Antagonist

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] This 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 from 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 of treating or preventing anemia (see, e.g., WO2016 / 098079, US2016 / 0176956A1). See also WO2017 / 191437, which discloses combinations of anti-BMP6 antagonists with erythropoietin enhancers (ESAs).

[0003] Anemia is a major disease affecting 25% of the world's population, or more than 1.7 billion people, particularly pregnant women, newborns, and children. More than 40% of anemias reflect dysfunction in the homeostatic control of iron intake, storage, and reuse. This dysregulation is a result of various chronic diseases, including infections (e.g., HIV, hepatitis), inflammation (e.g., rheumatoid arthritis), cancer, and kidney disease. The profound impact of diseases causing dysregulation of iron homeostasis can be seen in the United States, where 10% of the 40 million adults over 65 suffer from anemia, and one-third of these are caused by chronic disorders.

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

[0005] The Hump gene encodes hepcidin, a 25-amino acid peptide hormone produced by the liver. Hepcidin primarily functions by regulating the flow of iron from cells by modulating the amount of ferroportin, an iron transporter present on the cell surface of cells involved in iron transport. Hepcidin interacts with ferroportin, which is mainly expressed in macrophages and duodenal intestinal cells, causing its internalization and degradation (Ganz and Nemeth, 2011; Ramey et al., 2010). BMP6 induces hepcidin expression through mechanisms involving several receptors and cofactors. These include BMP class I and II receptors, matryptase-2, neogenin, HFE, and transferrin receptors 1 and 2, which are essential for inducing Hump expression and are members of the RGM family of repulsion guidance molecules, such as RGMc (hemomoduvelin, HJV) and RGMb (DRAGON). The SMAD pathway is thought to be triggered by the initial interaction between BMP6 and class IBMP receptors, possibly involving HJV. This leads to association with autophosphorylated BMPRII and activation of BMPRI phosphorylation, initiating the SMAD cascade. Phosphorylated SMAD1, 5, and 8, and ultimately SMAD4, translocate to the nucleus and interact with BMP response elements in the Hump gene regulatory region, inducing hepcidin expression. In mice, liver-specific disruption of SMAD4 signaling molecules or type I receptors Alk2 and ALk3 reduces hepcidin expression, similar to BMP6 knockout, confirming that these factors are also part of the related 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, [Overview of the Initiative] [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 the CDRH3 sequence of the VH domain including SEQ ID NO: 114.

[0007] An antibody or fragment comprising a binding site that specifically binds to BMP6, wherein the binding site comprises the VH domain containing SEQ ID NO: 114, or an amino acid that is 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 containing SEQ ID NO: 123.

[0009] An antibody or fragment comprising a binding site that specifically binds to BMP6, wherein the binding site comprises a VL domain containing SEQ ID NO: 123, or an amino acid that is 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 containing SEQ ID NO: 116, or amino acids that are at least 70% identical thereto, and / or (ii) a light chain sequence containing SEQ ID NO: 125, or amino acids that are at least 70% identical thereto.

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

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

[0013] In the second configuration, the present invention is The present invention provides an antibody or fragment that specifically binds to BMP6 and contains the CDRH3 sequence of the anti-BMP6 antibody described in the present invention, or the CDRH3 sequence containing 3, 2, or 1 amino acid substitutions (multiple substitutions are possible).

[0014] In the third configuration, the present invention is The present invention provides an antibody or fragment that specifically binds to BMP6 and contains a VH domain containing 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 an antibody or fragment containing the sequence containing 3, 2, or 1(s) amino acid substitutions.

[0015] In the fourth configuration, the present invention is The present invention provides an antibody or fragment containing a binding site that specifically binds to BMP6, wherein the binding site contains 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 that are at least 70% identical thereto.

[0016] In the fifth configuration, the present invention is The present invention provides an antibody or fragment containing a binding site that specifically binds to BMP6, the binding site comprising a VL domain encoded by a nucleotide sequence derived from a recombination of the human VL gene segment and JL gene segment, the VL gene segment being selected from IGKV3-20, IGKV1-5, and IGKV3-15.

[0017] In the sixth configuration, the present invention is The present invention provides an antibody or fragment that specifically binds to BMP6 and contains the CDRL3 sequence of the anti-BMP6 antibody of the present invention, comprising 3, 2, or 1 amino acid substitution(s).

[0018] In the seventh configuration, the present invention is The present invention provides antibodies or fragments containing a VL domain containing the CDRL3 (and optionally 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 the said sequence(s) containing 3, 2, or 1 amino acid substitutions(s).

[0019] In the eighth configuration, the present invention is The present invention provides an antibody or fragment containing a binding site that specifically binds to BMP6, wherein the binding site contains 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 that are at least 70% identical thereto.

[0020] In the ninth configuration, the present invention is The present invention provides an antibody or fragment that specifically binds to BMP6 and contains 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 containing amino acids that are at least 70% identical thereto.

[0021] In the tenth configuration, the present invention is The present invention provides an antibody or fragment that specifically binds to BMP6 and contains 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 containing amino acids that are at least 70% identical thereto.

[0022] In the eleventh configuration, the present invention is The present invention provides an antibody or fragment that specifically binds to a human BMP6 epitope that is identical to the epitope to which the antibody of the present invention (e.g., CL-58838) binds.

[0023] In the twelfth configuration, the present invention is This invention provides an antibody or fragment that competes with the antibody of the present invention in terms of binding to human BMP6.

[0024] In the 13th configuration, the present invention is The present invention provides an anti-BMP6 antibody or fragment for treating or preventing BMP6-mediated diseases or conditions (optionally, anemia) in a subject.

[0025] In the 14th configuration, the present invention is The present invention provides a combination of a certain amount of anti-BMP6 antibody or fragment and a certain amount of ESA (optionally including multiple doses of the antibody and / or ESA), wherein the antibody or fragment is one of the present invention.

[0026] In the 15th configuration, the present invention is The present invention provides for the use of antibodies, fragments, or combinations in the manufacture of pharmaceuticals for administration to a subject to treat or prevent BMP6-mediated diseases or conditions, or, optionally, anemia.

[0027] In the 16th configuration, the present invention is The present invention provides a method for treating or preventing a BMP6-mediated disease or condition (optionally, anemia) in a subject, the method comprising administering a therapeutically effective amount of the present invention antibody, fragment, or combination to the subject, thereby treating or preventing the BMP6-mediated disease or condition.

[0028] In the 17th configuration, the present invention is The present invention provides a pharmaceutical composition comprising an antibody, fragment, or combination thereof, and a pharmaceutically acceptable excipient, diluent, or carrier.

[0029] In the 18th configuration, the present invention is The present invention provides nucleic acids encoding the VH domain and / or VL domain of an antibody or fragment.

[0030] In the 19th configuration, the present invention is The present invention provides a VH domain containing 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 a nucleic acid encoding amino acids that are at least 70% identical thereto.

[0031] In the 20th configuration, the present invention provides the following: (a) A nucleotide sequence that is at least 70% identical to the sequence of sequence number 115, 520, or 521, and / or (b) Nucleic acids containing a nucleotide sequence that is at least 70% identical to sequence 124, 522, or 523 (e.g., in host cells, e.g., CHO, HEK293, or Cos cells).

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

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

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

[0035] In the 21st configuration, the present invention provides the following: (a) A nucleotide sequence that is at least 70% identical to the sequence of sequence number 115, 520, or 521, and / or (b) A combination of first and second nucleic acids (e.g., in host cells, e.g., CHO or HEK293 or Cos cells) each containing 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 Sequence ID No. 115, and / or (b) A combination of first and second nucleic acids, each containing a nucleotide sequence that is at least 70% identical to the sequence of Sequence ID No. 124 (e.g., in a host cell, e.g., CHO or HEK293 or Cos cell).

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

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

[0039] In the 22nd configuration, the present invention is The present invention provides nucleic acids encoding the heavy and / or light chains of an antibody or fragment.

[0040] In the 23rd configuration, the present invention is This invention provides a nucleic acid encoding a heavy chain containing an amino acid sequence that is at least 70% identical to sequence number 116.

[0041] In the 24th configuration, the present invention is This invention provides a nucleic acid encoding a light chain containing an amino acid sequence that is at least 70% identical to sequence number 125.

[0042] In configuration 25, the present invention provides the following: (a) A nucleotide sequence that is at least 70% identical to the 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 host cells, e.g., CHO or HEK293 or Cos cells) containing a nucleotide sequence that is at least 70% identical to the 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 sequence numbers 512, 514, 516, 518 and 519, and / or (b) A nucleic acid (e.g., in a host cell, e.g., CHO or HEK293 or Cos cell) having a nucleotide sequence that is at least 70% identical to a sequence selected from sequence numbers 513, 515, and 517.

[0044] (a) Nucleotide sequences that are at least 70% identical to Sequence ID No. 512, and / or (b) A nucleic acid containing a nucleotide sequence that is at least 70% identical to SEQ ID NO: 513 (e.g., in host cells, e.g., CHO or HEK293 or Cos cells).

[0045] (a) Nucleotide sequences that are at least 70% identical to Sequence ID No. 516, and / or (b) A nucleic acid containing a nucleotide sequence that is at least 70% identical to SEQ ID NO: 517 (e.g., in host cells, e.g., CHO or HEK293 or Cos cells).

[0046] (a) Nucleotide sequences that are at least 70% identical to Sequence ID No. 518, and / or (b) A nucleic acid containing a nucleotide sequence that is at least 70% identical to SEQ ID NO: 513 (e.g., in host cells, e.g., CHO or HEK293 or Cos cells).

[0047] (a) Nucleotide sequences that are at least 70% identical to Sequence ID No. 519, and / or (b) A nucleic acid containing a nucleotide sequence that is at least 70% identical to SEQ ID NO: 513 (e.g., in host cells, e.g., CHO or HEK293 or Cos cells).

[0048] (a) Nucleotide sequences that are at least 70% identical to Sequence ID No. 518, and / or (b) A nucleic acid containing a nucleotide sequence that is at least 70% identical to SEQ ID NO: 517 (e.g., in host cells, e.g., CHO or HEK293 or Cos cells).

[0049] (a) Nucleotide sequences that are at least 70% identical to Sequence ID No. 519, and / or (b) A nucleic acid containing a nucleotide sequence that is at least 70% identical to SEQ ID NO: 517 (e.g., in host cells, e.g., CHO or HEK293 or Cos cells).

[0050] (a) Nucleotide sequences that are at least 70% identical to Sequence ID No. 514, and / or (b) A nucleic acid containing a nucleotide sequence that is at least 70% identical to SEQ ID NO: 515 (e.g., in host cells, e.g., CHO or HEK293 or Cos cells).

[0051] (a) Nucleotide sequences that are at least 70% identical to Sequence ID No. 516, and / or (b) A nucleic acid containing a nucleotide sequence that is at least 70% identical to SEQ ID NO: 517 (e.g., in host cells, e.g., CHO or HEK293 or Cos cells).

[0052] (a) Nucleotide sequences that are at least 70% identical to Sequence ID No. 518, and / or (b) Nucleic acids containing a nucleotide sequence that is at least 70% identical to sequence numbers 513, 515, or 517 (e.g., in host cells, e.g., CHO or HEK293 or Cos cells).

[0053] (a) Nucleotide sequences that are at least 70% identical to Sequence ID No. 519, and / or (b) Nucleic acids containing a nucleotide sequence that is at least 70% identical to sequence numbers 513, 515, or 517 (e.g., in host cells, e.g., CHO or HEK293 or Cos cells).

[0054] In the 26th configuration, the present invention is We provide a vector containing nucleic acids (multiple nucleic acids are possible), and optionally the vector is either a CHO or HEK293 vector.

[0055] In configuration 27, the present invention is Provides host cells containing nucleic acids(s) or vectors.

[0056] In configuration 28, the present invention is This specification provides antibodies, fragments, combinations, vectors, host cells, uses, or methods described herein.

[0057] In configuration 29, the present invention provides the following: An antibody or fragment that specifically binds to bone morphogenetic proteins (BMPs) for use in a method of treating or preventing a disease or condition caused by a hemoduvelin (HJV)-deficient BMP-BMP receptor (BMPR) complex in a human or animal subject, the method comprising administering the antibody or fragment to a subject to inhibit the formation of the complex and / or inhibit the induction of intracellular signaling by such 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 bone morphogenetic protein (BMP) for use in a method for treating or preventing hemoduverin-independent anemia or osteoporosis in a human or animal subject, the method comprising administering the antibody or fragment to a subject to inhibit the formation of a hemoduverin (HJV)-deficient BMP-BMP receptor (BMPR) complex and / or inhibit the induction of intracellular signaling by such complex in the subject, thereby treating or preventing HJV-independent anemia or osteoporosis.

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

[0061] [Figure 1] Establishment of assay windows using the HepG2 humpluciferase reporter gene cell line and various human BMP ligands as accelerators. The function of the Red Firefly luciferase gene under the control of human hepcidin promoter regulators in HepG2 cells was tested by stimulating cells with various human BMP proteins (R&D Systems or Peprotech) at increased concentrations. The assay performance was tested in two different medium settings: MEM (A) containing 1% FBS and hybridoma medium (B, see Example 1 for details) containing 25% MEM. [Figure 2] The HepG2 humplusiferase reporter gene cell assay window was evaluated using various human or mouse BMP6 ligands added at higher concentrations as accelerators (R&D Systems or Peprotech). 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 humplusiferase reporter gene cell assay window was evaluated using MEM containing 1% FBS, diluted in hybridoma maintenance medium (HMM) (see Example 1 for further details and reagents), and commercially available mouse anti-BMP6 monoclonal antibodies MAB507(A) and MAB2365(B) from R&D Systems, with a fixed concentration of 1 nM human (Peprotech) or mouse BMP6 (R&D Systems). [Figure 4]Serum titer of the KM089 immunization regime was determined using a reverse DELFIA® assay (Perkin Elmer) in serum containing IgG antibodies added at various serial dilutions using five bmp6- / - Kymice. Anti-mouse IgG (goat anti-mouse IgG, Southern Biotech 1030-01) was captured via the Fc domain, followed by incubation with biotinated BMP6 and detection 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 + 3x standard deviation of all replications. KMBM codes in the legend refer to Kymouse® bmp6- / - animals. [Figure 5] Purified human BMP6 (Peprotech) at 2 μg / lane was separated by SDS-PAGE, stained with Coomassie Blue (A) under reducing (R) or non-reducing (NR) conditions, and Western blots from the gels were blotted onto membranes and then probed as labels with purified human anti-BMP6 antibody. The conjugated antibody was detected by anti-human kappa light chain-horseradish peroxidase (HRP) and enhanced chemiluminescence peroxidase (CL-58838 and antibody A), or by anti-human Fc-alkaline phosphatase (AP) and phosphatase 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitroblue tetrazolium (NBT) colorimetric substrate conversion (antibody B). [Figure 6-1]Receptor dimerization assays 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 (Peprotech120-06 SEQ ID NO: 2) generated a chemiluminescent signal. Human anti-BMP6 IgG4 antibody CL-58838 and anti-BMP6 antibodies A, 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 dilution). Representative data from 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 profiles of CL-58838 IgG4 (SEQ ID NOs. 116 and 125) purified 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 the humplusiferase reporter gene in HepG2 cells was investigated by adding CL-58838 in increasing amounts. As a positive control, relevant BMP-specific antibodies (all R&D Systems) were added to suppress the activation of the relevant BMPs. [Figure 7-2] See Figure 7-1. [Figure 7-3] See Figure 7-1. [Figure 8-1] Plotted transferrin saturation % (TSAT) in normal rats after a single intravenous injection of anti-BMP-6 or isotyped IgG control antibody, as plotted from Table 12 (for Figures 8A-D), Table 13 (for Figures 8E-G), and Table 14 (for Figures 8H and I). [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] Transferrin saturation % (TSAT) results 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] Transferrin saturation % (TSAT) results in normal rats after 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 comparison with 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 over time [h] as IgG [ng / ml]. [Figure 11-2] See Figure 11-1. [Figure 12-1] Results from a rat PG-PS model of ACD. Transferrin saturation TSAT (A), hemoglobin [g / dL] (B), MCH [pg] (C) after the start of treatment (*ρ<0.05, **p<0.01, ***p<0.001), and serum hepcidin levels [ng / mL] at week 0 (D), week 1 (E), and week 2 (F). [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 administration of CL-58838 and antibodies A and B (A) at 3 mg / kg, and CL-58838 (B) at various doses in cynomolgus monkeys, and plasma hepcidin levels after a single intravenous administration of CL-58838 and antibodies A and B (C) at 3 mg / kg, and CL-58838 (D) at various doses in cynomolgus monkeys. [Figure 13-2] See Figure 13-1. [Figure 14]PK profiles of CL-58838 after a single intravenous injection at various doses in cynomolgus monkeys (A), and a comparison of 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 associated with CL-58838 based on V region use and CDRH3 (Table 6). The top row shows the germline sequences of the IMGT V region genes IGHV3-11 and IGKV3-20 encoded by bmp6- / -Kymouse used in this invention. Amino acid positions that differ from the germline V region sequences of the selected antibody sequences below are enclosed in boxes. Antibodies selected for in vivo evaluation are shown in bold (Table 6). [Figure 16] The homogeneous time-resolved FRET (HTRF) assay demonstrates the antibody's ability to compete with CL-58838 (10 nM), which is highly related to CL-58838 and labeled to bind to human BMP6 present at 32 nM, based on the CDRH antibody identified by V-region use and NGS sequencing (Table 6). Competing antibodies were added as a concentration range of 11 dilution points, starting from a final concentration of 3 μM. [Figure 17-1] Experimental configuration and results obtained from Example 17. Graphs showing the experimental time course (A), decision plots for rat inclusion and EPO application (B), hemoglobin [g / dL] (C), MCV [fL] (D), MCH [pg] (E), and applied EPO dose [%] (F). ANCOVA analysis was used for the change from baseline at week 4 using Fisher's exact method (F), with baseline as a covariate (C-E). Data are shown 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 configuration and results of Example 18. Graphs showing the time course of the experiment (A), hemoglobin [g / dL] (B), MCV [fL] (C), MCH [pg] (D), hepatic hump mRNA level [multiple expression], plasma iron level [μg / dL] (F), and TSAT value [%]. Analysis of variance with Dunnett correction for multiple comparisons against EPO was applied. Comparison results for EPO vs. EPO+CL-58838 are shown. 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 obtained from Example 19. Hemoglobin levels [g / dL] (A) comparing treatment groups after administration of 1 μg / kg EPO in combination with different CL-58838 concentrations, hemoglobin levels [g / dL] (B) after administration of 10 mg / kg CL-58838 in combination with different EPO doses, MCV levels [fL] (C) and TSAT [%] (D) of mice after administration of 1 μg / kg EPO in combination with different CL-58838 concentrations, MCV levels [fL] (E) of mice after administration of 1 mg / kg CL-58838 in combination with different EPO doses, and hepatic hump mRNA levels [multiple expression] (F) across all treatment groups. Analysis of variance with Dunnett correction for multiple comparisons against group II was applied. Data are shown 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. [Modes for carrying out the invention]

[0062] definition Unless otherwise defined herein, scientific and technical terms shall have the meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms. The singular terms “a,” “an,” and “the” refer to multiple objects unless the context explicitly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context explicitly indicates otherwise. Methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, but preferred methods and materials are described below. The abbreviation “eg” is derived from the Latin “exempli gratia” and is used herein to indicate non-limiting examples. Thus, the abbreviation “eg” is synonymous with the term “for example.” In the specification and claims, the term “about” is used, for example, to modify the amount, concentration, volume, processing temperature, processing time, yield, flow rate, pressure, and similar values, as well as the ranges thereof, of components in a composition used in describing embodiments of the present disclosure. The term “about” refers to variations in quantity that may occur, for example, through typical measurement and handling procedures for making a compound, composition, concentrate, or formulation for use, through accidental errors in these procedures, through differences in purity in the manufacturing, source, or starting material or component used to carry out the method, and similar direct considerations. The term “about” also encompasses different amounts due to aging of a formulation having a particular initial concentration or mixture, and different amounts by mixing or processing a formulation having a particular initial concentration or mixture. Where modified by the term “about,” the appended claims include these quantities and their equivalents.

[0063] As used herein, “administer” or “dosage” means the act of injecting or otherwise physically delivering an extracorporeal substance (e.g., the 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 its symptoms are treated, the administration of the substance typically occurs after the onset of the disease or its symptoms. When a disease or its symptoms are prevented, the administration of the substance typically occurs before the onset of the disease or its symptoms.

[0064] The terms “antibody,” “immunoglobulin,” or “Ig” may be used interchangeably herein and mean an immunoglobulin molecule that recognizes a target such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combination thereof, and specifically binds to it at at least one antigen-recognizing site within the variable region of the immunoglobulin molecule. As used herein, the term “antibody” includes intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), multispecific antibodies such as single-chain Fv(scFv) variants, bispecific antibodies (including double-binding antibodies), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing the antigen-determining portion of an antibody, and any other modified immunoglobulin molecule containing an antigen-recognizing site, insofar as it exhibits the desired biological activity. The term “antibody” may also refer to a Y-shaped glycoprotein having a molecular weight of about 150 kDa, consisting of four polypeptide chains (two light (L) chains and two heavy (H) chains). There are five mammalian Ig heavy chain isotypes, represented by the Greek letters alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ). The heavy chain type defines the antibody class, namely IgA, IgD, IgE, IgG, and IgM, respectively. The γ and α 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, λ and κ. The “variable region” or “variable domain” of an antibody refers to the amino-terminal domain of the antibody’s heavy or light chain. The variable domains of the heavy and light chains are respectively called “V H " and "V L These domains can be expressed as follows: These domains are generally the most variable parts of an antibody (compared to other antibodies of the same class) and contain the antigen-binding site. 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 a light chain.

[0065] The antibodies described herein may be oligoclonal antibodies, polyclonal antibodies, monoclonal antibodies (including full-length monoclonal antibodies), camelized antibodies, chimeric antibodies, CDR-transplant antibodies, multispecific antibodies, bispecific antibodies (including double-conjugated antibodies), catalytic antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, and anti-idiotype antibodies, which may include antibodies that can 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. The antibodies may be of any species origin. The antibodies described herein may be naked or conjugated with other molecules such as toxins or radioisotopes.

[0066] The terms “antigen-binding site,” “antigen-binding domain,” “antigen-binding region,” and “antigen-binding fragment,” and similar terms, refer to the portion of an antibody (e.g., a complementarity-determining region (CDR)) that contains amino acid residues that interact with the antigen and confer its specificity and affinity to the antigen to the binder. The antigen-binding region may originate from any animal species, such as 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 Fv(scFv), single-chain antibodies, single-chain domain antibodies, domain antibodies, Fv fragments, Fab fragments, F(ab') fragments, F(ab')2 fragments, antibody fragments exhibiting desired biological activity, disulfide-stabilized variable regions (dsFv), dimeric variable regions (diabodies), anti-idiotype (anti-Id) antibodies (e.g., anti-Id antibodies against antibodies), intracellular antibodies, 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 antigen-binding sites. Digestion of antibodies with the enzyme papain yields two identical antigen-binding fragments, also known as "Fab" fragments and "Fc" fragments, which do not possess antigen-binding activity but have the ability to crystallize. As used herein, “Fab” refers to a fragment of an antibody containing one constant domain and one variable domain, each of the heavy and light chains. The term “Fc region” as used herein is used to define the C-terminal region of the immunoglobulin heavy chain, including the native sequence Fc region and variant Fc regions. “Fc fragment” refers to the carboxyl-terminal portions of both H chains held together by a disulfide. The effector function of an antibody is determined by the sequence within the Fc region, which is also recognized by Fc receptors (FcRs) found on certain types of cells. Digestion of an antibody by the enzyme pepsin results in an F(ab')2 fragment, in which the two arms of the antibody molecule remain bound, and which contains two antigen-binding sites. The F(ab')2 fragment has the ability to crosslink antigens.

[0068] The term “derived from recombination of” in relation to gene segments is readily apparent to those skilled in the art, understanding that B cells recombinate their variable region gene segments to produce coding sequences for variable domains. For example, the term “derived from recombination of human VH gene segment, DH gene segment and JH gene segment” refers to the recombination of one human VH gene segment in which one DH 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 features of the process, and the resulting recombined VDJ sequence may include the addition, substitution, or deletion of one or more nucleotides not composed of germline V, D, and J sequences (e.g., p-addition and / or n-addition). Equivalents are those of the Vκ and Jκ gene segments in the case of the kappa light chain variable domain, and Vλ and Jλ in the case of the lambda light chain variable domain. Any post-translational modifications are intended to be further encompassed within the variable domain.

[0069] "Fv," as used herein, refers to the smallest fragment of an antibody that possesses both an antigen recognition site and an antigen binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain associated by strong non-covalent or covalent bonds. In this configuration, the three CDRs of each variable domain interact to form V H -V L This defines the antigen-binding sites 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 the Fv containing only the three antigen-specific CDRs) has the ability to recognize and bind to the 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 substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical except for the possibility of native mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Monoclonal antibodies are highly specific and directed to a single antigenic determinant or epitope. In contrast, polyclonal antibody preparations typically contain different antibodies directed to 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-stranded (scFv) variants, fusion proteins containing antibody moieties, and any other modified immunoglobulin molecules containing antigen recognition sites. 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 as used herein may include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species belonging to a particular antibody class or subclass, while the remaining chain(s) are identical or homologous to a corresponding sequence in an antibody derived from another species belonging to a different antibody class or subclass, as well as fragments of such antibodies that exhibit desired biological activity.

[0072] The term "humanized antibody" refers to a subset of chimeric antibodies in which a "hypervariable region" derived from a non-human immunoglobulin (donor antibody) replaces residues derived from the hypervariable region in a human immunoglobulin (recipient antibody). Generally, a humanized antibody contains substantially all of at least one, typically two, variable domains, all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the framework region is of a human immunoglobulin sequence, however, the framework region may contain one or more substitutions that improve antibody performance, such as binding affinity, isomerization, or immunogenicity.

[0073] The term "bispecific antibody" refers to an antibody that exhibits specificity for two target molecules, such as DVD-Ig (see DiGiammarino et al., "Design and generation of DVD-Ig(trademark) molecules for dual-specific targeting", Meth.Mo.Biol., 2012, 889, 145-156) and mAb. 2 (See WO2008 / 003103, mAb) 2(Type descriptions are incorporated herein by reference), FIT-Ig (WO2015 / 103072, description of the FIT-Ig skeleton 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, Intracellular Antibody, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, Triple Body, Mini Antibody, Mini Body, TriBi Mini Body, scFv-CH3 This includes, but is not limited to, formats such as KIH, scFv-CH-CL-scFv, F(ab')2-scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCab, ImmTAC, knobs-in-holes, knobs-in-holes with a common light chain, knobs-in-holes 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 an overview of the bispecificity format, see Spiess, C., et al., Mol. Immunol. (2015).In another embodiment, the bispecific molecule is from a scaffold selected from another non-Ig format, such as a T cell receptor binding domain, an immunoglobulin superfamily domain, an agnathan variable lymphocyte receptor, a fibronectin domain (e.g., Adnectin™), an antibody constant domain (e.g., CH3 domain, e.g., CH2 and / or CH3 of Fcab™) (the constant domain is not a functional CH1 domain), scFv, (scFv)2, sc-diabody, scFab, centyrin and an epitope binding domain (Evibody™) derived from CTLA-4, a lipocalin domain, protein A such as the Z domain of protein A (e.g., Affibody™ or SpA), an A domain (e.g., Avimer™ or Maxibody™), a heat shock protein (such as an epitope binding domain derived from GroEI and GroES), a transferrin domain (e.g., transbody), an ankyrin repeat protein (e.g., DARPin™), a peptide aptamer, a C-type lectin domain (e.g., Tetranectin™), human γ-crystallin or human ubiquitin (affilin), a PDZ domain, a scorpion venom, and an antibody fused to a Kunitz-type domain of a human protease inhibitor.

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

[0075] In another embodiment, the bispecific antibody is a "dual binding antibody". As used herein, "dual binding antibody" means that both antigen binding domains are VH / V L These are bispecific antibodies formed by pairs, 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, Intracellular Antibody, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, Triple Body, Mini Antibody This includes, but is not limited to, bodies, 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 regions of antibody variable domains whose sequences are hypervariable and / or form structurally defined loops. Generally, the antigen-binding site of an antibody consists of six hypervariable regions (V H There are three (CDRH1, CDRH2, CDRH3) and V L It contains three (CDRL1, CDRL2, CDRL3) in the heavy and light chains of the antibody. These regions 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, USD Department of Health and Human Services). CDRs can be defined using other systems 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(plural) is used here to refer to one or more of these regions. Those skilled in the art can easily compare different nomenclature systems and determine whether a particular sequence can be defined as a CDR.

[0077] A “human antibody” is an antibody that possesses the 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, which determines the presence of a 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 more readily and / or for a longer period with greater affinity and binding strength than it would to other targets. In one embodiment, for example, when measured by radioimmunoassay (RIA), the degree of antibody binding to an unrelated target is less than about 10% of the degree of antibody binding to the target.

[0078] An antibody or fragment thereof that specifically binds to the hBMP6 antigen may cross-react with the relevant antigen. Preferably, an antibody or fragment thereof that specifically binds to the hBMP6 antigen does not cross-react with other antigens (however, optionally, it may cross-react with BMP6 of different species, e.g., rhesus monkey or mouse). An antibody or fragment thereof that specifically binds to the hBMP6 antigen may 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 the BBMP6 antigen if, when determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), it binds to the hBMP6 antigen with a higher affinity than to any cross-reactive antigen. Typically, a specific or selective reaction is accompanied by at least 2x background signal or noise, more typically more than 10x background (e.g., more than 15x, more than 20x, more than 50x, or more than 100x). For a discussion on antibody specificity, see, for example, pages 332-336 of Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York.

[0079] The term "aliphatic amino acid" means that the R group of an amino acid is nonpolar and hydrophobic. Hydrophobicity increases as the number of carbon atoms in the hydrocarbon chain increases. 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 R group of an amino acid 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 R group of an amino acid contains nitrogen and is basic at a neutral pH. Histidine, lysine, and arginine are basic amino acids.

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

[0083] The term "acidic amino acid" means that the R group of an amino acid 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 R group of an amino acid contains an amide group. Asparagine and glutamine are amide amino acids.

[0085] As used herein, “Approval Number” and “Marketing Approval Number” refer to numbers issued by a regulatory body when an agency determines that a particular medical product and / or composition may be sold and / or launched in the agency’s jurisdiction. As used herein, “Regulatory Body” refers to one of the agencies responsible for evaluating the safety and efficacy of a medical product and / or composition, and for controlling the launch / marketing of such product and / or composition 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 bodies. Other non-exclusive examples include the SDA, MPA, MHPRA, IMA, ANMAT, Hong Kong Department of Health-Drug Office, CDSCO, Medsafe, and KFDA.

[0086] As used herein, “buffer” refers to a chemical agent capable of absorbing a specific amount of acid or base without undergoing a significant change 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 administered with a therapeutic agent. Such pharmaceutical carriers may be sterile solutions, such as water, and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, or sesame oil. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solution, as well as aqueous solutions of dextrose and glycerol, can also be used as liquid carriers, particularly for injections.

[0088] The terms "chemotherapeutic agents" or "chemotherapy" typically refer to therapeutic agents whose primary purpose is to destroy cancer cells by inhibiting their ability to grow or proliferate. Many different types of chemotherapeutic agents exist, with over 50 approved chemotherapeutic agents 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 inhibit cell growth and proliferation by interfering with DNA production. An example of an antimetabolite is 5-fluorouracil (5-FU). Antitumor antibiotics are derived from natural substances such as fungi in the soil. They interfere with essential cellular functions, including the production of DNA and cellular proteins. Doxorubicin and bleomycin belong to this group of chemotherapeutic agents. Plant alkaloids prevent cells from dividing normally. Vinblastine and vincristine are plant alkaloids obtained from the periwinkle plant. Steroid hormones slow the growth of several hormone-dependent cancers. For example, tamoxifen is used to treat breast cancer that is dependent on the hormone estrogen for growth. DNA damage response (DDR) inhibitors, such as PARP inhibitors, block the DNA repair mechanism after single-strand 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, vincreistine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycin, esperamicin (see U.S. Patent 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 This is described in Ed. (MacMillan Publishing Co., 1985). Another example of chemotherapeutic agents is a class of toxins conjugated with antibodies, including, but not limited to, pyrrolobenzodiazepines, maytansanoids, and calicheamicin. Other suitable toxins and / or chemotherapeutic agents are known to those skilled in the art.

[0090] As used herein, the term “composition” is intended to encompass any product containing, optionally, a specified amount of a specified component (e.g., the antibody of the present invention), and any product resulting directly or indirectly from a combination of the specified components in optionally specified amounts.

[0091] As used herein, the terms “comprising” or “comprises” are used in reference to antibodies, fragments, uses, compositions, methods, and their respective components that are essential to the method or composition, but may include unspecified elements, whether essential or not.

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

[0093] As used herein, the term “essentially derived from” refers to elements required for a given embodiment. The term allows for the presence of elements that do not materially affect the basic, novel, or functional features of that embodiment.

[0094] As used herein, in the context of polypeptides, the term “derivative” includes polypeptides containing the amino acid sequence of the hBMP6 polypeptide, fragments of the hBMP6 polypeptide, or antibodies or fragments that specifically bind to the hBMP6 polypeptide, modified by the introduction of substitutions, deletions, or additions of amino acid residues. As used herein, the term “derivative” also includes antibodies that specifically bind to the hBMP6 polypeptide, fragments of the hBMP6 polypeptide, or chemically modified, for example, by covalent bonding of any type of molecule to the polypeptide. For example, but not limited to, hBMP6 polypeptides, fragments of the hBMP6 polypeptide, or hBMP6 antibodies may be chemically modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins. Derivatives are modified in a manner different from the native or starting peptide or polypeptide in either the type or location of the bound molecule. Derivatives further include deletions of one or more chemical groups naturally present on the peptide or polypeptide. hBMP6 polypeptides, hBMP6 polypeptide fragments, or derivatives of hBMP6 antibodies may be chemically modified by chemical modifications using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formulation, and tunicamycin metabolic synthesis. Furthermore, hBMP6 polypeptides, hBMP6 polypeptide fragments, or derivatives of hBMP6 antibodies may contain one or more non-classical amino acids. Polypeptide derivatives have the same 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 the following: antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC)-mediated response, Fc-mediated phagocytosis or antibody-dependent cytophagosis (ADCP), and antibodies that recirculate via FcRn receptors.

[0096] "Effective dose" refers to the amount effective in the dosage and duration required to achieve the desired effect, including therapeutic or prophylactic outcomes. "Therapeutic effective dose" refers to the minimum concentration required to achieve a measurable improvement or prevention of a particular disorder. The therapeutic effective dose as used herein may vary depending on factors such as the patient's disease state, age, sex, and weight, as well as the antibody's ability to elicit a desired response in the individual. The therapeutic effective dose is also the amount at which the therapeutically beneficial effect outweighs the toxic or adverse effects of the antibody. "Prophylactic effective dose" refers to the amount effective in the dosage and duration required to achieve the desired prophylactic effect. In some embodiments, the effective dose of the antibody of the present invention ranges from about 0.1 mg / kg (mg of antibody per kg of body weight of the subject) to about 100 mg / kg. In certain embodiments, the effective dose of the 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 within these). In some embodiments, when used herein, “effective dose” also refers to the amount of the antibody of the present invention to achieve a specified result (e.g., inhibition of the biological activity of hBMP6 in cells).

[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 and has antigenic or immunogenic activity in animals, preferably mammals, most preferably humans, and can elicit an immune response. An immunogenic epitope is a portion of a polypeptide that elicits an antibody response in animals. An antigenic epitope is a portion of a polypeptide to which an antibody specifically binds, as determined by any method well known in the art, for example, by the immunoassays described herein. Antigenic epitopes are not necessarily immunogenic. Epitopes typically consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural properties and specific charge properties. The polypeptide region contributing to the epitope may be a sequence of amino acids in the polypeptide, or the epitope may be assembled from two or more non-contiguous regions of the polypeptide. Epitopes are not necessarily three-dimensional surface features of an antigen. In certain embodiments, the hBMP6 epitope is a three-dimensional surface feature of the hBMP6 polypeptide (e.g., in the trimer form of the hBMP6 polypeptide). In other embodiments, the hBMP6 epitope is a linear feature of the hBMP6 polypeptide (e.g., in the trimer or monomeric form of the hBMP6 polypeptide). The antibodies provided herein may specifically bind to the epitope of the monomeric (denatured) form of hBMP6, to the epitope of the trimeric (natural) form of hBMP6, or to the epitopes of both the monomeric (denatured) and trimeric (natural) forms of hBMP6. In certain embodiments, the antibodies provided herein specifically bind to the epitope of the trimeric form of hBMP6 but not to the monomeric form of hBMP6.

[0098] As used herein, the term “excipient” generally refers to inert substances used as diluents, vehicles, preservatives, binders, or stabilizers for drugs, including, but not limited to, proteins (e.g., serum albumin), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylates), surfactants (e.g., SDS, polysorbates, nonionic surfactants), sugars (e.g., sucrose, maltose, trehalose), and polyols (e.g., mannitol, sorbitol). See also Remington's Pharmaceutical Sciences (1990), Mack Publishing Co., Easton, Pa., which is incorporated herein by reference in its entirety.

[0099] As used herein, in the context of peptides or polypeptides, the term “fragment” refers to a peptide or polypeptide containing an amino acid sequence less than the full length. Such fragments may arise, for example, from amino-terminus truncation, carboxy-terminus truncation, and / or internal deletion of residues from an amino acid sequence. Fragments may also arise, 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 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues of the amino acid sequence of the hBMP6 polypeptide or an antibody that specifically binds to the hBMP6 polypeptide. In certain embodiments, the hBMP6 polypeptide fragment or the antibody that specifically binds to the 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 its fragments and variants, combined with a buffer, and the polypeptide is not bound to the cell surface or cell membrane. Therefore, the term “free” can refer to a polypeptide that is surface-expressible (i.e., contains one or more transmembrane or membrane-binding domains) but is not currently expressed on the cell surface or bound to a protein expressed on the cell surface. Free polypeptides can also refer to free recombinant, native, or unbound polypeptides. In the context of phage display, free antigens can be selected in solution (referred to herein as “soluble selection”) or adsorbed to a surface, for example, to 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 typically part of an antibody (e.g., a non-anti-BMP6 antigen antibody)). The term “fusion,” when used in relation to BMP6 or anti-BMP6 antibodies, 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 the BMP6 or anti-BMP6 antibody. In certain embodiments, the fusion protein comprises the VH domain, VL domain, VH CDR (one, two, or three VH CDRs), and / or VL CDR (one, two, or three VL CDRs) of the BMP6 antibody, and the fusion protein specifically binds to the BMP6 epitope.

[0102] When used in relation to antibodies, the term “heavy chain” refers to five distinct types, called alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the heavy chain constant domain. These distinct types of heavy chains are well known, from which five classes of antibodies arise: 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 allele variants are accessible on publicly available databases such as IMGT, ENSEMBL Swiss-Prot, and Uniprot. Allele variants can also be identified in various genome sequencing projects. In one embodiment, the antibodies and antibody fragments disclosed herein are human IGGH1 * 01 (Sequence IDs 340, 341, and 537), IHG1 * 02 (Sequence IDs 340, 341, and 537), IHG1 * 03 (Sequence IDs 523 and 524), IHG1 * 04 (SEQ ID NOs. 525 and 526), ​​and IGGH1 * The heavy chain is encoded by an IgG1 constant region allele, including but not limited to 05 (SEQ ID NOs: 340, 341, and 537). In one embodiment, the antibodies and antibody fragments disclosed herein are human IgG1 * 01 (Sequence IDs 527 and 528), IGGG2 * 02 (Sequence IDs 529 and 530), IHG2 * 03 (Sequence IDs 527 and 528), IHG2 * 04 (Sequence IDs 531 and 532), IHG2 * 05 (Sequence IDs 527 and 528), and IGGG2 *The present invention relates to proteins encoded by IgG2 constant region alleles, including but not limited to 06 (SEQ ID NOs. 533 and 534). In one embodiment, the antibodies and antibody fragments disclosed herein relate to human IgG2 * 01, IGGH3 * 02, IGG3 * 03, IGGH3 * 04, IGGH3 * 05, IGG3 * 06, IGG3 * 07, IGG3 * 08, IGG3 * 09, IGGH3 * 10. IGG3 * 11. IGG3 * 12. IGGH3 * 13. IGG3 * 14. IGGH3 * 15. IGG3 * 16. IGG3 * 17. IGGH3 * 18, and IGGH3 * The present invention comprises proteins encoded by IgG3 constant region alleles, including but not limited to 19. In one embodiment, the antibody or antibody fragment disclosed herein is human IgG4 * 01 (see, for example, the sequence listing in this specification), IGGH4 * 02 (see, for example, the sequence listing in this specification), IHG4 * 03 (see, for example, the sequence listings in this specification), and IGGH4 *The present invention comprises a protein encoded by an IgG4 constant region allele, including but not limited to 04 (see, for example, the sequence listing herein). In another example, the heavy chain is a deficient IgG isotype, e.g., deficient IgG4. In certain embodiments, the antibody of the present invention comprises a human gamma 4 constant region. In another embodiment, the heavy chain constant region does not bind to the Fc-γ receptor and includes, for example, the Leu235Glu mutation. In another embodiment, the heavy chain constant region includes the Ser228Pro mutation to increase stability. In another embodiment, the heavy chain constant region is IgG4-PE (see, for example, the sequence listing herein). In another embodiment, the antibodies and antibody fragments disclosed herein are mouse IGGH1 * 01 or IGGH1 * The mouse IgG1 constant region comprises a heavy chain constant region encoded by an allele, including but not limited to 02. In one embodiment, the antibodies and antibody fragments disclosed herein are mouse IGGH2A * 01, IGGH2A * 02, IGGH2B * 01, IGGH2B * 02, IGGH2C * 01, IGGH2C * 02, or IGGH2C * The mouse IgG2 constant region comprises a heavy chain constant region encoded by an allele, including but not limited to 03. In one embodiment, the antibody or antibody fragment disclosed herein is mouse IgG3 * This includes proteins encoded by mouse IgG3 constant region alleles, including but not limited to 01.

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

[0104] As used herein, the term “host cell” refers to a specific target cell transfected with a nucleic acid molecule, and its offspring or potential offspring. Such offspring 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 the incorporation of the nucleic acid molecule into the host cell genome. The term “combined” in the context of administering other therapies refers to the use of two or more therapies. The use of the term “combined” does not restrict the order in which therapies are administered to a subject with the disease. The first therapy may be administered before (e.g., 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 administration of the second therapy to a subject who has, will have, or is susceptible to a BMP6-mediated disease, simultaneously with, or after (e.g., 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 administration of the second therapy to a subject who has, will have, or is susceptible to a BMP6-mediated disease. Any additional therapies may be administered in any order along with other additional therapies. In certain embodiments, the antibody of the present invention may be administered in combination with one or more therapies (e.g., therapies other than the antibody of the present invention currently administered to prevent, treat, manage, and / or improve BMP6-mediated diseases). Non-limiting examples of therapies that may be administered in combination with the antibody of the present invention include analgesics, anesthetics, antibiotics, or immunomodulators, or any other agents listed in the United States Pharmacopeia and / or the Medical Practitioners' Information Sheet.

[0105] As used herein, “injection device” means a device designed to administer an injection, and an injection includes the step of temporarily fluidizing the injection device to human tissue, typically subcutaneous tissue. An injection further includes administering a certain amount of liquid drug into the tissue and separating or removing the injection device from the tissue. In some embodiments, the injection device may be an intravenous device or IV device, which is a type of injection device used when the target tissue is blood in the circulatory system, for example, a venous vessel. Common but non-limiting examples of injection devices are needles and syringes.

[0106] As used herein, “instructions” means any document, printed material, or graphic representation on the immediate container of an article, such as a document displayed on a vial containing a pharmaceutically active agent, or a detailed representation of the composition and use of the product of interest contained in a kit containing the composition of interest. Instructions describe a method of administration or treatment intended to be carried out.

[0107] "Isolated" or "purified" antibodies or proteins are identified, separated, and / or recovered from the components of their production environment (e.g., natural or recombinant). For example, an antibody or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody originates, or, if chemically synthesized, substantially free of chemical precursors or other chemicals. The term "substantially free of cellular material" includes antibody preparations in which the antibody is separated from the cellular components of the isolated or recombinantly produced cell. Thus, substantially free of cellular material antibodies include antibody preparations having less than about 30%, 20%, 10%, or 5% (dry weight) of heterogeneous 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., the culture medium occupies less than about 20%, 10%, or 5% of the volume of the protein preparation. If the antibody is produced by chemosynthesis, the antibody is also preferably substantially free of chemical precursors or other chemicals, i.e., the antibody is separated from the chemical precursors or other chemicals involved in the synthesis of the protein. Therefore, such antibody preparations contain approximately 30%, 20%, 10%, or less than 5% (dry weight) of chemical precursors or compounds other than the antibody of interest. In preferred embodiments, the antibody of the present invention is 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 or antigen-binding region of an antibody (Kabat et al., (1971) Ann. NY Acad. Sci., 190:382-391 and Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242). In the case of the heavy chain variable region, the hypervariable region is typically in the range of amino acid positions 31-35 for CDR1, amino acid positions 50-65 for CDR2, and amino acid positions 95-102 for CDR3.

[0109] As used herein, “labeled” or “labeled” means a detectable portion of a polypeptide, such as radiolabeling, fluorescent labeling, enzymatic labeling, chemiluminescence labeling, or the addition of 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, 131I may be included, fluorescent labels may include rhodamine, lanthanide phosphors, or FITC, and enzyme labels may include horseradish peroxidase, β-galactosidase, luciferase, or alkaline phosphatase. Further labeling may include, but not restrictively, enzymes such as glucose-6-phosphate dehydrogenase ("G6PDH"), alpha-D-galactosidase, glucose oxidase, glucose amylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, and peroxidase; dyes (e.g., cyanine dyes, e.g., Cy5(trademark), Cy5.5(trademark), or Cy7(trademark)); additional fluorescent labels or fluorescent 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-phthalaldehyde (phthaldehyde), and fluorescein; fluorophores such as lanthanide cryptoates and chelates, e.g., europium (Perkin). Elmer and Cisbio assays); include chemifluorescent labels or chemifluorescent materials such as isoluminol, luminol, and dioxetane; 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-bromdexyuridine; and toxin moieties selected from the group consisting of, for example, Pseudomonas exotoxin (PE or its cytotoxic fragments or variants), diphtheria toxin or its cytotoxic fragments or variants, botulinum toxin A, B, C, D, E, or F, lysine or its cytotoxic fragments, e.g., lysine A, abrin or its cytotoxic fragments, saporin or its cytotoxic fragments, pokeweed antiviral toxin or its cytotoxic fragments, and bryodin 1 or its cytotoxic fragments.

[0110] When used in relation to antibodies, the term “light chain” refers to immunoglobulin light chains, 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. In the human population, there are multiple light chain constant region alleles. The nucleotide and amino acid sequences of these allele variants are accessible in publicly available databases such as IMGT, ENSEMBL Swiss-Prot, and Uniprot. In one embodiment, the antibody or antibody fragment disclosed herein is IGKC * 01 (see, for example, the sequence listing in this specification), IGKC * 02 (see, for example, the sequence listings in this specification), IGKC * 03 (see, for example, the sequence listings in this specification), IGKC * 04 (see, for example, the sequence listings in this specification), and IGKC * The present invention comprises a protein encoded by a human κ constant region allele, including but not limited to 05 (see, for example, the sequence listing herein). In one embodiment, the antibody or antibody fragment disclosed herein is IGLC1 * 01 (see, for example, the sequence listing in this specification), IGLC1 * 02 (see, for example, the sequence listing in this specification), IGLC2 * 01 (see, for example, the sequence listing in this specification), IGLC2 * 02 (see, for example, the sequence listing in this specification), IGLC2 * 03 (see, for example, the sequence listing in this specification), IGLC3 * 01 (see, for example, the sequence listing in this specification), IGLC3 * 02 (see, for example, the sequence listing in this specification), IGLC3 * 03 (see, for example, the sequence listing in this specification), IGLC3 * 04 (see, for example, the sequence listing in this specification), IGLC6 * 01 (see, for example, the sequence listing in this specification), IGLC7 * 01 (see, for example, the sequence listing in this specification), IGLC7 * 02 (see, for example, the sequence listing in this specification), IGLC7* It includes proteins encoded by human λ constant region alleles including, but not limited to, 03 (see, for example, the Sequence Listing of this specification). In another embodiment, the antibodies and antibody fragments disclosed herein are IGKC * 01, IGKC * 03, or it includes a light chain constant region encoded by mouse κ constant region alleles including, but not limited to, IGKC * 03. In another embodiment, the antibodies and antibody fragments disclosed herein are IGLC1 * 01, IGLC2 * 01, or it includes a light chain constant region encoded by mouse λ constant region alleles including, but not limited to, IGLC3 * 01.

[0111] "Percent amino acid sequence identity (%)" and "homology" in relation to a peptide, polypeptide, or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to a specific peptide or polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity, introducing gaps if necessary, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved by various methods within the scope of the art, using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, or MEG ALIGN (trademark) (DNASTAR) software. In one embodiment, the homology % is about 70%. In one embodiment, the homology % is about 75%. In one embodiment, the homology % is about 80%. In one embodiment, the homology % is about 85%. In one embodiment, the homology % is about 90%. In one embodiment, the homology % is about 92%. In one embodiment, the 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] When used in connection with a biological substance, such as a nucleic acid molecule, polypeptide, or host cell, the term "native" or "natural" refers to that which is found in nature and has not been manipulated by humans.

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

[0114] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia, European Pharmacopeia, or other generally recognized pharmacopeia for use in animals, more specifically 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, etc.

[0116] As used herein, the terms "prevent", "preventing", and "prevention" refer to the complete or partial inhibition of the expression, recurrence, onset or metastasis of hBMP6-mediated diseases and / or related symptoms resulting from 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 domains or cytoplasmic domains found in its native or membrane-bound form. In one embodiment, the "soluble" form of BMP6 lacks both the transmembrane domain and the cytoplasmic domain.

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

[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 for the treatment, management, or amelioration of BMP6-mediated diseases and / or related symptoms. 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 a drug that is known to be useful or has been used or is currently used for the treatment, management, or amelioration of BMP6-mediated diseases or one or more symptoms related thereto. 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” means any protocol, method, and / or agent that can be used to prevent, manage, treat, and / or improve BMP6-mediated diseases (e.g., cancer). In certain embodiments, the term “therapy” means any biological therapy, supportive therapy, and / or other therapy known to those skilled in the art, such as healthcare professionals, that is useful for preventing, managing, treating, and / or improving BMP6-mediated diseases. The terms “to treat,” “to treat,” and “to treat” refer to the reduction or improvement 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 antibodies of the present invention). In certain embodiments, such terms refer to the reduction or inhibition of the binding of hBMP6 to BMP receptors 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 portions of the light and heavy chains, typically the approximately 120–130 amino acids at the amino-terminus of the heavy chain and the approximately 100–110 amino acids in the light chain, which exhibit widely different sequences across antibodies and are used in the binding and specificity of each particular antibody to its specific antigens. Sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while more highly conserved regions within the variable domains are called framework regions (FRs). BMP6 and the CDRs of the heavy chain are primarily responsible for antibody-antigen interactions. The amino acid position numbering used herein is based on Kabat et al. (1991) Sequences of proteins of immunological interest. (USD Department of Health and Human Services, Washington, DC) 5 th The EU index is followed, as described in ed. ("Kabat et al."). In a preferred embodiment, the variable region is the human variable region.

[0123] Definitions of common terms in cell biology and molecular biology can be found 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 based on, for example, 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), or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol. 152, SLBerger and ARKimmel 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 The procedures were carried out using standard methods, as described in 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), and all of these are incorporated herein by reference. Other terms are defined herein in the description of various aspects of the present invention.

[0125] BMP6 and iron Iron is essential for all forms of life and must be supplied from the environment; therefore, its availability and use in the body are strictly controlled. A key regulator of iron homeostasis is a 25-amino acid peptide hormone called hepcidin. Produced by the liver, hepcidin controls the expression of the iron transporter molecule ferroportin, thereby ensuring iron retention in the duodenal intestinal cells and macrophages, which are the major iron uptake and storage compartments. Hepcidin itself is regulated by iron levels through homeostatic regulatory mechanisms after the activation of the immune system and erythropoiesis during infection and / or inflammation. Importantly, hepcidin levels are elevated in chronic inflammatory states, infections, and certain cancers. Elevated hepcidin levels sequester iron in intestinal cells, macrophages, and hepatocytes, thereby suppressing hemoglobin synthesis and erythropoiesis. This can lead to anemia despite normal iron storage levels. Hepcidin gene expression is regulated by a soluble factor called BMP6 (bone morphogenetic protein 6). In the absence of BMP6, cytokines alone (or other BMPs) cannot overcome the deficiency of BMP6 signaling; therefore, BMP6 is considered a master regulator. Accordingly, we have noted that BMP6 is an important drug target for controlling abnormal dysregulation of iron homeostasis in anemia, such as in anemia of chronic disease (ACD).

[0126] BMP6 is a highly conserved soluble protein factor considered to be the “master” regulator of hepcidin production in mice and humans. Therefore, administration of BMP6 to mice increases hepcidin levels and decreases blood and serum iron, while BMP6 inhibitors do the opposite. In addition, 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 resulted in increased available iron levels 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. 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 antibody and fragment This invention provides various anti-BMP6 antibodies and fragments (such as Fab or scFv fragments), uses, methods, and combinations (e.g., with ESA). Examples are described in the numbered sections 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 a recombination of the human VH gene segment, DH gene segment, and JH gene segment, 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 DH and JH gene segments are human gene segments. For example, the VH gene segment is IGHV1-3, and the DH and JH gene segments are human gene segments. Optionally, the VH segment is human IGHV3-11. * The 01 gene segment is optional. Alternatively, the VH segment may be human IGHV1-3. * This is the 01 gene segment.

[0130] For example, specific binding is KD, K, as further described below. off and / or K on This is accompanied by a specific binding reaction. For example, specific binding is accompanied by a KD of 1 pM to 5 nM.

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

[0132] Refer to 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] Refer to Table 9, which shows 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. For example, an antibody or fragment may contain a CDRH3 sequence selected from SEQ ID NOs: 110, 113, 290, 293, 308, 311, 272, and 275. For example, an antibody or fragment may contain a CDRL3 sequence selected from SEQ ID NOs: 119, 122, 299, 302, 317, 320, 281, and 284. For example, an antibody or fragment may contain a CDRH3 sequence selected from SEQ ID NOs: 110, 113, 290, 293, 308, 311, 272, and 275, as well as 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 includes 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 includes an anti-BMP6 binding site, the binding site including a VH domain including SEQ ID NO: 110 paired with a VL domain including SEQ ID NO: 119. For example, the antibody or fragment includes an anti-BMP6 binding site, the binding site including a VH domain including SEQ ID NO: 113 paired with a VL domain including SEQ ID NO: 122.

[0135] For example, the antibody or fragment includes the CDRH3 sequence of an antibody selected from CL-58838, CL-58835, CL-58756, and CL-58722, and optionally the CDRL3 sequence of such selected antibody. For example, the antibody or fragment includes the CDRH1 and CDRH3 sequences of an antibody selected from CL-58838, CL-58835, CL-58756, and CL-58722, and optionally the CDRL2 sequence of such selected antibody. For example, the antibody or fragment includes 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 includes 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 contains an anti-BMP6 binding site, and the binding site contains a VH domain which includes the CDRH3 sequence of CL-58838 that pairs with the VL domain of CL-58838.

[0136] For example, the antibody or fragment includes an anti-BMP6 binding site, and the binding site includes a VH domain containing SEQ ID NOs. 114, 294, 312, or 276, which is optionally paired with a VL domain containing SEQ ID NOs. 123, 303, 321, or 285, respectively.

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

[0138] 2. The antibody or fragment according to 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 selected from.

[0140] 3. The antibody or fragment according to 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 selected from.

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

[0143] 5. An antibody or fragment that specifically binds to bone morphogenetic protein 6 (BMP6) and contains the VH domain of any anti-BMP6 antibody disclosed herein (for example, any antibody selected from the antibodies or fragments listed in Tables 4 to 11 herein), or an antibody or fragment containing the CDRH3 with 3, 2, or 1 amino acid substitution(s) (plural possible).

[0144] 6. An antibody or fragment (optionally as described in any of clauses 1 to 5) that specifically binds to bone morphogenetic protein 6 (BMP6) and comprises a VH domain containing 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 the sequence containing 3, 2 or 1 amino acid substitutions.

[0145] Optionally, the VH domain contains a CDRH3 sequence selected from sequence number 110 or 113, or the selected sequence contains 3, 2, or 1 amino acid substitutions.

[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 (ii) an antibody or fragment according to Clause 6, comprising (ii) a CDRH3 sequence of an antibody selected from CL-58650, CL-58679, CL-58680 and CL-58713, or said CDRH3 sequence comprising 3, 2 or 1 amino acid substitutions, and (ii) a CDRH1 sequence of the selected antibody, or said CDRH1 sequence comprising 3, 2 or 1 amino acid substitutions, etc.

[0147] Optionally, the VH domain of the antibody or fragment may include (a) the CDRH3 sequence of SEQ ID NO: 110 or 113, or the CDRH3 sequence containing 3, 2, or 1 amino acid substitutions, and (b) the CDRH1 sequence of SEQ ID NO: 108 or 111, or the CDRH1 sequence containing 3, 2, or 1 amino acid substitutions.

[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 (iv) an antibody or fragment according to Clause 6 or 7, comprising the CDRH3 sequence of an antibody selected from CL-58650, CL-58679, CL-58680 and CL-58713, or the CDRH3 sequence comprising 3, 2 or 1 amino acid substitutions, and (iv) the CDRH2 sequence of the selected antibody, or the CDRH2 sequence comprising 3, 2 or 1 amino acid substitutions, etc.

[0149] Optionally, the VH domain of the antibody or fragment may include (c) the CDRH3 sequence of SEQ ID NO: 110 or 113, or the CDRH3 sequence containing 3, 2, or 1 amino acid substitutions, and (d) the CDRH2 sequence of SEQ ID NO: 109 or 112, or the CDRH2 sequence containing 3, 2, or 1 amino acid substitutions.

[0150] Optionally, the VH domain of the antibody or fragment may include (e) the CDRH3 sequence of SEQ ID NO: 110 or 113, or a CDRH3 sequence containing 3, 2, or 1 amino acid substitutions; (f) the CDRH1 sequence of SEQ ID NO: 108 or 111, or a CDRH1 sequence containing 3, 2, or 1 amino acid substitutions; and (g) the CDRH2 sequence of SEQ ID NO: 109 or 112, or a CDRH2 sequence containing 3, 2, or 1 amino acid substitutions.

[0151] 9. Antibodies or fragments containing a binding site that specifically binds to bone morphogenetic protein 6 (BMP6), wherein the binding site 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, C An antibody or fragment (optionally as described in any of clauses 1 to 8) containing a VH domain comprising the amino acid sequence of the VH domain of an antibody selected from L-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713, or an antibody or fragment (optionally as described in any of clauses 1 to 8) containing amino acids that are 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 contains 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. An antibody or fragment according to any of clauses 1 to 9, comprising first and second copies of the VH domain.

[0155] For example, an antibody or fragment may include a binding site comprising a VH domain of the present invention that pairs with a VL domain of the present invention, and the binding site may be capable of specifically binding to BMP6 (e.g., mature BMP6, e.g., human and / or cynomolgus monkey BMP6). For example, an antibody or fragment may include two of such binding sites.

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

[0157] For example, the VL gene segment is IGKV3-20, for example IGKV3-20 * It is 01. For example, the VL gene segment is IGKV1-5, for example IGKV1-5 * 03. For example, the VL gene segment is IGKV3-15, for example IGKV3-15 * It is 01.

[0158] 12. The antibody or fragment described in Clause 11, wherein VL is 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 contains the CDRL3 sequence of an anti-BMP6 antibody as described in clause 11 or 12.

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

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

[0163] 16. An antibody or fragment (optionally as described in any of clauses 1 to 15) that specifically binds to bone morphogenetic protein 6 (BMP6) and comprises a VL domain containing a CDRL3 sequence selected from sequence number 119 or 122, or the selected CDRL3 sequence containing 3, 2, or 1 amino acid substitutions.

[0164] 17. An antibody or fragment (optionally as described in any of clauses 1 to 16) that specifically binds to bone morphogenetic protein 6 (BMP6) and comprises a VL domain containing the CDRL3 (and optionally 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 the said sequence containing 3, 2 or 1 amino acid substitutions.

[0165] Optionally, the VL domain includes a CDRL3 sequence selected from SEQ ID NO: 119 or 122, or a selected sequence containing 3, 2, or 1 amino acid substitutions, and / or optionally, the VH domain includes a CDRH3 sequence selected from SEQ ID NO: 119 or 122, or a selected sequence containing 3, 2, or 1 amino acid substitutions.

[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- The antibody or fragment according to Clause 17, comprising (ii) the CDRL3 sequence (and optionally CDRH3) of an antibody selected from 58680 and CL-58713, or each CDR3 sequence(s) containing 3, 2, or 1 amino acid substitution(s), and (ii) the CDRL1 (and optionally CDRH1) sequence of the selected antibody, or each CDR1 sequence(s) containing 3, 2, or 1 amino acid substitution(s).

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

[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 An antibody or fragment according to Clause 17 or 18, comprising (iv) a CDRL3 (and optionally CDRH3) sequence of an antibody selected from 680 and CL-58713, or each CDR3 sequence(or more) containing 3, 2, or 1 amino acid substitutions, and (iv) a CDRL2 (and optionally CDRH2) sequence of the selected antibody, or each CDR2 sequence(or more) containing 3, 2, or 1 amino acid substitutions.

[0169] Optionally, the VL domain of the antibody or fragment includes (c) the CDRL3 sequence of SEQ ID NO: 119 or 122, or the CDRL3 sequence containing 3, 2, or 1 amino acid substitutions, and (d) the CDRL2 sequence of SEQ ID NO: 118 or 121, or the CDRL2 sequence containing 3, 2, or 1 amino acid substitutions.

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

[0171] 20. Antibodies or fragments containing a binding site that specifically binds to bone morphogenetic protein 6 (BMP6), wherein the binding site 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, C An antibody or fragment (optionally as described in any of clauses 1 to 19) containing a VL domain comprising the amino acid sequence of the VL domain of an antibody selected from L-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713, or amino acids that are 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 contains 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. An antibody or fragment according to any of clauses 1 to 20, comprising first and second copies of the VL domain.

[0175] For example, the antibody or fragment includes a binding site comprising the VL domain of the present invention paired with a VH domain, and the binding site can specifically bind to BMP6 (e.g., mature BMP6, e.g., human and / or cynomolgus monkey BMP6). For example, the antibody or fragment includes two of such binding sites.

[0176] 22. An antibody or fragment (optionally as described in any of clauses 1 to 21) that specifically binds to bone morphogenetic protein 6 (BMP6) and contains 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 amino acid that is 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 as described in any of clauses 1 to 22) that specifically binds to bone morphogenetic protein 6 (BMP6) and contains 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 amino acid that is 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 the selected antibody, or amino acids that are 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 as described in any of Clauses 1 to 24) that specifically binds to a human BMP6 epitope which is identical to the epitope to which the antibody described in any of Clauses 1 to 24 binds.

[0183] 26. An antibody or fragment as described in Clause 25, in which the epitope is identified by an unrelated amino acid scan or X-ray crystallography.

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

[0185] In one embodiment, sequential amino acid substitutions of 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 scan) or another unrelated amino acid, may provide residues that, by mutation, reduce or eliminate the antibody's ability to recognize the antigen of interest. Binding can be evaluated using standard techniques such as, but are not limited to, SPR, HTRF, and ELISA (as described elsewhere herein). Other substitutions may be made to enhance binding disruption, such as altering the charge of the side chains of antigen sequence amino acids (e.g., lysine to glutamate) or exchanging polar and nonpolar residues (e.g., serine to leucine). Alanine scan or other amino substitution methods can be performed either using recombinant soluble antigens 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 may be used to determine contact residues between the antibody and antigen (i.e., to determine the epitope to which the antibody binds), and crystallography allows for the direct visualization of contact residues involved in antibody-antigen interactions. Alongside 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-chain peptides may be produced based on the antigen sequence, and antibody binding to these peptides may be evaluated using standard techniques such as SPR, HTRF, and ELISA (these are described elsewhere in this specification), but are not limited to these. Further investigation of epitopes may be provided by performing an alanine scan on any peptide showing binding. Instead of linear peptides, structural scans may be performed using the Pepscan technique (http: / / www.pepscan.com / ), which uses chemical binding of peptides to a backbone, and has been used to determine discontinuous epitopes on CD20-targeted 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, binding epitopes can be identified using limited protein digestion and mass spectrometric mapping. The antibody-antigen complex is digested by a protease, such as but not limited to trypsin. The digested complex peptide is compared to the digestion mass spectrometric mapping of the antibody and antigen alone to determine whether a particular epitope is protected by complex formation. To narrow down the individual amino acid residues involved in the interaction, competitive binding, which is a further action involving amino acid substitution, may be used (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] Therefore, in one embodiment, the contact residue of the epitope is identified using an irrelevant amino acid scan (e.g., an alanine scan). In another embodiment, the irrelevant amino acid scan (e.g., an alanine scan) is performed using techniques selected from SPR, HTRF, ELISA, X-ray crystallography, cryo-electron microscopy, and a combination of limited protein digestion and mass spectrometry. In one embodiment, the irrelevant amino acid scan (e.g., an alanine scan) is performed using HTRF. In another embodiment, the irrelevant 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 an epitope if the signal reduction is at least 25%. 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 an 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, the epitope contact residue is identified by X-ray crystallography. In another embodiment, the epitope contact residue is identified by cryo-electron microscopy. In yet another embodiment, the epitope contact residue is identified by a combination of limited protein digestion and mass spectrometry.

[0191] 27. The antibody or fragment according to Clause 26, wherein the contact residue of the epitope is defined by at least a tenfold reduction in affinity in an unrelated amino acid scan, e.g., an alanine scan, as determined by SPR.

[0192] In one embodiment, the affinity reduction is at least 15 times. In one embodiment, the affinity reduction is at least 20 times. In one embodiment, the affinity reduction is at least 30 times. In one embodiment, the affinity reduction is at least 40 times. In one embodiment, the affinity reduction is at least 50 times. In one embodiment, the affinity reduction is at least 100 times. SPR may be carried out as described herein.

[0193] 28. An antibody or fragment (optionally selected from any of the antibodies in clauses 1 to 27) that competes with any antibody in 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 these techniques and standard conditions, for example.

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

[0196] Optionally, conflicts regarding binding to hBMP6 are resolved using SPR. SPR may be performed as described herein.

[0197] 29. An antibody or fragment according to any of clauses 1 to 28 that specifically binds to human BMP6 containing SEQ ID NO: 562, and / or cynomolgus monkey BMP6 containing SEQ ID NO: 564, and / or rat BMP6 containing SEQ ID NO: 563.

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

[0199] For example, BMP6 as used herein is human, mouse, or cynomolgus monkey BMP6.

[0200] In one embodiment, the antibody or fragment binds to cynomolgus monkey 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 monkey 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 monkey 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 monkey 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).

[0201] In one embodiment, the antibody or fragment binds to cynomolgus monkey BMP6 with an affinity of up to twice its affinity for hBMP6. In another embodiment, the antibody or fragment binds to cynomolgus monkey BMP6 with an affinity of up to four times its affinity for hBMP6. In yet another embodiment, the antibody or fragment binds to cynomolgus monkey BMP6 with an affinity of up to five times its affinity for hBMP6. In yet another embodiment, the antibody or fragment binds to cynomolgus monkey BMP6 with an affinity of up to six times its affinity for hBMP6. In yet another embodiment, the antibody or fragment binds to cynomolgus monkey BMP6 with an affinity of up to eight times its affinity for hBMP6. In yet another embodiment, the antibody or fragment binds to cynomolgus monkey BMP6 with an affinity of up to ten times its affinity for hBMP6. As used herein, "hBMP6" refers to human BMP6, for example, human BMP6 disclosed herein, including sequence number 562.

[0202] In one embodiment, the antibody or fragment does not bind detectably to cynomolgus monkey BMP6. In another embodiment, the antibody or fragment does not bind detectably to mouse (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-inactive 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 deficient IgG1 (disabled IgG1). Optionally, the antibody or fragment comprises a mouse (e.g., mouse and / or rat) constant region. Optionally, the antibody or fragment comprises one of the heavy chain constant region sequences described herein.

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

[0206] 30. The antibody or fragment according to any of the clauses 1 to 29, wherein the antibody or fragment comprises a human constant region, for example, an IgG4 constant region or an IgG1 constant region.

[0207] For example, the constant region includes a heavy chain constant region, which contains the amino acid sequence of SEQ ID NOs: 429, 431, 433, 435, 437, 439, 440, 442, 444, 446, 448, 450, 454, or 456. Optionally, the heavy chain C region is an IHG1 C region containing the amino acid sequence of SEQ ID NOs: 429, 431, 433, 435, or 437. Optionally, the heavy chain C region is an IHG2 C region containing the amino acid sequence of SEQ ID NOs: 439, 440, 442, or 444. Optionally, the heavy chain C region is an IHG4 C region containing the amino acid sequence of SEQ ID NOs: 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 containing SEQ ID NOs: 451, 452, or 453.

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

[0209] 31. The antibody or fragment described in Clause 30, wherein the constant region is the IgG4-PE constant region.

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

[0211] The anti-BMP6 antibody or fragment described in the present invention comprises a 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 a deficient IgG1 as defined in the sequence listing of the present invention.

[0212] In other embodiments, the antibody or fragment is one 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-capable IgG1 constant region, optionally having ADCC and / or CDC activity. In one embodiment, the constant region is manipulated for enhancement of ADCC and / or CDC and / or ADCP. In another embodiment, the constant region is manipulated for enhancement of 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 IgG4 containing both the Leu235Glu and Ser228Pro mutations. This “IgG4-PE” heavy chain constant region (see sequence listing for an example) is effector null.

[0214] Alternative effector null human constant regions include IgG1 containing L235A and / or G237A mutations (e.g., LAGA, see sequence listing). * 01 is the allele, which is a deficient IgG1. In one embodiment, the antibody or antibody fragment disclosed herein comprises the IgG1 heavy chain constant region, and the sequence contains alanine at positions 235 and / or 237 (EU index numbering).

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

[0216] 32. An antibody or fragment (e.g., a bispecific antibody) as described in any of clauses 1 to 31, further comprising an antigen-binding site that optionally specifically binds to another target antigen (e.g., human hemoduvelin, a transferrin receptor (e.g., TFR2), or a BMP receptor (e.g., BMPRI or BMPRII), or 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 the agonist binding site of the other antigen. In another example, the further binding site is the antagonist binding site of the other antigen.

[0219] In one example, the further binding site is a binding site comprising an antibody binding site including VH and VL, a constant domain of the 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 double-conjugated antibody or fragment, or a fusion protein containing the antibody or fragment described in Clauses 1 to 31. The double-conjugated antibody has the meaning described above.

[0221] For example, the antibody, fragment, or fusion protein of clause 24 or 24a is DVD-Ig, mAb 2 FIT-Ig, mAb-dAb, Dock-Lock, SEEDbody, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, Intracellular Antibody, 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 , knob-in-hole, knob-in-hole with common light chain, common light chain and charge pair and FIT-Ig, e.g., mAb 2 This includes a bispecificity format selected from knob-in-holes having FIT-Ig.

[0222] In one embodiment, the dual specificity format is DVD-Ig, mAb 2FIT-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, Intracellular Antibody, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, Triple Body, Mini Antibody, Mini Body, TriBi Mini Body, scFv-CH3KIH, scFv- Selected from 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, scFv4-Ig, and zybody.

[0223] In one embodiment, the bispecificity format includes 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, intracellular antibody, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, triplebody, mini-antibody, minibody, TriBi minibody, and 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 Selected from IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, and zybodies, e.g., DVD-Ig, FIT-Ig, mAb-dAb, dock-lock, SEEDbody, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intracellular antibodies, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, diabody-CH3, minibody, 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, among others, as well as FIT-Ig, e.g., FIT-Ig.

[0224] In one embodiment, the dual specificity format is DVD-Ig, mAb 2mAb-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, Intracellular Antibody, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, Triple Body, Mini Antibody, Mini Body, TriBi Mini Body, 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 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 zybody, e.g., DVD-Ig, mAb 2 mAb-dAb, Dock-Lock, SEEDbody, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, Intracellular antibody, 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, in particular, mAb 2 , knob-in-hole, knob-in-hole having a common light chain, knob-in-hole having a common light chain and charge pair, and knob-in-hole having a common light chain, for example mAb 2 Selected from.

[0225] In one embodiment, the bispecificity 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, Intracellular Antibody, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, Triple Body, Mini Antibody, Mini Body, TriBi Mini Body, 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, intracellular antibodies, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, minibody, 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, and among others, selected from knob-in-hole, knob-in-hole with a common light chain, knob-in-hole with a common light chain and charge pair, and knob-in-hole with a common light chain.

[0226] 33. An 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. In an alternative, the subject is a non-human animal. In one example, the subject is an adult. In another example, the subject is an infant. In one example, the subject is a human CKD patient undergoing dialysis. In another example, the subject is a human with end-stage renal disease.

[0228] For example, the antibodies or fragments of the present invention are intended to treat or prevent diseases or conditions in subjects (e.g., humans) 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, Sjögren'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 include cardiovascular disease, atherosclerosis, systemic sclerosis-associated pulmonary fibrosis, cardiac fibrosis, PAH, tumorigenesis, tumor invasion, tumor metastasis, fibrotic diseases, and cancer-associated fibroblast formation (e.g., pancreatic cancer).

[0230] In one example, the disease or condition is in humans. In another example, the disease or condition is in animals.

[0231] For example, the antibodies or fragments of the present invention are for treating or preventing TIGIT-mediated diseases or conditions in humans, selected from, for example, 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 cancer), soft tissue sarcoma, hematological malignancies, such as Hodgkin's and non-Hodgkin's diseases, and diffuse large B-cell lymphomas (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 virus-induced cancers (e.g., cervical cancer and nasopharyngeal cancer) and soft tissue sarcoma).

[0232] For example, BMP6-mediated diseases or conditions are neurodegenerative diseases, disorders, or conditions selected from Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, primary progressive multiple sclerosis, secondary progressive multiple sclerosis, corticobasal degeneration, Rett syndrome, age-related macular degeneration and retinitis pigmentosa, anterior ischemic optic neuropathy, glaucoma, uveitis, depression, trauma-related stress or post-traumatic stress disorder, frontotemporal dementia, Lewy body dementia, mild cognitive impairment, posterior cortical atrophy, primary progressive aphasia and progressive supranuclear palsy or age-related dementia, and in particular, neurodegenerative diseases, disorders, or conditions selected from Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease and Huntington's disease, for example, Alzheimer's disease.

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

[0234] 34. The antibody or fragment described in Clause 33, which is administered to the subject simultaneously with or sequentially with an erythropoietin enhancer (ESA).

[0235] 35. A combination of a certain amount of anti-BMP6 antibody or fragment and a certain amount of ESA (for example, including multiple doses of the antibody and / or ESA), wherein the antibody or fragment is as described in any one of the clauses 1 to 34.

[0236] A medical kit is also provided which includes a first sterile container containing the combination, the amount of antibody or fragment, and a second sterile container containing the amount of ESA, and optionally instructions for using the combination to treat the target anemia.

[0237] In one example, the combination is intended to treat or prevent anemia in the subject, and the total dose of antibodies and the total dose of ESA are administered to the subject over four consecutive weeks in an X:Y ratio, where X is 10 to 2x10 6 And Y=4, for example, X is 10 ~ 2x10 6 The values ​​are micrograms and Y = 4 micrograms.

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

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

[0240] 36. (a) A method for preventing a decrease in the target blood hemoglobin level to less than 10 g / dL, comprising administering an antibody or fragment and an erythropoiesis-promoting agent (ESA) to the target, (b) A method for increasing blood hemoglobin levels to at least 10 g / dL in a subject suffering from anemia, comprising administering an antibody or fragment and an erythropoiesis-stimulating agent (ESA) to the subject, 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 an antibody or fragment and an erythropoiesis-stimulating agent (ESA) to the subject, wherein the anemia is treated or prevented. (d) A method for eliminating or reducing the need for iron administration or transfusion to a subject suffering from anemia, comprising administering an antibody or fragment and an erythropoiesis-stimulating agent (ESA) to the subject, such that the need is eliminated or reduced. (e) A method for treating or preventing anemia in a subject suffering from a microbial infection, comprising administering an anti-BMP6 antagonist and an erythropoiesis-promoting agent (ESA) to the subject. (f) A method for reducing the administration of erythropoiesis-stimulating agents (ESAs) to treat anemia in a subject suffering from anemia, comprising administering an antibody or fragment and an ESA, wherein the anemia is treated in the subject, or (g) A method for treating or reducing the risk of anemia in a subject who is anemic or at risk of anemia, comprising administering an antibody or fragment and a low dose of an erythropoiesis-stimulating agent (ESA) to the subject, wherein anemia is treated or the risk of anemia is reduced in the subject, the antibody, fragment or combination described in any one of Clauses 1 to 34 for use in such a method.

[0241] 37. ESA is a. Epoetin alfa administered at weekly doses of 1000, 1500, 2500, 5000, 11000, 18000, 34000, or less than 90000 units, optional for subjects who have previously received epoetin alfa treatment at weekly doses of <1500, 1500-2499, 2500-4999, 5000-10999, 11000-17999, 18000-33999, 34000-89999, or ≥90000 units, respectively. b. Darbepoetin alfa or Aranesp®, administered at weekly doses of less than 6.25, 10, 12.5, 20, 25, 40, 60, 100, or 200 mcg, optional subjects who have previously received treatment with darbepoetin alfa or Aranesp® at weekly doses of 6.25, 10, 12.5, 20, 25, 40, 60, 100, or 200 mcg, respectively, or c. Darbepoetin alfa or Aranesp®, administered in weekly doses of less than 6.25, 10, 20, 40, 60, 100, or 200 mcg, and optionally, subjects who have previously received epoetin alfa treatment at weekly doses of 1500-2499, 2500-4999, 5000-10999, 11000-17999, 18000-33999, 34000-89999, or ≥90000 units, as described in any one of clauses 1-34 and 36.

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

[0243] 39. Use of any of the antibodies, fragments, or combinations described in any of Clauses 1 to 38 in the manufacture of a medicinal product to be administered to a subject for the treatment or prevention of 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 the 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 may be any of those disclosed herein.

[0246] 41. If the BMP6-mediated disease or condition is anemia, use as described in Clause 39 or as described in Clause 40.

[0247] 42. Further therapies, including, at the discretion of administering further therapeutic agents, (a) Iron in the veins, (b) ESA (e.g., EPO), (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) BMP inhibitors (e.g., further anti-BMP antibodies or fragments) (e.g., BMP is BMP2, 4, 5, 6, 7, or 9), (i) Matryptase-2 (MTP2) agonists (e.g., matryptase-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 fusion), (p) GDF11 inhibitors, and (q) Myostatin inhibitors, an antibody, fragment, combination, use or method as described in any one of the clauses 33 to 41, selected from the group consisting of these.

[0248] Optionally, additional agents include Luspatercept® or Sotatercept®. Optionally, additional agents include TGF-β superfamily inhibitors. For example, additional agents include transforming growth factor beta superfamily type 1 activin receptor-like kinase (ALK) receptor inhibitors, ALK2 inhibitors, ALK3 inhibitors, ALK4 inhibitors, ALK5 inhibitors, or ALK7 inhibitors.

[0249] For example, additional drugs include IL-6 or IL-6R inhibitors, such as sarilumab, bovalilizumab, or tocilizumab (e.g., Kevzara® or Actemra®).

[0250] For example, further drugs include TNF-alpha or TNF-alpha receptor inhibitors, such as adalimumab, Humira®, Remicade®, or Enbrel® or Simponi®. In one embodiment, an NRf2 inhibitor improves the efficacy of an anti-BMP6 antibody or fragment by disrupting the iron feedback loop that induces more BMP6 expression in the target being treated.

[0251] This disclosure includes generic versions of branded drugs, and disclosures of these generic drugs are included herein by reference for possible uses in the present invention, for example, as part of a combination.

[0252] For example, a combination may include 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, where the BMP6 inhibitor includes the antibody or fragment of the present invention.

[0253] 43. A pharmaceutical composition comprising an antibody, fragment or combination described in any one of Clauses 1 to 38 and 42, and a pharmaceutically acceptable excipient, diluent or carrier, which is optionally combined with a further therapeutic agent selected from the 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 or secondary PAH), cerebral cavernous hemangioma (CCM) (e.g., familial 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, Sjögren'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 include cardiovascular disease, atherosclerosis, systemic sclerosis-associated pulmonary fibrosis, cardiac fibrosis, PAH, tumorigenesis, tumor invasion, tumor metastasis, fibrotic diseases, and cancer-associated fibroblast formation (e.g., pancreatic cancer).

[0257] 45. A pharmaceutical composition as described in Clause 43 or 44, combined with a label or instructions for use in treating and / or preventing a disease or condition in a human, wherein optionally the label or instructions include a marketing authorization number (e.g., an FDA or EMA authorization number), and optionally the kit includes an IV or injector containing an antibody or fragment.

[0258] 46. ​​Nucleic acids encoding the VH domain and / or VL domain of an antibody or fragment as described in any one of clauses 1 to 32.

[0259] 47. A VH domain containing 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 a nucleic acid encoding an amino acid that is 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 is provided that encodes a VH domain containing the amino acid sequence of SEQ ID NO: 114, or an amino acid that is 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%.

[0262] 48. A VL domain containing 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 a nucleic acid encoding an amino acid that is at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical thereto.

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

[0264] Optionally, a VL domain containing the amino acid sequence of SEQ ID NO: 123 is provided, or a nucleic acid encoding amino acids that are 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%.

[0265] 49. (a) Nucleotide sequences that are at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to sequences of sequence numbers 115, 520, or 521, and / or (b) Nucleic acids containing nucleotide sequences that are at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to sequence 124, 522, or 523 (e.g., in host cells, e.g., CHO or HEK293 or Cos cells).

[0266] The following alternatives are offered: (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., CHO or HEK293 or Cos cell) containing 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) Nucleic acids (e.g., in host cells, e.g., CHO or HEK293 or Cos cells) containing 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) Nucleic acids (e.g., in host cells, e.g., CHO or HEK293 or Cos cells) containing 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) Nucleotide sequences that are at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to sequences of sequence numbers 115, 520, or 521, and / or (b) A combination of first and second nucleic acids (e.g., in host cells, e.g., CHO or HEK293 or Cos cells) each containing 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.

[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 first and second nucleic acids, each containing a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the sequence of Sequence ID No. 124 (for example, in a host cell, e.g., CHO or HEK293 or Cos cell).

[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 first and second nucleic acids (e.g., in a host cell, e.g., CHO or HEK293 or Cos cell) each containing a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical to the sequence of Sequence 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 first and second nucleic acids (e.g., in a host cell, e.g., CHO or HEK293 or Cos cell) each containing a nucleotide sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical to the sequence of Sequence ID No. 523.

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

[0274] For example, regarding (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 example in this specification where % identity is mentioned, there is 100% identity.

[0276] 50. Nucleic acids encoding the heavy and / or light chains of an antibody or fragment as described in any one of Clauses 1 to 32.

[0277] 51. A nucleic acid encoding a heavy chain containing 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 containing an amino acid sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the sequence of sequence number 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 the 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) Nucleic acids (e.g., in host cells, e.g., CHO or HEK293 or Cos cells) containing nucleotide sequences that are at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical to selected sequences of antibodies selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, 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, for example, CL-58838. Alternatively, for example, in a host cell, such as a CHO, HEK293, or Cos cell, the first nucleic acid comprises (a) and the second nucleic acid comprises (b).

[0283] All nucleic acids of the present invention as described herein are expressible in host cells, such as CHO, HEK293, or Cos cells, for example, to express variable domains or chains of the antibodies or fragments of the present invention.

[0284] For example, the following is provided: (a) Nucleotide sequences that are at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical to sequences selected from sequence numbers 512, 514, 516, 518 and 519, and / or (b) Nucleic acids (e.g., in host cells, e.g., CHO or HEK293 or Cos cells) that contain nucleotide sequences that are at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical to sequences selected from SEQ ID NOs. 513, 515 and 517.

[0285] (a) Nucleotide sequences that are at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical to Sequence ID No. 512, and / or (b) Nucleic acids containing 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 host cells, e.g., CHO or HEK293 or Cos cells).

[0286] (a) Nucleotide sequences that are at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 514, and / or (b) Nucleic acids containing 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 host cells, e.g., CHO or HEK293 or Cos cells).

[0287] (a) Nucleotide sequences that are at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 516, and / or (b) Nucleic acids containing 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 host cells, e.g., CHO or HEK293 or Cos cells).

[0288] (a) Nucleotide sequences that are at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical to Sequence ID No. 518, and / or (b) Nucleic acids containing nucleotide sequences that are at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to sequence numbers 513, 515, or 517 (for example, in host cells, e.g., CHO or HEK293 or Cos cells).

[0289] (a) Nucleotide sequences that are at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 519, and / or (b) Nucleic acids containing nucleotide sequences that are at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical to sequence numbers 513, 515, or 517 (for example, in host cells, e.g., CHO or HEK293 or Cos cells).

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

[0291] For example, regarding (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 nucleic acids (or multiple nucleic acids) (for example, any one of the nucleic acids described in any of clauses 46 to 53), wherein the vector is optionally a CHO or HEK293 vector.

[0293] 55. A host cell containing nucleic acid(s) (for example, nucleic acid(s) as described in any one of clauses 46-53) or the vector of clause 54.

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

[0295] For example, VH, DH, and JH are IGHV1-3, IGHD3-10, and IGHJ4 (e.g., IGHV1-3 * 01, IGHD3-10 * 01 and IGHJ4 * 02) is.

[0296] For example, VH, DH, and JH are IGHV1-3, IGHD3-10, and IGHJ3, for example, IGHV1-3 * 01, IGHD3-10 * 01 and IGHJ3 * It is 02.

[0297] For example, VH, DH, and JH are IGHV3-11, IGHD6-19, and IGHJ4, for example IGHV3-11 * 01, IGHD6-19 * 01 and IGHJ4 * It is 02.

[0298] For example, VH, DH, and JH are IGHV1-3, IGHD7-27, and IGHJ4, for example, IGHV1-3 * 01, IGHD7-27 * 02 and IGHJ4 * It is 02.

[0299] For example, VH, DH, and JH are IGHV1-3, IGHD4-23, and IGHJ4, for example, IGHV1-3 * 01, IGHD4-23 * 01 and IGHJ4 * It is 02.

[0300] For example, VH, DH, and JH are IGHV1-3, IGHD5-18, and IGHJ4, for example, IGHV1-3 * 01, IGHD5-18 * 01 and IGHJ4 * It is 02.

[0301] For example, VL and JL are IGKV1-5 and IGKJ1, e.g., IGKV1-5 * 03 and IGKJ1 * It is 01.

[0302] For example, VL and JL are IGKV3-20 and IGKJ1, e.g., IGKV3-20 * 01 and IGKJ1 * It is 01.

[0303] For example, VL and JL are IGKV3-15 and IGKJ3, e.g., IGKV3-15 * 01 and IGKJ3 * It is 01.

[0304] For example, VL and JL are IGKV3-20 and IGKJ3, e.g., IGKV3-20 * 01 and IGKJ3 * It is 01.

[0305] In one example, the antibody or fragment contains an HCDR3 length of 9, 10, 11, or 12 residues, e.g., 10 residues, e.g., 11 residues. In another example, the antibody or fragment contains an LCDR3 length of 7, 8, or 9 residues, e.g., 8 residues, e.g., 9 residues. In another example, each VH domain of the antibody or fragment contains 1 to 11 non-germ cell residues, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 non-germ cell residues. In another example, each VL domain of the antibody or fragment contains 3 to 8 non-germ cell residues, e.g., 3, 4, 5, 6, 7, or 8 non-germ cell residues.

[0306] In one embodiment, the CDR sequence described herein is determined according to Kabat. Alternatively, the CDR sequence is determined according to IMGT.

[0307] In one example, the selected antibodies are 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 another example, the selected antibody is CL-58838.

[0308] In one example, the selected antibodies include the heavy chains 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 another example, the selected antibodies include the heavy chain of CL-58838.

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

[0310] Optionally, the antibody is a four-chain antibody comprising a heavy chain dimer conjugated to 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 derived 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 derived from the same selected antibody. In one example, the heavy and light chains are derived from the same selected antibody, for example, any antibody disclosed in the sequence listing or the examples table herein.

[0311] In one example, the selected antibodies include the light chains 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 another example, the selected antibodies include the light chain of CL-58838.

[0312] In one example, the selected antibody contains 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 another example, the selected antibody contains the variable domain of CL-58838.

[0313] In one example, the selected antibody contains 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 another example, the selected antibody contains the VH domain of CL-58838.

[0314] In one example, the selected antibody contains 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 another example, the selected antibody contains the VH and VL domains of CL-58838.

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

[0316] In one example, the binding site includes a VH / VL pair that specifically binds to human BMP6 (e.g., human BMP6 containing or consisting of the bolded sequence of Sequence ID No. 1 in the sequence listing herein). In one example, the antibody or fragment contains two copies (e.g., two and two or fewer) of the binding site.

[0317] In one example, the antibody or fragment contains an HCDR3 length of 9–12 residues and / or an LCDR3 length of 7–9 residues. In one example, the antibody or fragment contains an HCDR3 length of 9, 10, 11 or 12 residues, e.g., 10 residues, e.g., 11 residues. In one example, the antibody or fragment contains an LCDR3 length of 7, 8 or 9 residues, e.g., 8 residues, e.g., 9 residues. In one example, each VH domain of the antibody or fragment contains 1–11 non-germ cell residues, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 non-germ cell residues. In one example, each VL domain of the antibody or fragment contains 3–8 non-germ cell residues, e.g., 3, 4, 5, 6, 7 or 8 non-germ cell residues.

[0318] Optionally, an 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 containing 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] At will, the amino acid substitutions are conservative amino acid substitutions, and at will, each conservative substitution belongs to one of the groups (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), and tryptophan (W).

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

[0321] Optionally, any BMP6 in this specification is human BMP6, e.g., hBMP6 (Peprotech 120-06), in an in vitro study.

[0322] For example, the antibody or fragment of the present invention is, for instance, 5 x 10 6 M -1 ×s -1 , or approximately 5x10 6 M -1 ×s -1 It binds to human BMP6 at a Ka of 4 or 5 s. For example, the antibody or fragment of the present invention binds to human BMP6 at a Ka of 4 or 5 s. -1 , or about 4 or 5 seconds -1 It binds to human BMP6 with a Kd of . 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 contains heavy chains, each heavy chain containing or consisting of the amino acid sequence of SEQ ID NO: 116, and each light chain containing or consisting of the amino acid sequence of SEQ ID NO: 125.

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

[0325] Alternative antibody or fragment: In any of its configurations, the present invention may relate to an antibody or fragment (a substitute antibody or fragment) as follows:

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

[0327] Optionally (Option 2), the antibody or fragment is: a. Each heavy chain containing the VH domain amino acid sequence of SEQ ID NO: 402 or 565, and each light chain containing the VL domain amino acid sequence of SEQ ID NO: 410, or b. Each heavy chain containing the VH domain amino acid sequence of SEQ ID NO: 418, and each light chain containing the VH domain amino acid sequence of SEQ ID NO: 426, c. Optionally, the heavy chain is human gamma-1 (e.g., IHG1) * 01), Gamma-4 (e.g., IGGH4) * 01 or IGGH4 * 01-PE) Contains the constant region, or the amino acid sequence of SEQ ID NOs. 429, 437, 446, 454, or 456.

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

[0329] The human IgG heavy chain gene naturally encodes lysine at the C-terminus. 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. Since 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 formulation. Accordingly, IgG antibodies, constant regions, or heavy chains shown herein to terminate at a C-terminus with G may, alternatively, be provided in a form terminated with GK (i.e., a resin bound to the C-terminal side of the indicated G).

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

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

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

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

[0334] Option 2b: For example, the antibody or fragment contains a heavy chain VH domain, each VH containing or comprising the amino acid sequence of SEQ ID NO: 565, and each light chain VL domain containing or comprising the amino acid sequence of SEQ ID NO: 410.

[0335] In one example, the antibody or fragment contains a heavy chain VH domain, each VH containing or comprising the amino acid sequence of SEQ ID NO: 114, and each light chain VL domain containing or comprising 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 one or more mature versions of these sequences.

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

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

[0339] As used herein, “inhibit,” “inhibit,” and “to inhibit” refer to the ability of an antagonist (e.g., an antibody or a fragment thereof) to bind to an epitope (e.g., of hBMP6) that partially or completely prevents the binding of another antigen. When the epitope to which the antagonist binds completely blocks the ligand binding site, ligand binding is completely blocked (this may be physical blockage in the case of overlapping epitopes, or steric blockage if the antagonist is large enough to prevent ligand binding to its own epitope), and the ligand is not removed from circulation. Thus, the concentration of circulating ligand may appear to increase. When the epitope to which the antagonist binds partially blocks the ligand binding site, the ligand may be able to bind, but only weakly (in the case of partial inhibition), or it may bind in an orientation different from the innate binding interaction. In this case, some of the ligand may be removed from circulation, but not as much as when the ligand binding site is 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 in this specification and in Mathis (1995) Clinical Chemistry 41(9), 1391-1397. Inhibition can also be measured by flow cytometry, in which the receptor is expressed on cells, or by ELISA, in which the receptor is adsorbed to a plate.

[0340] Optionally, the antibody contains a VH domain encoded by a VDJ region sequence, where VDJ is derived from recombination of the VH gene segment, D gene segment, and JH gene segment, and VH is the human germline (i)VH1-3, (ii)VH2-5, or (iii)VH3-15 gene segment. Additionally or alternatively, optionally, the antibody contains a VL domain encoded by a VJ region sequence, where VJ is derived from recombination of the VL gene segment and JL gene segment, and VL is the 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 a stronger affinity (lower KD as determined by SPR) than it binds to BMP7, and / or optionally, binds to BMP6 with a stronger affinity than it binds to BMP5.

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

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

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

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

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

[0347] In one example, the KD is 5–15 pM (e.g., 10 pM) (or around that). In another example, the KD is 2–5 nM (e.g., 3 nM) (or around that). In yet another example, the KD is 100–400 pM (e.g., 140 or 390 pM) (or around that).

[0348] In one example, an antibody (e.g., as Fab) or fragment is, (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 (e.g., human BMP6) off ) has.

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

[0350] In one example, an antibody (e.g., as Fab) or fragment is, (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) 1 x 10 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 The on-velocity (K) for coupling BMP6 (e.g., human BMP6) on ) has.

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

[0352] As provided in the provisions or other embodiments herein, the anti-BMP6 antibody or fragment is determined by surface plasmon resonance to have a K content of less than 50 nM, less than 40 nM, or less than 30 nM. D BMP6 having a chromosome that can bind to, for example, human BMP6. In another embodiment, the anti-BMP6 antibody or fragment has a K content of less than 20 nM, less than 15 nM, or less than 10 nM as determined by surface plasmon resonance. D BMP6 having a Kn, for example, human BMP6, can bind to it. The anti-BMP6 antibody or fragment has a Kn 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. DBMP6 having such a property, for example, human BMP6, can bind to it. D This 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 This is within the range of 0.01 to 1 nM, or within the range of 0.05 to 2 nM, or within the range of 0.05 to 1 nM. D This may relate to hBMP6, cynomolugus monkey (i.e., "cyno") BMP6, and / or mouse BMP6.

[0354] In another embodiment, the anti-BMP6 antibody described herein contains about 0.5–10 μM, for example, about 1–8 μM or about 1–7 μM of K. ON It has a velocity (for example, measured by SPR at 25°C or 37°C). In another embodiment, K ON The speed is approximately 1 to 5 μM, for example, approximately 1 μM, approximately 1.5 μM, approximately 2 μM, approximately 2.5 μM, or approximately 3 μM. In another embodiment, K ON The speeds are approximately 3.5 μM, 4 μM, 4.5 μM, 5 μM, or 5.5 μM.

[0355] In another embodiment, the anti-BMP6 antibody described herein is present in a concentration of about 0.01 to 100 mM, for example, about 0.1 to 50 mM or about 0.5 to 50 mM of K. OFF It has a velocity (for example, at 25°C or 37°C, measured by SPR). In another embodiment, K OFF The velocity is approximately 0.5–10 mM, or approximately 0.5–10 mM, for example, approximately 1 mM, approximately 2 mM, approximately 3 mM, approximately 4 mM, or approximately 5 mM. In another embodiment, K OFF The velocities are approximately 0.6mM, 0.7mM, 0.8mM, or 0.9mM.

[0356] The present invention also provides the following methods (or antibodies or fragments of the present invention for use in such methods): A method for treating anemia in a subject, wherein the method is (a) On the first day (D0), administer an anti-BMP6 antibody or fragment as the target. (b) for at least three consecutive weeks, the administration of multiple doses of an erythropoietin enhancer (ESA) during the period starting from D0, wherein the subject's blood hemoglobin (Hb) concentration increases from the baseline concentration of D0 throughout the entire period. (c) over the entire duration of the period, (i) The Hb concentration is greater than or equal to 100% 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 above baseline.

[0357] Optionally, antibodies, fragments, or combinations inhibit iron release by human hepatocytes, for example, in an in vitro assay or in humans. Those skilled in the art will be familiar with standard assays such as those referenced in the examples herein.

[0358] Optionally, antibodies, fragments, or combinations are intended to treat or prevent BMP6-mediated diseases or conditions as disclosed herein in humans by inhibiting iron release by human hepatocytes. Optionally, antibodies, fragments, or combinations are intended to treat or prevent human anemia, PAH, or fibrosis by inhibiting iron release by human hepatocytes.

[0359] Optionally, antibodies, fragments, or combinations are intended to treat or prevent BMP6-mediated diseases or conditions as disclosed herein in humans by inhibiting Hump gene expression in human hepatocytes. Optionally, antibodies, fragments, or combinations are intended to treat or prevent human anemia, PAH, or fibrosis by inhibiting Hump gene expression in human hepatocytes.

[0360] Optionally, antibodies, fragments, or combinations are intended to treat or prevent BMP6-mediated diseases or conditions as disclosed herein in humans by inhibiting hepcidin or its expression in human hepatocytes. Optionally, antibodies, fragments, or combinations are intended to treat or prevent human anemia, PAH, or fibrosis by inhibiting hepcidin or its expression in human hepatocytes.

[0361] Selectively, antibodies, fragments, or combinations are intended to treat or prevent BMP6-mediated diseases or conditions as disclosed herein in humans by inhibiting BMP6 activation of Hump gene expression in humans (e.g., in their hepatocytes). Selectively, antibodies, fragments, or combinations are intended to treat or prevent anemia, PAH, or fibrosis in humans by inhibiting BMP6 activation of Hump gene expression in humans (e.g., in their hepatocytes).

[0362] Optionally, antibodies, fragments, or combinations are intended to treat or prevent BMP6-mediated diseases or conditions as disclosed herein in humans by inhibiting HJV-mediated activation of Hump gene expression in humans (e.g., in their hepatocytes). Optionally, antibodies, fragments, or combinations are intended to treat or prevent anemia, PAH, or fibrosis in humans by inhibiting HJV-mediated activation of Hump gene expression in humans (e.g., in their hepatocytes). In one example, an antibody or fragment competitively inhibits the binding of HJV to BMP6 in vitro and / or in humans. In vitro competition can be determined, for example, by standard SPR or ELISA.

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

[0364] Optionally, antibodies, fragments, or combinations are intended to treat or prevent BMP6-mediated diseases or conditions as disclosed herein in humans by inhibiting BMP binding in humans (e.g., in their hepatocytes). Optionally, antibodies, fragments, or combinations are intended to treat or prevent anemia, PAH, or fibrosis in humans by inhibiting BMP binding in humans (e.g., in their hepatocytes). In one example, an antibody or fragment forms an HJV-BMP6 complex in which HJV binds to an epitope in contact with BMP6, thereby activating Hump gene expression in human hepatocytes.

[0365] In one example, the human cells are HepG2 cells in vitro. Further details are provided in the examples herein. In one example, inhibition is inhibition in an in vitro HepG2 cell assay, determined, for example, by inhibition of a reporter gene under the control of one or more human hump regulators in vitro. For example, the regulators include responsive 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 the activation of BMP in reporter gene expression in the assay, for example, neutralization is at least 20, 30, 40, 50, 60, 70, 80, 90 or 95%, or complete neutralization.

[0366] In one example, the antibody or fragment competes with a reference antibody for binding to BMP6, as determined in vitro using a labeled reference antibody, for example, by SPR, ELISA, or in a HepG2 assay (e.g., 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 is complete inhibition of binding. For example, the reference antibody is MAb507 or MAb2365, and for example, the reference antibody is a surrogate antibody (e.g., Option 1 or Option 2 antibody).

[0367] For example, one of the BMP6s herein is human BMP6 (Peprotech#120-06) (SEQ ID NO: 2). For example, one of the BMP6s herein is any other human BMP6 disclosed herein.

[0368] In one example, the antibody or fragment is determined by surface plasmon resonance (SPR) at, for example, room temperature or RTP, as shown in 1x10⁻¹⁰ samples. -4 S -1 Binding to human BMP6 is performed at the following off-rates. See the examples.

[0369] For example, the antibodies for binding to human BMP6 were 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 It competes with reference antibodies selected from 680, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650 and CL-58756, for example, CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102 and CL-57945, for example, CL-58722, CL-58713, CL-58680, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650 and CL-58756.

[0370] Alternatively, the reference antibody according to this specification may be 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 may be CL-58838.

[0371] In one example, an antibody competes with a reference antibody for binding to human BMP6, and the reference antibody contains a VH amino acid sequence selected from, for example, 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, and / or the reference antibody contains a VH amino acid sequence selected from, for example, SEQ ID NOs: 24, 42, 114, 132, 96, 78, and 60, and / or the reference antibody contains a VH amino acid sequence selected from, for example, SEQ ID NOs: 276, 384, 366, 348, 294, 330, and 312, and / or the reference antibody contains a VH amino acid sequence selected from, for example, SEQ ID NOs: 33, 51, 123, 141, 105, 87, 69, 267, 249, 231, 213 The VL amino acid sequences include those selected from 195, 177, 159, 285, 393, 375, 357, 303, 339, and 321, for example, those selected from SEQ ID NOs. 33, 51, 123, 141, 105, 87, and 69, for example, those selected from SEQ ID NOs. 285, 393, 375, 357, 303, 339, and 321 (e.g., each included). 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 binds preferentially to human BMP6 rather than human BMP5 and / or human BMP7. Binding or competition can be determined, for example, by SPR or ELISA, as is known to those skilled in the art.

[0372] In one example, an antibody competes with a reference antibody for binding to human BMP6, and the reference antibody is selected from the sequence listing herein or from the table of examples, or from 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 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 contains the VH amino acid sequence of the VH of the antibody, and / or the reference antibody contains 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, the antibody of the present invention preferentially binds to human BMP6 rather than human BMP5 and / or human BMP7. Binding or competition can be determined, for example, by SPR or ELISA, as is known to those skilled in the art.

[0373] For example, the antibodies or fragments of the present invention include 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-7 Includes VH and VL domains of 5519, CL-75511, CL-75506, CL-75500, CL-75496, CL-75194, CL-75183, CL-58722, CL-58713, CL-58680, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650, or CL-58756.

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

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

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

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

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

[0379] For example, the antibody or fragment of the present invention is human gene segment 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) comprises a VH domain encoded by a nucleotide sequence which is a recombinant of 02). Additionally or alternatively, optionally, the antibody or fragment of the present invention comprises human gene segments IGKV3-20 and IGKJ1 (e.g., IGKV3-20 * 01 and IGKJ1 * It contains a VL domain encoded by a nucleotide sequence that is a recombinant of 01). Additionally or alternatively, optionally, the antibody or fragment of the present invention contains HCDR3 of CL-58835.

[0380] Optionally, the antibodies or fragments of the present invention are 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, and CL-7549. 6, comprising HCDR3 of an antibody selected from CL-75194, CL-75183, CL-58722, CL-58713, CL-58680, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650, and CL-58756, for example, 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 HCDR1 and / or HCDR2 of the selected antibody.

[0381] Optionally, the antibodies or fragments of the present invention are 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, and CL-7549. 6, comprising HCDR1 of an antibody selected from CL-75194, CL-75183, CL-58722, CL-58713, CL-58680, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650, and CL-58756, for example, 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 antibodies or fragments of the present invention are 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, and CL-7549. 6, comprising HCDR2 of an antibody selected from CL-75194, CL-75183, CL-58722, CL-58713, CL-58680, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650, and CL-58756, for example, 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 HCDR1 and / or HCDR3 of the selected antibody.

[0383] Optionally, the antibodies or fragments of the present invention are 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 The present invention includes the VH of an antibody selected from 0, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650 and CL-58756, for example, CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102 and CL-57945, for example, 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 includes the VL of the selected antibody.

[0384] Optionally, the antibodies or fragments of the present invention are 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 The present invention includes the VL of an antibody selected from 0, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650 and CL-58756, for example, CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102 and CL-57945, for example, 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 includes the VH of the selected antibody.

[0385] Optionally, the antibodies or fragments of the present invention are 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 The present invention comprises the heavy chain of an antibody selected from 0, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650 and CL-58756, for example, CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102 and CL-57945, for example, 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 antibodies or fragments of the present invention are 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 The present invention comprises the light chain of an antibody selected from 0, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650 and CL-58756, for example, CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102 and CL-57945, for example, 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 selected antibody is CL-58835.

[0388] Optionally, the antibody of the present invention includes the human IgG4 constant region.

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

[0390] Contact amino acid residues involved in antibody-antigen interactions, such as BMP6, can be determined by various methods known to those skilled in the art.

[0391] In one embodiment, when an antibody recognizes a linear epitope, short-chain peptides may be produced based on the antigen sequence, and the binding of antibodies to these peptides can be evaluated using standard techniques.

[0392] In one embodiment, the binding epitope can be identified using limited protein digestion and mass spectrophotometric measurements.

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

[0394] In another embodiment, the anti-BMP6 antibody (and fragment) described herein provides 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 approximately 100 μg / mL, or in the range of approximately 100–350 μg / mL. In another embodiment, the expression level exceeds approximately 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 approximately 100 μg / mL, or in the range of approximately 100–350 μg / mL. In yet another embodiment, the expression level exceeds approximately 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 approximately 100 μg / mL, or in the range of approximately 100–350 μg / mL. In another embodiment, the expression level is greater than approximately 350 μg / mL. For example, the antibody comprises either one of the VH and VL domains 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 approximately 0.5 mL to 3 mL, for example, approximately 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 expressed in CHO cells, e.g., 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 expressed in CHO cells, e.g., 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 expressed in CHO cells, e.g., CHO-E7 EBNA cells.

[0398] In any of these expression systems, the expression level is approximately 100 μg / mL to approximately 1500 μg / mL, for example, approximately 100 μg / mL to approximately 1000 μg / mL, or approximately 200 μg / mL to approximately 1000 μg / mL, or approximately 350 μg / mL to approximately 1000 μg / mL. In any of these expression systems, the lower limit of expression may be approximately 100 μg / mL, approximately 200 μg / mL, approximately 300 μg / mL, or approximately 400 μg / mL. In another embodiment, the lower limit of expression may be approximately 500 μg / mL, approximately 600 μg / mL, approximately 700 μg / mL, or approximately 800 μg / mL. In any of these expression systems, the upper limit of expression may be approximately 2000 μg / mL, approximately 1800 μg / mL, approximately 1600 μg / mL, or approximately 1500 μg / mL. In another embodiment, the upper limit of expression may 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, for example, the Lonza X-Ceed® system. In the Lonza expression system, expression can be performed in amounts of approximately 30 mL to 2 L, for example, 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 and optionally without any helper plasmid. In the Lonza expression system, the anti-BMP6 antibody (or fragment) can be expressed at levels of approximately 1 g / L, or approximately 900 mg / L, or approximately 800 mg / L, or approximately 700 mg / L. In another embodiment, in the Lonza expression system, the anti-BMP6 antibody (or fragment) can be expressed at levels of approximately 600 mg / L, or approximately 500 mg / L, or approximately 400 mg / L. In the Lonza expression system, the anti-BMP6 antibody (or fragment) can be expressed at levels of approximately 400 mg / L to approximately 2 g / L, for example, approximately 500 mg / L to approximately 1.5 g / L, or approximately 500 mg / L to approximately 1 g / L. In another embodiment, the expression level exceeds 1 g / L. In another embodiment, the anti-BMP6 antibody provides an improved half-life compared to 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 antibody-antigen interactions can be determined by a variety of methods known to those skilled in the art, such as alanine scanning, protein crystal structure analysis, mass spectrophotometric measurement, or any other techniques that would be apparent to those skilled in the art.

[0403] In one embodiment, the enumerated CDR includes one amino acid substitution, which may be a conservative amino acid substitution. In one embodiment, the enumerated CDR includes two amino acid substitutions, which may be conservative amino acid substitutions. In one embodiment, the enumerated CDR includes three amino acid substitutions, which may be conservative amino acid substitutions. In one embodiment, the enumerated CDR includes four amino acid substitutions, which may be conservative amino acid substitutions. In one embodiment, the enumerated CDR includes five amino acid substitutions, which may be conservative amino acid substitutions. In one embodiment, the enumerated CDR includes six amino acid substitutions, which may be conservative amino acid substitutions.

[0404] Amino acid substitutions include modifications in which an amino acid is replaced by a different native amino acid residue. Such substitutions may be classified as “conservative,” in which case the amino acid residue contained in the polypeptide is replaced by another native 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 may also be “non-conservative,” in which an amino acid residue present in the peptide is replaced by an amino acid having different properties, such as a native amino acid of a different group (e.g., a charged or hydrophobic amino acid is replaced with alanine), or a native amino acid is replaced with a non-conventional amino acid.

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

[0406] combination The antibody or fragment of the present invention may constitute a combination therapy with an ESA for the treatment or prevention of anemia, particularly moderate to severe anemia (i.e., indicated by blood hemoglobin less than 9.5 g / dL). Such a combination may be effective in treating anemia such as ACD (anemia of chronic disease), inflammation, or infection, and this combination therapy may produce a maintenance and increase in blood hemoglobin concentration that is statistically significant compared to the use of anti-BMP6 antibody alone. Furthermore, such an effect may be durable for several weeks (even after a single dose of anti-BMP6 antibody has been administered). In addition, the combination therapy of the present invention is useful for the treatment of anemia that conserves ESA, i.e., allows treatment with lower doses of ESA than the standard dose. This is useful in terms of the potentially harmful side effects of ESA. The present invention may also be useful for the treatment of anemia in subjects that are refractory to ESA or 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 a range. Therefore, the present invention is useful in reducing the need for iron or blood transfusion therapy.

[0407] This invention is useful for the treatment of anemia in the context of inflammatory diseases and microbial infections.

[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 an anti-BMP6 antagonist and an erythropoiesis-promoting agent (ESA) to the subject.

[0409] 2. A method for preventing a decrease in the blood hemoglobin level of a target to less than 10 g / dL, comprising administering an anti-BMP6 antagonist and an erythropoiesis-promoting agent (ESA) to the target.

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

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

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

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

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

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

[0417] 9. A method for treating anemia or reducing the risk of anemia in a subject who is anemic or at risk of developing anemia, comprising administering an anti-BMP6 antagonist and a low dose of an erythropoiesis-stimulating agent (ESA) to the subject, 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 subjects who are anemic or at risk of developing anemia, comprising administering an anti-BMP6 antagonist and an ESA simultaneously or sequentially to the subject. a. On day 0, administer the antagonist to the target, and by day 7 (for example, day 1), administer the ESA to the target, or b. On day 0, administer the ESA to the target, and by day 7 (for example, day 1), administer the antagonist to the target, or c. On day 0, administer the antagonist and ESA simultaneously, or d. On day 0, the subject has already received ESA, and on day 0, the antagonist is administered to the subject, 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 in which, on the 14th day or thereafter, the blood hemoglobin level in the subject is at least 10 g / dL, and the anemia is treated or prevented.

[0419] 11. A combination therapy for use in any method or regimen of claims 1 to 10 for treating or preventing anemia in a subject, wherein the combination is a. Anti-BMP6 antagonist, b. ESA, and c. Optional combination therapy, including instructions for use in a method or regimen.

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

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

[0422] Where “anti-BMP6 antagonist” is referred to herein, the antagonist may be any anti-BMP6 antibody or fragment disclosed herein, such as an alternative antibody or fragment (as described elsewhere herein), or an antibody or fragment as claimed, or as referred to 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 is (a) On the first day (D0), administer an anti-BMP6 antibody or fragment as the target. (b) The administration of multiple doses of ESA over a period of at least three consecutive weeks, starting from D0, wherein the subject's blood Hb concentration increases from the baseline concentration of D0 over the entire period. As a result, (i) Throughout the entire period, the Hb concentration is greater than or equal to 100% 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 above baseline throughout the entire period.

[0424] The embodiments of the present invention are as follows, and these embodiments (and all unnumbered paragraphs) can be combined with any other constructs, examples, features, embodiments, or provisions of the present invention described herein, and the antagonist (e.g., an anti-BMP6 antibody or fragment) or ESA of the present invention can be provided for use in the methods of the following embodiments (or can be used in the methods of the following embodiments). 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 an anti-BMP6 antagonist and an erythropoiesis-promoting agent (ESA) to the subject.

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

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

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

[0428] In one example of any embodiment, the subject suffers from moderate or severe anemia prior to administration of 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 an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA) to the subject, 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 an anti-BMP6 antagonist and an erythropoiesis-stimulating agent (ESA) to the subject, wherein the anemia is treated or prevented.

[0431] For example, an inflammatory disease or condition is selected from a group consisting of microbial infections (e.g., bacterial infections) or inflammation of rheumatoid arthritis. For example, anemia is inflammatory anemia (also known as chronic disease anemia, ACD).

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

[0433] In one embodiment, the method involves reducing the dosage of iron (e.g., weekly, bi-weekly, or monthly dosage) or the frequency of administration.

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

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

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

[0437] The dose is lower than the standard dose typically used to treat or reduce anemia in a subject, such as a human or adult, such as a male or female. Typical doses for treatment or prevention will be readily apparent 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 isotonic sodium chloride / sodium citrate buffer are supplied in multiple strengths. Each 1 mL vial contains 2,000, 3,000, 4,000, or 10,000 units of epoetin alfa, human albumin (2.5 mg), citrate (0.06 mg), sodium chloride (5.9 mg), and sodium citrate (5.8 mg) in USP (pH 6.9 ± 0.3), which is sterile water for injection. The single-dose 1 mL vial, formulated with isotonic sodium chloride / sodium phosphate buffer, contains 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 dihydrogen phosphate (1.8 mg), and monohydrate phosphate (1.2 mg) in USP (pH 6.9 ± 0.3) for injection. The multi-dose 2 mL vial contains 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 1 mL of USP (pH 6.1 ± 0.3) for injection. The 1 mL vial, intended for multiple doses, contains 20,000 units of epoetin alfa, 2.5 mg of human albumin, 1% benzyl alcohol, 8.2 mg of sodium chloride, 0.11 mg of citric acid, and 1.3 mg of sodium citrate per 1 mL of USP (pH 6.1 ± 0.3) for injection. In one example of the present invention, ESA is administered as one of these formulations.

[0439] 10. ESA a. Epoetin alfa administered at weekly doses of 1000, 1500, 2500, 5000, 11000, 18000, 34000, or less than 90000 units, optional for subjects who have previously received epoetin alfa treatment at weekly doses of <1500, 1500-2499, 2500-4999, 5000-10999, 11000-17999, 18000-33999, 34000-89999, or ≥90000 units, respectively. b. Darbepoetin alfa or Aranesp®, administered at weekly doses of less than 6.25, 10, 12.5, 20, 25, 40, 60, 100, or 200 mcg, optional subjects who have previously received treatment with darbepoetin alfa or Aranesp® at weekly doses of 6.25, 10, 12.5, 20, 25, 40, 60, 100, or 200 mcg, respectively, or c. Darbepoetin alfa or Aranesp®, administered in weekly doses of less than 6.25, 10, 20, 40, 60, 100, or 200 mcg, and optionally, subjects who have previously received epoetin alfa treatment at weekly doses of 1500-2499, 2500-4999, 5000-10999, 11000-17999, 18000-33999, 34000-89999, or ≥90000 units, according to any of the methods in any of embodiments 1 to 9.

[0440] 11. The anti-BMP6 antagonist is an antibody, and is administered in a total dose of 30 mg / kg or less (e.g., 0.1 to 30 mg / kg) every 1, 2, or 3 weeks, or monthly, every 2 months, or every 3 months, according to any of the methods in embodiments 1 to 10. Administration may be, for example, intravenously or subcutaneously, and the subjects are human beings such as adults.

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

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

[0443] 13. Any method or regimen of embodiment 1 to 12, wherein an anti-BMP6 antagonist and an ESA are administered to the subject at intervals not exceeding 7 days.

[0444] 14. Any one of the methods or regimens described in aspects 1 to 13, wherein the method or regimen maintains a blood hemoglobin level in the subject above 10 g / dL.

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

[0446] 16. The method or regimen of embodiment 14 or 15, wherein an anti-BMP6 antagonist and an ESA are administered to the subject at intervals not exceeding one day.

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

[0448] 18. Any method or regimen according to aspects 1 to 17, which prevents the target blood hemoglobin level from decreasing to less than 10, 10.5, 11, 11.5, 12, 12.5, or 13 g / dL (for example, on day 14).

[0449] 19. Any method or regimen according to embodiment 1 to 18, wherein the subject's blood hemoglobin level rises to at least 10, 10.5, 11, 11.5, 12, 12.5, or 13 g / dL (for example, on day 14).

[0450] 20. Any method or regimen of aspects 1 to 19, which prevents moderate or severe anemia in the subject (for example, on day 14).

[0451] 21. The target is, a. You have an inflammatory disease or condition, or b. Are you suffering from an infectious disease? c. Do you have kidney disease? d. Whether you have HIV or are receiving treatment for HIV, e. I have cancer. A method or regimen in any of embodiments 1 to 20 for treating or preventing moderate or severe anemia in a subject.

[0452] For example, the subjects may be those suffering from HIV infection, hepatitis, rheumatoid arthritis, chronic kidney disease, or end-stage renal disease. For example, the infection may be a Gram-negative bacterial infection. For example, the infection may be a Gram-positive bacterial infection.

[0453] HIV-infected individuals treated with antiretroviral therapy may develop anemia. Therefore, the present invention may be useful for treating or preventing anemia in such patients. For example, a method or regimen may treat or prevent anemia in HIV-infected individuals treated with antiretroviral therapy, for example, by administering zidovudine at a dose of less than 4200 mg / week.

[0454] Cancer patients treated with anti-cancer chemotherapy (e.g., immunotherapy, such as administering immune checkpoint inhibitors to targets such as anti-CTLA4, anti-PD-L1, anti-TIGIT, anti-ICOS, or anti-PD1 antibodies) 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 person with cancer. In the art, ESAs such as erythropoietin are typically administered intravenously or subcutaneously to such patients initially at a dose of 150 units / kg three times a week, and alternatively, subcutaneously at 40,000 units once a week until the course of chemotherapy is completed. In one example, the present invention treats or prevents anemia in human cancer patients by administering an ESA to a person intravenously or subcutaneously at a dose of less than 150 units / kg three times a week, or at a total weekly dose of less than 450 units / kg, or subcutaneously at a dose of less than 40,000 units per week.

[0455] ESA therapy is used in the art to reduce the need for red blood cell (RBC) transfusions in patients, such as those undergoing surgery. Therefore, ESA therapy is used in human patients with preoperative hemoglobin levels greater than 10 g / dL but less than 13 g / dL who are at high risk of pre- and postoperative blood loss due to surgeries such as elective surgery, non-cardiac surgery, and non-vascular surgery. ESA is administered subcutaneously at a dose of 300 units / kg once daily for 15 consecutive days (10 days before surgery, on 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. For example, the present invention treats or prevents anemia in human surgical patients, and the ESA is administered to humans at a dose of 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 in a total 15-day dose of less than 4500 units / kg, or in 3 to 5 or 4 doses of less than 600 units / kg, for example, 21 days, 14 days, 1 day, and 7 days before surgery and on the day of surgery.

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

[0457] 23. Any method or regimen of embodiments 1 to 22, wherein the subject is a mammal.

[0458] 24. Combination therapy for use in any of the methods or regimens described in Embodiments 1 to 23 for treating or preventing anemia in a subject, wherein the combination is a. Anti-BMP6 antagonist, b. ESA, and c. Optional combination therapy, including instructions for use in a method or regimen.

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

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

[0461] 27. A combination of an anti-inflammatory agent and one of the antagonists described in embodiments 24 to 26.

[0462] 28. A method, regimen, combination, or antagonist according to any of embodiments 1 to 27, wherein the antagonist comprises an anti-BMP6 antibody binding site, for example, the antagonist is an antibody or an anti-BMP6 trap.

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

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

[0465] 30. One of the methods or regimens described in embodiments 1 to 23, 28, and 29, wherein an anti-inflammatory agent is administered to the target.

[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 erythrocyte production. In one embodiment, the present invention can reduce the need for the simultaneous and common use of intravenous iron or blood transfusions in ACD patients. Additionally or alternatively, the present invention can reduce the doses that form the basis of standard treatment with ESAs (e.g., EPO), or make patients who are unresponsive (or have low responsiveness) to ESAs (e.g., EPO) responsive to co-administration of ESAs with an anti-BMP6 antagonist. 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 prevalent in patients with uncontrolled hypertension or with pure red cell aplasia (PRCA, a type of anemia) resulting from the administration of ESAs (e.g., darbepoetin alfa such as Arenesp®, or epoetin alfa such as Epogen® or Procrit®).

[0467] Therefore, in one embodiment of the present invention, the subject (e.g., a human) is i. If the patient is refractory or unresponsive to ESA (e.g., darbepoetin alfa or epoetin alfa), ii. To have, or currently have, hypertension (e.g., uncontrolled hypertension), iii. The patient has or is suffering from pure red cell aplasia (for example, caused by administration of an ESA such as darbepoetin alfa or epoetin alfa).

[0468] The "refractory" nature of drug therapies such as ESA therapy is readily apparent to those skilled in the art and means, for example, that the patient is ESA-resistant or has a low response to ESA (i.e., below the mean response), and that the anemia is not effectively treated by standard therapy using ESA.

[0469] ESAs are typically used to maintain hemoglobin at the lowest possible levels, which results in minimizing blood transfusions and best meeting the patient's needs. As described above, the present invention, in its various configurations, aspects, examples, and embodiments, is useful for the treatment of anemia that conserves ESAs, i.e., allows treatment with ESAs at lower doses than the standard dose. This is useful in terms of the potentially harmful side effects of ESAs. Tables A-D provide relevant information on this point. [Table 1]

[0470] In one example, the subjects are chronic kidney disease (CKD) patients who are not undergoing dialysis. In another example, the subjects are chronic kidney disease (CKD) patients who are undergoing dialysis. In yet another example, the subjects are chemotherapy patients (for example, those receiving or currently receiving chemotherapy for cancer). [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 the occurrence or risk of one or more adverse events listed in Table B, e.g., "common," "more common," or "very common" adverse events, in a subject.

[0472] In one embodiment, the present invention provides a method of ESA therapy for 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 “common,” “more common,” or “very common” side effects) is reduced. In one example, the therapy is for the treatment of anemia. In another example, the therapy is for the prevention of anemia. In another example, the anemia is moderate or severe anemia. [Table 3]

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

[0474] In one embodiment, the present invention provides a method of ESA therapy for a subject who is anemic or at risk of developing 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 side effects of one or more ESAs listed in Table C (e.g., reduced overall survival and / or increased risk of tumor progression or recurrence, provided the subject is a patient with breast cancer, non-small cell lung cancer, head and neck cancer, lymphoid cancer, or cervical cancer, or has a risk of cardiovascular or thromboembolic reactions such as stroke) is reduced. In one example, the therapy is for the treatment of anemia. In one example, the therapy is for the prevention of anemia. In one example, the anemia is moderate anemia. In one example, the anemia is moderate to severe anemia. In one example, the anemia is 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 agents (ESAs) to treat anemia in a subject suffering from anemia, comprising administering an anti-BMP6 antagonist and the ESA, wherein the anemia is treated in the subject.

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

[0477] In these examples, the ESA a. Epoetin alfa administered at weekly doses of 1000, 1500, 2500, 5000, 11000, 18000, 34000, or less than 90000 units, optional for subjects who have previously received epoetin alfa treatment at weekly doses of <1500, 1500-2499, 2500-4999, 5000-10999, 11000-17999, 18000-33999, 34000-89999, or ≥90000 units, respectively. b. Darbepoetin alfa or Aranesp®, administered at weekly doses of less than 6.25, 10, 12.5, 20, 25, 40, 60, 100, or 200 mcg, optional subjects who have previously received treatment with darbepoetin alfa or Aranesp® at weekly doses of 6.25, 10, 12.5, 20, 25, 40, 60, 100, or 200 mcg, respectively, or c. Darbepoetin alfa or Aranesp®, administered in weekly doses of less than 6.25, 10, 20, 40, 60, 100, or 200 mcg, optional for subjects who have previously received epoetin alfa treatment at weekly doses of 1500-2499, 2500-4999, 5000-10999, 11000-17999, 18000-33999, 34000-89999, or ≥90000 units, respectively.

[0478] For example, ESA (i) Epoietin alfa, administered in weekly doses 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, The target is humans, for example, adults.

[0479] In one example, blood hemoglobin levels may rise to or remain above 10 g / dL.

[0480] In one example, the subjects are adults. In another example, the subjects are infants. In yet another example, the subjects are human CKD patients undergoing dialysis treatment. In yet another example, the subjects are humans with end-stage renal disease.

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

[0482] Examples of erythropoiesis-stimulating agents (ESAs) include 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 present invention is any one of these or a combination of two or more of them.

[0483] For example, ESA may contain or consist of recombinant erythropoietin selected from, for instance, the following table. 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] One example of an ESA is a hypoxia-inducible factor-prolyl hydroxylase (HIF-PH) inhibitor, such as roxadastat or FG-4592. HIF is a major 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 work by mimicking the body's natural response to anemia. This allows for controlled, adaptive stimulation of the erythropoiesis system in the body. Activation of this entire system results in both increased red blood cell (RBC) production and improved stabilization of iron supply in the bone marrow, ensuring proper uptake of iron into hemoglobin necessary for such RBC production. This adaptive simulation closely resembles the natural response induced when a person ascends to high altitude. At higher altitudes, lower 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 the 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 the prolyl hydroxylase enzyme, which promotes the degradation of HIFα proteins. When degradation is inhibited, levels of these HIFα proteins increase within cells. These HIFs are key protein mediators that enable the body and all its individual cells to adapt to changes in oxygen levels. Both HIFα proteins are produced constancy, and their levels in cells are regulated by the activity of HIF-PH enzymes that target HIFα proteins for degradation. HIF1α helps cells survive in very low oxygen conditions, while HIF2α helps cells and the body adapt to moderate changes in oxygen, 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 moves to the cell nucleus. When bound 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, just as a decrease in oxygen increases HIFα levels. Sustained stabilization of HIFα (due to stays at higher altitudes or daily administration of HIF-PH inhibitors) leads to increased hemoglobin and RBC levels in order to increase the amount of oxygen circulating in the blood.

[0487] An example of an anti-BMP6 antibody is MAB507, commercially available from R&D Systems (monoclonal mouse IgG2B, clone #74219). Other suitable antibodies are disclosed in US8980582, WO2016 / 098079, and US2016 / 0176956A1, whose disclosure (as well as the sequences of the antibodies, variable regions, and CDRs explicitly contained herein) is incorporated herein by reference for possible 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 disclosed in US8980582, and these sequences are expressly incorporated herein by reference for 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 disclosed in US8980582, and these sequences are expressly incorporated herein by reference for 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 disclosed in US8980582, and these sequences are expressly incorporated herein by reference for 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 LCVR and HCVR, where LCVR is the polypeptide of SEQ ID NO: 9 and 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 LCVR and HCVR, where LCVR is the polypeptide of SEQ ID NO: 9 and 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 LCVR and HCVR, where LCVR is the polypeptide of SEQ ID NO: 9 and HCVR is the polypeptide of SEQ ID NO: 11. The SEQ ID NOs in this paragraph are disclosed in US8980582, and these sequences are expressly incorporated herein by reference for 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 LCVR and HCVR, where LCVR is the polypeptide of SEQ ID NO: 9 and 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 LCVR and HCVR, where LCVR is the polypeptide of SEQ ID NO: 9 and 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 LCVR and HCVR, where LCVR is the polypeptide of SEQ ID NO: 9 and 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 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), where LC is the polypeptide of SEQ ID NO: 12 and 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 LC and HC, where LC is the polypeptide of SEQ ID NO: 12 and 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 LC and HC, where LC is the polypeptide of SEQ ID NO: 12 and HC is the polypeptide of SEQ ID NO: 14. The SEQ ID NOs in this paragraph are disclosed in US8980582, and these sequences are expressly incorporated herein by reference for 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, where each light chain is the polypeptide of SEQ ID NO: 12 and each heavy chain is the 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, where each light chain is the polypeptide of SEQ ID NO: 12 and each heavy chain is the polypeptide of SEQ ID NO: 14. The SEQ ID NOs in this paragraph are disclosed in US8980582, and these sequences are expressly incorporated herein by reference for 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 the 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, e.g., an ESA and / or an anti-inflammatory agent. Suitable anti-inflammatory agents may be antibodies or antibody fragments, e.g., 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] For example, an anti-BMP6 antagonist, antibody, or fragment is determined by a common method known in the art, for instance, using a surface plasmon resonance (SPR) biosensor at 37°C, resulting in a measurement of 1 × 10⁶. -8 Less than M, preferably 1 × 10 -9 It binds to BMP6 with a KD of less than M.

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

[0498] Terms such as “treatment,” “to treat,” and “to treat” are intended to include slowing or restoring the progression of a disorder such as anemia, moderate anemia, severe anemia, or decreased blood hemoglobin. These terms also include improving, alleviating, reducing, 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 eliminated completely. Subject or patient refers to a mammal that would benefit from inhibition of BMP-6 activity, preferably a human having a disease, disorder, or condition (e.g., anemia or at risk of anemia). The term “prevention” is, for example, reducing the risk of a disease or condition such as anemia.

[0499] The pharmaceutical compositions comprising the ESA, anti-BMP6 antagonist antibody, or antigen-binding fragments, combinations, or similar thereof of the present invention may be administered via parenteral routes (e.g., subcutaneous, intravenous, intraperitoneal, intramuscular, or transdermal). Administration may be performed on a target alone or in single or multiple doses in combination with pharmaceutically acceptable carriers and / or diluents. The pharmaceutical compositions, combinations, or antagonists of the present invention may be administered by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 1919). thIt can be prepared by (ed. 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 embodiment, the subject has moderate or severe anemia prior to administration of the BMP6 antagonist, and the moderate or severe anemia is treated. In one embodiment, the subject has moderate anemia prior to administration, and after treatment, the subject has mild anemia or is anemia-free. In one embodiment, the subject has severe anemia prior to administration, and after treatment, the subject has mild, moderate anemia or is anemia-free. In one embodiment, after treatment, the subject has mild anemia or is anemia-free, and is not moderate or severe anemia. In another embodiment, after treatment, the subject is anemia-free. In one embodiment, the subject has a blood hemoglobin level of less than 9.5 g / dL prior to 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 to be a condition in which hemoglobin levels fall 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. If hemoglobin levels are between 9.5 and 13.0 g / dL, it is considered mild anemia. A hemoglobin level of 8.0-9.5 g / dL is considered moderate anemia. • A hemoglobin concentration of less than 8.0 g / dL is considered severe anemia.

[0503] In one example, hemoglobin levels are measured at sea level or equivalent.

[0504] In one embodiment, the subject is a human male, e.g., an adult or an infant. In another embodiment, the subject is a human female, e.g., an adult or an infant, e.g., a non-pregnant or pregnant woman. In one example, the human is a dialysis patient. The infant may be a human being older than one month.

[0505] One example of a method is to eliminate or reduce the need for iron administration or transfusions in an anemic subject, for instance, by reducing the dosage or frequency of iron administration to the subject.

[0506] The present invention may involve administering an anti-BMP6 antagonist and an ESA simultaneously or sequentially. For example, the antagonist and ESA may be 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 illustrated herein, administration of the antagonist and ESA may be effective at intervals of 7 days or less (e.g., 1 day or less). For example, the anti-BMP6 antagonist and ESA may be administered to the subject at intervals of 10, 14, 21, or 28 days or less.

[0507] In one example, ESA is administered two, three, or four times a week. In another example, ESA is administered one, two, three, or four times a month, or over an eight-week period. In one example, ESA (e.g., epoetin alfa) is administered weekly in total doses 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. In yet another example, ESA is administered one, two, three, or four times a month, or over an eight-week period. In one example, an 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 are administered intravenously or subcutaneously to the subject.

[0509] In one example, anemia is observed in patients who are receiving or have received zidovudine treatment.

[0510] Optionally, any configuration of the present invention is also (a) For example, increasing or maintaining elevated blood iron levels in order to treat or reduce the risk of anemia, (b) Treating iron deficiency, (c) To treat or reduce the risk of anemia due to chronic inflammation (ACI), (d) To treat or reduce the risk of anemia of chronic disease (ACD), (e) To treat or reduce the risk of anemia associated with cancer, kidney condition, or GvHD. (f) To increase blood or serum iron levels, (g) Increasing the reticulocyte count, (h) Increasing the red blood cell count, (i) to increase hemoglobin, and (j) for the purpose of increasing the hematocrit of the subject (e.g., human).

[0511] In one embodiment, the present invention is for regulating (e.g., increasing) erythrocyte production in a subject.

[0512] In one embodiment, the subjects are humans containing the BMP6 gene SNP rs111588693, which may correlate with an increased tendency towards anemia.

[0513] For example, anemia can be cancer anemia or anemia of chronic disease (ACD), such as anemia of chronic kidney disease (CKD). Certain chronic diseases, such as cancer, kidney disease, and autoimmune disorders, can lead to ACD if excessive inflammatory cytokines cause dysregulation of iron homeostasis, reduced erythropoiesis, and decreased lifespan of red blood cells. Hepcidin has been identified as a key hormone involved in iron homeostasis, and high levels of hepcidin are associated with iron-restricted erythropoiesis seen in ACD. BMP-6 has been shown to increase hepcidin expression. For example, the present invention relates to reducing or maintaining reduced hepcidin levels in a subject.

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

[0515] In one example, the subjects have a chronic disease such as cancer (e.g., hematological malignancies or solid tumors), kidney disease, autoimmune disorders, or chemotherapy-induced anemia. In another example, the subjects (e.g., humans) have one or more of the following conditions: CKD, diabetic neuropathy, cardiovascular disease, and retinopathy.

[0516] In one example, the anemia is hepcidin-related iron-restrictive anemia. In another example, the anemia is iron-refractory iron-deficiency anemia (IRIDA). In one embodiment, IRIDA is caused by a defect in the TMPRSS6 gene in question, for example, IRIDA is caused by a TMPRSS6 gene mutation (e.g., SNPs such as rs855791, rs2543519, rs2235324, or rs1421312).

[0517] One example is a method for treating or preventing Sjögren'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 (e.g., a human).

[0519] In one embodiment, the present invention provides the use of anti-BMP6 antagonists and ESAs for the manufacture of pharmaceuticals. In a further embodiment, the present invention provides the use of anti-BMP6 antagonists and ESAs for the manufacture of pharmaceuticals for the treatment or prevention of anemia, for example, moderate to severe anemia. In another embodiment, the present invention provides the use of anti-BMP6 antagonists and ESAs for the manufacture of pharmaceuticals for the treatment of anemia of chronic disease. In another embodiment, the present invention provides the use of anti-BMP6 antagonists and ESAs for the manufacture of pharmaceuticals for the treatment of anemia of chronic kidney disease. In another embodiment, the present invention provides the use of anti-BMP6 antagonists and ESAs for the manufacture of pharmaceuticals for the treatment of anemia of cancer. In one embodiment, the present invention provides the use of anti-BMP6 antagonists and ESAs for the manufacture of pharmaceuticals for the treatment of IRIDA. In further embodiments, the present invention provides the use of anti-BMP6 antagonists and ESAs for the manufacture of pharmaceuticals for the treatment of IRIDA, which is caused by TMPRSS6 gene mutations (e.g., SNPs such as rs855791, rs2543519, rs2235324, or rs1421312). In one embodiment, the present invention provides the use of anti-BMP6 antagonists and ESAs for the manufacture of pharmaceuticals 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 constructs, clauses, paragraphs, examples, features, or aspects of the present invention described herein, and an antagonist (e.g., an anti-BMP6 antibody or fragment) or ESA of the present invention can be provided for use in the methods of the following embodiments (or can be used in the methods of the methods described herein).

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

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

[0523] In one example, the antagonist includes or consists of an anti-BMP6 antibody or fragment, e.g., a human, humanized, or chimeric antibody. As an alternative to the antibody or fragment, different BMP6 antagonists, e.g., anti-BMP6 trap or HJV-Fc, are intended by the present invention.

[0524] 2. The antagonist contains or consists of an anti-BMP6 antibody or fragment thereof, and the method is: (a) On the first day (D0), administer an anti-BMP6 antibody or fragment as the target. (b) The administration of multiple doses of ESA over a period of at least three consecutive weeks, starting from D0, wherein the subject's blood Hb concentration increases from the baseline concentration of D0 over the entire period. As a result, (i) Throughout the entire period, the Hb concentration is greater than or equal to 100% of the baseline Hb concentration, and during the period, the Hb concentration reaches at least 120% of the baseline, and / or (ii) The antagonist according to Embodiment 1, wherein the Hb concentration increases by at least 1 g / dl above baseline over the entire duration of the period.

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

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

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

[0528] 4. A combination of Embodiment 3, wherein the method comprises obtaining a single dose from the amount of antibody or fragment, the single dose being administered to a subject in D0, and obtaining multiple doses of the ESA, with at least one dose being administered weekly from D0.

[0529] In any embodiment of this specification, for example, the initial dose of ESA is administered to D0.

[0530] 5. A combination of Embodiments 3 or 4, wherein the antibody or fragment is composed of a pharmaceutical composition, and the antibody or fragment is mixed with a dose of ESA for administration to the subject in D0.

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

[0532] 7. The antagonist comprises or consists of an erythropoietin enhancer (ESA) (for example, composed of multiple doses of said ESA), and the method is: (a) On the first day (D0), administer an anti-BMP6 antibody or fragment as the target. (b) for at least three consecutive weeks, administering multiple doses of the ESA during the period starting from D0, such that the subject's blood hemoglobin (Hb) concentration increases from the baseline D0 concentration over the entire period. Such (i) Throughout the entire period, the Hb concentration is greater than or equal to 100% of the baseline Hb concentration, and during the period, the Hb concentration reaches at least 120% of the baseline, and / or (ii) The antagonist according to Embodiment 1, wherein the Hb concentration increases by at least 1 g / dl above baseline over the entire duration of the period.

[0533] 8. (iii) Any one antagonist, combination, or kit of Embodiments 2 to 7, wherein the Hb concentration on the last day of the continuous weekly period is at least 120% of the Hb concentration on the 7th day immediately preceding the last day.

[0534] 9. An 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. An antagonist, combination, or kit according to any one of embodiments 2 to 9, wherein the consecutive weekly period consists of 3 or 4 consecutive weekly periods.

[0536] 11. During the period, any one antagonist, combination, or kit of Embodiments 2 to 10 (e.g., Embodiment 10) causes the Hb concentration to increase above baseline to a range of 1 to 8 g / dl.

[0537] In any embodiment of this specification, for example, during the period, the Hb concentration increases above the baseline to an increase in the range of 1–3, 2.5, 2, 1.5, or 1.25 g / dl. For example, the Hb concentration increases to an increase of 1–2 g / dl.

[0538] 12. Any one antagonist, combination, or kit of Embodiments 2 to 11, comprising a period of 3 or 4 consecutive weeks, wherein the Hb concentration reaches at least 150% of the baseline at the end of the period.

[0539] 13. The period consists of 3 or 4 consecutive weeks. (a) Throughout the entire period, the Hb concentration is 110% or greater than the baseline Hb concentration, and during the period, the Hb concentration reaches at least 150% of the baseline, and / or (b) Any one of the antagonists, combinations, or kits of Embodiments 2 to 12, in which the Hb concentration increases by at least 1 g / dl above baseline over the entire duration of the period, and during the period, the Hb concentration reaches an increase of 1 to 8 g / dl above baseline. For example, the Hb concentration reaches an increase of 1 to 2 g / dl.

[0540] 14. An antagonist, combination, or kit from any one of Embodiments 2 to 13, in which the antibody or fragment is administered to D0 during the said period.

[0541] 15. An antagonist, combination, or kit of Embodiment 14, wherein the antibody or fragment is administered to the subject as a single dose to D0.

[0542] In any embodiment of this specification, for example, the antibody or fragment is administered to the subject as a single dose in D0, and the single dose is administered to the subject in one or more aliquots.

[0543] 16. Any one antagonist, combination, or kit from Embodiments 2 to 15, in which the initial ESA dose is administered on D0 or within 2 days thereafter.

[0544] 17. An antagonist, combination, or kit of any one of Embodiments 2 to 16, in which the ESA dose is administered immediately after D0 on day 4 to 9 (e.g., day 7).

[0545] 18. An antagonist, combination, or kit of any one of Embodiments 2 to 17, in which the ESA dose is administered immediately after D0 on day 12 to 16 (e.g., day 14).

[0546] 19. An antagonist, combination, or kit of any one of Embodiments 2 to 18, in which the ESA dose is administered immediately after D0 on day 19 to 23 (e.g., day 21).

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

[0548] 20. An antagonist, combination, or kit from any one of Embodiments 2 to 19, in which an equivalent amount of four ESA doses is administered during the period.

[0549] In this specification, “equivalent amount” means that multiple aliquots of ESA can be administered (e.g., on the same day or sequentially), and each aliquot corresponds to the total dose of ESA. For example, the ESA is darbepoetin alfa or Aranesp®, and the dose is in the range of 15–100 mcg (micrograms) or 30–100 mcg. For example, the ESA is epoetin alfa, and the dose is in the range of 3000–30000 units (i.e., units refer to International Units, also known as IU, UI, IE, ME, NE in various languages).

[0550] Generally, as used herein, doses (e.g., of antibodies, fragments, or ESAs) may be administered in one or more aliquots (e.g., on the same day, simultaneously, or within a 30, 1, or 24-hour time frame).

[0551] 21. An antagonist, combination, or kit of any one of Embodiments 2-20, wherein the ESA is administered to the subject during the first and second weeks, respectively, following the initial ESA dose.

[0552] 22. An antagonist, combination, or kit of any one of Embodiments 2-21, wherein the ESA is administered to the subject during the first, second, and third weeks, respectively, following the initial ESA dose.

[0553] 23. An antagonist, combination, or kit of Embodiment 21 or 22, wherein the ESA is administered as a single dose at the end of each such week, and optionally, the period consists of 3 or 4 weeks beginning from D0.

[0554] In one example of the embodiment, the period consists of four weeks starting from D0, and the anemia is treated in the fourth week.

[0555] 24. An antagonist, combination, or kit according to any one of Embodiments 2 to 23, wherein during the period, four doses or less of the ESA and optionally a single dose of the antibody or fragment are administered to the target.

[0556] 25. During the said period (which is four consecutive weeks), the total dose of antibodies and the total volume of ESA are administered to the subject in a ratio of X:Y, where X is 10 to 2x10 6 and any one of the antagonists, combinations, or kits of embodiments 2 to 24, where Y=4.

[0557] For example, the total weekly dose of ESA (e.g., if the subject is human) is 10 or 15–80, 100, 200 or 300 mcg (micrograms). For example, the total weekly dose is 10–80, 15–80, or 30–80 mcg. For example, the ESA includes or consists of darbepoetin alfa, epoetin alfa, or any other ESA disclosed herein. For example, each dose (or weekly dose) of ESA is administered to the subject in the range of 1.5–2 mcg / kg of ESA.

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

[0559] 26. Any one of the antagonists, combinations, or kits of Embodiments 2 to 25, wherein the Hb concentration at the end of the period (e.g., 3 or 4 consecutive weeks) is at least 130% of the Hb concentration in a control patient of the same type of anemia who received an anti-BMP6 antibody or fragment in the same dosing regimen as the subject, except for patients who did not receive ESA during the period.

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

[0561] 28. Any one of the antagonists, combinations, or kits of Embodiments 2 to 27, wherein the mean corpuscular hemoglobin (MCH) at the end of the period is at least 109% of the MCH in a control patient of the same type of anemia who received anti-BMP6 antibody or fragment in the same dosing regimen as the subject, except for patients who did not receive ESA during the period.

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

[0563] 29. Any one of the antagonists, combinations, or kits 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 a control patient of the same type of anemia who received the ESA in the same dosing regimen as the subject, except for patients who did not receive the anti-BMP6 antibody or fragment during the period. Optionally, the control patient 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 in the same doses, respectively). Optionally, X is 10-2x10 5 , 2x10 4 or 2x10 3 That is the case.

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

[0565] 31. Any one of the antagonists, combinations, or kits of Embodiments 2 to 30, wherein the mean erythrocyte hemoglobin (MCH) at the end of the period (e.g., 3 or 4 consecutive weeks) is at least 119% of the MCH in a control patient of the same type of anemia who received the ESA in the same dosing regimen as the subject, except for patients who did not receive anti-BMP6 antibody or fragments during the period.

[0566] 32. An antagonist, combination, or kit of Embodiment 31 in which the MCH at the end of the period is significantly higher than that of the control at the end of the period, as determined by a p-value of p<0.0001.

[0567] 33. The subject in D0 is suffering from anemia of a chronic disease (ACD), and optionally, the anemia is associated with chronic inflammation (e.g., the subject suffers from arthritis) or bacterial infection (e.g., streptococcal infection), and the subject is a patient with chronic kidney disease (CKD), one of the antagonists, combinations, or kits from Embodiments 2 to 32.

[0568] 34. Anemia in the subject at the end of the period is less severe than D0, one of the antagonists, combinations, or kits of Embodiments 2 to 33.

[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 the reference antibody to bind BMP6, and the reference antibody is mAb507 (R&D Systems) or a. Each heavy chain containing or consisting of the amino acid sequence of SEQ ID NO: 1 or 2, and each light chain containing or consisting of the amino acid sequence of SEQ ID NO: 3, b. An antagonist, combination, or kit of any one of Embodiments 2 to 34, comprising an antibody containing or consisting of each heavy chain containing or consisting of the amino acid sequence of SEQ ID NO: 4, and each light chain containing or consisting of the amino acid sequence of SEQ ID NO: 5.

[0571] The competition described herein can be determined, for example, by SPR (e.g., at 37°C, pH 7.6, optionally as Fab), by ELISA, by fluorescent cell sorting (FACS), or by homogeneous time-resolved fluorescence (HTRF) assay. SPR may be performed using Biacore®, Proteon®, or other standard SPR techniques. 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 may be any anti-BMP6 antibody disclosed in WO2016 / 098079 (sequence and disclosure relating to such antibody incorporated herein for potential use in the present invention).

[0573] 37. An antagonist, combination, or kit of any one of Embodiments 2 to 36, wherein the antibody or fragment competes with the 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 further sequences selected from the group consisting of SEQ ID NOs: 7-19. These further sequences can be used as the peptide itself, as part of a larger peptide (e.g., including SEQ ID NO: 6), or as part of a BMP6 protein (e.g., wild-type human BMP6 or recombinant BMP6, including 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, the antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 15. For example, the antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 16. For example, the antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 17. For example, the antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 18. For example, the antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 19. The sequence ID numbers in this paragraph and the embodiments herein are disclosed in WO2017 / 191437, which is expressly incorporated herein by reference.

[0575] 38. An antibody or fragment competitively inhibits the binding of soluble hemoduvelin (HJV) to BMP6, comprising any one antagonist, combination, or kit from Embodiments 2 to 37.

[0576] 39. An antagonist, combination, or kit from any one of Embodiments 1 to 38, wherein the antibody or fragment does not competitively inhibit the binding of soluble hemoduvelin (HJV) to BMP6 (as determined, for example, by SPR, HTRF, or ELISA).

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

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

[0579] 42. An antibody or fragment that binds to BMP6 with a stronger affinity (lower KD as determined by SPR) than it binds to BMP7, and optionally, one of the antagonists, combinations, or kits from Embodiments 2 to 41 that binds to BMP6 with a stronger affinity than it binds to BMP5.

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

[0581] 43. An antagonist, combination, or kit of any one of Embodiments 2 to 42, wherein an antibody or fragment binds to a human BMP6 sequence containing SEQ ID NO: 6.

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

[0583] In one example, an antibody (e.g., as Fab) or fragment is, (a) 2, 3, 4, 5 or 10 pM to 3, 4 or 5 nM, (b) 1~10 pM~5 nM, (c) 10 pM ~ 3, 4 or 5 nM, (d) 50 or 80 pM to 200 nM, (e) 50 or 80 pM to 150 nM, (f) Has affinity (KD) for binding BMP6 at 50 or 80 pM to 100 nM.

[0584] In one example, the KD is 5–15 pM (e.g., 10 pM) (or around that). In another example, the KD is 2–5 nM (e.g., 3 nM) (or around that). In yet another example, the KD is 100–400 pM (e.g., 140 or 390 pM) (or around that).

[0585] 45. Antibodies are 1 x 10 -5 ~1 × 10 -3 S -1 Off-speed (K) for joining BMP6 off The antagonist, combination, or kit of any one of Embodiments 2 to 44 (e.g., Embodiment 44), having ) and optionally, binding is determined by SPR using the Fab of the antibody at 37°C, pH 7.6.

[0586] In one example, an antibody (e.g., as Fab) or fragment is, (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-speed (K) for joining BMP6 off ) has.

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

[0588] 46. ​​Antibodies are 1 x 105 ~1 × 10 7 M -1 S -1 On-speed (K) for coupling BMP6 on The antagonist, combination, or kit of any one of Embodiments 2 to 45 (e.g., Embodiments 44 and / or 45), wherein binding is optionally determined by SPR using the Fab of the antibody at 37°C, pH 7.6.

[0589] In one example, an antibody (e.g., as Fab) or fragment is, (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) 1 x 10 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-speed (K) for coupling BMP6 on ) has.

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

[0591] 47. (a) The period consists of three or four consecutive weeks, (i) Throughout the entire period, the Hb concentration is 110% or greater than the baseline Hb concentration, and during the period, the Hb concentration reaches at least 120% of the baseline, and / or (ii) Throughout the entire period, the Hb concentration increased by at least 1 g / dl above baseline, and during the period, the Hb concentration increased by in the range of 1 to 8 g / dl above baseline. (b) The dose of the ESA is administered at least twice during the first two or three weeks of the period. (c) The antibody or fragment binds to BMP6 with a stronger affinity than it binds to BMP7 (lower KD as determined by SPR), and optionally binds to BMP6 with a stronger affinity than it binds to BMP5 (optionally binds to BMP6 with a stronger affinity than it binds to BMP2, 4, 5, and 9, respectively), (d) The antibody or fragment competes with the reference antibody to bind BMP6, and the reference antibody is mAb507 (R&D Systems) or I. Each heavy chain containing or consisting of the amino acid sequence of SEQ ID NO: 1 or 2, and each light chain containing or consisting of the amino acid sequence of SEQ ID NO: 3, II. An antagonist, combination, or kit of any one of Embodiments 2 to 46, comprising an antibody containing or consisting of each heavy chain containing or consisting of the amino acid sequence of SEQ ID NO: 4, and each light chain containing 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 (using 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 the HTRF assay. For example, in the HTRF assay, the antibody of the present invention is a labeled antibody that is pre-incubated with human BMP6 and then combined with an unlabeled reference antibody (by part I or II), and competition between antibodies is detected by the assay. In one example, the assay uses the AlexaFluor® 647 labeled antibody of the present invention. Alternatively, human BMP6 is labeled (for example, using AlexaFluor® 647, the test antibody is labeled with biotin to bind to Eu3+ cryptotate-streptavidin, and the reference antibody is unlabeled).

[0594] Optionally, the anti-BMP6 antibody (test antibody) of the present invention competes with a reference antibody in an HTRF assay to bind to human BMP6 (or to the same human BMP6 epitope as the reference antibody), and the assay allows energy transfer between the donor and acceptor using the test antibody, directly or indirectly labeled with a donor (e.g., Eu3+ cryptotate) or an acceptor fluorophore (e.g., AlexaFluor® 647), and a target BMP6 labeled with either the donor or acceptor fluorophore, thereby producing and detecting a fluorescence signal. In one example, where AlexaFluor® 647 labeling is used, competition is detected by a reduction of at least 20% in the fluorescence signal at 665 nM when the test antibody is in the presence of the reference antibody and when the reference antibody is not present. Optionally, the reduction in the signal at 665 nM is at least 20, 30, 40, 50, 60, 70, 80, or 90%.

[0595] Optionally, an anti-BMP6 antibody (test antibody) competes with the reference antibody in an HTRF assay for binding to human BMP6 (or to the same human BMP6 epitope as the reference antibody), the reference antibody comprising each heavy chain containing the amino acid sequence of SEQ ID NO: 1 or 2, and each light chain containing the amino acid sequence of SEQ ID NO: 3, the assay enables energy transfer between the donor and acceptor using the test antibody directly or indirectly labeled with a donor label (e.g., Eu3+ cryptotate) or acceptor fluorophore label (e.g., AlexaFluor® 647), and human BMP6 labeled with either the acceptor fluorophore or the donor, respectively, and the competition between the antibodies is detected by at least a 20% reduction in the fluorescence signal when the test antibody is in the presence of the reference antibody and when it is not. For example, the test antibody is directly or indirectly labeled with AlexaFluor® 647, and competition is detected by a reduction of at least 20% in the fluorescence signal at 665 nM when the test antibody is present and when it is not. Optionally, the signal reduction at 665 nM is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0596] Optionally, an anti-BMP6 antibody (test antibody) also competes with a reference antibody in an HTRF assay to bind to human BMP6 (or to the same human BMP6 epitope as the reference antibody), the reference antibody comprising each heavy chain containing the amino acid sequence of SEQ ID NO: 4 and each light chain containing the amino acid sequence of SEQ ID NO: 5, the assay enables energy transfer between the donor and acceptor using the test antibody directly or indirectly labeled with, for example, a donor label (e.g., Eu3+ cryptotate) or an acceptor fluorophore label (e.g., AlexaFluor® 647), and human BMP6 labeled with either the acceptor fluorophore or the donor, respectively, and the competition between the antibodies is detected by at least a 20% reduction in the fluorescence signal when the test antibody is in the presence of the reference antibody and when the reference antibody is not present. For example, the test antibody is directly or indirectly labeled with AlexaFluor® 647, and competition is detected by a reduction of at least 20% in the fluorescence signal at 665 nM when the test antibody is present and when it is not. Optionally, the signal reduction at 665 nM is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0597] For example, in part (b), the dose of the ESA is administered two, three, or four times during the first three weeks of the period.

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

[0599] 48. a. Treat ACD in the subject, b. To treat or prevent moderate or severe anemia in the subjects, c. To treat or prevent anemia in subjects who have an inflammatory disease or condition, d. Eliminate or reduce the need for iron administration or blood transfusions for the subject. e. To treat or prevent anemia in subjects who have a microbial infection, f. One antagonist, combination, or kit from any of Embodiments 1 to 47 for reducing the administration of ESA to a subject.

[0600] 49. A method for treating anemia in a subject, wherein the method is (a) On the first day (D0), administer an anti-BMP6 antibody or fragment as the target. (b) for at least three consecutive weeks, the administration of multiple doses of an erythropoietin enhancer (ESA) during the period starting from D0, wherein the subject's blood hemoglobin (Hb) concentration increases from the baseline concentration of D0 throughout the entire period. For the entire duration of that period, (i) The Hb concentration is greater than or equal to 100% 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 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. An antagonist, combination, kit, or method of any of Embodiments 1 to 50, wherein the anemia is moderate or severe.

[0603] 52. An anti-osteogenic 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, wherein the method comprises administering an anti-BMP6 antagonist and an erythropoiesis-promoting agent (ESA) to the subject.

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

[0605] 53. An anti-osteogenic protein 6 (BMP6) antagonist for use in a method to prevent a decrease in the blood hemoglobin level of a subject to less than 10 g / dL, wherein the method comprises administering an anti-BMP6 antagonist and an erythropoiesis-promoting agent (ESA) to the subject.

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

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

[0608] 56. An anti-osteogenic protein 6 (BMP6) antagonist for use in a method of eliminating or reducing the need for iron administration or transfusion in a subject suffering from anemia, wherein the method comprises administering an anti-BMP6 antagonist and an erythropoiesis-promoting agent (ESA) to the subject, thereby eliminating or reducing the need for such administration.

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

[0610] 58. An anti-osteogenic protein 6 (BMP6) antagonist for use in a method for reducing the administration of erythropoiesis-stimulating agents (ESAs) for the treatment of anemia in a subject suffering from anemia, wherein the method comprises administering the anti-BMP6 antagonist and the ESA, and the anemia is treated in the subject.

[0611] 59. An anti-osteogenic protein 6 (BMP6) antagonist for use in a method of treating anemia or reducing the risk of anemia in subjects who are anemic or at risk of anemia, wherein the method comprises administering an anti-BMP6 antagonist and a low dose of an erythropoiesis-stimulating agent (ESA) to the subject, thereby treating anemia or reducing the risk of anemia in the subject.

[0612] 60. An antagonist according to any one of embodiments 52 to 59, wherein the antagonist is one of the embodiments described in any one of embodiments 1 to 1, 2, 7 to 48 and 51.

[0613] 61. An antagonist of any one of embodiments 52 to 59, wherein the antagonist is any other described in any other embodiment 52 to 59.

[0614] 62. ESA a. Epoetin alfa administered at weekly doses of 1000, 1500, 2500, 5000, 11000, 18000, 34000, or less than 90000 units, optional for subjects who have previously received epoetin alfa treatment at weekly doses of <1500, 1500-2499, 2500-4999, 5000-10999, 11000-17999, 18000-33999, 34000-89999, or ≥90000 units, respectively. b. Darbepoetin alfa or Aranesp®, administered at weekly doses of less than 6.25, 10, 12.5, 20, 25, 40, 60, 100, or 200 mcg, optional subjects who have previously received treatment with darbepoetin alfa or Aranesp® at weekly doses of 6.25, 10, 12.5, 20, 25, 40, 60, 100, or 200 mcg, respectively, or c. Darbepoetin alfa or Aranesp®, administered in weekly doses of less than 6.25, 10, 20, 40, 60, 100, or 200 mcg, and optionally, subjects who have previously received epoetin alfa treatment at weekly doses of 1500-2499, 2500-4999, 5000-10999, 11000-17999, 18000-33999, 34000-89999, or ≥90000 units, respectively, an antagonist, combination, kit, or method of any of Embodiments 1-61.

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

[0616] 64. Anti-BMP6 antagonists and / or ESAs for use in therapeutic regimens for treating or preventing anemia in subjects who are anemic or at risk of developing anemia, comprising administering the anti-BMP6 antagonist and the ESA simultaneously or sequentially to the subject. a. On day 0, administer the antagonist to the target, and by day 7 (for example, day 1), administer the ESA to the target, or b. On day 0, administer the ESA to the target, and by day 7 (for example, day 1), administer the antagonist to the target, or c. On day 0, administer the antagonist and ESA simultaneously, or d. On day 0, the subject has already received ESA, and on day 0, the antagonist is administered to the subject, or e. On day 0, the subject has already received an antagonist, and on day 0, the subject is administered an ESA. As a result, on day 14 or thereafter, the blood hemoglobin level in the subject is at least 10 g / dL, and the anemia is treated or prevented by an anti-BMP6 antagonist and / or ESA.

[0617] 65. An 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 ESA are administered to the subject at intervals not exceeding 7 days.

[0619] 67. An antagonist and / or ESA according to Embodiment 64, 65, or 66, wherein the regimen maintains a blood Hb level in the subject above 10 g / dL.

[0620] 68. An antagonist and / or ESA according to any one of embodiments 64 to 67, wherein the method maintains or incr...

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 includes CDRH1 containing SEQ ID NO: 108, CDRH2 containing SEQ ID NO: 109, and CDRH3 containing SEQ ID NO: 110; and the VL domain includes CDRL1 containing SEQ ID NO: 117, CDRL2 containing SEQ ID NO: 118, and CDRL3 containing SEQ ID NO: 119, (b) The VH domain includes CDRH1 containing SEQ ID NO: 111, CDRH2 containing SEQ ID NO: 112, and CDRH3 containing SEQ ID NO: 113; and the VL domain includes CDRL1 containing SEQ ID NO: 120, CDRL2 containing SEQ ID NO: 121, and CDRL3 containing SEQ ID NO: 122, (c) The VH domain includes CDRH1 containing SEQ ID NO: 288, CDRH2 containing SEQ ID NO: 289, and CDRH3 containing SEQ ID NO: 290; and the VL domain includes CDRL1 containing SEQ ID NO: 297, CDRL2 containing SEQ ID NO: 298, and CDRL3 containing SEQ ID NO: 299, or (d) The VH domain includes CDRH1 containing SEQ ID NO: 291, CDRH2 containing SEQ ID NO: 292, and CDRH3 containing SEQ ID NO: 293; and the VL domain includes CDRL1 containing SEQ ID NO: 300, CDRL2 containing SEQ ID NO: 301, and CDRL3 containing SEQ ID NO: 302, Antibody or fragment, (ii) Erythropoietin enhancers (ESAs) and A kit that includes this.

2. The VH domain of the antibody or fragment is encoded by a nucleotide sequence derived from a recombination of the human VH gene segment, DH gene segment and JH gene segment, (a) The VH gene segment is IGHV3-11, (b) The DH gene segment is IGHD6-19 and / or (c) The kit according to claim 1, wherein the JH gene segment is IGHJ4.

3. The antibody or fragment is (i) A VH domain containing SEQ ID NO: 110 paired with a VL domain containing SEQ ID NO: 119, or (ii) A VH domain containing SEQ ID NO: 113 paired with a VL domain containing SEQ ID NO: 122, or (iii) A VH domain containing SEQ ID NO: 290 paired with a VL domain containing SEQ ID NO: 299, or (iv) The kit according to claim 1 or 2, comprising a VH domain containing SEQ ID NO: 293 paired with a VL domain containing SEQ ID NO:

302.

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

5. The kit according to any one of claims 1 to 4, wherein the antibody or fragment comprises first and second copies of the VH domain.

6. The VL domain of the antibody or fragment is encoded by a nucleotide sequence derived from a recombination of the human VL gene segment and the JL gene segment, (a) The VL gene segment is IGKV3-20 and / or (b) The kit according to any one of claims 1 to 5, wherein the JL gene segment is IGKJ1.

7. The kit according to any one of claims 1 to 6, wherein the antibody or fragment comprises first and second copies of the VL domain.

8. The kit according to any one of claims 1 to 7, wherein the antibody or fragment comprises a human constant region, optionally an IgG4 constant region, or an IgG1 constant region.

9. The kit according to 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. The kit according to any one of claims 1 to 9, further comprising an antigen-binding site that specifically binds to another target antigen (optionally human hemoduvelin, transferrin receptor, or BMP receptor) or to BMP6 and another BMP (optionally BMP2, 4, 7, or 9).

11. The kit according to any one of claims 1 to 10, comprising multiple doses of the antibody or a fragment thereof and / or ESA.

12. The kit according to any one of claims 1 to 11, wherein the ESA is recombinant erythropoietin.

13. The kit according to any one of claims 1 to 12, wherein the ESA is one or more of epoetin alpha, Epogen®, Dynepo®, Eprex®, erythropoietin, darbepoetin alpha, Aranesp®, epoetin beta, NeoRecormon®, methoxypolyethylene glycol-epoetin beta, Mircera®, or Procrit®.

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

15. The kit according to any one of claims 1 to 14, comprising a further additional therapeutic agent.

16. The kit according to claim 15, wherein the additional therapeutic agent is for the treatment, management or improvement of BMP6-mediated diseases.

17. The additional therapeutic agent is (a) Iron in the veins, (b) ESA (EPO optional), (c) ActRIIa inhibitors, (d) ActRIIb inhibitors, (e) IL-6 or IL-6 receptor inhibitors (optional anti-IL-6 or IL-6 receptor antibodies), (f) TNF-alpha or TNF-alpha receptor inhibitor (optional anti-TNF-alpha or TNF-alpha receptor antibody), (g) HJV inhibitors (optional anti-HJV antibodies), (h) BMP antagonist (optionally further anti-BMP antibody or fragment), optionally the BMP being BMP2, 4, 5, 6, 7, or 9. (i) Matryptase-2 (MTP2) agonist (optional matryptase-2 (MTP2) agonist antibody), (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 (optional activin receptor Fc fusion), (p) GDF11 inhibitors, and (q) A kit according to claim 14 or 15, selected from the group consisting of (q) a myostatin inhibitor.

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

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

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

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