BMP7, and optionally with a stronger affinity than BMP5.

[0580] Optionally, the antibody or fragment binds to BMP6 with greater affinity than it binds to each of BMP2, 4, 5 and 9.

[0581] 43. The antagonist, combination or kit of any one of embodiments 2-42, wherein the antibody or fragment binds to a human BMP6 sequence comprising SEQ ID NO:6.

[0582] 44. The antagonist, combination, or kit of any one of embodiments 2-43, wherein the antibody has an affinity (KD) for binding BMP6 of 1 pM to 5 nM, and optionally, binding is determined by SPR using the Fab of the antibody at 37°C, pH 7.6.

[0583] In one example, the antibody (e.g., as a Fab) or fragment may be (a) 2, 3, 4, 5, or 10 pM to 3, 4, or 5 nM; (b) 1-10 pM-5 nM, (c) 10 pM to 3, 4, or 5 nM; (d) 50 or 80 pM to 200 nM; (e) 50 or 80 pM to 150 nM, or (f) have an affinity (KD) for binding BMP6 of 50 or 80 pM to 100 nM;

[0584] In one example, the KD is (or is about) 5-15 pM (e.g., 10 pM). In one example, the KD is (or is about) 2-5 nM (e.g., 3 nM). In one example, the KD is (or is about) 100-400 pM (e.g., 140 or 390 pM).

[0585] 45. Antibody is 1x10 -5 ~1×10 -3 S -1 Off-rate (K) for binding BMP6 off ), and optionally binding is determined by SPR using the Fab of the antibody at 37°C, pH 7.6.

[0586] In one example, the antibody (e.g., as a Fab) or fragment may be (a) 1x10-5 ~5×10 -4 S -1 , (b) 1x10 -5 ~6×10 -4 S -1 , (c)1x10 -5 ~7×10 -4 S -1 , (d) 1x10 -5 ~8×10 -4 S -1 , (e) 2x10 -5 ~1×10 -3 S -1 , (f)2x10 -5 ~5×10 -4 S -1 , (g)2x10 -5 ~6×10 -4 S -1 , (h)2x10 -5 ~7×10 -4 S -1 ,or (i) 2x10 -5 ~8×10 -4 S -1 Off-rate (K) for binding BMP6 off )

[0587] In one example, K off is 5x10 -4 S -1 (or about) (e.g., when the KD is 2 nM to 400 pM (or about), when the KD is 2 to 5 nM (e.g., 3 nM) (or about), or when the KD is 100 to 400 pM (e.g., 140 or 390 pM) (or about). off is 3x10 -5 S -1 is (or is about) 5 to 15 pM (e.g., 10 pM) (for example, when the KD is (or is about) 5 to 15 pM (e.g., 10 pM)).

[0588] 46. Antibody is 1x105 ~1×10 7 M -1 S -1 On-rate (K) for binding BMP6 on 46. The antagonist, combination, or kit of any one of embodiments 2 to 45 (e.g., embodiment 44 and / or 45), wherein the antibody has a Fab fragment (Fab fragment) of 2-46.

[0589] In one example, the antibody (e.g., as a Fab) or fragment may be (a) 1x10 5 ~1x10 6 M -1 S -1 , (b) 1x10 5 ~2x10 6 M -1 S -1 , (c)1x10 5 ~3x10 6 M -1 S -1 , (d) 1x10 5 ~4x10 6 M -1 S -1 , (e) 1x10 5 ~5x10 6 M -1 S -1 , (f)2x10 5 ~5x10 6 M -1 S -1 , (g)3x10 5 ~5x10 6 M -1 S -1 , (h)4x10 5 ~5x10 6 M -1 S -1 , (i) 5x10 5 ~5x10 6 M -1 S -1 ,or (j)6x10 5 ~5x10 6 M -1 S -1 On-rate (K) for binding BMP6 on )

[0590] In one example, K on is 1 or 2x10 -5 M -1 S -1 is (or is about) (e.g., when the KD is 2 to 5 nM (e.g., 3 nM)). on 1-4, 1, 2, 3 or 4x10 -6 M -1 S -1 (or about the same) (e.g., when the KD is 5 to 400 pM (e.g., 140 or 390 pM) or 5 to 15 pM (e.g., 10 pM)).

[0591] 47. (a) The period consists of three or four consecutive weeks; (i) for the entire period, the Hb concentration is equal to or greater than 110% of the baseline Hb concentration, and during the period, the Hb concentration reaches at least 120% of the baseline; and / or (ii) the Hb concentration increases by at least 1 g / dl over the entire period, and during the period, the Hb concentration reaches an increase over baseline in the range of 1 to 8 g / dl; (b) the dose of ESA is administered at least twice during the first 2 or 3 weeks of the period; (c) the antibody or fragment binds to BMP6 with a stronger affinity (lower KD as determined by SPR) than it binds to BMP7, and optionally binds to BMP6 with a stronger affinity than BMP5 (optionally binds to BMP6 with a stronger affinity than it binds to each of BMP2, 4, 5, and 9); (d) The antibody or fragment competes with a reference antibody for binding BMP6, the reference antibody being mAb507 (R&D Systems) or I. each heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or 2, and each light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 3, or II. The antagonist, combination, or kit of any one of embodiments 2 to 46, which is an antibody comprising each heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 4, and each light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 5.

[0592] Optionally, in part I, the heavy chain consists of the amino acid sequence of SEQ ID NO: 1, and the light chain consists of the amino acid sequence of SEQ ID NO: 3. Optionally, in part I, the heavy chain consists of the amino acid sequence of SEQ ID NO: 2, and the light chain consists of the amino acid sequence of SEQ ID NO: 3. Optionally, in part II, the heavy chain consists of the amino acid sequence of SEQ ID NO: 4, and the light chain consists of the amino acid sequence of SEQ ID NO: 5.

[0593] In one example (based on the antibody used in Example 2 below), in part (d), the anti-BMP6 antibody of the present invention is an antibody that competes with the reference antibody of Part I or Part II in an HTRF assay. For example, in an HTRF assay, the antibody of the present invention is a labeled antibody that is preincubated with human BMP6 and then combined with an unlabeled reference antibody (based on Part I or II), and competition between the antibodies is detected by the assay. In one example, the assay uses an AlexaFluor™ 647-labeled antibody of the present invention. Alternatively, human BMP6 is labeled (e.g., when using AlexaFluor™ 647, the test antibody is labeled with biotin to bind to Eu3+ cryptate-streptavidin, and the reference antibody is unlabeled).

[0594] Optionally, the anti-BMP6 antibody of the present invention (test antibody) competes with a reference antibody in an HTRF assay to bind to human BMP6 (or to bind to the same epitope of human BMP6 as the reference antibody), and the assay uses a test antibody directly or indirectly labeled with a donor (such as Eu3+ cryptate) or an acceptor fluorophore (such as AlexaFluor™ 647), and a target BMP6 labeled with either a donor or an acceptor fluorophore, allowing energy transfer between the donor and the acceptor, thereby producing and detecting a fluorescent signal. In one example, when an AlexaFluor™ 647 label is used, competition is detected by a reduction of at least 20% in the fluorescent signal at 665 nM when the test antibody is in the presence of the reference antibody compared to when the reference antibody is absent. Optionally, the reduction in the signal at 665 nM is at least 20, 30, 40, 50, 60, 70, 80, or 90%.

[0595] Optionally, the anti-BMP6 antibody (test antibody) competes with a reference antibody in an HTRF assay to bind to human BMP6 (or to bind to the same epitope of human BMP6 as the reference antibody), wherein the reference antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 or 2, and a light chain comprising the amino acid sequence of SEQ ID NO: 3, and the assay uses a test antibody directly or indirectly labeled with a donor label (e.g., Eu3+ cryptate) or an acceptor fluorophore label (e.g., AlexaFluor™ 647), and human BMP6 labeled with either an acceptor fluorophore or a donor, respectively, to allow energy transfer between the donor and acceptor, and the competition between the antibodies is detected by a reduction of at least 20% in the fluorescent signal when the test antibody is in the presence of the reference antibody compared to when the reference antibody is absent. For example, the test antibody is directly or indirectly labeled with AlexaFluor™ 647, and competition is detected by at least a 20% reduction in the fluorescent signal at 665 nM when the test antibody is in the presence of the reference antibody versus when the reference antibody is absent. Optionally, the reduction in signal at 665 nM is at least 20, 30, 40, 50, 60, 70, 80, or 90%.

[0596] Optionally, the anti-BMP6 antibody (test antibody) also competes with a reference antibody in an HTRF assay to bind to human BMP6 (or to bind to the same epitope of human BMP6 as the reference antibody), the reference antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 5, and the assay uses, for example, a test antibody directly or indirectly labeled with a donor label (e.g., Eu3+ cryptate) or an acceptor fluorophore label (e.g., AlexaFluor™ 647), and human BMP6 labeled with either an acceptor fluorophore or a donor, respectively, to allow energy transfer between the donor and acceptor, and the competition between the antibodies is detected by a reduction of at least 20% in the fluorescent signal when the test antibody is in the presence of the reference antibody compared to when the reference antibody is absent. For example, the test antibody is directly or indirectly labeled with AlexaFluor™ 647, and competition is detected by at least a 20% reduction in the fluorescent signal at 665 nM when the test antibody is in the presence of the reference antibody versus when the reference antibody is absent. Optionally, the reduction in signal at 665 nM is at least 20, 30, 40, 50, 60, 70, 80, or 90%.

[0597] For example, in part (b), doses of ESA are administered during the first 3 weeks of the period, or 2, 3 or 4 times during the period.

[0598] Alternatively, the reference antibody is any of the anti-BMP6 antibodies disclosed in WO2016 / 098079 (the sequences and disclosure relating to such antibodies incorporated herein for potential use in the present invention).

[0599] 48. a. Treating ACD in a subject; b. Treating or preventing moderate or severe anemia in a subject; c. Treating or preventing anemia in a subject, wherein the subject is suffering from an inflammatory disease or condition; d. Eliminates or reduces the need for iron administration or blood transfusions in a subject; e. Treating or preventing anemia in a subject, wherein the subject is suffering from a microbial infection; f. The antagonist, combination or kit of any one of embodiments 1-47 for reducing the administration of an ESA to a subject.

[0600] 49. A method of treating anemia in a subject, the method comprising: (a) on day 1 (D0), administering an anti-BMP6 antibody or fragment to a subject; (b) administering, for at least three consecutive weeks, multiple doses of an erythropoietin-stimulating agent (ESA) for said period beginning on D0, wherein said subject's blood hemoglobin (Hb) concentration is increased from the baseline concentration on D0 throughout said period; so that throughout the period, (i) the Hb concentration is equal to or greater than 100% of the baseline Hb concentration, during which the Hb concentration reaches at least 120% of baseline; and / or (ii) the Hb concentration increases by at least 1 g / dl above baseline.

[0601] 50. The method of embodiment 49, wherein the method, antibody fragment or ESA is as described in any one of embodiments 2 to 48.

[0602] 51. The antagonist, combination, kit or method of any of embodiments 1-50, wherein the anemia is moderate or severe anemia.

[0603] 52. An anti-bone morphogenetic protein 6 (BMP6) antagonist for use in a method for maintaining a blood hemoglobin (Hb) level of at least 10 g / dL in a subject, the method comprising administering to the subject an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA).

[0604] Any anti-BMP6 antibody or fragment of the present invention can be used as an anti-bone morphogenetic protein 6 (BMP6) antagonist.

[0605] 53. An anti-bone morphogenetic protein 6 (BMP6) antagonist for use in a method for preventing a decrease in a subject's blood hemoglobin level to below 10 g / dL, the method comprising administering to the subject an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA).

[0606] 54. An anti-bone morphogenetic protein 6 (BMP6) antagonist for use in a method for increasing blood hemoglobin to a level of at least 10 g / dL in a subject suffering from anemia, the method comprising administering to the subject an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA), wherein the anemia is treated.

[0607] 55. An anti-bone morphogenetic protein 6 (BMP6) antagonist for use in a method for treating or preventing anemia in a subject suffering from an inflammatory disease or condition, wherein the method comprises administering to the subject an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA), wherein the anemia is treated or prevented.

[0608] 56. An anti-bone morphogenetic protein 6 (BMP6) antagonist for use in a method for eliminating or reducing the need for iron administration or blood transfusions in a subject suffering from anemia, the method comprising administering to the subject an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA), wherein the need is eliminated or reduced.

[0609] 57. An anti-bone morphogenetic protein 6 (BMP6) antagonist for use in a method for treating or preventing anemia in a subject suffering from a microbial infection, the method comprising administering to the subject an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA).

[0610] 58. An anti-bone morphogenetic protein 6 (BMP6) antagonist for use in a method for reducing the administration of an erythropoiesis-stimulating agent (ESA) to a subject suffering from anemia for treating anemia, wherein the method comprises administering an anti-BMP6 antagonist and the ESA, and wherein anemia is treated in the subject.

[0611] 59. An anti-bone morphogenetic protein 6 (BMP6) antagonist for use in a method for treating anemia or reducing the risk of anemia in a subject suffering from or at risk of anemia, the method comprising administering to the subject an anti-BMP6 antagonist and a low dose of an erythropoiesis-stimulating agent (ESA), wherein the anemia is treated or the risk of anemia is reduced in the subject.

[0612] 60. The antagonist of any one of embodiments 52-59, wherein the antagonist is as defined in any one of embodiments 1-1, 2, 7-48 and 51.

[0613] 61. The antagonist of any one of embodiments 52-59, wherein the antagonist is as described in any other of embodiments 52-59.

[0614] 62.ESA epoetin alfa administered at a weekly dose of less than 1000, 1500, 2500, 5000, 11000, 18000, 34000, or 90000 units, and optionally the subject has previously received epoetin alfa therapy at <1500, 1500-2499, 2500-4999, 5000-10999, 11000-17999, 18000-33999, 34000-89999, or ≥ 90000 units per week, respectively; b. Darbepoetin alfa or Aranesp®, administered at a weekly dose of less than 6.25, 10, 12.5, 20, 25, 40, 60, 100, or 200 mcg, and optionally, the subject has previously received weekly darbepoetin alfa or Aranesp® treatment at 6.25, 10, 12.5, 20, 25, 40, 60, 100, or 200 mcg, respectively; or c. The antagonist, combination, kit or method of any of embodiments 1-61, wherein the darbepoetin alfa or Aranesp® is administered at a weekly dose of less than 6.25, 10, 20, 40, 60, 100, or 200 mcg, and optionally the subject has previously received 1500-2499, 2500-4999, 5000-10999, 11000-17999, 18000-33999, 34000-89999, or ≧90000 Units of epoetin alfa therapy per week, respectively.

[0615] 63. The antagonist, combination, kit or method of any of embodiments 1-62, wherein the anti-BMP6 antagonist is an antibody and each dose is administered at a total of 30 mg / kg or less.

[0616] 64. An anti-BMP6 antagonist and / or ESA for use in a therapeutic regimen method for treating or preventing anemia in a subject suffering from or at risk of anemia, comprising administering to the subject an anti-BMP6 antagonist and an ESA simultaneously or sequentially; a. On day 0, the subject is administered an antagonist, and by day 7 (e.g., on day 1), the subject is administered an ESA; or b. On day 0, an ESA is administered to the subject, and by day 7 (e.g., on day 1), an antagonist is administered to the subject; or c. On day 0, the antagonist and ESA are administered simultaneously to the subject; or d. On day 0, the subject is already receiving an ESA, and on day 0, the subject is administered an antagonist; or e. On day 0, the subject has already received an antagonist, and on day 0, the subject is administered an ESA; The anti-BMP6 antagonist and / or ESA, whereby on day 14 or thereafter, the subject has a blood hemoglobin level of at least 10 g / dL, and the anemia is treated or prevented.

[0617] 65. The antagonist and / or ESA according to embodiment 64, further described in any one of embodiments 1-1, 2, 7-48 and 51.

[0618] 66. The antagonist and / or ESA according to embodiment 64 or 65, wherein the anti-BMP6 antagonist and the ESA are administered to the subject at intervals not exceeding 7 days.

[0619] 67. The antagonist and / or ESA of embodiment 64, 65 or 66, wherein the regimen maintains blood Hb levels in the subject above 10 g / dL in the subject.

[0620] 68. The antagonist and / or ESA described in any one of embodiments 64 to 67, wherein the method maintains or increases blood hemoglobin levels in the subject to at least 10 g / dL on at least 13 or 14 days after the subject receives the anti-BMP6 antagonist and ESA.

[0621] 69. The antagonist and / or ESA according to embodiment 67 or 68, wherein the anti-BMP6 antagonist and the ESA are administered to the subject at intervals of not more than 1 day.

[0622] 70. The antagonist and / or ESA according to any one of embodiments 64 to 69, wherein the anti-BMP6 antagonist and the ESA are administered to the subject simultaneously.

[0623] 71. The antagonist and / or ESA according to any one of embodiments 64-70, wherein the subject's blood hemoglobin level is prevented from falling below 10 g / dL (e.g., on day 14).

[0624] 72. The antagonist and / or ESA according to any one of embodiments 64-71, wherein the subject's blood hemoglobin is increased to a level of at least 10 g / dL (e.g., on day 14).

[0625] 73. The antagonist and / or ESA according to any one of embodiments 64-72, wherein moderate or severe anemia is prevented in the subject (e.g., on day 14).

[0626] 74. The target a. suffers from an inflammatory disease or condition, or b. Have an infectious disease? c. Have kidney disease, d. Have HIV or are undergoing treatment for HIV; or e. have cancer, The antagonist, combination, kit, ESA or method of any of embodiments 1-73, wherein anemia is treated or prevented in a subject.

[0627] 75. The antagonist, combination, kit, ESA or method of any of embodiments 1-74, wherein the subject is a mammal.

[0628] 76. The antagonist, combination, kit, ESA or method of any of embodiments 1-75, in combination with an anti-inflammatory agent, or wherein an anti-inflammatory agent is administered to the subject.

[0629] 77. The antagonist, combination, kit, ESA or method of any of embodiments 1-76, wherein the ESA is erythropoietin.

[0630] In one example, the subject is suffering from chronic kidney disease (CKD). See "KDIGO Clinical Practice Guideline for Anemia in Chronic Kidney Disease," Kidney International Supplements (2012) 2,279; doi:10.1038 / kisup.2012.37, which describes the stages of chronic kidney disease (stages 1-5), diagnosis, CKD nomenclature, Hb levels and ranges for humans of various ages, and ESA...

Claims

1. (i) An antibody or fragment that specifically binds to bone morphogenetic protein 6 (BMP6), the antibody or fragment comprises a VH domain and a VL domain; (a) the VH domain comprises a CDRH1 comprising SEQ ID NO: 108, a CDRH2 comprising SEQ ID NO: 109, and a CDRH3 comprising SEQ ID NO: 110; and the VL domain comprises a CDRL1 comprising SEQ ID NO: 117, a CDRL2 comprising SEQ ID NO: 118, and a CDRL3 comprising SEQ ID NO: 119; (b) the VH domain comprises a CDRH1 comprising SEQ ID NO: 111, a CDRH2 comprising SEQ ID NO: 112, and a CDRH3 comprising SEQ ID NO: 113; and the VL domain comprises a CDRL1 comprising SEQ ID NO: 120, a CDRL2 comprising SEQ ID NO: 121, and a CDRL3 comprising SEQ ID NO: 122; (c) the VH domain comprises a CDRH1 comprising SEQ ID NO:288, a CDRH2 comprising SEQ ID NO:289, and a CDRH3 comprising SEQ ID NO:290; and the VL domain comprises a CDRL1 comprising SEQ ID NO:297, a CDRL2 comprising SEQ ID NO:298, and a CDRL3 comprising SEQ ID NO:299, or (d) the VH domain comprises a CDRH1 comprising SEQ ID NO:291, a CDRH2 comprising SEQ ID NO:292, and a CDRH3 comprising SEQ ID NO:293; and the VL domain comprises a CDRL1 comprising SEQ ID NO:300, a CDRL2 comprising SEQ ID NO:301, and a CDRL3 comprising SEQ ID NO:

302. an antibody or fragment; (ii) an erythropoietin-stimulating agent (ESA); and Kit including: Claim 2: The VH domain of the antibody or fragment is encoded by a nucleotide sequence derived from the recombination of a human VH gene segment, a D gene segment, and a JH gene segment; (a) the VH gene segment is IGHV3-11; (b) the DH gene segment is IGHD6-19, and / or (c) the JH gene segment is IGHJ4.

3. The antibody or fragment thereof (i) a VH domain comprising SEQ ID NO: 110 paired with a VL domain comprising SEQ ID NO: 119; or (ii) a VH domain comprising SEQ ID NO: 113 paired with a VL domain comprising SEQ ID NO: 122; or (iii) a VH domain comprising SEQ ID NO: 290 paired with a VL domain comprising SEQ ID NO: 299; or (iv) a VH domain comprising SEQ ID NO: 293 paired with a VL domain comprising SEQ ID NO:

302.

4. The antibody or fragment thereof (a) a VH domain comprising an amino acid sequence at least 70% identical to SEQ ID NO: 114 and / or a VL domain comprising an amino acid sequence at least 70% identical to SEQ ID NO: 123; (b) a VH domain comprising SEQ ID NO: 114 and / or a VL domain comprising SEQ ID NO: 123; (c) a heavy chain amino acid sequence that is at least 70% identical to SEQ ID NO: 116 and / or a light chain amino acid sequence that is at least 70% identical to SEQ ID NO: 125; (d) a heavy chain amino acid sequence comprising SEQ ID NO: 116 and / or a light chain amino acid sequence comprising SEQ ID NO: 125; (e) a VH domain comprising an amino acid sequence at least 70% identical to SEQ ID NO: 294 and / or a VL domain comprising an amino acid sequence at least 70% identical to SEQ ID NO: 303; (f) a VH domain comprising SEQ ID NO: 294 and / or a VL domain comprising SEQ ID NO: 303; (g) a heavy chain amino acid sequence that is at least 70% identical to SEQ ID NO: 296 and / or a light chain amino acid sequence that is at least 70% identical to SEQ ID NO: 305; or (h) a heavy chain amino acid sequence comprising SEQ ID NO: 296 and / or a light chain amino acid sequence comprising SEQ ID NO: 305; The kit according to any one of claims 1 to 3, comprising:

5. A kit described in any one of claims 1 to 4, wherein the antibody or fragment comprises a first and a second copy of the VH domain.

6. The antibody or fragment thereof, wherein the VL domain is encoded by a nucleotide sequence derived from the recombination of a human VL gene segment and a JL gene segment; (a) the VL gene segment is IGKV3-20, and / or (b) the JL gene segment is IGKJ1.

7. A kit described in any one of claims 1 to 6, wherein the antibody or fragment comprises a first and a second copy of the VL domain.

8. The kit of claim 1, wherein the antibody or fragment comprises a human constant region, optionally an IgG4 constant region or an IgG1 constant region.

9. The kit described in claim 8, wherein the constant region is an IgG4-PE constant region, and optionally, the constant region comprises the amino acid sequence of SEQ ID NO:

454.

10. A kit described in any one of claims 1 to 9, wherein the antibody or fragment further comprises an antigen binding site that specifically binds to another target antigen (optionally human hemojuvelin, transferrin receptor or BMP receptor), or that binds to BMP6 and another BMP (optionally BMP2, 4, 7 or 9).

11. A kit described in any one of claims 1 to 10, comprising multiple doses of the antibody or fragment thereof and / or ESA.

12. A kit described in any one of claims 1 to 11, wherein the ESA is recombinant erythropoietin.

13. A kit described in any one of claims 1 to 12, wherein the ESA is one or more of epoetin alfa, Epogen (registered trademark), Dynepo (registered trademark), Eprex (registered trademark), erythropoietin, darbepoetin alfa, Aranesp (registered trademark), epoetin beta, NeoRecormon (registered trademark), methoxypolyethylene glycol-epoetin beta, Mircera (registered trademark), or Procrit (registered trademark).

14. A kit described in any one of claims 1 to 13, wherein the antibody or fragment is contained in a first sterilized container and the ESA is contained in a second sterilized container.

15. A kit described in any one of claims 1 to 14, further comprising an additional therapeutic agent.

16. The kit described in claim 15, wherein the additional therapeutic agent is for treating, managing or ameliorating a BMP6-mediated disease.

17. The additional therapeutic agent: (a) intravenous iron, (b) ESA (optionally EPO), (c) an ActRIIa inhibitor; (d) an ActRIIb inhibitor; (e) an IL-6 or IL-6 receptor inhibitor (optionally an anti-IL-6 or IL-6 receptor antibody); (f) a TNF alpha or TNF alpha receptor inhibitor (optionally an anti-TNF alpha or TNF alpha receptor antibody); (g) an HJV inhibitor (optionally an anti-HJV antibody); (h) a BMP antagonist (optionally a further anti-BMP antibody or fragment), optionally wherein the BMP is BMP 2, 4, 5, 6, 7, or 9; (i) a matriptase-2 (MTP2) agonist (optionally a matriptase-2 (MTP2) agonist antibody); (j) HIF-PH inhibitors, (k) transferrin receptor 2 (TFR2) inhibitors; (l) HFE inhibitors, (m) an NRf2 inhibitor, (n) transforming growth factor beta superfamily type 1 activin receptor-like kinase (ALK) receptor inhibitors; (o) an activin receptor inhibitor (optionally an activin receptor Fc fusion); (p) a GDF11 inhibitor, and (q) a myostatin inhibitor.

18. A kit according to any one of claims 1 to 17, further comprising a label or instructions for use in treating and / or preventing a BMP6-mediated condition or disease, optionally wherein the disease or condition is selected from anemia, pulmonary arterial hypertension (PAH), cerebral cavernous hemangioma (CCM), restless legs syndrome (RLS), cancer, cancer metastasis, systemic sclerosis, Sjogren's syndrome, endothelial-mesenchymal transition (EndoMT), cardiovascular disease, atherosclerosis, systemic sclerosis-associated pulmonary fibrosis and cardiac fibrosis.

19. The kit of claim 18, wherein the label or instructions include a marketing authorization number (optionally, an FDA or EMA approval number).

20. The kit of any one of claims 1 to 19, comprising an IV or injection device containing the antibody or fragment.