Antagonists

Abstract

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

Description

Technical Field

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

[0002] Bone morphogenetic protein 6 (BMP6) is a key regulator of hepcidin, a small peptide secreted by the liver that is a major regulator of iron metabolism in mammals. Anti-BMP6 antagonists, such as antibodies, are being developed for use in methods for treating or preventing anemia (see, e.g., WO2016098079, US20160176956A1). See also WO2017191437, which discloses a combination of an anti-BMP6 antagonist and an erythropoietin stimulating agent (ESA).

[0003] Anemia is a serious disease, affecting 25% of the global population, or more than 1.7 billion people, specifically pregnant women, newborns and children. More than 40% of anemias reflect dysfunction in the homeostatic control of iron uptake, storage and recycling. The disorder is the result of a variety of chronic diseases including infection (e.g. HIV, hepatitis), inflammation (e.g. rheumatoid arthritis), cancer and kidney disease. The huge impact of diseases that lead to iron homeostasis disorders can be seen in the United States, where 10% of the 40 million adults aged >65 years suffer from anemia and one-third of these are caused by chronic conditions.

[0004] The standard of care focuses on blood transfusions and ESAs such as EPO or (Amgen, Inc) treatment.

[0005] The hamp gene encodes hepcidin, a 25-amino acid peptide hormone produced by the liver. Hepcidin acts primarily by controlling the efflux of iron from cells by regulating the amount of the iron transport protein ferroportin present on the cell surface of cells involved in iron transport. Hepcidin interacts with ferroportin, which is primarily expressed on macrophages and duodenal enterocytes, leading to the internalization and degradation of ferroportin (Ganz and Nemeth, 2011; Ramey et al., 2010). BMP6 triggers hepcidin expression in a mechanism involving several receptors and cofactors. These receptors include BMP class I and class II receptors, which are necessary to trigger hamp expression, as well as members of the repulsive guidance molecule family RGM such as RGMc (hemojuvelin, HJV) and RGMb (DRAGON), protease-2, neogenin, HFE, and transferrin receptors 1 and 2. The SMAD pathway may be triggered by the initial interaction of BMP6 with class I BMP receptors that may also involve HJV. This leads to the binding of autophosphorylated BMPR II and the activation of BMPRI phosphorylation, thereby triggering the SMAD cascade. Phosphorylated SMAD1, 5 and 8 and finally SMAD4 translocate to the nucleus to interact with the BMP response element in the hamp gene control region to trigger hepcidin expression. In mice, liver-specific destruction of SMAD4 signaling molecules or type I receptors Alk2 and ALk3 reduces hepcidin expression, similar to BMP6 knockout, confirming that the factor is also part of the relevant path (Steinbicker et al., 2011; Wang et al., 2005).

[0006] Ganz, T., Nemeth, E., 2011. The Hepcidin-Ferroportin System as a Therapeutic Target in Anemias and Iron Overload Disorders. Hematology, 2011, 538-542.

[0007] 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.

[0008] 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.

[0009] Wang, R.-H., Li, C., Xu, X., Zheng, Y., Xiao, C., Zerfas, P., Cooperman, S., Eckhaus, M., Rouault, T., Mishra, L., 2005. A role of SMAD4 in iron metabolism through the positive regulation of hepcidin expression. Cell Metab. 2, 399-409. Summary of the invention

[0010] The present invention provides the following:

[0011] 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, wherein the VH domain comprises a CDRH3 sequence including the VH domain of SEQ ID NO: 114.

[0012] An antibody or fragment comprising a binding site that specifically binds to BMP6, wherein the binding site comprises a VH domain comprising SEQ ID NO: 114 or an amino acid having at least 70% identity thereto.

[0013] An antibody or fragment comprising a binding site that specifically binds BMP6, wherein the binding site comprises a VL domain, wherein the VL domain comprises a CDRL3 sequence comprising the VL domain of SEQ ID NO: 123.

[0014] An antibody or fragment comprising a binding site that specifically binds to BMP6, wherein the binding site comprises a VL domain comprising SEQ ID NO: 123 or an amino acid having at least 70% identity thereto.

[0015] An antibody or fragment that specifically binds to bone morphogenetic protein 6 (BMP6) and comprises (i) a heavy chain amino acid sequence comprising SEQ ID NO: 116 or an amino acid having at least 70% identity thereto; and / or (ii) a light chain sequence comprising SEQ ID NO: 125 or an amino acid having at least 70% identity thereto.

[0016] An antibody or fragment thereof (i) specifically binds to a human BMP6 antigenic determinant that is consistent with the antigenic determinant bound by the antibody of the present invention; and / or (ii) competes with the antibody of the present invention for binding to human BMP6.

[0017] The present invention also provides the following configuration.

[0018] In the first configuration, The present invention provides:

[0019] An antibody or fragment comprising a binding site that specifically binds to bone morphogenetic protein 6 (BMP6), wherein the binding site comprises a VH domain encoded by a nucleotide sequence derived from the recombination of a human VH gene segment, a DH gene segment, and a JH gene segment, wherein the VH gene segment is selected from IGHV3-11 and IGHV1-3.

[0020] In the second configuration, The present invention provides:

[0021] An antibody or fragment which specifically binds to BMP6 and comprises the CDRH3 sequence of the anti-BMP6 antibody according to the present invention, or the CDRH3 sequence comprises 3, 2 or 1 amino acid substitutions.

[0022] In the third configuration, The present invention provides:

[0023] An antibody or fragment that specifically binds to BMP6 and comprises a VH domain, wherein the VH domain comprises the CDRH3 sequence of an antibody selected from the group consisting of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713; or the sequence comprises 3, 2 or 1 amino acid substitutions.

[0024] In the fourth configuration, The present invention provides:

[0025] An antibody or fragment comprising a binding site that specifically binds to BMP6, wherein the binding site comprises a VH domain, and the VH domain comprises the amino acid sequence of the VH domain of an antibody selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713; or an amino acid having at least 70% identity thereto.

[0026] In the fifth configuration, The present invention provides:

[0027] An antibody or fragment comprising a binding site that specifically binds to BMP6, wherein the binding site comprises a VL domain encoded by a nucleotide sequence derived from the recombination of a human VL gene segment and a JL gene segment, wherein the VL gene segment is selected from IGKV3-20, IGKV1-5 and IGKV3-15.

[0028] In the sixth configuration, The present invention provides:

[0029] An antibody or fragment that specifically binds to BMP6 and comprises the CDRL3 sequence of the anti-BMP6 antibody of the present invention, wherein the CDRL3 sequence comprises 3, 2 or 1 amino acid substitutions.

[0030] In the seventh configuration, The present invention provides:

[0031] An antibody or fragment that specifically binds to BMP6 and comprises a VL domain comprising the CDRL3 (and optionally CDRH3) sequence of an antibody selected from the group consisting of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713; or each of the sequences comprises 3, 2 or 1 amino acid substitutions.

[0032] In the eighth configuration, The present invention provides:

[0033] 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 the amino acid sequence of the VL domain of an antibody selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713; or an amino acid having at least 70% identity thereto.

[0034] In the ninth configuration, The present invention provides:

[0035] An antibody or fragment that specifically binds to BMP6 and comprises the heavy chain amino acid sequence of an antibody selected from the group consisting of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713; or an amino acid having at least 70% identity thereto.

[0036] In the tenth configuration, The present invention provides:

[0037] An antibody or fragment that specifically binds to BMP6 and comprises the light chain amino acid sequence of an antibody selected from the group consisting of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713; or an amino acid having at least 70% identity thereto.

[0038] In the eleventh configuration, The present invention provides:

[0039] An antibody or fragment thereof specifically binds to a human BMP6 antigenic determinant, wherein the human BMP6 antigenic determinant is consistent with the antigenic determinant bound by the antibody of the present invention (eg, CL-58838).

[0040] In the twelfth configuration, The present invention provides:

[0041] An antibody or fragment that competes with the antibody of the present invention for binding to human BMP6.

[0042] In the thirteenth configuration, The present invention provides:

[0043] An anti-BMP6 antibody or fragment of the present invention for use in treating or preventing a BMP6-mediated disease or condition (optionally anemia) in an individual.

[0044] In the fourteenth configuration, The present invention provides:

[0045] A combination of an amount of an anti-BMP6 antibody or fragment and an amount of an ESA (optionally comprising multiple doses of said antibody and / or ESA), wherein said antibody or fragment is according to the present invention.

[0046] In the fifteenth configuration, The present invention provides:

[0047] A use of an antibody, fragment or combination of the invention for the manufacture of a medicament for administration to an individual to treat or prevent a BMP6-mediated disease or condition, optionally anemia.

[0048] In the sixteenth configuration, The present invention provides:

[0049] A method of treating or preventing a BMP6-mediated disease or condition (optionally anemia) in an individual, the method comprising administering to the individual a therapeutically effective amount of an antibody, fragment or combination of the invention, wherein the BMP6-mediated disease or condition is thereby treated or prevented.

[0050] In the seventeenth configuration, The present invention provides:

[0051] A pharmaceutical composition comprising the antibody, fragment or combination of the present invention and a pharmaceutically acceptable excipient, diluent or carrier.

[0052] In the eighteenth configuration, The present invention provides:

[0053] A nucleic acid encoding the VH domain and / or VL domain of an antibody or fragment of the invention.

[0054] In the nineteenth configuration, The present invention provides:

[0055] A nucleic acid encoding a VH domain comprising the amino acid sequence of the VH domain of an antibody selected from the group consisting of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713; or an amino acid having at least 70% identity thereto.

[0056] In the twentieth configuration, The present invention provides:

[0057] A nucleic acid (e.g., in a host cell such as a CHO or HEK293 or Cos cell) comprising

[0058] (a) a nucleotide sequence having at least 70% identity to the sequence of SEQ ID NO: 115, 520 or 521; and / or

[0059] (b) a nucleotide sequence having at least 70% identity to the sequence of SEQ ID NO: 124, 522 or 523.

[0060] A nucleic acid (e.g., in a host cell such as a CHO or HEK293 or Cos cell) comprising

[0061] (a) a nucleotide sequence having at least 70% identity to the sequence of SEQ ID NO: 115; and / or

[0062] (b) a nucleotide sequence having at least 70% identity to the sequence of SEQ ID NO:124.

[0063] A nucleic acid (e.g., in a host cell such as a CHO or HEK293 or Cos cell) comprising

[0064] (a) a nucleotide sequence having at least 70% identity to the sequence of SEQ ID NO: 520; and / or

[0065] (b) a nucleotide sequence having at least 70% identity to the sequence of SEQ ID NO:522.

[0066] A nucleic acid (e.g., in a host cell such as a CHO or HEK293 or Cos cell) comprising

[0067] (a) a nucleotide sequence having at least 70% identity to the sequence of SEQ ID NO: 521; and / or

[0068] (b) a nucleotide sequence having at least 70% identity to the sequence of SEQ ID NO:523.

[0069] In the twenty-first configuration, The present invention provides:

[0070] A combination of a first nucleic acid and a second nucleic acid (e.g., in a host cell such as a CHO or HEK293 or Cos cell), each comprising

[0071] (a) a nucleotide sequence having at least 70% identity to the sequence of SEQ ID NO: 115, 520 or 521; and / or

[0072] (b) a nucleotide sequence having at least 70% identity to the sequence of SEQ ID NO: 124, 522 or 523.

[0073] A combination of a first nucleic acid and a second nucleic acid (e.g., in a host cell such as a CHO or HEK293 or Cos cell), each comprising

[0074] (a) a nucleotide sequence having at least 70% identity to the sequence of SEQ ID NO: 115; and / or

[0075] (b) a nucleotide sequence having at least 70% identity to the sequence of SEQ ID NO:124.

[0076] A combination of a first nucleic acid and a second nucleic acid (e.g., in a host cell such as a CHO or HEK293 or Cos cell), each comprising

[0077] (a) a nucleotide sequence having at least 70% identity to the sequence of SEQ ID NO: 520; and / or

[0078] (b) a nucleotide sequence having at least 70% identity to the sequence of SEQ ID NO:522.

[0079] A combination of a first nucleic acid and a second nucleic acid (e.g., in a host cell such as a CHO or HEK293 or Cos cell), each comprising

[0080] (a) a nucleotide sequence having at least 70% identity to the sequence of SEQ ID NO: 521; and / or

[0081] (b) a nucleotide sequence having at least 70% identity to the sequence of SEQ ID NO:523.

[0082] In the twenty-second configuration, The present invention provides:

[0083] A nucleic acid encoding the heavy chain and / or light chain of an antibody or fragment of the invention.

[0084] In the twenty-third configuration, The present invention provides:

[0085] A nucleic acid encoding a heavy chain comprising an amino acid sequence at least 70% identical to SEQ ID NO:116.

[0086] In the twenty-fourth configuration, The present invention provides:

[0087] A nucleic acid encoding a light chain comprising an amino acid sequence at least 70% identical to SEQ ID NO: 125.

[0088] In the twenty-fifth configuration, The present invention provides:

[0089] A nucleic acid (e.g., in a host cell such as a CHO or HEK293 or Cos cell) comprising

[0090] (a) a nucleotide sequence having at least 70% identity to a selected heavy chain sequence of an antibody selected from the group consisting of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, and CL-58713; and / or

[0091] (b) a nucleotide sequence having at least 70% identity to a selected sequence of an antibody selected from the group consisting of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713.

[0092] A nucleic acid (e.g., in a host cell such as a CHO or HEK293 or Cos cell) comprising

[0093] (a) a nucleotide sequence having at least 70% identity to a sequence selected from the group consisting of SEQ ID NOs: 512, 514, 516, 518 and 519; and / or

[0094] (b) a nucleotide sequence having at least 70% identity to a sequence selected from the group consisting of SEQ ID NOs: 513, 515 and 517.

[0095] A nucleic acid (e.g., in a host cell such as a CHO or HEK293 or Cos cell) comprising

[0096] (a) a nucleotide sequence that is at least 70% identical to SEQ ID NO: 512; and / or

[0097] (b) a nucleotide sequence that is at least 70% identical to SEQ ID NO:513.

[0098] A nucleic acid (e.g., in a host cell such as a CHO or HEK293 or Cos cell) comprising

[0099] (a) a nucleotide sequence that is at least 70% identical to SEQ ID NO: 516; and / or

[0100] (b) a nucleotide sequence that is at least 70% identical to SEQ ID NO:517.

[0101] A nucleic acid (e.g., in a host cell such as a CHO or HEK293 or Cos cell) comprising

[0102] (a) a nucleotide sequence that is at least 70% identical to SEQ ID NO: 518; and / or

[0103] (b) a nucleotide sequence that is at least 70% identical to SEQ ID NO:513.

[0104] A nucleic acid (e.g., in a host cell such as a CHO or HEK293 or Cos cell) comprising

[0105] (a) a nucleotide sequence that is at least 70% identical to SEQ ID NO: 519; and / or

[0106] (b) a nucleotide sequence that is at least 70% identical to SEQ ID NO:513.

[0107] A nucleic acid (e.g., in a host cell such as a CHO or HEK293 or Cos cell) comprising

[0108] (a) a nucleotide sequence that is at least 70% identical to SEQ ID NO: 518; and / or

[0109] (b) a nucleotide sequence that is at least 70% identical to SEQ ID NO:517.

[0110] A nucleic acid (e.g., in a host cell such as a CHO or HEK293 or Cos cell) comprising

[0111] (a) a nucleotide sequence that is at least 70% identical to SEQ ID NO: 519; and / or

[0112] (b) a nucleotide sequence that is at least 70% identical to SEQ ID NO:517.

[0113] A nucleic acid (e.g., in a host cell such as a CHO or HEK293 or Cos cell) comprising

[0114] (a) a nucleotide sequence that is at least 70% identical to SEQ ID NO: 514; and / or

[0115] (b) a nucleotide sequence that is at least 70% identical to SEQ ID NO:515.

[0116] A nucleic acid (e.g., in a host cell such as a CHO or HEK293 or Cos cell) comprising

[0117] (a) a nucleotide sequence that is at least 70% identical to SEQ ID NO: 516; and / or

[0118] (b) a nucleotide sequence that is at least 70% identical to SEQ ID NO:517.

[0119] A nucleic acid (e.g., in a host cell such as a CHO or HEK293 or Cos cell) comprising

[0120] (a) a nucleotide sequence that is at least 70% identical to SEQ ID NO: 518; and / or

[0121] (b) a nucleotide sequence having at least 70% identity to SEQ ID NO: 513, 515 or 517.

[0122] A nucleic acid (e.g., in a host cell such as a CHO or HEK293 or Cos cell) comprising

[0123] (a) a nucleotide sequence that is at least 70% identical to SEQ ID NO: 519; and / or

[0124] (b) a nucleotide sequence having at least 70% identity to SEQ ID NO: 513, 515 or 517.

[0125] In the twenty-sixth configuration, The present invention provides:

[0126] A vector comprising the nucleic acid; optionally wherein the vector is a CHO or HEK293 vector.

[0127] In the twenty-seventh configuration, The present invention provides:

[0128] A host cell comprising the nucleic acid or the vector.

[0129] In the twenty-eighth configuration, The present invention provides:

[0130] An antibody, fragment, combination, vector, host cell, use or method as described herein.

[0131] In the twenty-ninth configuration, The present invention provides:

[0132] An antibody or fragment that specifically binds to a bone morphogenetic protein (BMP) for use in a method for treating or preventing a disease or condition caused by a hemojuvelin (HJV)-deficient BMP-BMP receptor (BMPR) complex in a human or animal individual, wherein the method comprises administering the antibody or fragment to the individual to inhibit the formation of the complex and / or inhibit the triggering of intracellular signaling by the complex in the individual, thereby treating or preventing an HJV-independent BMP-BMPR-mediated disease or condition.

[0133] An antibody or fragment that specifically binds to bone morphogenetic protein (BMP) for use in a method for treating or preventing HJV-independent anemia or osteoporosis in a human or animal individual, wherein the method comprises administering the antibody or fragment to the individual to inhibit the formation of a hemojuvelin (HJV)-deficient BMP-BMP receptor (BMPR) complex and / or inhibit the triggering of intracellular signaling by the complex in the individual, thereby treating or preventing HJV-independent anemia or osteoporosis.

[0134] An antibody or fragment that specifically binds to a bone morphogenetic protein (BMP) for use in a method for treating or preventing hemojuvelin (HJV)-independent anemia or osteoporosis in a human or animal subject, wherein the method comprises administering the antibody to the subject, thereby treating or preventing HJV-independent anemia or osteoporosis.

[0135] The invention also provides methods for treating a disease or condition such as anemia or osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0136] Figure 1 :

[0137] The assay window was established using the HepG2 hamp luciferase reporter cell line and various human BMP ligands as stimulants. The function of the red firefly luciferase gene under the control of the human hepcidin promoter regulatory element in HepG2 cells was tested by stimulating cells with various human BMP proteins (R&D Systems or Peprotech) added in increasing concentrations. The performance of the assay was tested in two different culture medium settings, namely MEM containing 1% FBS (A) and fusion tumor culture medium containing 25% MEM (B; see Example 1 for details).

[0138] Figure 2 :

[0139] The HepG2 hamp luciferase reporter cell assay window was evaluated using various human or mouse BMP6 ligands added in increasing concentrations as stimulators (R&D Systems or Peprotech). BMP6 was diluted in MEM containing 1% FBS. 25% HMM was used with a total assay volume of 60 μl (see Example 1 for more details and reagents used).

[0140] Figure 3 :

[0141] The HepG2 hamp luciferase reporter cell assay window was evaluated using human BMP6 (Peprotech) or mouse BMP6 (R&D Systems) at a fixed concentration of 1 nM in MEM containing 1% FBS and commercial mouse anti-BMP6 monoclonal antibodies MAB507 (A) and MAB2365 (B) from R&D Systems diluted in hybridoma maintenance medium (HMM); (see Example 1 for more details and reagents).

[0142] Figure 4 :

[0143] Five BMP6- / - kymics were used to reverse Serum titer determination of immunization protocol KM089 was performed by assay (Perkin Elmer), in which anti-mouse IgG (goat anti-mouse IgG; Southern Biotech 1030-01) was used to capture IgG antibodies contained in serum added in various serial dilutions via the Fc domain, followed by incubation with biotinylated BMP6, and detection using DELFIA Eu-N1 europium-labeled streptavidin (Perkin Elmer). The cutoff value (any signal below the value was judged as negative) was defined as the negative control mean + 3x standard deviation of all replicates. The KMBM code in the legend refers to individual Kymouse TM bmp6- / - animals.

[0144] Figure 5 :

[0145] 2 μg / lane purified human BMP6 (Peprotech) was separated by SDS-PAGE and stained with Coomassie blue (A) under reducing (R) and non-reducing (NR) conditions, and the protein blot from the gel was blotted onto the membrane, followed by detection with purified human anti-BMP6 antibodies as markers. Bound antibodies were detected by enhanced chemiluminescence with anti-human kappa light chain-horseradish peroxidase (HRP) and peroxidase (CL-58838 and antibody A) or with anti-human Fc-alkaline phosphatase (AP) and phosphatase 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitro blue tetrazolium (NBT) colorimetric substrate conversion (antibody B).

[0146] Figure 6 :

[0147] Receptor dimerization assay based on U2OS cells transfected with BMPRIA (ALK3, CD292) and BMPRII (T-ALK; panel A) or BMPRIA (ALK6) and BMPRII (T-ALK; panel B). Chemiluminescent signal generated by ligand-driven dimerization with BMP6 (Peprotech 120-06 SEQ ID NO: 2). Human anti-BMP6 IgG4 antibody CL-58838 and anti-BMP6 antibodies A, B (A), human anti-BMP6 IgG4 antibodies 1-8 (B) and murine monoclonal antibody MAB507 (R&D Systems; A and B) were added in increasing concentrations (11-point dilution). Representative data of two experiments in each case. Appropriate isotype control IgG was used in both experiments.

[0148] Figure 7 :

[0149] Cross-reactivity profile of purified CL-58838 IgG4 (SEQ ID NO: 116 and SEQ ID NO: 125) with human BMP2 (A), BMP4 (B), BMP5 (C), BMP7 (D) and BMP9 (E; details described in Example 7). The effect of anti-BMP6 antibody CL-58838 on BMP-driven activation of the hamp luciferase reporter gene in HepG2 cells was investigated by adding increasing amounts of CL-58838. As a positive control, relevant BMP-specific antibodies (all from R&D Systems) were added to interfere with the activation of the relevant BMP.

[0150] Figure 8 :

[0151] After a single intravenous injection of anti-BMP-6 or isotype IgG control antibody, the Figure 8 AD), Table 13 (for Figure 8EG) and Table 14 (for Figure 8 H and I) Plotted transferrin saturation % (TSAT) in normal rats.

[0152] Fig. 9 :

[0153] Results of transferrin saturation % (TSAT) in normal rats after a single intravenous injection of different doses of CL-58838 (A) or 1 mg / kg of CL-58838 and Antibody A (B; Table 15).

[0154] Fig.10 :

[0155] Results of % transferrin saturation (TSAT) in normal rats after a single subcutaneous injection of different doses of CL-58838 (A) or 1 mg / kg of CL-58838 and antibodies A and B (B; Table 16).

[0156] Fig.11 :

[0157] Pharmacokinetic (PK) profiles of human anti-BMP6 IgG4 antibody CL-58838 after a single intravenous injection at different doses (A) or compared with antibody A at a dose of 1 mg / kg (B). PK profiles after a single subcutaneous (sc) injection of different doses of CL-58838 (C) or 1 mg / kg doses of antibodies A and B (D). Results are plotted as IgG [ng / ml] IgG over time [hours (h)].

[0158] Fig.12 :

[0159] Results of the rat PG-PS model of ACD. Transferrin saturation TSAT (A), hemoglobin [g / dL] (B), MCH [pg] (C), and serum hepcidin content [ng / mL] at week 0 (D), week 1 (E), and week 2 (F) after the start of treatment (*ρ < 0.05, **p < 0.01, ***p < 0.001).

[0160] Fig.13 :

[0161] Transferrin saturation % (TSAT) after a single intravenous administration of 3 mg / kg of CL-58838 and antibodies A and B (A) and different doses of CL-58838 (B) in cynomolgus monkeys, and plasma hepcidin levels after a single intravenous administration of 3 mg / kg of CL-58838 and antibodies A and B (C) and different doses of CL-58838 (D) in cynomolgus monkeys.

[0162] Fig.14 :

[0163] PK profiles of CL-58838 after a single intravenous injection at different doses in cynomolgus monkeys (A) and comparison of PK profiles of CL-58838 at 3 mg / kg with antibodies A and B (B).

[0164] Fig.15 :

[0165] Amino acid sequence alignment of antibody Vh and Vk sequences (Table 6) highly related to CL-58838 based on V region usage and CDRH3. The top line shows the germline sequences of the IMGT V region genes IGHV3-11 and IGKV3-20 encoded in the bmp6- / -Kymouse used in the present invention. The amino acid positions that differ from the germline V region sequences in the selected antibody sequences below are boxed. The antibodies selected for in vivo evaluation are shown in bold (Table 6).

[0166] Fig.16 :

[0167] Homogeneous time-resolved FRET (HTRF) assays showed the ability of V-region based CDRH antibodies (Table 6) to compete with labeled CL-58838 (10 nM) for binding to human BMP6 present at 32 nM using antibodies highly related to CL-58838 and identified by NGS sequences. Competing antibodies were added in a series of concentrations starting at 3 μM final concentration over an 11-point dilution range.

[0168] Fig.17 :

[0169] Experimental setup and results obtained from Example 17. Graphical illustration of the experimental time course (A), decision diagram for inclusion of rats and administration of EPO (B), hemoglobin [g / dL] (C), MCV [fL] (D), MCH [pg] (E) and EPO dose administered [%] (F). ANCOVA analysis of changes from baseline at week 4 using baseline as a covariate (CE) and using Fishers exact test (F). Data are shown as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).

[0170] Fig.18 :

[0171] Experimental setup and results of Example 18. Graphical illustration of the experimental time course (A), hemoglobin [g / dL] (B), MCV [fL] (C), MCH [pg] (D), liver Hamp mRNA content [expression fold], plasma iron content [μg / dL] (F) and TSAT values ​​[%]. Analysis of variance with Dunnett correction for multiple comparisons with EPO was applied. Comparison results of EPO and EPO + CL-58838 are shown. Data are shown as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).

[0172] Fig.19 :

[0173] Results obtained from Example 19. Comparison of hemoglobin values ​​[g / dL] (A) of the treatment groups after administration of 1 μg / kg EPO in combination with different CL-58838 concentrations, hemoglobin values ​​[g / dL] (B) after administration of 10 mg / kg CL-58838 in combination with different EPO doses, MCV values ​​[fL] (C) and TSAT [%] (D) of mice after administration of 1 μg / kg EPO in combination with different CL-58838 concentrations, MCV values ​​[fL] (E) of mice after administration of 1 mg / kg CL-58838 in combination with different EPO doses, liver Hamp mRNA content [fold expression] (F) in all treatment groups. ANOVA with Dunnett correction for multiple comparisons with Group II was applied. Data are shown as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001). DETAILED DESCRIPTION

[0174] definition

[0175] Unless otherwise defined herein, scientific and technical terms shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0176] Unless the context clearly indicates otherwise, the singular terms "a", "an" and "the" include plural referents. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "for example" is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Therefore, the abbreviation "for example" is synonymous with the term "for example".

[0177] In this specification and claims, the term "about" is used to modify, for example, the amount, concentration, volume, process temperature, process time, yield, flow rate, pressure and the like of ingredients in the composition, as well as the range thereof, to describe embodiments of the present disclosure. The term "about" refers to, for example, typical measurements and processing procedures for preparing compounds, compositions, concentrates, or using formulations; through unintentional errors in these procedures; through differences in the manufacture, source or purity of the starting materials or ingredients used to implement the method, and similar considerations that may occur. The term "about" also encompasses amounts that differ due to aging of formulations with a specific initial concentration or mixture, and amounts that differ due to mixing or processing formulations with a specific initial concentration or mixture. In the case of being modified by the term "about", the appended claims include equivalents of the amounts.

[0178] As used herein, "administering" or "administering" refers to the act of injecting or otherwise physically delivering a substance present in vitro (e.g., an anti-hBMP6 antibody provided herein) into a patient's body, such as by mucosal, intradermal, intravenous, intramuscular delivery and / or any other physical delivery method described herein or known in the art. When treating a disease or its symptoms, the substance is typically administered after the onset of the disease or its symptoms. When preventing a disease or its symptoms, the administration of the substance is typically performed before the onset of the disease or its symptoms.

[0179] The terms "antibody", "immunoglobulin" or "Ig" are used interchangeably herein and refer to an immunoglobulin molecule that recognizes and specifically binds to a target such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing via at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab') 2The term "antibody" may also refer to a Y-shaped glycoprotein with a molecular weight of about 150 kDa, which is composed of four polypeptide chains: two light (L) chains and two heavy (H) chains. There are five types of mammalian Ig heavy chain isotypes, represented by the Greek letters α, δ, ε, γ and μ. The types of heavy chains define the categories of antibodies, i.e., IgA, IgD, IgE, IgG and IgM, respectively. The γ and α classes are further divided into subclasses based on the differences in constant domain sequence and function, such as IgG1, hIgG2, mIgG2A, mIgG2B, IgG3, IgG4, IgA1 and IgA2. 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 heavy chain or light chain of an antibody. The variable domains of the heavy chain and light chain can be referred to as "V H ” and “V L ". The domain is usually the most variable part of an antibody (relative to other antibodies of the same class) and contains the antigen binding site. An example of an antibody is a heavy chain-only (ie, H2) antibody, which comprises a dimer of heavy chains (5'-VH-(optional hinge)-CH2-CH3-3') and no light chains.

[0180] The antibodies described herein may be oligoclonal antibodies, polyclonal antibodies, monoclonal antibodies (including full-length monoclonal antibodies), camelized antibodies, chimeric antibodies, CDR-grafted antibodies, multispecific antibodies, bispecific antibodies (including dual-binding antibodies), catalytic antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, anti-idiotypic antibodies, including antibodies that can be labeled in soluble or bound form and fragments, variants or derivatives thereof, alone or in combination with other amino acid sequences provided by known techniques. The antibodies may be from any species. The antibodies described herein may be naked antibodies or combined with other molecules such as toxins, radioisotopes, etc.

[0181] The terms "antigen binding site," "antigen binding domain," "antigen binding region," "antigen binding fragment," and similar terms refer to the portion of an antibody comprising the amino acid residues that interact with an antigen and confer specificity and affinity to the binding agent for the antigen (e.g., the complementarity determining region (CDR)). The antigen binding region may be derived from any animal species, such as rodents (e.g., rabbits, rats, or hamsters) and humans. Preferably, the antigen binding region will be of human origin.

[0182] The antigen-binding fragments described herein may include single-chain Fv (scFv), single-chain antibodies, single-domain antibodies, domain antibodies, Fv fragments, Fab fragments, F(ab') fragments, F(ab') 2 Fragments, antibody fragments exhibiting the desired biological activity, disulfide-stabilized variable regions (dsFv), dimeric variable regions (diabodies), anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies of antibodies), intrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from any of the above antibody fragments and antigenic determinant binding fragments. Specifically, the 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 produces two identical antigen-binding fragments, also known as "Fab" fragments, and "Fc" fragments that do not have antigen binding activity but have the ability to crystallize. When used herein, "Fab" refers to an antibody fragment that includes one constant domain and one variable domain of each of the heavy and light chains. The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including a native sequence Fc region and a variant Fc region. "Fc fragment" refers to the carboxyl terminal portion of the two H chains held together by disulfide bonds. The effector functions of an antibody are determined by sequences in the Fc region, which is also recognized by Fc receptors (FcR) found on certain types of cells. Digestion of antibodies with the enzyme pepsin produces F(ab') 2 A fragment in which the two arms of an antibody molecule remain linked and contains two antigen-binding sites. 2 The fragments have the ability to cross-link antigens.

[0183] The term "derived from the recombination of..." in relation to gene segments is obvious to those skilled in the art and is understood to be a B cell recombining its variable region gene segments to produce the coding sequence of the variable domain. For example, "derived from the recombination of human VH gene segments, DH gene segments and JH gene segments" involves a human VH gene segment being recombined with a DH gene segment and a JH gene segment to form a rearranged VDJ sequence encoding a heavy chain antibody variable domain. Joining and somatic hypermutation may also be features of the process, whereby the resulting recombinant VDJ sequence includes one or more nucleotide additions, substitutions or deletions (e.g., p additions and / or n additions) not included in the germline V, D and J sequences. For the κ light chain variable domain, the equivalents are referred to as Vκ and Jκ gene segments, and for the λ light chain variable domain, as Vλ and Jλ. It is meant that any post-translational modifications may be additionally encompassed in the variable domain.

[0184] As used herein, "Fv" refers to the smallest fragment of an antibody that retains both antigen recognition and antigen binding sites. The region consists of a dimer of one heavy chain and one light chain variable domain in tight non-covalent or covalent association. In this configuration, the three CDRs of each variable domain interact to define V H -V L The antigen binding site on the surface of the dimer. A total of six CDRs give the antibody antigen binding specificity. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, but its affinity is lower than that of the entire binding site.

[0185] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic determinant or antigenic determinant. In contrast, polyclonal antibody preparations typically include different antibodies directed against different antigenic determinants (or antigenic determinants). The term "monoclonal antibody" as used herein encompasses intact and full-length monoclonal antibodies as well as antibody fragments (e.g., Fab, Fab', F(ab') 2 , Fv), single chain (scFv) mutants, fusion proteins comprising antibody portions, and any other modified immunoglobulin molecules comprising antigen recognition sites. In addition, "monoclonal antibodies" refer to these antibodies prepared in a variety of ways, including but not limited to fusion tumors, phage selection, recombinant expression, and transgenic animals. The monoclonal antibodies 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 or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as antibody fragments exhibiting the desired biological activity.

[0186] The term "humanized antibody" refers to a subset of chimeric antibodies in which a "hypervariable region" from a non-human immunoglobulin (donor antibody) replaces residues from a hypervariable region in a human immunoglobulin (acceptor antibody). In general, a humanized antibody will include substantially all of at least one and usually two variable domains, in which 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 regions are those of a human immunoglobulin sequence, although the framework regions may include one or more substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc.

[0187] The term "bispecific antibody" refers to an antibody that contains specificities for two target molecules, and includes, but is not limited to, the following formats: DVD-Ig (see DiGiammarino et al., "Design and Generation of DVD-Ig for Bispecific Targeting"). TM Molecule (Design and generation of DVD-Ig TM molecules for dual-specific targeting), "Meth. Mo. Biol., 2012, 889, 145-156), mAb 2 (See WO2008 / 003103, mAb 2 format is incorporated herein by reference), FIT-Ig (see WO2015 / 103072, the description of the FIT-Ig scaffold is incorporated herein by reference), mAb-dAb, dock and lock, Fab arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, kappa lambda body, orthogonal Fab, single chain diabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, single chain diabody, single chain diabody-CH3, diabody-CH3, triabody, minibody, minibody, TriBi minibody, scFv-CH3 KIH, scFv-CH-CL-scFv, F(ab′)2-scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCab, ImmTAC, knobs-in-holes, knobs-in-holes with common light chain, knobs-in-holes 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. For a review of bispecific formats, see Spiess, C. et al., Mol. Immunol. (2015). In another embodiment, the bispecific molecule comprises an antibody fused to another non-Ig format, such as a T cell receptor binding domain; an immunoglobulin superfamily domain; a jawless variable lymphocyte receptor; a fibronectin domain (e.g., AdnectinTM ); antibody constant domains (e.g. CH 3 Domains, e.g., Fcab TM CH 2 and / or CH 3 ), where the constant domain is not a functional CH 1 Domain; scFv; (scFv) 2 ; Single-chain bifunctional antibody; scFab; Centyrin and derived from CTLA-4 (Evibody TM ) of the scaffold; a lipocalin domain; a protein A, such as the Z domain of protein A (e.g., Affibody TM or SpA); A domain (e.g. Avimer TM or Maxibody TM ); heat shock proteins (such as antigen-binding domains derived from GroEI and GroES); transferrin domains (such as trans-bodies); ankyrin repeat proteins (such as DARPins TM ); peptide aptamers; C-type lectin domains (e.g. Tetranectin TM ); human γ-crystallin or human ubiquitin (affilin); a PDZ domain; a scorpion toxin; and a Kunitz-type domain of a human protease inhibitor.

[0188] In one embodiment, the bispecific antibody is a mAb 2 mAb 2 Contains VH and V from intact antibodies L Domains fused to modified constant regions (called "Fcabs") that have been engineered to form antigen binding sites. Fcabs / mAbs are described in more detail in WO2008 / 003103 2 The technology behind the format and mAb 2 The description of the form is incorporated herein by reference.

[0189] In another embodiment, the bispecific antibody is a "dual binding antibody". As used herein, the term "dual binding antibody" is a bispecific antibody in which the two antigen binding domains are composed of V and H / V LPair formation, and include FIT-Ig (see WO2015 / 103072, which is incorporated herein by reference), mAb-dAb, docking and locking, Fab arm exchange, SEEDbodv, Triomab, LUZ-Y, Fcab, kappa lambda body, orthogonal Fab, single chain diabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, single chain diabody, single chain diabody-CH3, diabody-CH3, triabody, minibody, minibody, scFv-CH 3 KIH,scFv-CH-CL-scFv,F(ab′) 2-sc Fv, 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 and scFv4-Ig.

[0190] The term "hypervariable region", "CDR region" or "CDR" refers to the region of the antibody variable domain that is hypervariable in sequence and / or forms a structurally defined loop. In general, the antigen binding site of an antibody includes six hypervariable regions: V H Three of them (CDRH1, CDRH2, CDRH3) and V LThe three (CDRL1, CDRL2, CDRL3) of the heavy and light chains of the antibody. These regions of the heavy and light chains of the antibody confer antigen-binding specificity to the antibody. The CDRs can be defined according to the Kabat system (see Kabat, EA et al., 1991, "Sequences of Proteins of Immunological Interest", 5th edition, NIH publication number 91-3242, U.S. Department of Health and Human Services). Other systems can be used to define CDRs, including the system designed by Chothia et al. (see Chothia, C. and Lesk, AM, 1987, "Canonical structures for the hypervariable regions of immunoglobulins", Journal of Molecular Biology (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 usually contain 3 heavy chain CDRs and 3 light chain CDRs. The terms CDR or CDRs are used herein to refer to one or more of these regions. Those skilled in the art can easily compare different naming systems and determine whether a particular sequence can be defined as a CDR.

[0191] "Human antibodies" are antibodies having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human being and / or prepared using any technology for preparing human antibodies, and specifically excludes humanized antibodies comprising non-human antigen binding residues. The term "specific binding" refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, which determines the presence of a target in the presence of a heterogeneous population of molecules including biomolecules. For example, an antibody that specifically binds to a target (which may be an antigenic determinant) is an antibody that binds to the target with greater affinity, avidity, more readily and / or with longer duration than it binds to other targets. In one embodiment, the degree of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA).

[0192] Antibodies or fragments thereof that specifically bind to the hBMP6 antigen may cross-react with related antigens. Preferably, antibodies or fragments thereof that specifically bind to the hBMP6 antigen do not cross-react with other antigens (but may optionally cross-react with BMP6 of different species, such as rhesus monkeys or mice). TM Or other techniques known to those skilled in the art identify antibodies or fragments thereof that specifically bind to hBMP6 antigen.Antibody or its fragment binds specifically to BMP6 antigen, and when it binds to hBMP6 antigen, it has a higher affinity than any cross-reactive antigen measured using experimental techniques, such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA).Generally, specific or selective reactions will be at least twice of background signal or noise, and more generally more than 10 times (such as more than 15 times, more than 20 times, more than 50 times or more than 100 times) of background.For discussion of antibody specificity, see, for example, Paul (Paul) ed., 1989, Fundamental Immunology (Fundamental Immunology), second edition, Raven Press, New York (New York), pp. 332-336.

[0193] The term "aliphatic amino acid" refers to an amino acid whose R group is nonpolar and hydrophobic. The hydrophobicity increases with the number of C atoms in the hydrocarbon chain. Glycine, alanine, valine, leucine and isoleucine are aliphatic amino acids.

[0194] 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.

[0195] The term "hydroxyl-containing amino acid" means that the amino acid R group contains a hydroxyl group and is hydrophilic. Serine, cysteine, threonine and methionine are hydroxyl-containing amino acids.

[0196] The term "basic amino acid" means that the amino acid R group contains nitrogen and is basic at neutral pH. Histidine, lysine, and arginine are basic amino acids.

[0197] The term "cyclic amino acid" means that the amino acid R group has an aliphatic ring structure. Proline is the only cycloaliphatic amino acid.

[0198] The term "acidic amino acid" means that the amino acid R group is polar and negatively charged at physiological pH. Aspartic acid and glutamic acid are acidic amino acids.

[0199] The term "amide amino acid" means that the amino acid R group contains an amide group. Asparagine and glutamine are amide amino acids.

[0200] As used herein, "license number" or "marketing authorization number" refers to a number issued by a regulatory agency that determines that a specific medical product and / or composition can be marketed and / or offered for sale in the region under the jurisdiction of the agency. As used herein, "regulatory agency" refers to one of the agencies responsible for evaluating the safety and efficacy of, for example, medical products and / or compositions and controlling the sale / marketing of the products and / or compositions in a given region. The Food and Drug Administration (FDA) of the United States and the European Medicines Agency (EPA) in Europe are only two examples of such regulatory agencies. Other non-limiting examples may include SDA, MPA, MHPRA, IMA, ANMAT, Hong Kong Department of Health Pharmaceutical Affairs, CDSCO, Medsafe and KFDA.

[0201] As used herein, "buffer" refers to a chemical agent that is able to absorb a certain amount of acid or base without undergoing a drastic change in pH.

[0202] As used herein, the term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which a therapeutic agent is administered. The pharmaceutical carrier may be a sterile liquid, such as water and oil, including oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions may also be used as liquid carriers, specifically for injectable solutions.

[0203] The term "chemotherapeutic agent" or "chemotherapy" refers to a therapeutic agent whose primary purpose is to destroy cancer cells, usually by interfering with the growth or reproduction ability of tumor cells. There are many different types of chemotherapeutic agents, of which more than 50 approved chemotherapeutic drugs are available. Chemotherapeutic drugs can be classified based on their mode of action. Alkylating drugs kill cancer cells by directly attacking the genetic material of genes, DNA. Cyclophosphamide is an alkylating drug. Antimetabolites interfere with the production of DNA and keep cells from growing and reproducing. An example of an antimetabolite is 5-fluorouracil (5-FU). Antitumor antibiotics are made from natural substances such as fungi in the soil. They interfere with important cell functions, including the production of DNA and cell proteins. Doxorubicin and bleomycin belong to this group of chemotherapeutic drugs. Plant alkaloids prevent cells from dividing normally. Vinblastine and vincristine are plant alkaloids obtained from the periwinkle plant. Steroid hormones slow the growth of some hormone-dependent cancers. For example, tamoxifen is used to treat breast cancers that depend on the hormone estrogen for growth. DNA damage response (DDR) inhibitors, such as PARP inhibitors, block the DNA repair mechanism following single-strand or double-strand breaks.

[0204] Examples of chemotherapeutic agents include Adriamycin, Cranberry, 5-fluorouracil, Cytarabine (Ara-C), Cyclophosphamide, Thiotepa, Taxotere (docetaxel), Busulfan, Cytoxin, Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, and Cyclophosphamide. antrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycins, esperamicins (see U.S. Pat. No. 4,675,187), melphalan and other related nitrogen mustards. Suitable toxins and chemotherapeutic agents are described in Remington's Pharmaceutical Sciences, 19th edition (Mack Publishing Co. 1995); and Goodman and Gilman, The Pharmacological Basis of Therapeutics, 7th edition (MacMillan Publishing Co. 1985). Another example of a chemotherapeutic agent is the class of antibody-conjugated toxins, including but not limited to pyrrolobenzodiazepines, maytansanoids, calicheamicins, etc. Other suitable toxins and / or chemotherapeutic agents are known to those skilled in the art.

[0205] As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients (eg, antibodies of the invention), optionally in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients, optionally in the specified amounts.

[0206] As used herein, the term "comprising" refers to antibodies, fragments, uses, compositions, methods and corresponding components thereof that are essential to the method or composition, but still include unspecified elements, whether essential or not.

[0207] The term "consisting of" refers to the antibodies, fragments, uses, compositions, methods and corresponding components thereof as described herein, which do not include any elements not listed in the description of the embodiment.

[0208] As used herein, the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of elements that do not substantially affect the basic and novel or functional characteristics of the embodiment.

[0209] In the context of polypeptides, the term "derivative" as used herein includes polypeptides comprising hBMP6 polypeptides, hBMP6 polypeptide fragments, or antibodies or fragments of amino acid sequences that specifically bind hBMP6 polypeptides that are changed by introducing amino acid residue substitutions, deletions or additions. The term "derivative" as used herein also includes hBMP6 polypeptides, hBMP6 polypeptide fragments, or antibodies that specifically bind hBMP6 polypeptides that have been chemically modified, for example, by covalent attachment of any type of molecule to a polypeptide. For example, but not as a limitation, hBMP6 polypeptides, hBMP6 polypeptide fragments or hBMP6 antibodies can be chemically modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, connection to cell ligands or other proteins, etc. Derivatives are modified in a manner different from naturally occurring or starting peptides or polypeptides in the type or position of the connecting molecule. Derivatives further include the absence of one or more chemical groups that are naturally present in a peptide or polypeptide. The derivatives of hBMP6 polypeptide, hBMP6 polypeptide fragment or hBMP6 antibody can be chemically modified by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. In addition, the derivatives of hBMP6 polypeptide, hBMP6 polypeptide fragment or hBMP6 antibody may contain one or more than one non-classical amino acid. The polypeptide derivatives have similar or identical functions to the hBMP6 polypeptide, hBMP6 polypeptide fragment or hBMP6 antibody described herein.

[0210] As used herein, the term "effector function" (or "effector activation") refers to one or more of antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC)-mediated responses, Fc-mediated phagocytosis or antibody-dependent cellular phagocytosis (ADCP), and antibody recycling via the FcRn receptor.

[0211] "Effective amount" refers to an amount that effectively achieves the desired effect (including therapeutic or preventive results) within the necessary dosage and time period. "Therapeutically effective amount" refers to the minimum concentration required to achieve a measurable improvement or prevention of a specific condition. The therapeutically effective amount herein may vary according to factors such as the disease state, the age, sex and weight of the patient, and the ability of the antibody to elicit the desired response in the individual. The therapeutically effective amount is also the amount in which the toxic or deleterious effects of the antibody exceed the therapeutically beneficial effects. "Preventively effective amount" refers to an amount that effectively achieves the desired preventive results within the necessary dosage and time period. In some embodiments, the effective amount of an antibody of the present invention is about 0.1 mg / kg (mg antibody / kg individual body weight) to about 100 mg / kg. In certain embodiments, the effective amount of the antibody provided 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 range therein). In some embodiments, "effective amount" as used herein also refers to the amount of the antibody of the present invention to achieve a specified result (e.g., inhibit the hBMP6 biological activity of cells).

[0212] As used herein, the term "antigenic determinant" refers to a local area on the surface of an antigen such as an hBMP6 polypeptide or an hBMP6 polypeptide fragment, which is capable of binding to one or more than one antigen binding region of an antibody, and has antigenic or immunogenic activity in animals, preferably mammals, and most preferably humans, which can trigger an immune response. An antigenic determinant with immunogenic activity is a part of a polypeptide that triggers an antibody response in an animal. An antigenic determinant with antigenic activity is a part of a polypeptide that is specifically bound by an antibody, as determined by any method known in the art, such as by an immunoassay as described herein. Antigenic antigenic determinants are not necessarily immunogenic. Antigenic determinants are generally composed of chemically active surface groups of molecules such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics and specific charge characteristics. The polypeptide region that contributes to the antigenic determinant may be continuous amino acids of the polypeptide, or the antigenic determinant may be aggregated by two or more non-continuous regions of the polypeptide. The antigenic determinant may or may not be a three-dimensional surface feature of the antigen. In certain embodiments, the hBMP6 antigenic determinant is a three-dimensional surface feature of the hBMP6 polypeptide (e.g., in the form of a trimer of the hBMP6 polypeptide). In other embodiments, the hBMP6 antigenic determinant is a linear feature of the hBMP6 polypeptide (e.g., in a trimer or monomeric form of the hBMP6 polypeptide). The antibodies provided herein can specifically bind to an antigenic determinant in the monomeric (denatured) form of hBMP6, an antigenic determinant in the trimer (native) form of hBMP6, or a monomeric (denatured) form and a trimer (native) form of hBMP6. In a specific embodiment, the antibodies provided herein specifically bind to an antigenic determinant in the trimer form of hBMP6, but do not specifically bind to the monomeric form of hBMP6.

[0213] The term "excipient" as used herein refers to an inert substance generally used as a diluent, vehicle, preservative, binder or drug stabilizer, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, octanoates, etc.), surfactants (e.g., SDS, polysorbates, nonionic surfactants, etc.), sugars (e.g., sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.). See also Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa., which is incorporated herein by reference in its entirety.

[0214] In the context of a peptide or polypeptide, the term "fragment" as used herein refers to a peptide or polypeptide that contains less than the full-length amino acid sequence. The fragment may, for example, result from truncation at the amino terminus, truncation at the carboxyl terminus, and / or internal deletions of residues from the amino acid sequence. Fragments may, for example, result from alternative RNA splicing or in vivo protease activity. In certain embodiments, the BMP6 fragment includes 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 the antibody that specifically binds to the hBMP6 antigen. In a specific embodiment, the hBMP6 polypeptide fragment or the antibody that specifically binds to the hBMP6 antigen retains at least 1, at least 2 or at least 3 functions of the polypeptide or antibody.

[0215] The term "free" can refer to a polypeptide combined with a buffer, such as BMP6 or a fragment and its variant, wherein the polypeptide is not combined with a cell surface or a cell membrane. Thus, the term "free" can refer to being able to surface express (i.e. including one or more than one transmembrane domain or membrane-bound domain), but not expressed on the cell surface or in the present state The polypeptide of the protein expressed on the cell surface. Free polypeptides may also refer to free recombinant or natural or uncombined polypeptides. In the context of phage presentation, free antigens can be selected in a solution (referred to herein as "soluble selection") or adsorbed to a surface, such as adsorbed to the surface of a 96-well plate (referred to herein as "bio-panning selection").

[0216] The term "fusion protein" as used herein 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 usually a part of an antibody (e.g., a non-anti-BMP6 antigen antibody)). When used with BMP6 or anti-BMP6 antibodies, the term "fusion" refers to the connection of a peptide or polypeptide or its fragment, variant and / or derivative to a heterologous peptide or polypeptide. Preferably, the fusion protein retains the biological activity of BMP6 or anti-BMP6 antibodies. In certain embodiments, the fusion protein comprises a BMP6 antibody VH domain, a VL domain, a VH CDR (one, two or three VHCDRs) and / or a VL CDR (one, two or three VL CDRs), wherein the fusion protein specifically binds to a BMP6 antigenic determinant.

[0217] When used with respect to antibodies, the term "heavy chain" refers to five different types of amino acid sequences based on heavy chain constant domains, referred to as α, δ, ε, γ and μ. The different types of heavy chains are well known and produce five classes of antibodies, IgA, IgD, IgE, IgG and IgM, respectively, including four subclasses of IgG, i.e. IgG1, IgG2, IgG3 and IgG4. Preferably, the heavy chain is a human heavy chain. In the human population, there are multiple heavy chain constant region alleles of each immunoglobulin or immunoglobulin subclass. The nucleotide and amino acid sequences of the allelic variant can be obtained on publicly available databases such as IMGT, ENSEMBL Swiss-Prot and Uniprot. Allelic variants can also be identified in various genome sequencing projects. In one embodiment, the antibodies and antibody fragments disclosed herein comprise a heavy chain encoded by an IgG1 constant region allele, including but not limited to human IGHG1*01 (Seq ID Nos: 340, 341, and 537), IGHG1*02 (Seq ID Nos: 340, 341, and 537), IGHG1*03 (Seq ID Nos: 523 and 524), IGHG1*04 (Seq ID Nos: 525 and 526), ​​and IGHG1*05 (Seq ID Nos: 340, 341, and 537). In one embodiment, the antibodies and antibody fragments disclosed herein comprise a protein encoded by an IgG2 constant region allele, including but not limited to human IGHG2*01 (Seq ID Nos: 527 and 528), IGHG2*02 (Seq ID Nos: 529 and 530), IGHG2*03 (Seq ID Nos: 527 and 528), IGHG2*04 (Seq ID Nos: 531 and 532), IGHG2*05 (Seq ID Nos: 527 and 528), and IGHG2*06 (Seq ID Nos: 533 and 534). In one embodiment, the antibodies or antibody fragments disclosed herein comprise a protein encoded by an IgG3 constant region allele, including but not limited to human IGHG3*01, IGHG3*02, IGHG3*03, IGHG3*04, IGHG3*05, IGHG3*06, IGHG3*07, IGHG3*08, IGHG3*09, IGHG3*10, IGHG3*11, IGHG3*12, IGHG3*13, IGHG3*14, IGHG3*15, IGHG3*16, IGHG3*17, IGHG3*18 and IGHG3*19.In one embodiment, the antibodies or antibody fragments disclosed herein comprise a protein encoded by an IgG4 constant region allele, including but not limited to human IGHG4*01 (see, e.g., the sequence listing herein), IGHG4*02 (see, e.g., the sequence listing herein), IGHG4*03 (see, e.g., the sequence listing herein), and IGHG4*04 (see, e.g., the sequence listing herein). In another example, the heavy chain is a disabled IgG isotype, such as a disabled IgG4. In certain embodiments, the antibodies of the present invention comprise a human γ4 constant region. In another embodiment, the heavy chain constant region does not bind to the Fc-γ receptor and, for example, comprises a Leu235Glu mutation. In another embodiment, the heavy chain constant region comprises a Ser228Pro mutation to increase stability. In another embodiment, the heavy chain constant region is IgG4-PE (see, e.g., the sequence listing herein). In another embodiment, the antibodies and antibody fragments disclosed herein comprise a heavy chain constant region encoded by a murine IgG1 constant region allele, including but not limited to mouse IGHG1*01 or IGHG1*02. In one embodiment, the antibodies and antibody fragments disclosed herein comprise a heavy chain constant region encoded by a murine IgG2 constant region allele, including but not limited to mouse IGHG2A*01, IGHG2A*02, IGHG2B*01, IGHG2B*02, IGHG2C*01, IGHG2C*02, or IGHG2C*03. In one embodiment, the antibodies or antibody fragments disclosed herein comprise a protein encoded by a murine IgG3 constant region allele, including but not limited to mouse IGHG3*01.

[0218] The term "host" as used herein refers to an animal, preferably a mammal, and most preferably a human.

[0219] As used herein, the term "host cell" refers to a specific individual cell transfected with a nucleic acid molecule and the offspring or potential offspring of such a cell. The offspring of such a cell may be different from the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in the offspring or the integration of the nucleic acid molecule into the host cell genome.

[0220] In the case of applying other therapies, the term "combination" refers to the use of more than one therapy. The use of the term "combination" does not limit the order of individual therapy for disease. The first therapy can be applied to the individual suffering from, having or susceptible to BMP6-mediated diseases before the second therapy (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), while or afterwards (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) is applied. Any additional therapy can be applied in any order with other additional therapies. In certain embodiments, the antibodies of the invention may be administered in combination with one or more than one therapy (e.g., a therapy other than an antibody of the invention that is currently administered for the prevention, treatment, management, and / or amelioration of a BMP6-mediated disease). Non-limiting examples of therapies that may be administered in combination with the antibodies of the invention include analgesics, anesthetics, antibiotics, or immunomodulators, or any other agent listed in the U.S. Pharmacopoeia and / or Physician's Desk Reference.

[0221] As used herein, "injection device" refers to a device designed to perform an injection, which includes the step of temporarily fluidly connecting the injection device to human tissue (usually subcutaneous tissue). The injection further includes applying a certain amount of liquid medicine to 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 an injection device used when the target tissue is blood in the circulatory system (e.g., blood in a vein). Common but non-limiting examples of injection devices are needles and syringes.

[0222] As used herein, "instructions" refers to written, printed or graphic matter displayed on the immediate container of an article, such as written material displayed on a vial containing a pharmaceutically active agent, or details regarding the composition and use of a related product included in a kit containing the related composition. The instructions set forth the intended method of treatment to be administered or performed.

[0223] An "isolated" or "purified" antibody or protein is an antibody or protein that has been identified, separated and / or recovered from a component of its production environment (e.g., natural or recombinant). For example, an antibody or protein is substantially free of cellular material or other contaminating proteins from a cell or tissue source from which the antibody is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The term "substantially free of cellular material" includes antibody preparations in which the antibody is separated from cellular components of the cell from which it is isolated or recombinantly produced. Thus, antibodies substantially free of cellular material include antibody preparations having less than about 30%, 20%, 10% or 5% (dry weight) of heterologous proteins (also referred to herein as "contaminating proteins"). When the antibody is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium accounts for less than about 20%, 10% or 5% of the volume of the protein preparation. When the antibody is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals involved in protein synthesis. Thus, the antibody preparation has less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the relevant antibody. In a preferred embodiment, the antibodies of the invention are isolated or purified.

[0224] The term "Kabat numbering" and similar terms are recognized in the art and refer to a numbering system for amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the heavy chain variable region of an antibody or antigen-binding portion thereof (Kabat et al., (1971) Ann. NY Acad. Sci., 190: 382-391; and Kabat et al., (1991) Protein Sequences of Interest in Immunology, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable region is generally within the range of amino acid positions 31 to 35 of CDR1, amino acid positions 50 to 65 of CDR2, and amino acid positions 95 to 102 of CDR3.

[0225] As used herein, "label" or "labeled" refers to the addition of a detectable moiety to a polypeptide, such as a radioactive label, a fluorescent label, an enzyme label, a chemiluminescent label, or a biotinylated group or gold. Radioisotopes or radionuclides may include 3 H. 14 C. 15 N. 35 S. 90 Y. 99 Tc, 115 In, 125 I. 131I, fluorescent labels may include rhodamine, lanthanide phosphors or FITC, and enzyme labels may include horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase. Other labels include (by way of illustration and not limitation): enzymes such as glucose-6-phosphate dehydrogenase ("G6PDH"), α-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, and peroxidase; dyes (e.g., cyanine dyes, such as Cy5 TM 、Cy5.5 TM or Cy7 TM ); additional fluorescent labels or agents include, for example, fluorescein and its derivatives, fluorescent dyes, GFP (GFP is "green fluorescent protein"), other fluorescent proteins (e.g., mCherry, mTomato), dansyl, umbelliferone, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde and fluorescamine; fluorophores, such as cryptates and chelates of lanthanides, such as europium (as assayed by PerkinElmer and Cisbio); chemiluminescent labels or agents, such as isoluminol, luminol and dioxetanes; sensitizers; coenzymes; enzyme substrates; particles, such as latex or carbon particles; metal sols; microcrystals; liposomes; cells, etc., which can be further labeled with dyes, catalysts or other detectable groups; molecules such as biotin, digoxygenin or 5-bromodeoxyuridine; toxin parts, such as toxin parts selected from the following groups: Pseudomonas exotoxin (PE or its cytotoxic fragments or mutants), diphtheria toxin or its cytotoxic fragments or mutants, botulinum toxin A, B, C, D, E or F, ricin or its cytotoxic fragments such as ricin A, abrin toxin or its cytotoxic fragments, saporin or its cytotoxic fragments, American pokeweed antiviral toxin or its cytotoxic fragments, and isoflavone 1 or its cytotoxic fragments.

[0226] When used with respect to antibodies, the term "light chain" refers to immunoglobulin light chains, of which there are two types, λ and κ, in mammals. 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 the allele variants can be obtained in publicly available databases such as IMGT, ENSEMBL, Swiss-Prot and Uniprot. In one embodiment, antibodies or antibody fragments disclosed herein include proteins encoded by human κ constant region alleles, including but not limited to IGKC*01 (see, e.g., sequence table herein), IGKC*02 (see, e.g., sequence table herein), IGKC*03 (see, e.g., sequence table herein), IGKC*04 (see, e.g., sequence table herein) and IGKC*05 (see, e.g., sequence table herein). In one embodiment, the antibodies or antibody fragments disclosed herein comprise a protein encoded by a human lambda constant region allele, including but not limited to IGLC1*01 (see, e.g., the sequence listing herein), IGLC1*02 (see, e.g., the sequence listing herein), IGLC2*01 (see, e.g., the sequence listing herein), IGLC2*02 (see, e.g., the sequence listing herein), IGLC2*03 (see, e.g., the sequence listing herein), IGLC3*01 (see, e.g., the sequence listing herein), IGLC3*02 (see, e.g., the sequence listing herein), IGLC3*03 (see, e.g., the sequence listing herein), IGLC3*04 (see, e.g., the sequence listing herein), IGLC6*01 (see, e.g., the sequence listing herein), IGLC7*01 (see, e.g., the sequence listing herein), IGLC7*02 (see, e.g., the sequence listing herein), IGLC7*03 (see, e.g., the sequence listing herein). In another embodiment, the antibodies and antibody fragments disclosed herein comprise a light chain constant region encoded by a mouse kappa constant region allele, including but not limited to IGKC*01, IGKC*03 or IGKC*03. In another embodiment, the antibodies and antibody fragments disclosed herein comprise a light chain constant region encoded by a mouse lambda constant region allele, including but not limited to IGLC1*01, IGLC2*01 or IGLC3*01.

[0227] "Percentage (%) amino acid sequence identity" and "homology" with respect to peptide, polypeptide or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage sequence identity and not considering any conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be achieved in various ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEG ALIGN. TM (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%.

[0228] The terms "naturally occurring" or "native" when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, etc., refer to those found in nature and not subject to human manipulation.

[0229] As used herein, "packaging" refers to how the components are organized and / or confined into a unit suitable for distribution and / or use. The packaging may include, for example, boxes, bags, syringes, ampoules, vials, tubes, clamshell packaging, barriers and / or containers to maintain sterility, labeling, etc.

[0230] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the US Pharmacopeia, European Pharmacopeia, or other generally recognized pharmacopeia, for use in animals, and more particularly in humans.

[0231] As used herein, the terms "polynucleotide," "nucleotide," "nucleic acid," "nucleic acid molecule" and other similar terms are used interchangeably and include DNA, RNA, mRNA, and the like.

[0232] As used herein, the term "prevention" refers to the complete or partial inhibition of the development, recurrence, onset or spread of an hBMP6-mediated disease and / or its associated 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 antibodies of the invention).

[0233] The term "soluble" refers to a polypeptide that exists in a native or membrane-bound form, such as BMP6 and variants or fragments thereof, which lack one or more than one transmembrane or cytoplasmic domain. In one embodiment, the "soluble" form of BMP6 lacks a transmembrane domain and a cytoplasmic domain.

[0234] The term "subject" or "patient" refers to any animal, including, but not limited to, mammals. As used herein, the term "mammal" refers to any vertebrate that nurses its young and gives birth to live young (eumammalia or placental mammals) or lays eggs (metatheria or non-placental mammals). Examples of mammalian species include, but are not limited to, humans and other primates, including non-human primates, such as chimpanzees and other apes and monkey species; farm animals, such as cattle, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, including rodents, such as mice, rats (including cotton rats), and guinea pigs; birds, including domestic, wild, and game birds, such as chickens, turkeys and other gallinaceous, ducks, geese, and the like.

[0235] As used herein, "substantially all" means 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%.

[0236] As used herein, the term "therapeutic agent" refers to any agent that can be used to treat, manage or improve a BMP6-mediated disease and / or symptoms associated therewith. In certain embodiments, the term "therapeutic agent" refers to an antibody of the present invention. In certain other embodiments, the term "therapeutic agent" refers to an agent other than an antibody of the present invention. Preferably, the therapeutic agent is an agent that is known to be useful for or has been or is currently used to treat, manage or improve a BMP6-mediated disease or one or more than one symptom associated therewith. In a specific embodiment, the therapeutic agent is a fully human anti-BMP6 antibody, such as a fully human anti-BMP6 monoclonal antibody.

[0237] As used herein, the term "therapy" refers to any regimen, method and / or agent that can be used to prevent, manage, treat and / or improve a BMP6-mediated disease (e.g., cancer). In certain embodiments, the term "therapy" refers to biological therapy, supportive therapy and / or other therapies that can be used to prevent, manage, treat and / or improve a BMP6-mediated disease known to those skilled in the art, such as a physician.

[0238] The term "treatment" refers to the reduction or improvement of the progression, severity and / or duration of hBMP6-mediated diseases (e.g., cancer) caused by the administration of one or more than one therapy (including but not limited to the administration of one or more than one preventive or therapeutic agent, such as an antibody of the present invention). In a specific embodiment, the term refers to the reduction or inhibition of the binding of hBMP6 to BMP receptors or HJVs, and / or the inhibition or reduction of one or more than one symptom associated with BMP6-mediated diseases such as anemia.

[0239] The term "variable region" or "variable domain" refers to a portion of a light chain and a heavy chain, generally about 120 to 130 amino acids at the amino terminal end of the heavy chain and about 100 to 110 amino acids in the light chain, which are widely different in sequence in the antibody and are used for the binding and specificity of each specific antibody to its specific antigen. The variability of the sequence is concentrated in those regions called complementary determining regions (CDRs), while the more highly conserved regions in the variable domains are called framework regions (FRs). The CDRs of BMP6 and heavy chains are primarily responsible for the interaction of antibodies with antigens. The numbering of amino acid positions used herein is according to the EU index, as in Kabat et al., (1991) Protein Sequences of Interest in Immunology. (U.S. Department of Health and Human Services, Washington, DC) 5th edition ("Kabat et al."). In a preferred embodiment, the variable region is a human variable region.

[0240] Definitions of common terms in cell biology and molecular biology can be found in “The Merck Manual of Diagnosis and Therapy”, 19th 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 Publishing, 2003 (ISBN 10: 0763766321). Published by Publishers, Inc., 1995 (ISBN 1-56081-569-8); and Current Protocols in Protein Sciences, 2009, Wiley Intersciences, Coligan et al., eds.

[0241] Unless otherwise indicated, the present invention was performed using standard procedures as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (4th edition), 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, ed. S. L. Berger and AR Kimmel, Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (Johnson, MD). E. Coligan et al., eds., John Wiley and Sons, Inc.); Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et al., eds., John Wiley and Sons, Inc.); and Culture of Animal Cells: A Manual of Basic Technique, 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, eds., Academic Press Inc., 1st edition, 1998), all of which are incorporated herein by reference in their entirety.

[0242] Other terms are defined herein in the description of various aspects of the invention.

[0243] BMP6 and Iron

[0244] Since iron is the basis of all life forms and must come from the environment, its availability and dosage in the body are strictly controlled. The key regulator of iron homeostasis is a 25-amino acid peptide hormone called hepcidin. Hepcidin is produced by the liver and allows the main iron uptake and storage compartments, namely duodenal enterocytes and macrophages, to retain iron by controlling the expression of the iron transporter molecule membrane ferroportin. After the immune system is activated during infection and / or inflammation and through erythropoiesis, hepcidin itself is regulated by iron content via a homeostatic control mechanism. Importantly, hepcidin levels are increased in chronic inflammatory conditions, infections, and certain cancers. Elevated hepcidin levels chelate iron in enterocytes, macrophages, and hepatocytes, thereby inhibiting hemoglobin synthesis and erythropoiesis. This leads to anemia despite normal iron reserves. Hepcidin gene expression is controlled by a soluble factor called BMP6 (bone morphogenetic protein 6). BMP6 is considered a major regulator because, in the absence of BMP6, individual cytokines (or other BMPs) cannot overcome the defects of BMP6 signals. Therefore, the present inventors focused on BMP6 as a key drug target for controlling dysregulated iron homeostasis in anemia, for example, in anemia of chronic disease (ACD).

[0245] BMP6 is a highly conserved soluble protein factor that is considered a "main" regulator of hepcidin production in mice and humans. Therefore, the administration of BMP6 to mice increases hepcidin content and reduces blood and serum iron, while inhibitors of BMP6 are the opposite. In addition, knockout or human mutations of the mouse BMP6 gene within the BMP6 pathway support the key role of BMP6 in controlling hepcidin and blood and serum iron content. In addition, preclinical and clinical validation of targeting BMP6 comes from increasing available iron content by administering anti-BMP6 antibodies to rodents or cynomolgus monkeys or using HJV-Fc (FMX-8, Ferrumax Inc) to neutralize BMP6 in Phase I studies. Reference Andriopoulos Jr.B, Corradini E, Xia Y, FaasseSA, 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 metabolism. Nature Genet. 41(4), 482-487; WO2016098079 and US8980582.

[0246] Anti-BMP6 antibodies and fragments

[0247] The present invention provides various anti-BMP6 antibodies and fragments (such as Fab or scFv fragments), uses, methods and combinations (eg, in combination with ESA). Examples are listed in the following numbered clauses.

[0248] 1. An antibody or fragment comprising a binding site that specifically binds to bone morphogenetic protein 6 (BMP6), wherein the binding site comprises a VH domain encoded by a nucleotide sequence derived from the recombination of a human VH gene segment, a DH gene segment, and a JH gene segment, wherein the VH gene segment is selected from IGHV3-11 and IGHV1-3.

[0249] For example, the VH gene segment is IGHV3-11 and the DH gene segment and the JH gene segment are human gene segments. For example, the VH gene segment is IGHV1-3 and the DH gene segment and the JH gene segment are human gene segments. Optionally, the VH segment is a human IGHV3-11*01 gene segment. Alternatively, optionally, the VH segment is a human IGHV1-3*01 gene segment.

[0250] In one example, the specific binding has a KD, K off and / or K on In one example, the specific binding has a KD of 1 pM to 5 nM.

[0251] Those skilled in the art are familiar with databases and other sources of antibody gene segments for humans and other species. For example, the IMGT database (www.IMGT.org) is a suitable source, such as the version as of September 1, 2018.

[0252] Reference is made to Tables 7 and 8, which show antibodies based on IGHV1-3. Surprisingly, the human VH gene segments produce anti-BMP6 antibodies with desired anti-BMP6 properties, such as those described in, for example, the Examples.

[0253] With reference to Table 9, antibodies based on IGHV3-11 are shown. Surprisingly, the human VH gene segments produce anti-BMP6 antibodies with desired anti-BMP6 properties, such as those described in, for example, the Examples. Thus, for example, the antibody or fragment comprises a CDRH3 sequence selected from SEQ ID NOs: 110, 113, 290, 293, 308, 311, 272, and 275. For example, the antibody or fragment comprises a CDRL3 sequence selected from SEQ ID NOs: 119, 122, 299, 302, 317, 320, 281, and 284. For example, the antibodies or fragments comprise a CDRH3 sequence selected from SEQ ID NOs: 110, 113, 290, 293, 308, 311, 272 and 275 and a CDRL3 sequence selected from SEQ ID NOs: 119, 122, 299, 302, 317, 320, 281 and 284, respectively.

[0254] For example, the antibody or fragment comprises a CDRH3 sequence selected from SEQ ID NOs: 110 and 113; and a CDRL3 sequence selected from SEQ ID NOs: 119 and 122. For example, the antibody or fragment comprises an anti-BMP6 binding site, wherein the binding site comprises a VH domain comprising SEQ ID NO: 110 paired with a VL domain comprising SEQ ID NO: 119. For example, the antibody or fragment comprises an anti-BMP6 binding site, wherein the binding site comprises a VH domain comprising SEQ ID NO: 113 paired with a VL domain comprising SEQ ID NO: 122.

[0255] For example, the antibody or fragment comprises a CDRH3 sequence of an antibody selected from CL-58838, CL-58835, CL-58756 and CL-58722 and optionally a CDRL3 of the selected antibody. For example, the antibody or fragment comprises a CDRH1 and CDRH3 sequence of an antibody selected from CL-58838, CL-58835, CL-58756 and CL-58722 and optionally a CDRL2 of the selected antibody. For example, the antibody or fragment comprises a CDRH1 and CDRH2 sequence of an antibody selected from CL-58838, CL-58835, CL-58756 and CL-58722. For example, the antibody or fragment comprises a CDRH2 and CDRH3 sequence of an antibody selected from CL-58838, CL-58835, CL-58756 and CL-58722. For example, the antibody or fragment comprises an anti-BMP6 binding site, wherein the binding site comprises a VH domain comprising the CDRH3 sequence of CL-58838 paired with a VL domain of CL-58838.

[0256] For example, the antibody or fragment comprises an anti-BMP6 binding site, wherein the binding site comprises a VH domain comprising SEQ ID NO: 114, 294, 312, or 276, optionally paired with a VL domain comprising SEQ ID NO: 123, 303, 321, or 285, respectively. For example, the antibody or fragment comprises an anti-BMP6 binding site, wherein the binding site comprises a VH domain comprising SEQ ID NO: 114, paired with a VL domain comprising SEQ ID NO: 123.

[0257] For example, the antibody or fragment comprises an anti-BMP6 binding site, wherein the binding site comprises a VH domain of an antibody selected from CL-58838, CL-58835, CL-58756 and CL-58722, optionally paired with a VL domain of the selected antibody. For example, the antibody or fragment comprises an anti-BMP6 binding site, wherein the binding site comprises a VH domain of CL-58838, paired with a VL domain of CL-58838.

[0258] 2. An 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.

[0259] Optionally, the DH gene segment is selected from IGHD3-10*01, IGHD6-19*01, IGHD7-27*02, IGHD4-23*01, IGHD5-18*01, IGHD3-22*01 and IGHD3-16*02.

[0260] 3. An 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.

[0261] Optionally, the JH gene segment is selected from IGHJ3*02, IGHJ4*02 and IGHJ5*02.

[0262] 4. An antibody or fragment which specifically binds to bone morphogenetic protein 6 (BMP6) and comprises the CDRH3 sequence of an anti-BMP6 antibody according to any preceding clause.

[0263] 5. An antibody or fragment that specifically binds to bone morphogenetic protein 6 (BMP6) and comprises a VH domain, wherein the VH domain comprises the CDRH3 sequence of any anti-BMP6 antibody disclosed herein (e.g., any antibody selected from the antibodies or fragments listed in any one of Tables 4 to 11 herein), or the CDRH3 sequence comprises 3, 2 or 1 amino acid substitutions.

[0264] 6. An antibody or fragment (optionally according to any preceding clause) which specifically binds bone morphogenetic protein 6 (BMP6) and comprises a VH domain comprising the CDRH3 sequence of an antibody selected from the group consisting of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713; or said sequence comprises 3, 2 or 1 amino acid substitutions.

[0265] Optionally, the VH domain comprises a CDRH3 sequence selected from SEQ ID NO: 110 or 113, or the selected sequence comprises 3, 2 or 1 amino acid substitutions.

[0266] 7. An antibody or fragment according to clause 6, wherein the VH domain comprises (i) a CDRH3 sequence of an antibody selected from the group consisting of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713; or the CDRH3 sequence comprises 3, 2 or 1 amino acid substitutions; and (ii) a CDRH1 sequence of the selected antibody; or the CDRH1 sequence comprises 3, 2 or 1 amino acid substitutions.

[0267] Optionally, the VH domain of the antibody or fragment comprises (a) a CDRH3 sequence of SEQ ID NO: 110 or 113; or the CDRH3 sequence comprises 3, 2 or 1 amino acid substitutions; and (b) a CDRH1 sequence of SEQ ID NO: 108 or 111, or the CDRH1 sequence comprises 3, 2 or 1 amino acid substitutions.

[0268] 8. An antibody or fragment according to clause 6 or 7, wherein the VH domain comprises (iii) a CDRH3 sequence of an antibody selected from the group consisting of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713; or said CDRH3 sequence comprises 3, 2 or 1 amino acid substitutions; and (iv) a CDRH2 sequence of said selected antibody; or said CDRH2 sequence comprises 3, 2 or 1 amino acid substitutions.

[0269] Optionally, the VH domain of the antibody or fragment comprises (c) a CDRH3 sequence of SEQ ID NO: 110 or 113; or the CDRH3 sequence comprises 3, 2 or 1 amino acid substitutions; and (d) a CDRH2 sequence of SEQ ID NO: 109 or 112, or the CDRH2 sequence comprises 3, 2 or 1 amino acid substitutions.

[0270] Optionally, the VH domain of the antibody or fragment comprises (e) a CDRH3 sequence of SEQ ID NO: 110 or 113; or the CDRH3 sequence comprises 3, 2 or 1 amino acid substitutions; (f) a CDRH1 sequence of SEQ ID NO: 108 or 111, or the CDRH1 sequence comprises 3, 2 or 1 amino acid substitutions; and (g) a CDRH2 sequence of SEQ ID NO: 109 or 112, or the CDRH2 sequence comprises 3, 2 or 1 amino acid substitutions.

[0271] 9. An antibody or fragment (optionally according to any preceding clause) comprising a binding site that specifically binds to bone morphogenetic protein 6 (BMP6), wherein the binding site comprises a VH domain comprising a protein selected from the group consisting of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-7550 6. The amino acid sequence of the VH domain of the antibodies of 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 having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0272] For example, the consistency is at least 85%. For example, the consistency is at least 90%. For example, the consistency is at least 95%.

[0273] Optionally, the VH domain of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 114, or a heavy chain variable domain amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 114. For example, the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0274] 10. An antibody or fragment according to any preceding clause, comprising a first and a second copy of said VH domain.

[0275] In one example, the antibody or fragment comprises a binding site comprising a VH domain of the invention paired with a VL domain of the invention, wherein the binding site is capable of specifically binding to BMP6 (e.g., mature BMP6, such as human and / or cynomolgus monkey BMP6). For example, the antibody or fragment comprises both of these binding sites.

[0276] 11. An antibody or fragment (optionally according to any of the preceding clauses) 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 the recombination of a human VL gene segment and a JL gene segment, wherein the VL gene segment is selected from IGKV3-20, IGKV1-5 and IGKV3-15.

[0277] For example, the VL gene segment is IGKV3-20, such as IGKV3-20*01. For example, the VL gene segment is IGKV1-5, such as IGKV1-5*03. For example, the VL gene segment is IGKV3-15, such as IGKV3-15*01.

[0278] 12. The antibody or fragment according to clause 11, wherein the VL is Vκ and the JL gene segment is a human gene segment selected from IGKJ1 and IGKJ3.

[0279] Optionally, the JL gene segment is selected from IGKJ1*01 and IGKJ3*01.

[0280] 13. An antibody or fragment which specifically binds to bone morphogenetic protein 6 (BMP6) and comprises the CDRL3 sequence of the anti-BMP6 antibody according to clause 11 or 12.

[0281] 14. An antibody or fragment (optionally according to any of the preceding clauses) which specifically binds to bone morphogenetic protein 6 (BMP6) and comprises a VL domain, wherein the VL domain comprises the CDRL3 sequence of any anti-BMP6 antibody disclosed herein (e.g., any antibody selected from the antibodies or fragments listed in any one of Tables 4 to 11 herein), or the selected CDRL3 sequence comprises 3, 2 or 1 amino acid substitutions.

[0282] 15. An antibody or fragment according to clause 14, comprising a VH domain comprising the CDRH3 sequence of said selected antibody.

[0283] 16. An antibody or fragment (optionally according to any preceding clause) which specifically binds bone morphogenetic protein 6 (BMP6) and comprises a VL domain comprising a CDRL3 sequence selected from SEQ ID NO: 119 or 122, or the selected CDRL3 sequence comprises 3, 2 or 1 amino acid substitutions.

[0284] 17. An antibody or fragment (optionally according to any preceding clause) which specifically binds bone morphogenetic protein 6 (BMP6) and comprises a VL domain comprising the CDRL3 (and optionally CDRH3) sequence of an antibody selected from the group consisting of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713; or each of said sequences comprises 3, 2 or 1 amino acid substitutions.

[0285] Optionally, the VL domain comprises a CDRL3 sequence selected from SEQ ID NO: 119 or 122, or the selected sequence comprises 3, 2 or 1 amino acid substitutions, and / or optionally, the VH domain comprises a CDRH3 sequence selected from SEQ ID NO: 119 or 122, or the selected sequence comprises 3, 2 or 1 amino acid substitutions.

[0286] 18. An antibody or fragment according to clause 17, wherein the VL domain comprises (i) a polypeptide selected from the group consisting of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL- or said CDR3 sequences each comprise 3, 2 or 1 amino acid substitutions; and (ii) the CDRL1 (and optionally CDRH1) sequence of said selected antibodies; or said CDR1 sequences each comprise 3, 2 or 1 amino acid substitutions.

[0287] Optionally, the VL domain of the antibody or fragment comprises (a) a CDRL3 sequence of SEQ ID NO: 119 or 122; or the CDRL3 sequence comprises 3, 2 or 1 amino acid substitutions; and (b) a CDRL1 sequence of SEQ ID NO: 117 or 120, or the CDRL1 sequence comprises 3, 2 or 1 amino acid substitutions.

[0288] 19. An antibody or fragment according to clause 17 or 18, wherein the VL domain comprises (iii) selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75320, CL-75539, CL-75565, CL-75 / 14, or said CDR3 sequences each comprise 3, 2 or 1 amino acid substitutions; and (iv) the CDRL2 (and optionally CDRH2) sequence of said selected antibodies; or said CDR2 sequences each comprise 3, 2 or 1 amino acid substitutions.

[0289] Optionally, the VL domain of the antibody or fragment comprises (c) a CDRL3 sequence of SEQ ID NO: 119 or 122; or the CDRL3 sequence comprises 3, 2 or 1 amino acid substitutions; and (d) a CDRL2 sequence of SEQ ID NO: 118 or 121, or the CDRL2 sequence comprises 3, 2 or 1 amino acid substitutions.

[0290] Optionally, the VL domain of the antibody or fragment comprises (e) a CDRL3 sequence of SEQ ID NO: 119 or 122; or the CDRL3 sequence comprises 3, 2 or 1 amino acid substitutions; (f) a CDRL1 sequence of SEQ ID NO: 117 or 120, or the CDRL1 sequence comprises 3, 2 or 1 amino acid substitutions; and (g) a CDRL2 sequence of SEQ ID NO: 118 or 121, or the CDRL2 sequence comprises 3, 2 or 1 amino acid substitutions.

[0291] 20. An antibody or fragment (optionally according to any preceding clause) comprising a binding site that specifically binds to bone morphogenetic protein 6 (BMP6), wherein the binding site comprises a VL domain comprising a protein selected from the group consisting of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-755 06, 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 having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0292] For example, the consistency is at least 85%. For example, the consistency is at least 90%. For example, the consistency is at least 95%.

[0293] Optionally, the VL domain of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 123, or a heavy chain variable domain amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 123. For example, the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0294] 21. An antibody or fragment according to any preceding clause, comprising a first and a second copy of said VL domain.

[0295] In one example, the antibody or fragment comprises a binding site comprising a VL domain of the invention paired with a VH domain, wherein the binding site is capable of specifically binding to BMP6 (e.g., mature BMP6, such as human and / or cynomolgus monkey BMP6). For example, the antibody or fragment comprises both of the binding sites.

[0296] 22. An antibody or fragment (optionally according to any of the preceding clauses) which specifically binds to bone morphogenetic protein 6 (BMP6) and comprises a member selected from the group consisting of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75300, CL-75506, CL-75520, C The heavy chain amino acid sequence of the antibodies of L-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713, or an amino acid having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0297] For example, the consistency is at least 85%. For example, the consistency is at least 90%. For example, the consistency is at least 95%.

[0298] 23. An antibody or fragment (optionally according to any of the preceding clauses) which specifically binds to bone morphogenetic protein 6 (BMP6) and comprises a member selected from the group consisting of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, C The light chain amino acid sequence of the antibodies of L-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713, or an amino acid having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0299] For example, the consistency is at least 85%. For example, the consistency is at least 90%. For example, the consistency is at least 95%.

[0300] 24. An antibody or fragment according to clause 23, comprising the light chain amino acid sequence of the selected antibody; or an amino acid having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0301] For example, the consistency is at least 85%. For example, the consistency is at least 90%. For example, the consistency is at least 95%.

[0302] 25. An antibody or fragment (optionally according to any preceding clause) which specifically binds to an antigenic determinant of human BMP6 identical to an antigenic determinant bound by an antibody according to any preceding clause.

[0303] 26. The antibody or fragment according to clause 25, wherein the antigenic determinant is identified by unrelated amino acid scanning or by X-ray crystallography.

[0304] The contact amino acid residues involved in the interaction between an antibody and an antigen can be determined by various methods known to those skilled in the art.

[0305] In one embodiment, the amino acids of the antigen sequence are sequentially replaced (using standard molecular biology techniques to mutate the DNA encoding the antigen sequence), in the case of BMP6, with alanine (also known as alanine scanning) or another unrelated amino acid substitution, which can provide the following residues, whose mutations reduce or eliminate the ability of the antibody to recognize the antigen. Binding can be assessed using standard techniques, such as but not limited to SPR, HTRF, ELISA (which are described elsewhere herein). Other substitutions can be made to enhance the disruption of binding, such as changing the charge on the amino acid side chains of the antigen sequence (e.g., lysine to glutamic acid), switching polar and non-polar residues (e.g., serine to leucine). Alanine scanning or other amino substitution methods can be performed with recombinant soluble antigens, or when the target is a cell membrane target, directly on the cell using transient or stable expression of the mutant form.

[0306] In one embodiment, protein crystallography can be used to determine the contact residues between the antibody and the antigen (i.e., determine the antigenic determinant bound by the antibody), and crystallography allows direct visualization of the contact residues involved in the antibody-antigen interaction. In addition to standard X-ray crystallography, cryo-electron microscopy has been used to determine the contact residues between the antibody and the HIV capsid protein (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)).

[0307] In one embodiment, if the antibody recognizes a linear antigenic determinant, short peptides based on the antigenic sequence can be generated, and the binding of the antibody to these peptides can be assessed using standard techniques, such as, but not limited to, SPR, HTRF, ELISA (described elsewhere herein). Further investigation of the antigenic determinant can be provided by alanine scanning of any peptide that shows binding. As an alternative to linear peptides, conformational scanning using chemical attachment of peptides on a scaffold can be performed using the Pepscan technique (http: / / www.pepscan.com / ), which has been used to determine discontinuous antigenic determinants on CD20 targeting antibodies (Niederfellner, Gerhard et al., "Epitope characterization and crystal structure of GA101 provide insights into the molecular basis for type I / II distinction of CD20 antibodies.", Blood, 118.2, (2011), 358-367).

[0308] In one embodiment, limited proteolytic digestion and mass spectrometry can be used to identify binding antigenic determinants. Antibody-antigen complexes are digested by proteases, such as but not limited to trypsin. Digested complex peptides are compared with antibody-only and antigen-only digestion mass spectrometry spectrophotometry to determine whether specific antigenic determinants are protected by complexation. Further studies involving amino acid substitutions and competitive binding can then be used to narrow the scope to individual amino acid residues involved in the interaction (see, for example, Suckau, Detlev et al., "Molecular epitope identification by limited proteolysis of an immobilized antigen-antibody complex and mass spectrometric peptide mapping.", Proceedings of the National Academy of Sciences, 87.24, (1990), 9848-9852).

[0309] Thus, in one embodiment, the contact residues of the antigenic determinant are identified using an unrelated amino acid scan (e.g., an alanine scan). In another embodiment, an unrelated amino acid scan (e.g., an alanine scan) is performed using a technique selected from a combination of SPR, HTRF, ELISA, X-ray crystallography, cryo-electron microscopy, and limited proteolytic digestion and mass spectrometry. In one embodiment, an unrelated amino acid scan (e.g., an alanine scan) is performed using HTRF. In one embodiment, an unrelated amino acid scan (e.g., an alanine scan) is performed using ELISA.

[0310] When carrying out alanine scanning with ELISA or HTRF, if the signal is reduced to at least 25%, the amino acid residue is identified as contributing to the antigenic determinant. In one embodiment, the signal is reduced to at least 30%. In one embodiment, the signal is reduced to at least 35%. In one embodiment, the signal is reduced to at least 40%. In one embodiment, the signal is reduced to at least 45%. In one embodiment, the signal is reduced to at least 50%. In one embodiment, the signal is reduced to at least 55%. In one embodiment, the signal is reduced to at least 60%. In one embodiment, the signal is reduced to at least 70%. In one embodiment, the signal is reduced to at least 75%. In one embodiment, the signal is reduced to at least 80%. In one embodiment, the signal is reduced to at least 85%. In one embodiment, the signal is reduced to at least 90%.

[0311] When alanine scanning is performed using SPR, an amino acid residue is identified as contributing to an antigenic determinant if the affinity is reduced by at least 10 times. In one embodiment, the affinity is reduced by at least 15 times. In one embodiment, the affinity is reduced by at least 20 times. In one embodiment, the affinity is reduced by at least 30 times. In one embodiment, the affinity is reduced by at least 40 times. In one embodiment, the affinity is reduced by at least 50 times. In one embodiment, the affinity is reduced by at least 100 times.

[0312] In one embodiment, the contact residues of the antigenic determinant are identified by X-ray crystallography. In one embodiment, the contact residues of the antigenic determinant are identified by cryo-electron microscopy. In one embodiment, the contact residues of the antigenic determinant are identified by a combination of limited proteolytic digestion and mass spectrometry.

[0313] 27. An antibody or fragment according to clause 26, wherein the contact residues of the antigenic determinant are defined by at least a 10-fold decrease in affinity in an unrelated amino acid scan, such as an alanine scan as determined by SPR.

[0314] In one embodiment, the affinity is reduced by at least 15 times. In one embodiment, the affinity is reduced by at least 20 times. In one embodiment, the affinity is reduced by at least 30 times. In one embodiment, the affinity is reduced by at least 40 times. In one embodiment, the affinity is reduced by at least 50 times. In one embodiment, the affinity is reduced by at least 100 times.

[0315] SPR can be performed as described herein.

[0316] 28. An antibody or fragment (optionally according to any preceding clause) which competes with the antibody according to any preceding clause for binding to human BMP6.

[0317] Optionally, competition is determined by surface plasmon resonance (SPR) or ELISA. For example, those skilled in the art will be familiar with such techniques and standard conditions.

[0318] In one embodiment, the antibody or fragment competes with hBMP6 (or its fusion protein) (e.g., in a dose-dependent manner) for binding to hBMP6 expressed on the cell surface. In one embodiment, the antibody or fragment competes with hBMP6 (or its fusion protein) (e.g., in a dose-dependent manner) for binding to soluble hBMP6.

[0319] Optionally, competition for binding to hBMP6 is performed using SPR. SPR can be performed as described herein.

[0320] 29. An antibody or fragment according to any preceding clause, which specifically binds to human BMP6 comprising SEQ ID NO:562; and / or cynomolgus monkey BMP6 comprising SEQ ID NO:564; and / or rat BMP6 comprising SEQ ID NO:563.

[0321] Optionally, the antibody or fragment of the invention specifically binds to the amino acid sequence of SEQ ID NO: 562. Optionally, the antibody or fragment of the invention specifically binds to the amino acid sequence of SEQ ID NO: 563. Optionally, the antibody or fragment of the invention specifically binds to the amino acid sequence of SEQ ID NO: 564.

[0322] In one example, the BMP6 herein is human, mouse or cynomolgus monkey BMP6.

[0323] In one embodiment, the antibody or fragment binds to cynomolgus monkey BMP6 with an affinity of less than 1nM (e.g., 1nM to 0.01pM, or 1nM to 0.1pM, or 1nM to 1pM). In one embodiment, the antibody or fragment binds to cynomolgus monkey BMP6 with an affinity of less than 10nM (e.g., 10nM to 0.01pM, or 10nM to 0.1pM, or 10nM to 1pM). In one embodiment, the antibody or fragment binds to cynomolgus monkey BMP6 with an affinity of less than 0.1nM (e.g., 0.1nM to 0.01pM, or 0.1nM to 0.1pM, or 0.1nM to 1pM). In one embodiment, the antibody or fragment binds to cynomolgus monkey BMP6 with an affinity of less than 0.01nM (e.g., 0.011nM to 0.01pM or 0.01nM to 0.1pM).

[0324] In one embodiment, the binding affinity of the antibody or fragment to cynomolgus monkey BMP6 is within 2 times of its binding affinity to hBMP6. In one embodiment, the binding affinity of the antibody or fragment to cynomolgus monkey BMP6 is within 4 times of its binding affinity to hBMP6. In one embodiment, the binding affinity of the antibody or fragment to cynomolgus monkey BMP6 is within 5 times of its binding affinity to hBMP6. In one embodiment, the binding affinity of the antibody or fragment to cynomolgus monkey BMP6 is within 6 times of its binding affinity to hBMP6. In one embodiment, the binding affinity of the antibody or fragment to cynomolgus monkey BMP6 is within 8 times of its binding affinity to hBMP6. In one embodiment, the binding affinity of the antibody or fragment to cynomolgus monkey BMP6 is within 10 times of its binding affinity to hBMP6.

[0325] "hBMP6" herein is human BMP6, such as human BMP6 disclosed herein comprising SEQ ID NO:562.

[0326] In one embodiment, the antibody or fragment does not detectably bind to cynomolgus monkey BMP6. In one embodiment, the antibody or fragment does not detectably bind to murine (eg, mouse and / or rat) BMP6.

[0327] In one embodiment, the antibody or fragment has a binding affinity to murine (e.g., mouse and / or rat) BMP6 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 has a binding affinity to murine BMP6 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 has a binding affinity to murine BMP6 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 has a binding affinity to murine BMP6 of less than 0.01 nM (e.g., 0.011 nM to 0.01 pM or 0.01 nM to 0.1 pM).

[0328] Optionally, the antibody or fragment comprises an effector-enabled or effector-disabled constant region, such as a human constant region, such as an effector-null human constant region, such as an IgG4 constant region or an IgG1 constant region, optionally wherein the constant region is IgG4-PE, or IgG1 is disabled. Optionally, the antibody or fragment comprises a murine (e.g., mouse and / or rat) constant region. Optionally, the antibody or fragment comprises any heavy chain constant region sequence described herein.

[0329] Optionally, the constant region has CDC and / or ADCC activity.

[0330] 30. An antibody or fragment according to any preceding clause, wherein the antibody or fragment comprises a human constant region, such as an IgG4 constant region or an IgG1 constant region.

[0331] For example, the constant region comprises a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 429, 431, 433, 435, 437, 439, 440, 442, 444, 446, 448, 450, 454 or 456. Optionally, the heavy chain constant region is an IGHG1 constant region comprising an amino acid sequence of SEQ ID NO: 429, 431, 433, 435 or 437. Optionally, the heavy chain constant region is an IGHG2 constant region comprising an amino acid sequence of SEQ ID NO: 439, 440, 442 or 444. Optionally, the heavy chain constant region is an IGHG4 constant region comprising an amino acid sequence of SEQ ID NO: 446, 448, 450, 454 or 456, preferably SEQ ID NO: 454, preferably SEQ ID NO: 456. In one example, the heavy chain constant region is encoded by a nucleic acid comprising SEQ ID NO: 451, 452 or 453.

[0332] In one example (optionally in addition to a heavy chain region according to the above paragraph), the constant region comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 458, 460, 462, 464, 466, 468, 470, 473, 476, 478, 480, 482, 484, 486, 488 or 490. Optionally, the light chain constant region is an IGKC constant region comprising the amino acid sequence of SEQ ID NO: 458, 460, 462, 464 or 466, preferably SEQ ID NO: 458. Optionally, the light chain constant region is an IGLC constant region comprising the amino acid sequence of SEQ ID NO: 468, 470, 473, 476, 478, 480, 482, 484, 486, 488 or 490.

[0333] 31. The antibody or fragment according to clause 30, wherein the constant region is an IgG4-PE constant region.

[0334] Optionally, the antibody or fragment comprises a heavy chain constant region, wherein the constant region comprises the amino acid sequence of SEQ ID NO:454.

[0335] An anti-BMP6 antibody or fragment according to the invention may comprise a constant region, such as a human constant region, for example an effector null human constant region, such as an IgG4 constant region or an IgG1 constant region, optionally wherein the constant region is IgG4-PE, or a disabled IgG1 as defined in the sequence listing herein.

[0336] In other embodiments, the antibody or fragment is any isotype or constant region as defined herein. In one embodiment, the constant region is a wild-type human IgG1. For example, the constant region is an effector-enabled IgG1 constant region, optionally with ADCC and / or CDC activity. In one embodiment, the constant region is designed to enhance ADCC and / or CDC and / or ADCP. In another embodiment, the constant region is designed to enhance effector function.

[0337] The IgG4 constant region may be any IgG4 constant region amino acid sequence or encoded by any nucleic acid sequence in the sequence list herein. The heavy chain constant region may be an IgG4 comprising a Leu235Glu mutation and a Ser228Pro mutation. The "IgG4-PE" heavy chain constant region (see sequence list for examples) is effector-free.

[0338] The alternative effector empty human constant region is a disabled IgG1, which is an IgG1*01 allele (e.g., LAGA, see sequence listing) comprising L235A and / or G237A mutations. In one embodiment, the antibodies or antibody fragments disclosed herein comprise an IgG1 heavy chain constant region, wherein the sequence contains alanine at position 235 and / or 237 (EU index numbering).

[0339] The performance of Fc-mediated effects may be enhanced by engineering the Fc domain by any technique apparent to those skilled in the art. In another embodiment, the antibodies and fragments disclosed herein may comprise a triple mutation (M252Y / S254T / T256E) that enhances binding to FcRn.

[0340] 32. An antibody or fragment (e.g., a bispecific antibody) according to any of the preceding clauses, further comprising an antigen binding site that specifically binds to another target antigen (e.g., human hemojuvelin, transferrin receptor (e.g., TFR2) or BMP receptor (e.g., BMPRI or BMPRII)) or binds to BMP6 and another BMP (e.g., BMP2, 4, 7 or 9).

[0341] For example, the bispecific antibody specifically binds to BMP6 and BMP2. For example, the bispecific antibody specifically binds to BMP6 and HJV. For example, the bispecific antibody specifically binds to BMP6 and BMPRI (i.e., BMPR1, such as BMPR1A or BMPR1B). For example, the bispecific antibody specifically binds to BMP6 and BMPRII. For example, the bispecific antibody specifically binds to BMP6 and BMP2. For example, the bispecific antibody specifically binds to BMP6 and BMP4. For example, the bispecific antibody specifically binds to BMP6 and BMP9. For example, the bispecific antibody specifically binds to BMP6 and BMP7. For example, the bispecific antibody specifically binds to BMP6 and TFR2.

[0342] In one example, the additional binding site is an agonist binding site for the other antigen. In one example, the additional binding site is an antagonist binding site for the other antigen.

[0343] In one example, the additional binding site is an antibody binding site comprising VH and VL; a binding site comprised by the constant domains of an antibody (e.g., an 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.

[0344] For example, the antibody or fragment is a bispecific antibody or fragment. For example, the antibody or fragment is a dual binding antibody or fragment, or a fusion protein comprising an antibody or fragment thereof as defined in any of the preceding clauses. Dual binding antibody has the meaning as described above.

[0345] In one example, the antibody, fragment or fusion protein of clause 24 or 24a comprises a bispecific format selected from the group consisting of: DVD-Ig, mAb 2 , FIT-Ig, mAb-dAb, docking and locking, SEEDbody, single-chain diabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, single-chain diabody, single-chain diabody-CH 3 , bifunctional antibody-CH 3 , miniantibodies, knobs into holes, knobs into holes with common light chains, knobs into holes with common light chains and charge pairs, charge pairs, charge pairs with common light chains, specifically mAbs 2 , knob-in-hole, knob-in-hole with common light chain, knob-in-hole with common light chain and charge pair, and FIT-Ig, such as mAb 2 and FIT-Ig.

[0346] In one embodiment, the bispecific format is selected from DVD-Ig, mAb 2 , FIT-Ig, mAb-dAb, docking and locking, Fab arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, κλ body, orthogonal Fab, single-chain diabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, single-chain diabody, single-chain diabody-CH 3 , bifunctional antibody-CH 3 , trifunctional antibody, mini antibody, mini antibody, TriBi mini antibody, scFv-CH 3 KIH,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 zybody.

[0347] In one embodiment, the bispecific format is selected from DVD-Ig, FIT-Ig, mAb-dAb, docking and locking, Fab arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, κλ body, orthogonal Fab, single chain diabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, single chain diabody, single chain diabody-CH 3 , bifunctional antibody-CH 3 , trifunctional antibody, mini antibody, mini antibody, TriBi mini antibody, scFv-CH 3 KIH,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 zybody, such as DVD-Ig, FIT-Ig, mAb-dAb, dock and lock, SEEDbody, single-chain diabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, single-chain diabody, single-chain diabody-CH 3 , bifunctional antibody-CH3 , miniantibodies, 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, specifically knob-in-hole, knob-in-hole with common light chain, knob-in-hole with common light chain and charge pair, and FIT-Ig, for example FIT-Ig.

[0348] In one embodiment, the bispecific format is selected from DVD-Ig, mAb 2 , mAb-dAb, docking and locking, Fab arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, κλ body, orthogonal Fab, single-chain diabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, single-chain diabody, single-chain diabody-CH 3 , bifunctional antibody-CH 3 , trifunctional antibody, mini antibody, mini antibody, TriBi mini antibody, scFv-CH 3 KIH,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 zybody, such as DVD-Ig, mAb 2 , mAb-dAb, docking and locking, SEEDbody, single-chain diabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, single-chain diabody, single-chain diabody-CH 3 , bifunctional antibody-CH 3 , miniantibodies, knobs into holes, knobs into holes with common light chains, knobs into holes with common light chains and charge pairs, charge pairs, charge pairs with common light chains, specifically mAbs 2, knob-in-hole, knob-in-hole with common light chain and charge pair, and knob-in-hole with common light chain, such as mAb 2 .

[0349] In one embodiment, the bispecific format is selected from DVD-Ig, mAb-dAb, dock and lock, Fab arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, κλ body, orthogonal Fab, single chain diabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, single chain diabody, single chain diabody-CH 3 , bifunctional antibody-CH 3 , trifunctional antibody, mini antibody, mini antibody, TriBi mini antibody, scFv-CH 3 KIH,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, KIHIgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig and zybody, e.g., DVD-Ig, mAb-dAb, dock and lock, SEEDbody, single-chain diabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, single-chain diabody, single-chain diabody-CH 3 , bifunctional antibody-CH 3 , miniantibodies, 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, specifically knob-in-hole, knob-in-hole with common light chain and charge pair, and knob-in-hole with common light chain.

[0350] 33. An anti-BMP6 antibody or fragment as defined in any preceding clause for use in treating or preventing a BMP6-mediated disease or condition (eg, anemia) in a subject.

[0351] 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 one example, the subject is a child. In one example, the subject is a human CKD patient undergoing dialysis treatment. In one example, the subject is a human with end-stage renal disease.

[0352] In one example, the antibodies or fragments herein are used to treat or prevent a disease or condition in an individual (e.g., a human) selected from anemia, pulmonary arterial hypertension (PAH) (e.g., primary PAH or secondary PAH), cerebral cavernous malformation (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 ( Syndrome, endothelial-mesenchymal transition (EndoMT), cardiovascular disease, atherosclerosis, systemic sclerosis-related pulmonary fibrosis and cardiac fibrosis.

[0353] Examples of diseases or conditions mediated by EndoMT are cardiovascular disease, atherosclerosis, systemic sclerosis-associated pulmonary fibrosis, cardiac fibrosis, PAH, tumor formation, tumor invasion, tumor metastasis, fibrotic diseases and the generation of cancer-associated fibroblasts (e.g. in pancreatic cancer).

[0354] In one example, the disease or condition is in a human. In one example, the disease or condition is in an animal.

[0355] In one example, the antibodies or fragments of the invention are used to treat or prevent a TIGIT-mediated disease or condition in humans, e.g., selected from a neoplastic or non-neoplastic disease, a chronic viral infection, and a malignancy, 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, virally induced cancers (e.g., cervical cancer and nasopharyngeal carcinoma), soft tissue sarcoma, hematological malignancies such as Hodgkin's disease and non-Hodgkin's disease and diffuse large B-cell lymphoma (e.g., melanoma, Merkel cell carcinoma, non-small cell lung cancer (squamous and non-squamous), renal cell carcinoma, bladder cancer, head and neck squamous cell carcinoma, and mesothelioma or, for example, virally induced cancers (e.g., cervical cancer and nasopharyngeal carcinoma) and soft tissue sarcoma).

[0356] In one example, the BMP6-mediated disease or condition is a neurodegenerative disease, disorder or condition, such as 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, a retinal degeneration disorder selected from age-related macular degeneration and retinitis pigmentosa; anterior ischemic optic neuropathy, glaucoma, uveitis, depression, trauma-related stress or post-traumatic stress disorder, frontotemporal dementia, Lewy body dementia, mild cognitive impairment, posterior cortical atrophy, primary progressive aphasia and progressive supranuclear palsy or age-related dementia, specifically, a neurodegenerative disease, disorder or condition selected from Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease and Huntington's disease, such as Alzheimer's disease.

[0357] In one example, the antibodies, fragments, combinations of the invention are administered intravenously to a subject; or are used for intravenous administration to a subject. In one example, the antibodies, fragments, combinations of the invention are administered subcutaneously to a subject; or are used for subcutaneous administration to a subject.

[0358] 34. The antibody or fragment according to clause 33, wherein the antibody or fragment is administered to the individual simultaneously or sequentially with an erythropoietin stimulating agent (ESA).

[0359] 35. A combination of an amount of an anti-BMP6 antibody or fragment and an amount of an ESA (eg comprising multiple doses of said antibody and / or ESA), wherein said antibody or fragment is according to any one of clauses 1 to 34.

[0360] Also provided is a medical kit comprising the combination, a first sterile container comprising the amount of the antibody or fragment, and a second sterile container comprising the amount of the ESA, and optionally instructions for using the combination to treat anemia in an individual.

[0361] In one example, the combination is for treating or preventing anemia in an individual, wherein the total dose of the antibody and the total dose of the ESA are administered to the individual at a ratio of X:Y over a period of 4 consecutive weeks, wherein X is 10 to 2×10 6 and Y=4, for example, X is 10 to 2×10 6 micrograms and Y=4 micrograms.

[0362] In one example, treatment increases (or is used to increase) one, more or all of Hb concentration, mean corpuscular hemoglobin (MCH) and transferrin saturation in an individual. Those skilled in the art will be familiar with the parameters and how to determine them, for example, using one or more than one serum sample of an individual. For example, transferrin saturation (measured as a percentage) is the value of serum iron divided by total iron binding capacity.

[0363] In one example, the subject suffers from anemia of chronic disease (ACD) at the start of treatment, and optionally wherein the anemia is associated with chronic inflammation (e.g., the subject suffers from arthritis) or bacterial infection (e.g., streptococcal infection), or wherein the subject is a chronic kidney disease (CKD) patient.

[0364] 36. An antibody, fragment or combination according to any one of clauses 1 to 34 for use in a method of:

[0365] (a) preventing a subject's blood hemoglobin level from decreasing to less than 10 g / dL, the method comprising administering to the subject the antibody or fragment and an erythropoiesis stimulating agent (ESA);

[0366] (b) raising blood hemoglobin to a level of at least 10 g / dL in an individual suffering from anemia, the method comprising administering to the individual the antibody or fragment and an erythropoiesis stimulating agent (ESA), wherein the anemia is treated;

[0367] (c) treating or preventing anemia in an individual suffering from an inflammatory disease or condition, the method comprising administering to the individual the antibody or fragment and an erythropoiesis stimulating agent (ESA), wherein the anemia is treated or prevented;

[0368] (d) eliminating or reducing the need to administer iron or blood transfusion to an individual suffering from anemia, the method comprising administering to the individual the antibody or fragment and an erythropoiesis stimulating agent (ESA), wherein the need is eliminated or reduced;

[0369] (e) treating or preventing anemia in an individual suffering from a microbial infection, the method comprising administering to the individual an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA);

[0370] (f) reducing the administration of an erythropoiesis stimulating agent (ESA) to an individual suffering from anemia to treat the anemia, the method comprising administering the antibody or fragment and the ESA, wherein the anemia in the individual is treated; or

[0371] (g) treating or reducing the risk of anemia in an individual suffering from anemia or at risk of anemia, the method comprising administering to the individual the antibody or fragment and a low dose of an erythropoiesis stimulating agent (ESA), wherein the anemia in the individual is treated or the risk of anemia is reduced.

[0372] 37. The antibody, fragment or combination according to any one of clauses 1 to 34 and 36, wherein the ESA is

[0373] a. epoetin alfa and administered at a weekly dose of less than 1000, 1500, 2500, 5000, 11000, 18000, 34000 or 90000 units, optionally wherein the individual has previously been treated with epoetin alfa at <1500, 1500 to 2499, 2500 to 4999, 5000 to 10999, 11000 to 17999, 18000 to 33999, 34000 to 89999 or ≥90000 units per week, respectively;

[0374] b. Darbepoetin alfa or and administered at a weekly dose of less than 6.25, 10, 12.5, 20, 25, 40, 60, 100 or 200 micrograms, optionally wherein the individual has previously received 6.25, 10, 12.5, 20, 25, 40, 60, 100 or 200 micrograms per week of darbepoetin alfa or treatment; or

[0375] c. Darbepoetin alfa or and administered at a weekly dose of less than 6.25, 10, 20, 40, 60, 100 or 200 micrograms, optionally wherein the individual has previously received 1500 to 2499, 2500 to 4999, 5000 to 10999, 11000 to 17999, 18000 to 33999, 34000 to 89999 or ≥90,000 units of Epoetin alfa per week, respectively.

[0376] 38. The antibody, fragment or combination according to any one of clauses 1 to 34, 36 and 37, for use in maintaining or increasing blood hemoglobin levels in said individual to at least 10 g / dL for at least 13 or 14 days after said individual has received said anti-BMP6 antibody or fragment and an ESA.

[0377] 39. Use of an antibody, fragment or combination as defined in any preceding clause in the manufacture of a medicament for administration to a subject to treat or prevent a BMP6-mediated disease or condition such as anaemia.

[0378] 40. A method for treating or preventing a BMP6-mediated disease or condition (e.g., anemia) in an individual, the method comprising administering to the individual a therapeutically effective amount of an antibody, fragment or combination as defined in any one of clauses 1 to 38, wherein the BMP6-mediated disease or condition is thereby treated or prevented.

[0379] The disease or condition can be any disease or condition disclosed herein.

[0380] 41. The use according to clause 39 or the method according to clause 40, wherein the BMP6-mediated disease or condition is anemia.

[0381] 42. The antibody, fragment, combination, use or method according to any one of clauses 33 to 41, further comprising administering to the individual another therapy, such as another therapeutic agent, optionally wherein the other therapeutic agent is selected from the group consisting of:

[0382] (a) Intravenous iron;

[0383] (b) ESA (e.g. EPO);

[0384] (c) ActRIIa inhibitors;

[0385] (d) ActRIIb inhibitors;

[0386] (e) IL-6 or IL-6 receptor inhibitors (e.g. anti-IL-6 or IL-6 receptor antibodies);

[0387] (f) TNF-α or TNF-α receptor inhibitors (e.g., anti-TNF-α or TNF-α receptor antibodies);

[0388] (g) HJV inhibitors (e.g., anti-HJV antibodies);

[0389] (h) a BMP inhibitor (eg, another anti-BMP antibody or fragment), for example, wherein the BMP is BMP2, 4, 5, 6, 7 or 9;

[0390] (i) MTP2 agonists (eg, MTP2 agonist antibodies);

[0391] (j) HIF-PH inhibitors;

[0392] (k) transferrin receptor 2 (TFR2) inhibitors;

[0393] (1) HFE inhibitors;

[0394] (m) NRf2 inhibitors;

[0395] (n) transforming growth factor β superfamily type I activin receptor-like kinase (ALK) receptor inhibitor;

[0396] (o) activin receptor inhibitors (e.g., activin receptor Fc fusions);

[0397] (p) GDF11 inhibitors; and

[0398] (q) Myostatin inhibitors.

[0399] Optionally, the other agent is Luspatercept TM Sotatercept TM Optionally, the other agent is a TGF-β superfamily inhibitor. In one example, the other agent is a transforming growth factor β superfamily type I activin receptor-like kinase (ALK) receptor inhibitor; an ALK2 inhibitor, an ALK3 inhibitor; an ALK4 inhibitor; an ALK5 inhibitor; or an ALK7 inhibitor.

[0400] In one example, the other agent is an IL-6 or IL-6R inhibitor, such as Sarilumab, Vobarilizumab, or tocilizumab (eg, or ).

[0401] In one example, the other agent is TNF-α or a TNF-α receptor inhibitor, such as adalimumab, or

[0402] In one embodiment, NRf2 inhibitors increase the efficacy of anti-BMP6 antibodies or fragments by breaking the more iron feedback loop, thereby inducing more BMP6 expression in the treated individual.

[0403] The present disclosure includes generic forms of brand name drugs, and the disclosures of such generic drugs are incorporated herein by reference for possible use in the present invention, for example as part of a combination.

[0404] In one example, the combination comprises an inhibitor of: BMP6 and HJV; or BMP6 and HFE; or BMP6 and TFR2; or BMP6 and BMP2; or BMP6 and BMP4; or BMP6 and ALK2, wherein the BMP6 inhibitor comprises an antibody or fragment of the invention.

[0405] 43. A pharmaceutical composition comprising an antibody, fragment or combination as defined in any one of clauses 1 to 38 and 42 and a pharmaceutically acceptable excipient, diluent or carrier and optionally in combination with another therapeutic agent selected from those described above (eg in clause 42).

[0406] 44. A pharmaceutical composition according to clause 43 for use in the treatment and / or prevention of a BMP6-mediated condition or disease, such as anemia.

[0407] Suitable diseases and conditions include anemia, pulmonary arterial hypertension (PAH) (e.g., primary PAH or secondary PAH), cerebral cavernous malformation (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-related pulmonary fibrosis, and cardiac fibrosis.

[0408] Examples of diseases or conditions mediated by EndoMT are cardiovascular disease, atherosclerosis, systemic sclerosis-associated pulmonary fibrosis, cardiac fibrosis, PAH, tumor formation, tumor invasion, tumor metastasis, fibrotic diseases and the generation of cancer-associated fibroblasts (e.g. in pancreatic cancer).

[0409] 45. A pharmaceutical composition according to clause 43 or 44, in combination with a label or instructions for use for treating and / or preventing said disease or condition in humans; optionally wherein said label or instructions comprises a marketing authorization number (e.g., an FDA or EMA authorization number); optionally wherein said kit comprises an IV or injection device comprising said antibody or fragment.

[0410] 46. ​​A nucleic acid encoding the VH domain and / or VL domain of an antibody or fragment as defined in any one of clauses 1 to 32.

[0411] 47. A nucleic acid encoding a VH domain comprising the amino acid sequence of a VH domain of an antibody selected from the group consisting of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, and CL-58713; or an amino acid having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0412] For example, the consistency is at least 85%. For example, the consistency is at least 90%. For example, the consistency is at least 95%.

[0413] Optionally, a nucleic acid is provided that encodes a VH domain comprising the amino acid sequence of SEQ ID NO: 114, or an amino acid having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity 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%.

[0414] 48. A nucleic acid encoding a VL domain comprising the amino acid sequence of a VL domain of an antibody selected from the group consisting of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, and CL-58713; or an amino acid having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0415] Optionally, the nucleic acid also encodes a VH domain comprising the amino acid sequence of the VH domain of the selected antibody; or an amino acid having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity 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%.

[0416] Optionally, a nucleic acid is provided that encodes a VL domain comprising the amino acid sequence of SEQ ID NO: 123, or an amino acid having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity 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%.

[0417] 49. A nucleic acid (e.g., in a host cell, such as a CHO or HEK293 or Cos cell) comprising

[0418] (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 115, 520 or 521; and / or

[0419] (b) 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.

[0420] In the alternative, provide:

[0421] A nucleic acid (e.g., in a host cell, such as a CHO or HEK293 or Cos cell) comprising

[0422] (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

[0423] (b) 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.

[0424] A nucleic acid (e.g., in a host cell, such as a CHO or HEK293 or Cos cell) comprising

[0425] (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

[0426] (b) 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.

[0427] A nucleic acid (e.g., in a host cell, such as a CHO or HEK293 or Cos cell) comprising

[0428] (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

[0429] (b) 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.

[0430] A combination of a first nucleic acid and a second nucleic acid (e.g., in a host cell, such as a CHO or HEK293 or Cos cell), each comprising

[0431] (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 115, 520 or 521; and / or

[0432] (b) 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.

[0433] A combination of a first nucleic acid and a second nucleic acid (e.g., in a host cell, such as a CHO or HEK293 or Cos cell), each comprising

[0434] (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

[0435] (b) 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.

[0436] A combination of a first nucleic acid and a second nucleic acid (e.g., in a host cell, such as a CHO or HEK293 or Cos cell), each comprising

[0437] (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

[0438] (b) 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.

[0439] A combination of a first nucleic acid and a second nucleic acid (e.g., in a host cell, such as a CHO or HEK293 or Cos cell), each comprising

[0440] (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

[0441] (b) 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.

[0442] For example, for (a), the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0443] For example, for (b), the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0444] Herein, where any instance of % identity is mentioned, in an example there is 100% identity.

[0445] 50. A nucleic acid encoding the heavy and / or light chain of an antibody or fragment as defined in any one of clauses 1 to 32.

[0446] 51. A nucleic acid encoding a heavy chain comprising an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 116.

[0447] For example, the consistency is at least 85%. For example, the consistency is at least 90%. For example, the consistency is at least 95%.

[0448] 52. A nucleic acid encoding a light chain comprising an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 125.

[0449] For example, the consistency is at least 85%. For example, the consistency is at least 90%. For example, the consistency is at least 95%.

[0450] 53. A nucleic acid (e.g., in a host cell, such as a CHO or HEK293 or Cos cell) comprising

[0451] (a) a nucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a selected heavy chain sequence of an antibody selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713; and / or

[0452] (b) a nucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a selected sequence of an antibody selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713.

[0453] Preferably, the selected antibody in (a) and (b) is the same antibody, such as CL-58838. In an alternative, the first nucleic acid comprises (a) and the second nucleic acid comprises (b), such as in a host cell, such as a CHO or HEK293 or Cos cell.

[0454] All nucleic acids of the invention herein can be expressed in host cells such as CHO or HEK293 or Cos cells, such as for expressing the variable domains or chains of the antibodies or fragments of the invention.

[0455] For example, provide:

[0456] A nucleic acid (e.g., in a host cell, such as a CHO or HEK293 or Cos cell) comprising

[0457] (a) a nucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a sequence selected from SEQ ID NO: 512, 514, 516, 518 and 519; and / or

[0458] (b) a nucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a sequence selected from SEQ ID NOs: 513, 515 and 517.

[0459] A nucleic acid (e.g., in a host cell, such as a CHO or HEK293 or Cos cell) comprising

[0460] (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:512; and / or

[0461] (b) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:513.

[0462] A nucleic acid (e.g., in a host cell, such as a CHO or HEK293 or Cos cell) comprising

[0463] (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:514; and / or

[0464] (b) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:515.

[0465] A nucleic acid (e.g., in a host cell, such as a CHO or HEK293 or Cos cell) comprising

[0466] (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:516; and / or

[0467] (b) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:517.

[0468] A nucleic acid (e.g., in a host cell, such as a CHO or HEK293 or Cos cell) comprising

[0469] (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:518; and / or

[0470] (b) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:513, 515 or 517.

[0471] A nucleic acid (e.g., in a host cell, such as a CHO or HEK293 or Cos cell) comprising

[0472] (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:519; and / or

[0473] (b) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:513, 515 or 517.

[0474] For example, for (a), the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0475] For example, for (b), the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0476] 54. A vector comprising a nucleic acid (eg, a nucleic acid according to any one of clauses 46 to 53); optionally wherein the vector is a CHO or HEK293 vector.

[0477] 55. A host cell comprising a nucleic acid (eg, a nucleic acid according to any one of clauses 46 to 53) or a vector according to clause 54.

[0478] Optionally, the VH gene segment is selected from IGHV1-3*01 and IGHV3-11*01. Optionally, the VL gene segment is selected from IGKV1-5*03, IGKV3-20*01 and IGKV3-15*01.

[0479] In one example, the VH, DH and JH are IGHV1-3, IGHD3-10 and IGHJ4 (eg, IGHV1-3*01, IGHD3-10*01 and IGHJ4*02).

[0480] In one example, the VH, DH and JH are IGHV1-3, IGHD3-10 and IGHJ3, such as IGHV1-3*01, IGHD3-10*01 and IGHJ3*02.

[0481] In one example, the VH, DH and JH are IGHV3-11, IGHD6-19 and IGHJ4, such as IGHV3-11*01, IGHD6-19*01 and IGHJ4*02.

[0482] In one example, the VH, DH and JH are IGHV1-3, IGHD7-27 and IGHJ4, such as IGHV1-3*01, IGHD7-27*02 and IGHJ4*02.

[0483] In one example, the VH, DH and JH are IGHV1-3, IGHD4-23 and IGHJ4, such as IGHV1-3*01, IGHD4-23*01 and IGHJ4*02.

[0484] In one example, the VH, DH and JH are IGHV1-3, IGHD5-18 and IGHJ4, such as IGHV1-3*01, IGHD5-18*01 and IGHJ4*02.

[0485] In one example, the VL and JL are IGKV1-5 and IGKJ1, such as IGKV1-5*03 and IGKJ1*01.

[0486] In one example, the VL and JL are IGKV3-20 and IGKJ1, such as IGKV3-20*01 and IGKJ1*01.

[0487] In one example, the VL and JL are IGKV3-15 and IGKJ3, such as IGKV3-15*01 and IGKJ3*01.

[0488] In one example, the VL and JL are IGKV3-20 and IGKJ3, such as IGKV3-20*01 and IGKJ3*01.

[0489] In one example, the antibody or fragment comprises a HCDR3 length of 9, 10, 11 or 12 residues, such as 10 residues, such as 11 residues. In one example, the antibody or fragment comprises a LCDR3 length of 7, 8 or 9 residues, such as 8 residues, such as 9 residues. In one example, each VH domain of the antibody or fragment comprises 1-11 non-germline residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 non-germline residues. In one example, each VL domain of the antibody or fragment comprises 3-8 non-germline residues, such as 3, 4, 5, 6, 7 or 8 non-germline residues.

[0490] In one embodiment, the CDR sequences herein are determined according to Kabat. In an alternative, the CDR sequences are determined according to IMGT.

[0491] In one example, the selected antibody is CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 or CL-58713. In one example, the selected antibody is CL-58838.

[0492] In one example, the selected antibody comprises a heavy chain of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, or CL-58713. In one example, the selected antibody comprises a heavy chain of CL-58838.

[0493] In one example, the heavy chain of the antibody or fragment of the present invention is human γ-1, γ-2, γ-3, γ-4, μ, δ, ε or α isotype, preferably γ isotype (e.g., IgG4 isotype). In one example, the light chain of the antibody or fragment of the present invention comprises a human κ constant region. Alternatively, in one example, the light chain of the antibody or fragment of the present invention comprises a human λ constant region.

[0494] Optionally, the antibody is a 4-chain antibody comprising a heavy chain dimer combined with a light chain dimer. In one example, the heavy chain comprises one or heavy chain CDR or CDR combination as disclosed herein and / or the light chain comprises one or heavy chain CDR or CDR combination as disclosed herein, such as from the same selected antibody. In one example, the heavy chain comprises a VH domain as disclosed herein and / or the light chain comprises a VL as disclosed herein, such as from the same selected antibody. In one example, the heavy chain and the light chain are from the same selected antibody, such as any antibody disclosed in the sequence table herein or the table in the embodiments herein.

[0495] In one example, the selected antibody comprises a light chain of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, or CL-58713. In one example, the selected antibody comprises a light chain of CL-58838.

[0496] In one example, the selected antibody comprises the variable domain of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, or CL-58713. In one example, the selected antibody comprises the variable domain of CL-58838.

[0497] In one example, the selected antibody comprises the VH domain of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, or CL-58713. In one example, the selected antibody comprises the VH domain of CL-58838.

[0498] In one example, the selected antibody comprises the VH and VL domains of CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680, or CL-58713. In one example, the selected antibody comprises the VH and VL domains of CL-58838.

[0499] Optionally, the VH segment is a human IGHV3-11 gene segment, for example, VH is encoded by a nucleotide sequence derived from the recombination of human IGHV3-11 and IGHJ4 (eg, human gene segments 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 the recombination of a human VL gene segment and a JL gene segment, wherein the VL gene segment is selected from IGKV1-5, IGKV3-20 and IGKV3-15. Optionally, VL is human IGKV3-20 (eg, IGKV3-20 * 01). Optionally, JL is IGKJ1 (eg, IGKJ1 * 01). For example, the VL is encoded by a nucleotide sequence derived from the recombination of human IGKV3-20 (eg, IGKV3-20*01) and human IGKJ1 (eg, IGKJ1*01).

[0500] In one example, the binding site comprises a VH / VL pair that specifically binds to human BMP6 (e.g., human BMP6 comprising or consisting of the bold sequence of SEQ ID NO: 1 in the sequence listing herein). In one example, the antibody or fragment comprises 2 (e.g., 2 and no more than 2) copies of the binding site.

[0501] In one example, the antibody or fragment comprises a HCDR3 length of 9-12 residues and / or the antibody or fragment comprises a LCDR3 length of 7-9 residues. In one example, the antibody or fragment comprises a HCDR3 length of 9, 10, 11 or 12 residues, such as 10 residues, such as 11 residues. In one example, the antibody or fragment comprises a LCDR3 length of 7, 8 or 9 residues, such as 8 residues, such as 9 residues. In one example, each VH domain of the antibody or fragment comprises 1-11 non-germline residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 non-germline residues. In one example, each VL domain of the antibody or fragment comprises 3-8 non-germline residues, such as 3, 4, 5, 6, 7 or 8 non-germline residues.

[0502] Optionally, the antibody or fragment competes with CL-58838 (e.g., CL-58838 in IgG form, such as IgG-PE) for binding to BMP6 (e.g., human BMP6, such as mature human BMP6, such as BMP6 comprising or consisting of the sequence of mature BMP6 disclosed in the sequence listing herein, i.e., the bold sequence of SEQ ID NO: 1), as determined by SPR.

[0503] Optionally, the amino acid substitutions are conservative amino acid substitutions, optionally wherein each conservative substitution is from group (1) to (6):

[0504] 1) Alanine (A), serine (S), threonine (T);

[0505] 2) Aspartic acid (D), glutamic acid (E);

[0506] 3) Asparagine (N), glutamine (Q);

[0507] 4) Arginine (R), Lysine (K);

[0508] 5) isoleucine (I), leucine (L), methionine (M), valine (V); and

[0509] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0510] Any SPR herein is, for example, surface plasmon resonance (SPR) at 37°C and pH 7.6.

[0511] Optionally, any BMP6 herein (eg, tested in vitro) is human BMP6, such as hBMP6 (Peprotech 120-06).

[0512] In one example, the antibodies or fragments of the invention are present at 5×10 6 M -1 xs -1 ; or about 5×10 6 M -1 xs -1 In one example, the antibodies or fragments of the invention bind to human BMP6 at a Ka of 4 or 5 s. -1 ; or about 4 or 5s -1 In one example, the antibody or fragment of the invention binds to human BMP6 at 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 a scFv.

[0513] In one example, the antibody comprises heavy chains, wherein each heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 116; and the light chains each comprise or consist of the amino acid sequence of SEQ ID NO: 125.

[0514] In one example, the antibody or fragment comprises a heavy chain VH domain, wherein each VH comprises or consists of the amino acid sequence of SEQ ID NO: 418; and the light chain VL domains each comprise or consist of the amino acid sequence of SEQ ID NO: 426.

[0515] Alternative antibodies or fragments:

[0516] In any configuration of the invention, the invention may relate to antibodies or fragments thereof (alternative antibodies or fragments) as follows.

[0517] Optionally (Option 1), the antibody or fragment comprises

[0518] a. each heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 403 or 566, and each light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 411; or

[0519] b. each heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 419, and each light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 427.

[0520] Optionally (option 2), the antibody or fragment comprises

[0521] a. each heavy chain comprising or consisting of a VH domain amino acid sequence of SEQ ID NO: 402 or 565, and each light chain comprising or consisting of a VL domain amino acid sequence of SEQ ID NO: 410; or

[0522] b. each heavy chain comprising or consisting of a VH domain amino acid sequence of SEQ ID NO: 418, and each light chain comprising or consisting of a VH domain amino acid sequence of SEQ ID NO: 426; and

[0523] c. Optionally, the heavy chain comprises a human γ-1 (eg, IGHG1*01) or γ-4 (eg, IGHG4*01 or IGHG4*01-PE) constant region, or the amino acid sequence of SEQ ID NO: 429, 437, 446, 454 or 456.

[0524] Optionally, the antibody or fragment competes with a reference antibody for binding to BMP6, wherein the reference antibody is mAb507 (R&D Systems) or an alternative antibody (e.g., an Option 1 or Option 2 antibody as defined herein). Competition can be by SPR or ELISA, for example, or in a functional assay such as an assay described herein (e.g., in the Examples). BMP6 can be human BMP6 (e.g., a mature BMP6 comprising the sequence of SEQ ID NO: 562), rat BMP6 (e.g., a mature BMP6 comprising the sequence of SEQ ID NO: 56), or cynomolgus monkey BMP6 (e.g., a mature BMP6 comprising the sequence of SEQ ID NO: 564).

[0525] Human IgG heavy chain genes naturally encode C-terminal lysine. The residue is mostly missing in antibodies isolated from serum, and is present in low but variable amounts on therapeutic antibodies expressed in mammalian cell culture systems. Since C-terminal lysine is naturally sheared in serum and it is unknown whether it affects overall antibody function, it can be removed from the heavy chain coding sequence to provide homologous "lysine shearing" heavy chains and thus homologous drug products. Therefore, any IgG antibody, constant region or heavy chain terminated with G at the C-terminus shown herein can be provided alternatively in the form of GK (i.e., lysine bonded to the C-terminal side of the G shown) termination.

[0526] Examples of options 1 and 2 are:

[0527] Option 1a: In one example, the antibody comprises a heavy chain, wherein each heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 403; and a light chain, wherein each light chain comprises or consists of the amino acid sequence of SEQ ID NO: 411.

[0528] Option 1b: In one example, the antibody comprises heavy chains, wherein each heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 566; and light chains, wherein each light chain comprises or consists of the amino acid sequence of SEQ ID NO: 411.

[0529] Option 1c: In one example, the antibody comprises a heavy chain, wherein each heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 419; and a light chain, wherein each light chain comprises or consists of the amino acid sequence of SEQ ID NO: 427.

[0530] Option 2a: In one example, the antibody or fragment comprises a heavy chain VH domain, wherein each VH comprises or consists of the amino acid sequence of SEQ ID NO: 402; and a light chain VL domain, wherein each VL comprises or consists of the amino acid sequence of SEQ ID NO: 410.

[0531] Option 2b: In one example, the antibody or fragment comprises a heavy chain VH domain, wherein each VH comprises or consists of the amino acid sequence of SEQ ID NO: 565, and a light chain VL domain, wherein each VL comprises or consists of the amino acid sequence of SEQ ID NO: 410.

[0532] In one example, the antibody or fragment comprises a heavy chain VH domain, wherein each VH comprises or consists of the amino acid sequence of SEQ ID NO: 114; and a light chain VL domain, wherein each VL comprises or consists of the amino acid sequence of SEQ ID NO: 123.

[0533] 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 any one or more of the mature forms of said.

[0534] Optionally, the antibody or fragment competitively inhibits the binding of soluble hemojuvelin (HJV) to BMP6. Optionally, the HJV herein is human HJV.

[0535] Optionally, the antibody or fragment does not competitively inhibit the binding of soluble hemojuvelin (HJV) to BMP6.

[0536] As used herein, "inhibition" etc. refers to the ability of an antagonist (e.g., an antibody or its fragment) to bind to an antigenic determinant (e.g., an antigenic determinant of hBMP6), which partially or completely blocks the binding of another antigen. If the antigenic determinant bound by the antagonist completely blocks the binding site of the ligand, the ligand is completely blocked from binding (it can be a physical blockage, in the case of overlapping antigenic determinants; or a spatial blockage, in which the antagonist is very large, so that it can prevent the ligand from binding to its unique antigenic determinant), and the ligand is not removed from the circulation. Therefore, the concentration of circulating ligands seems to increase. If the antigenic determinant bound by the antagonist partially blocks the binding site of the ligand, the ligand may be able to bind, but only weakly (in the case of partial inhibition), or with an orientation different from the natural binding interaction. In the case, some ligands can be removed from the circulation, but not as much as when the ligand binding site is completely free and available for binding. Therefore, inhibition refers to the physical interaction of a ligand with a receptor. Inhibition can be measured by HTRF, which is described in more detail elsewhere herein and in Mathis (1995) Clinical Chemistry 41(9), 1391-1397. Inhibition can also be measured by flow cytometry, where the receptor is expressed on cells, or by ELISA, where the receptor is adsorbed to a culture plate.

[0537] Optionally, the antibody comprises a VH domain encoded by a VDJ region sequence, wherein the VDJ is derived from the recombination of a VH gene segment, a D gene segment, and a JH gene segment, wherein the VH is a human germline (i) VH1-3, (ii) VH2-5, or (iii) VH3-15 gene segment. Additionally or additionally, optionally the antibody comprises a VL domain encoded by a VJ region sequence, wherein the VJ is derived from the recombination of a VL gene segment and a JL gene segment, wherein the VL is a human germline (iv) Vκ3-20, (v) Vλ3-1, (vi) Vκ1-17, or (vii) Vλ1-40.

[0538] Optionally, the antibody or fragment binds to BMP6 with a stronger affinity (lower KD as determined by SPR) than to BMP7; and / or optionally binds to BMP6 with a stronger affinity than to BMP5.

[0539] For example,

[0540] (a) the antibody or fragment binds to BMP6 with a stronger affinity (lower KD as determined by SPR) than to BMP7; and optionally binds to BMP6 with a stronger affinity than to BMP5; and

[0541] (b) the antibody or fragment competes with a reference antibody for binding to BMP6, wherein the reference antibody is mAb507 (R&D Systems) or an alternative antibody (eg, Option 1 antibody or Option 2 antibody).

[0542] Optionally, the antibody of the invention binds to BMP6 with an affinity (KD) of 1 pM to 5 nM, optionally wherein binding is determined by SPR using a Fab of said antibody at 37°C at pH 7.6.

[0543] Optionally, the dissociation rate (K) of the antibody binding to BMP6 off ) is 1×10 -5 to 1×10 -3 S -1 , optionally wherein binding is determined by SPR using a Fab of said antibody at 37°C at pH 7.6.

[0544] Optionally, the binding rate (K) of the antibody to BMP6 on ) is 1×10 5 to 1×10 7 M -1 S -1 , optionally wherein binding is determined by SPR using a Fab of said antibody at 37°C at pH 7.6.

[0545] In one example, the antibody (eg, in Fab form) or fragment binds to BMP6 (eg, human BMP6) with an affinity (KD) of

[0546] (a) 2, 3, 4, 5 or 10 pM to 3, 4 or 5 nM;

[0547] (b) 1-10 pM to 5 nM;

[0548] (c) 10 pM to 3, 4 or 5 nM;

[0549] (d) 50 or 80 pM to 200 nM;

[0550] (e) 50 or 80 pM to 150 nM; or

[0551] (f) 50 or 80 pM to 100 nM.

[0552] In one example, the KD is (or is about) 5-15 pM (e.g., 10 pM). In one example, the KD is (or is about) 2-5 nM (e.g., 3 nM). In one example, the KD is (or is about) 100-400 pM (e.g., 140 or 390 pM).

[0553] In one example, the antibody (eg, in Fab form) or fragment has a dissociation rate (K) of binding to BMP6 (eg, human BMP6). off )for

[0554] (a)1×10 -5 to 5×10 -4 S -1 ;

[0555] (b)1×10 -5 to 6×10 -4 S -1 ;

[0556] (c)1×10 -5 to 7×10 -4 S -1 ;

[0557] (d)1×10 -5 to 8×10 -4 S -1 ;

[0558] (e)2×10 -5 to 1×10 -3 S -1 ;

[0559] (f)2×10 -5 to 5×10 -4 S -1 ;

[0560] (g)2×10 -5 to 6×10 -4 S -1 ;

[0561] (h)2×10 -5 to 7×10 -4 S -1 ;or

[0562] (i)2×10 -5 to 8×10 -4 S -1 .

[0563] In one example, K off is (or about) 5×10 -4 S -1 (For example, when KD is (or is about) 2nM to 400pM; when KD is (or is about) 2-5nM (for example, 3nM); or when KD is (or is about) 100-400pM (for example, 140 or 390pM). In one example, K off is (or about) 3×10 -5 S -1 (For example, when KD is (or is about) 5-15 pM (eg, 10 pM)).

[0564] In one example, the binding rate (K) of the antibody (eg, in Fab form) or fragment to BMP6 (eg, human BMP6) is on )for

[0565] (a)1×10 5 to 1×10 6 M -1 S -1 ;

[0566] (b)1×10 5 to 2×10 6 M -1 S -1 ;

[0567] (c)1×10 5 to 3×10 6 M -1 S -1 ;

[0568] (d)1×10 5 to 4×10 6 M -1 S -1 ;

[0569] (e)1×10 5 to 5×106 M -1 S -1 ;

[0570] (f)2×10 5 to 5×10 6 M -1 S -1 ;

[0571] (g)3×10 5 to 5×10 6 M -1 S -1 ;

[0572] (h)4×10 5 to 5×10 6 M -1 S -1 ;

[0573] (i)5×10 5 to 5×10 6 M -1 S -1 ;or

[0574] (i)6×10 5 to 5×10 6 M -1 S -1 .

[0575] In one example, K on is (or is approximately) 1 or 2×10 -5 M -1 S -1 (For example, when KD is 2-5nM (for example, 3nM)). In one example, K on is (or is about) 1-4, 1, 2, 3 or 4×10 -6 M -1 S -1 (For example, when KD is (or is about) 5-400 pM (eg, 140 or 390 pM) or 5-15 pM (eg, 10 pM)).

[0576] As provided in the clauses or other aspects herein, the anti-BMP6 antibody or fragment can bind to BMP6, such as human BMP6, and its K D Less than 50 nM, less than 40 nM, less than 30 nM, as determined by surface plasmon resonance. In another embodiment, the anti-BMP6 antibody or fragment can bind to BMP6, such as human BMP6, with a K D Less than 20 nM, less than 15 nM, less than 10 nM, as determined by surface plasmon resonance. The anti-BMP6 antibody or fragment can bind to BMP6, such as human BMP6, with a KD 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. D It may 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.

[0577] In another embodiment, K D In the range of 0.01 to 1 nM, or in the range of 0.05 to 2 nM, or in the range of 0.05 to 1 nM. K D It may be about hBMP6, cynomolgus monkey (ie "cyno") BMP6 and / or mouse BMP6.

[0578] In another embodiment, the K of the anti-BMP6 antibodies described herein is ON The rate (e.g., as measured by SPR, e.g., at 25° C. or at 37° C.) is about 0.5 to 10 μM, e.g., about 1 to 8 μM or about 1 to 7 μM. In another embodiment, K ON The rate is about 1 to 5 μM, such as about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, or about 3 μM. In another embodiment, K ON The rate is about 3.5 μM, about 4 μM, about 4.5 μM, about 5 μM, or about 5.5 μM.

[0579] In another embodiment, the K of the anti-BMP6 antibodies described herein is OFF The rate (e.g., as measured by SPR, e.g., at 25° C. or at 37° C.) is about 0.01 to 100 mM, e.g., about 0.1 to 50 mM or about 0.5 to 50 mM. In another embodiment, K OFF The rate is about 0.5 to 10 mM, or about 0.5 to 10 mM, such as about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM. In another embodiment, K OFF The rate is about 0.6 mM, about 0.7 mM, about 0.8 mM, or about 0.9 mM.

[0580] The present invention also provides the following method (or the antibody or fragment of the present invention used in the method):

[0581] A method of treating anemia in an individual, the method comprising

[0582] (a) On the initial day (D 0 ), administering an anti-BMP6 antibody or fragment to the individual; and

[0583] (b) during a period of at least 3 consecutive weeks, the period commencing on D 0 , administering multiple doses of an erythropoietin stimulating agent (ESA), wherein throughout the duration of said period, the blood hemoglobin (Hb) concentration in said individual is reduced compared to D 0 The baseline concentration increased when

[0584] (c) such that, for the entire duration of the said period:

[0585] (i) the Hb concentration does not fall below 100% of the baseline Hb concentration; and the Hb concentration reaches at least 120% of the baseline during the period; and / or

[0586] (ii) Hb concentration increased by at least 1 g / dl compared to baseline.

[0587] Optionally, the antibody, fragment or combination inhibits iron release from human hepatocytes, for example, in an in vitro assay or in vivo in humans.One skilled in the art will be aware of standard assays, such as those mentioned in the Examples herein.

[0588] Optionally, the antibody, fragment or combination is used to treat or prevent a BMP6-mediated disease or condition as disclosed herein in a human by inhibiting iron release from human hepatocytes in the human body. Optionally, the antibody, fragment or combination is used to treat or prevent anemia, PAH or fibrosis in a human by inhibiting iron release from human hepatocytes in the human body.

[0589] Optionally, the antibody, fragment or combination is used to treat or prevent a BMP6-mediated disease or condition as disclosed herein in humans by inhibiting hamp gene expression in human hepatocytes in humans. Optionally, the antibody, fragment or combination is used to treat or prevent anemia, PAH or fibrosis in humans by inhibiting hamp gene expression in human hepatocytes in humans.

[0590] Optionally, the antibody, fragment or combination is used to treat or prevent a BMP6-mediated disease or condition as disclosed herein in a human by inhibiting hepcidin or its expression in human hepatocytes in the human body. Optionally, the antibody, fragment or combination is used to treat or prevent anemia, PAH or fibrosis in a human by inhibiting hepcidin or its expression in human hepatocytes in the human body.

[0591] Optionally, the antibody, fragment or combination is used to treat or prevent a disease or condition mediated by BMP6 as disclosed herein in humans by inhibiting BMP6 activation of hamp gene expression in humans (e.g., in their hepatocytes). Optionally, the antibody, fragment or combination is used to treat or prevent anemia, PAH or fibrosis in humans by inhibiting BMP6 activation of hamp gene expression in humans (e.g., in their hepatocytes).

[0592] Optionally, the antibody, fragment or combination is used to treat or prevent a disease or condition mediated by BMP6 as disclosed herein in humans, which is activated by HJV mediation by suppressing the expression of hamp genes in humans (e.g., in their hepatocytes). Optionally, the antibody, fragment or combination is used to treat or prevent anemia, PAH or fibrosis in humans, which is activated by HJV mediation by suppressing the expression of hamp genes in humans (e.g., in their hepatocytes). In one example, the antibody or fragment competitively inhibits HJV binding to BMP6 in vitro and / or in humans. In vitro competition can be determined by, for example, standard SPR or ELISA.

[0593] In one example, the antibody or fragment inhibits human BMP6-induced luciferase expression in HepG2 cells under the control of the hamp regulatory region in vitro.

[0594] Optionally, the antibody, fragment or combination is used to treat or prevent a BMP6-mediated disease or condition as disclosed herein in humans by inhibiting BMP binding in humans (e.g., in their hepatocytes). Optionally, the antibody, fragment or combination is used to treat or prevent anemia, PAH or fibrosis in humans by inhibiting BMP binding in humans (e.g., in their hepatocytes). In one example, the antibody or fragment binds to an antigenic determinant, wherein HJV contacts BMP6 to form an HJV-BMP6 complex, which can activate hamp gene expression in human hepatocytes.

[0595] In one example, the human cells are HepG2 cells in vitro. More details are provided in the embodiments herein. In one example, inhibition is inhibition in an in vitro HepG2 cell assay, for example, as determined by reporter gene inhibition under the control of one or more than one human hamp regulatory element in vitro. For example, the regulatory element comprises a response element to pSMAD (BMP) and pSTAT (IL6). In one example, human BMP6, cynomolgus monkey BMP6, rat BMP6 or mouse BMP6 are used for assay; and / or human HJV, cynomolgus monkey HJV, rat HJV or mouse HJV are used for assay. In one example, the reporter gene is a luciferase gene. In one example, the antibody or fragment neutralizes the BMP activation expressed by the reporter gene in the assay, for example, neutralization is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% or is completely neutralized.

[0596] In one example, the antibody or fragment competes with a reference antibody for binding to BMP6, e.g., as determined by SPR, ELISA, or in a HepG2 assay (e.g., a HepG2 assay as described herein), e.g., in vitro using a labeled reference antibody assay. In one example, competition reduces the binding by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or completely inhibits binding. For example, the reference antibody is MAb507 or MAb2365; e.g., the reference antibody is an alternative antibody (e.g., an option 1 or option 2 antibody).

[0597] In one example, the BMP6 herein is human BMP6 (Peprotech #120-06) (SEQ ID NO: 2). In one example, the BMP6 herein is any other human BMP6 disclosed herein.

[0598] In one example, the antibody or fragment has a dissociation rate of 1×10 -4 S -1 or lower, as determined by surface plasmon resonance (SPR), e.g., at room temperature or rtp. See Examples.

[0599] In one example, the antibody competes for binding to human BMP6 with a reference antibody selected from 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, C L-58722, CL-58713, CL-58680, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650 and CL-58756; for example, selected from CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102 and CL-57945; for example, selected from CL-58722, CL-58713, CL-58680, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650 and CL-58756.

[0600] In an alternative embodiment, the reference antibody herein is selected from CL-58838, CL-66833, CL-57931, CL-57945, CL-58102, CL-58252, CL-58851, CL-75183, CL-75500, CL-75506, CL-75520, CL-75539, CL-75565, CL-75714, CL-58722, CL-58835, CL-58756, CL-58650, CL-58679, CL-58680 and CL-58713. Optionally, the reference antibody is CL-58838.

[0601] In one example, the antibody competes with a reference antibody for binding to human BMP6, the reference antibody comprising a VH amino acid sequence selected from SEQ ID No: 24, 42, 114, 132, 96, 78, 60, 258, 240, 222, 204, 186, 168, 150, 276, 384, 366, 348, 294, 330 and 312C; for example, selected from SEQ ID No: 24, 42, 114, 132, 96, 78 and 60; for example, selected from SEQ ID No: 276, 384, 366, 348, 294, 330 and 312; and / or the reference antibody comprises (e.g., respectively comprises) a VH amino acid sequence selected from SEQ ID No: 33, 51, 123, 141, 105, 87, 69, 267, 249, 231, 213195, 177, 159, 285, 393, 375, 357, 303, 339 and 321; for example, selected from SEQ ID No: 33, 51, 123, 141, 105, 87 and 69; for example, selected from SEQ ID No: 285, 393, 375, 357, 303, 339 and 321 VL amino acid sequence. 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 known to those skilled in the art.

[0602] In one example, the antibody competes with a reference antibody for binding to human BMP6, the reference antibody comprising the VH amino acid sequence of the VH of the following antibody, the antibody being selected from the sequence listing herein; or from the table in the Examples; or from CL-66833, CL-57890, 42CL-57931, 114CL-58838, 132CL-58851, 96CL-58252, 78CL-58102, CL-57859, CL-58832, 60CL-57945, 258CL-75714, CL-75605, 240CL-75565, 222CL -75539, 204CL-75520, CL-75519, CL-75511, 186CL-75506, 168CL-75500, CL-75496, CL-75194, 150CL-75183, 276CL-58722, 384CL-58713, 366CL-58680, 348CL-58679, CL-58921, CL-58676, 294CL-58835, 330CL-58650 and 312CL-58756; and / or the reference antibody comprises the VL amino acid sequence of the VH of the selected antibody. In one example, the reference antibody is an IgG4 (e.g., IgG4-PE) antibody. In one example, the reference antibody is an IgG1 antibody. In one example, the antibody of the present invention is an IgG4 (e.g., IgG4-PE) antibody. In one example, the antibody of the invention is an IgG1 antibody. In one example, the antibody of the invention preferentially binds to human BMP6 over human BMP5 and / or human BMP7. Binding or competition can be determined, for example, by SPR or ELISA as known to those skilled in the art.

[0603] In one example, the antibodies or fragments of the invention comprise 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, The VH and VL domains of CL-75519, 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.

[0604] In one example, the antibody or fragment of the present invention comprises the VH and VL domains of CL-66833. In one example, the antibody or fragment of the present invention comprises the VH and VL domains of CL-57931. In one example, the antibody or fragment of the present invention comprises the VH and VL domains of CL-58838. In one example, the antibody or fragment of the present invention comprises the VH and VL domains of CL-58851. In one example, the antibody or fragment of the present invention comprises the VH and VL domains of CL-58252. In one example, the antibody or fragment of the present invention comprises the VH and VL domains of CL-58102. In one example, the antibody or fragment of the present invention comprises the VH and VL domains of CL-57945.

[0605] In one example, the selected antibody is CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, or CL-58102. In one example, the selected antibody is CL-66833. In one example, the selected antibody is CL-58838.

[0606] In one example, the selected antibody comprises the variable domain of CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, or CL-58102. In one example, the selected antibody comprises the variable domain of CL-66833. In one example, the selected antibody comprises the variable domain of CL-58838.

[0607] In one example, the selected antibody comprises the VH domain of CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, or CL-58102. In one example, the selected antibody comprises the VH domain of CL-66833. In one example, the selected antibody comprises the VH domain of CL-58838.

[0608] In one example, the selected antibody comprises the VH and VL domains of CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, or CL-58102. In one example, the selected antibody comprises the VH and VL domains of CL-66833. In one example, the selected antibody comprises the VH and VL domains of CL-58838.

[0609] In one example, an antibody or fragment of the invention comprises a VH domain encoded by a nucleotide sequence that is a recombinant of human gene segments IGHV3-11 and IGHJ4 (e.g., human gene segments IGHV3-11*01 and IGHJ4*02; IGHV3-11, IGHD6-19, IGHJ4; or IGHV3-11*01, IGHD6-19*01 and IGHJ4*02). Additionally or alternatively, optionally an antibody or fragment of the invention comprises a VL domain encoded by a nucleotide sequence that is a recombinant of human gene segments IGKV3-20 and IGKJ1 (e.g., IGKV3-20*01 and IGKJ1*01). Additionally or alternatively, optionally an antibody or fragment of the invention comprises the HCDR3 of CL-58835.

[0610] Optionally, the antibody or fragment of the invention comprises an antibody selected from the group consisting of CL-66833, CL-57890, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102, CL-57859, CL-58832, CL-57945, CL-75714, CL-75605, CL-75565, CL-75539, CL-75520, CL-75519, CL-75511, CL-75506, CL-7550 0, CL-75496, 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, HCDR3 of an antibody selected from CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102 and CL-57945. Optionally, the antibody or fragment of the invention comprises the HCDR1 and / or HCDR2 of the selected antibody.

[0611] Optionally, the antibody or fragment of the invention comprises an antibody selected from the group consisting of CL-66833, CL-57890, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102, CL-57859, CL-58832, CL-57945, CL-75714, CL-75605, CL-75565, CL-75539, CL-75520, CL-75519, CL-75511, CL-75506, CL-7550 0, CL-75496, 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, HCDR1 of an antibody selected from CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102 and CL-57945. Optionally, the antibody or fragment of the invention comprises the HCDR2 and / or HCDR3 of the selected antibody.

[0612] Optionally, the antibody or fragment of the invention comprises an antibody selected from the group consisting of CL-66833, CL-57890, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102, CL-57859, CL-58832, CL-57945, CL-75714, CL-75605, CL-75565, CL-75539, CL-75520, CL-75519, CL-75511, CL-75506, CL-7550 0, CL-75496, 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, HCDR2 of an antibody selected from CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102 and CL-57945. Optionally, the antibody or fragment of the invention comprises the HCDR1 and / or HCDR3 of the selected antibody.

[0613] Optionally, the antibody or fragment of the invention comprises a member selected from the group consisting of CL-66833, CL-57890, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102, CL-57859, CL-58832, CL-57945, CL-75714, CL-75605, CL-75565, CL-75539, CL-75520, CL-75519, CL-75511, CL-75506, CL-75500, CL-75496, CL-75194, CL-75183, CL-58722, CL- 58713, CL-58680, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650 and CL-58756; for example, the VH of an antibody selected from the group consisting of CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102 and CL-57945; for example, the VH of an antibody selected from the group consisting of 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 invention comprises the VL of said selected antibody.

[0614] Optionally, the antibody or fragment of the invention comprises a member selected from the group consisting of CL-66833, CL-57890, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102, CL-57859, CL-58832, CL-57945, CL-75714, CL-75605, CL-75565, CL-75539, CL-75520, CL-75519, CL-75511, CL-75506, CL-75500, CL-75496, CL-75194, CL-75183, CL-58722, CL- 58713, CL-58680, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650 and CL-58756; for example, the VL of an antibody selected from the group consisting of CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102 and CL-57945; for example, the VL of an antibody selected from the group consisting of 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 invention comprises the VH of said selected antibody.

[0615] Optionally, the antibody or fragment of the invention comprises a member selected from the group consisting of CL-66833, CL-57890, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102, CL-57859, CL-58832, CL-57945, CL-75714, CL-75605, CL-75565, CL-75539, CL-75520, CL-75519, CL-75511, CL-75506, CL-75500, CL-75496, CL-75194, CL-75183, CL-58722, CL- 58713, CL-58680, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650 and CL-58756; for example, a heavy chain of an antibody selected from the group consisting of CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102 and CL-57945; for example, a heavy chain of an antibody selected from the group consisting of 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 invention comprises a light chain of the selected antibody.

[0616] Optionally, the antibody or fragment of the invention comprises a member selected from the group consisting of CL-66833, CL-57890, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102, CL-57859, CL-58832, CL-57945, CL-75714, CL-75605, CL-75565, CL-75539, CL-75520, CL-75519, CL-75511, CL-75506, CL-75500, CL-75496, CL-75194, CL-75183, CL-58722, CL- 58713, CL-58680, CL-58679, CL-58921, CL-58676, CL-58835, CL-58650 and CL-58756; for example, a light chain of an antibody selected from the group consisting of CL-66833, CL-57931, CL-58838, CL-58851, CL-58252, CL-58102 and CL-57945; for example, a light chain of an antibody selected from the group consisting of 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 invention comprises the heavy chain of the selected antibody.

[0617] In one example, the selected antibody is CL-58835.

[0618] Optionally, the antibodies of the invention comprise a human IgG4 constant region.

[0619] Preferably, an antibody or fragment thereof that specifically binds hBMP6 will not cross-react with other antigens (but may optionally cross-react with different BMP6 species such as rhesus monkeys, cynomolgus monkeys or mice; and or may optionally cross-react with different BMPs such as BMP2, 4 or 9). For example, by immunoassay, BIAcore TM Or other techniques known to those skilled in the art, can identify antibodies or fragments thereof that specifically bind to BMP6 antigen. As measured using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), when the affinity of the antibody or its fragment combined with the hBMP6 antigen is higher than the affinity combined with any cross-reactive antigen, the antibody or its fragment specifically binds to the hBMP6 antigen. Typically, specific or selective reactions will be at least twice the background signal or noise and more typically more than 10 times the background. For discussion of antibody specificity, see, for example, Paul, 1989, Basic Immunology, Second Edition, Raven Press, New York, pp. 332-336.

[0620] The contact amino acid residues involved in the interaction between an antibody and an antigen, such as BMP6, can be determined by various methods known to those skilled in the art.

[0621] In one embodiment, if the antibody recognizes a linear antigenic determinant, short peptides based on the antigenic sequence can be generated and binding of the antibody to the peptide can be assessed using standard techniques.

[0622] In one embodiment, limited proteolytic digestion and mass spectrometry analysis can be used to identify binding antigenic determinants.

[0623] In one embodiment, the contact residues of the antigenic determinant are identified by X-ray crystallography. In one embodiment, the contact residues of the antigenic determinant are identified by cryo-electron microscopy. In one embodiment, the contact residues of the antigenic determinant are identified by a combination of limited proteolytic digestion and mass spectrometry.

[0624] In another embodiment, the anti-BMP6 antibodies (and fragments) described herein provide transient expression levels that are improved over other anti-BMP6 antibodies and fragments. Thus, in one embodiment, the anti-BMP6 antibodies (or fragments) are expressed in HEK293 cells (e.g., HEK293T cells) at an expression level of about 100 μg / mL, or in the range of about 100 to 350 μg / mL. In another embodiment, the expression level is greater than about 350 μμ / mL.

[0625] In another embodiment, the anti-BMP6 antibody (or fragment) is expressed in CHO cells (e.g., Expi-CHO cells) at an expression level of about 100 μg / mL, or in the range of about 100 to 350 μg / mL. In another embodiment, the expression level is greater than about 350 μg / mL.

[0626] In another embodiment, the anti-BMP6 antibody (or fragment) is expressed in CHO cells (e.g., Expi-CHO cells or CHO-E7 EBNA cells) at an expression level of about 100 μg / mL, or in the range of about 100 to 350 μg / mL. In another embodiment, the expression level is greater than about 350 μg / mL. The antibody, for example, comprises the VH and VL domains of any one of CL-58838 in the form of human IgG1 or human IgG4 (e.g., IgG4-PE).

[0627] In any of the expression systems, expression is carried out in a range of about 0.5 mL to 3 mL, for example, about 0.5 mL to 2 mL. In any of the expression systems, the anti-BMP6 antibody (or fragment) can be expressed from a pTT5 vector. In any of the expression systems, the anti-BMP6 antibody (or fragment) can be expressed in conjunction with a lipid transfection reagent, and can optionally be expressed in a CHO cell (e.g., an Expi-CHO cell). In any of the expression systems, the anti-BMP6 antibody (or fragment) can be expressed in conjunction with a PEI transfection reagent, and can optionally be expressed in a CHO cell (e.g., a CHO-E7 EBNA cell). In any of the expression systems, the anti-BMP6 antibody (or fragment) can be expressed in conjunction with an auxiliary plasmid (e.g., an AKT auxiliary plasmid), and can optionally be expressed in a CHO cell (e.g., a CHO-E7 EBNA cell).

[0628] In any of the expression systems, the expression amount is between about 100 μg / mL and about 1500 μg / mL, for example, between about 100 μg / mL and about 1000 μg / mL, or between about 200 μg / mL and about 1000 μg / mL, or between about 350 μg / mL and about 1000 μg / mL. In any of the expression systems, the lower limit of expression may be about 100 μg / mL, about 200 μg / mL, about 300 μg / mL, or about 400 μg / mL. In another embodiment, the lower limit of expression may be about 500 μg / mL, about 600 μg / mL, about 700 μg / mL, or about 800 μg / mL. In any of the expression systems, the lower limit of expression may be about 2000 μg / mL, about 1800 μg / mL, about 1600 μg / mL, or about 1500 μg / mL. In another embodiment, the lower limit of expression may be about 1250 μg / mL, about 1000 μg / mL, about 900 μg / mL, or about 800 μg / mL.

[0629] In another embodiment, the expression system is a Lonza expression system, such as the Lonza System. In the Lonza expression system, expression can be carried out in the range of about 30mL to 2L, for example, 50mL to 1L or 1L to 2L. In the Lonza expression system, the anti-BMP6 antibody (or fragment) can be expressed in combination with electroporation, and optionally in the absence of any auxiliary plasmid. In the Lonza expression system, the anti-BMP6 antibody (or fragment) can be expressed in an amount of about 1g / L, or about 900mg / L, or about 800mg / L or about 700mg / L. In another embodiment, in the Lonza expression system, the anti-BMP6 antibody (or fragment) can be expressed in an amount of about 600mg / L, or about 500mg / L, or about 400mg / L. In the Lonza expression system, the anti-BMP6 antibody (or fragment) can be between about 400mg / L and about 2g / L, for example, between about 500mg / L and about 1.5g / L, or between about 500mg / L and about 1g / L. In another embodiment, the expression level is above 1 g / L. In another embodiment, the anti-BMP6 antibody provides an improved half-life compared to other anti-BMP6 antibodies.

[0630] 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.

[0631] 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.

[0632] The contact amino acid residues involved in the interaction between the antibody and the antigen can be determined by various methods known to those skilled in the art, such as alanine scanning, protein crystallography, mass spectrometry analysis or any other technique obvious to those skilled in the art.

[0633] In one embodiment, the CDR comprises one amino acid substitution, which may be a conservative amino acid substitution. In one embodiment, the CDR comprises two amino acid substitutions, which may be conservative amino acid substitutions. In one embodiment, the CDR comprises three amino acid substitutions, which may be conservative amino acid substitutions. In one embodiment, the CDR comprises four amino acid substitutions, which may be conservative amino acid substitutions. In one embodiment, the CDR comprises five amino acid substitutions, which may be conservative amino acid substitutions. In one embodiment, the CDR comprises six amino acid substitutions, which may be conservative amino acid substitutions.

[0634] Amino acid substitutions include changes in which an amino acid is replaced by a different naturally occurring amino acid residue. The substitutions can be classified as "conservative", in which case the amino acid residue contained in the polypeptide is replaced by another naturally occurring amino acid with similar properties in terms of polarity, side chain functionality or size. The conservative substitutions are well known in the art. The substitutions encompassed by the present invention can also be "non-conservative", in which the amino acid residues present in the peptide are replaced by amino acids with different properties, such as naturally occurring amino acids from different groups (e.g., replacing a charged or hydrophobic amino acid with alanine), or, in which a naturally occurring amino acid is replaced with an unconventional amino acid.

[0635] In one embodiment, conservative amino acid substitutions are as described herein. For example, substitutions may be substitutions of 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, A with S, K or P. In another embodiment, conservative amino acid substitutions may be substitutions of Y with F, T with A or S, I with L or V, W with Y, M with L, N with D, G with A, T with A or S, D with N, I with L or V, F with Y or L, S with A or T, and A with S, G, T or V.

[0636] combination

[0637] The antibody or fragment of the present invention can be included in a combination therapy together with 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). The combination can effectively treat anemia such as ACD (anemia of chronic disease), inflammation or infection, and the combination therapy can produce maintenance and increase of blood hemoglobin concentration, which is statistically significant compared with the use of anti-BMP6 antibodies alone. In addition, the effect can last for several weeks (even after a single dose of anti-BMP6 antibodies). In addition, the combination therapy of the present invention can be used for ESA-saving anemia therapy, i.e., ESA treatment can be performed with ESA at a lower dose than the standard dose. In view of the potential harmful side effects of ESA, this is useful. The present invention can also be used for the treatment of anemia in ESA-refractory individuals or individuals who respond poorly to standard ESA therapy. The present invention can usefully maintain blood hemoglobin beyond the range of moderate to severe anemia and / or prevent blood hemoglobin from falling to this range. Therefore, the present invention can be used to reduce the need for iron or transfusion therapy.

[0638] The present invention can be used to treat anemia in the setting of inflammatory diseases and in the setting of microbial infections.

[0639] To this end, the present invention provides the following configurations 1-13:

[0640] 1. A method of maintaining a blood hemoglobin level of at least 10 g / dL in an individual, the method comprising administering to the individual an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA).

[0641] 2. A method of preventing a subject's blood hemoglobin level from falling to less than 10 g / dL, the method comprising administering to the subject an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA).

[0642] 3. A method of raising blood hemoglobin to at least 10 g / dL in an individual suffering from anemia, the method comprising administering to the individual an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA), wherein the anemia is treated.

[0643] 4. A method for treating or preventing moderate or severe anemia in an individual, the method comprising administering to the individual an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA), wherein the anemia is treated or prevented.

[0644] 5. A method of treating or preventing anemia in an individual suffering from an inflammatory disease or condition, the method comprising administering to the individual an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA), wherein the anemia is treated or prevented.

[0645] 6. A method of eliminating or reducing the need for administration of iron or blood transfusion to an individual suffering from anemia, the method comprising administering to the individual an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA), wherein the need is eliminated or reduced.

[0646] In one example, one, more or all of labile plasma iron (LPI), enhanced LPI (eLPI) and non-transferrin bound iron (NTBI) are reduced in an individual. In one example, one, more or all of labile plasma iron (LPI), enhanced LPI (eLPI) and non-transferrin bound iron (NTBI) are reduced in an individual.

[0647] 7. A method of treating or preventing anemia in an individual suffering from a microbial infection, the method comprising administering to the individual an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA).

[0648] 8. A method of reducing administration of an erythropoiesis stimulating agent (ESA) in an individual suffering from anemia to treat anemia, the method comprising administering an anti-BMP6 antagonist and the ESA, wherein the anemia in the individual is treated.

[0649] 9. A method for treating or reducing the risk of anemia in an individual suffering from anemia or at risk of anemia, the method comprising administering an anti-BMP6 antagonist and a low dose of an erythropoiesis stimulating agent (ESA) to the individual, wherein the anemia in the individual is treated or the risk of anemia is reduced.

[0650] 10. A therapeutic regimen for treating or preventing anemia in an individual suffering from anemia or at risk of anemia, the regimen comprising administering to the individual simultaneously or sequentially an anti-BMP6 antagonist and an ESA, wherein

[0651] a. administering the antagonist to the individual on day 0; and administering the ESA to the individual no later than day 7 (eg, day 1); or

[0652] b. administering the ESA to the individual on day 0; and administering the antagonist to the individual no later than day 7 (eg, day 1); or

[0653] c. administering to said individual said antagonist and said ESA simultaneously on day 0; or

[0654] d. on day 0 the individual has received the ESA, and on day 0 the antagonist is administered to the individual; or

[0655] e. on day 0, the individual has received the antagonist, and on day 0, the individual is administered the ESA;

[0656] Thereby, on or after day 14, the blood hemoglobin level in the individual is at least 10 g / dL, wherein the anemia is treated or prevented.

[0657] 11. A combination therapy for treating or preventing anemia in an individual, which is used in any of the methods or schemes of the aforementioned technical schemes, the combination comprising

[0658] a. Anti-BMP6 antagonist;

[0659] b. ESA; and

[0660] c. Optionally, instructions for use in the method or protocol.

[0661] 12. An anti-BMP6 antagonist for use in the method or regimen of any preceding configuration for treating or preventing anemia in an individual.

[0662] 13. An anti-bone morphogenetic protein 6 (BMP6) antagonist for use in a method of treating or preventing anemia in an individual, the method comprising administering the anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA) to the individual, wherein the anemia is treated or prevented.

[0663] When referring to an "anti-BMP6 antagonist" 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 any antibody or fragment as claimed or mentioned in the present statement or in the examples (such as in Tables 4 to 11).

[0664] In one aspect, the antagonist comprises or consists of an anti-BMP6 antibody or fragment, and the method comprises

[0665] (a) On the initial day (D 0 ) administering the anti-BMP6 antibody or fragment to the individual; and

[0666] (b) during a period of at least 3 consecutive weeks, the period commencing on D 0 , administering multiple doses of an ESA, wherein throughout the duration of the period, the blood Hb concentration in the individual is reduced compared to D 0 The baseline concentration increased when

[0667] So that:

[0668] (i) throughout the duration of the period, the Hb concentration does not fall below 100% of the baseline Hb concentration; and during the period, the Hb concentration reaches at least 120% of the baseline; and / or

[0669] (ii) the Hb concentration increases by at least 1 g / dl compared to baseline throughout the duration of the period.

[0670] Aspects of the invention are as follows, and the aspects (and any unnumbered paragraphs) may be combined with any other configuration, embodiment, feature, aspect or clause of the invention as described herein; the antagonists (e.g., anti-BMP6 antibodies or fragments) or ESAs of the invention may provide for (or may be used for) methods in the following aspects:

[0671] 1. A method of maintaining a blood hemoglobin level in an individual of at least 10, 10.5, 11, 11.5, 12, 12.5 or 13 g / dL, the method comprising administering to the individual an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA).

[0672] In one example, the Hb level in the individual is no greater than 11, 11.5, or 12 g / dl.

[0673] 2. A method of preventing a subject's blood hemoglobin level from falling to less than 10, 10.5, 11, 11.5, 12, 12.5 or 13 g / dL, the method comprising administering to the subject an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA).

[0674] 3. A method of increasing the blood hemoglobin level in an individual suffering from anemia to at least 10, 10.5, 11, 11.5, 12, 12.5 or 13 g / dL, the method comprising administering to the individual an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA), wherein the anemia is treated.

[0675] In an example of any aspect, the individual suffers from moderate or severe anemia prior to administration of the BMP6 antagonist. In one embodiment, the result of the method is that the individual has no anemia or has mild (not moderate or severe) anemia.

[0676] 4. A method of treating or preventing moderate or severe anemia in an individual, the method comprising administering to the individual an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA), wherein the anemia is treated or prevented.

[0677] 5. A method of treating or preventing anemia in an individual suffering from an inflammatory disease or condition, the method comprising administering to the individual an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA), wherein the anemia is treated or prevented.

[0678] In one example, the inflammatory disease or condition is selected from the group consisting of microbial infection (eg, bacterial infection) or inflammation of rheumatoid arthritis. In one example, the anemia is anemia of inflammation (also known as anemia of chronic disease, ACD).

[0679] 6. A method of eliminating or reducing the need for administration of iron or blood transfusion to an individual suffering from anemia, the method comprising administering to the individual an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA), wherein the need is eliminated or reduced.

[0680] In one embodiment, the method reduces the dosage (eg, weekly, biweekly, or monthly dosage) or frequency of administration of iron.

[0681] 7. A method of treating or preventing anemia in an individual suffering from a microbial (eg, bacterial) infection, the method comprising administering to the individual an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA).

[0682] 8. A method of reducing the administration of an erythropoiesis stimulating agent (ESA) to an individual suffering from anemia to treat anemia, the method comprising administering an anti-BMP6 antagonist and the ESA, wherein the anemia in the individual is treated.

[0683] 9. A method for treating or reducing the risk of anemia in an individual suffering from anemia or at risk of anemia, the method comprising administering an anti-BMP6 antagonist and a low dose of an erythropoiesis stimulating agent (ESA) to the individual, wherein the anemia in the individual is treated or the risk of anemia is reduced.

[0684] The dosage is lower than the standard dosage typically used to treat or reduce anemia in an individual (e.g., a human or adult, such as a male or female). Typical dosages for treatment or prevention are apparent to those skilled in the art. For example, see Aspect 10.

[0685] Epogen is typically formulated in vials of various formulations. Single-dose vials formulated with isotonic sodium chloride / sodium citrate buffer are available in various strengths. Each 1 mL vial contains 2000, 3000, 4000, or 10,000 units of epoetin alfa, albumin (human) (2.5 mg), citric acid (0.06 mg), sodium chloride (5.9 mg), and sodium citrate (5.8 mg) in Water for Injection USP (pH 6.9 ± 0.3). Single-dose 1 mL vials formulated with isotonic sodium chloride / sodium phosphate buffer contain 40,000 units of epoetin alfa, albumin (human) (2.5 mg), citric acid (0.0068 mg), sodium chloride (5.8 mg), sodium citrate (0.7 mg), disodium hydrogen phosphate anhydrous (1.8 mg), and sodium dihydrogen phosphate monohydrate (1.2 mg) in Water for Injection USP (pH 6.9 ± 0.3). The multiple-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 water for injection, USP (pH 6.1 ± 0.3). The multiple-dose 1 mL vial contains 20,000 units of Epoetin alfa, albumin (human) (2.5 mg), benzyl alcohol (1%), sodium chloride (8.2 mg), citric acid (0.11 mg), and sodium citrate (1.3 mg) per 1 mL of water for injection, USP (pH 6.1 ± 0.3). In one embodiment of the invention, ESA is administered as one of the formulations.

[0686] 10. The method according to any preceding aspect, wherein the ESA is

[0687] a. Epoetin alfa and administered at a weekly dose of less than 1000, 1500, 2500, 5000, 11000, 18000, 34000 or 90000 units, optionally wherein the individual has previously received <1500, 1500 to 2499, 2500 to 4999, 5000 to 10999, 11000 to 17999, 18000 to 33999, 34000 to 89999 or ≥90000 units of Epoetin alfa per week, respectively;

[0688] b. Darbepoetin alfa or and administered at a weekly dose of less than 6.25, 10, 12.5, 20, 25, 40, 60, 100 or 200 micrograms, optionally wherein the individual has previously received 6.25, 10, 12.5, 20, 25, 40, 60, 100 or 200 micrograms of darbepoetin alfa or treatment; or

[0689] c. Darbepoetin alfa or and administered at a weekly dose of less than 6.25, 10, 20, 40, 60, 100 or 200 micrograms, optionally wherein the individual has previously received 1500 to 2499, 2500 to 4999, 5000 to 10999, 11000 to 17999, 18000 to 33999, 34000 to 89999 or ≥90,000 units of weekly Epoetin alfa treatment, respectively.

[0690] 11. The method according to any of the preceding aspects, wherein the anti-BMP6 antagonist is an antibody and is administered at a total dose of no more than 30 mg / kg (e.g., 0.1 to 30 mg / kg) such as every 1 week, 2 weeks, or 3 weeks, or every month, 2 months, or 3 months. Administration can be, for example, intravenous or subcutaneous administration, and the individual is a human such as an adult.

[0691] 12. A therapeutic regimen for treating or preventing anemia in an individual suffering from or at risk of anemia, the regimen comprising administering to the individual simultaneously or sequentially an anti-BMP6 antagonist and an ESA, wherein

[0692] a. administering the antagonist to the individual on day 0; and no later than day 56, 28, 14 or 7 (eg, on day 1, 6 or 7), administering the ESA to the individual; or

[0693] b. administering the ESA to the individual on day 0; and administering the antagonist to the individual no later than day 56, 28, 14 or 7 (eg, on day 1, 6 or 7); or

[0694] c. administering to said individual said antagonist and said ESA simultaneously on day 0; or

[0695] d. on day 0 the individual has received the ESA, and on day 0 the antagonist is administered to the individual; or

[0696] e. on day 0, the individual has received the antagonist, and on day 0, the individual is administered the ESA;

[0697] Thereby, on day 14 or thereafter (e.g., on day 28, 56 or 70), the blood hemoglobin level in the individual is at least 10, 10.5, 11, 11.5, 12, 12.5 or 13 g / dL, wherein the anemia is treated or prevented.

[0698] Optionally, the antagonist is administered a second time no later than day 7 (eg, the antagonist is administered on day 6).

[0699] 13. The method or regimen according to any preceding aspect, wherein the anti-BMP6 antagonist and the ESA are administered to the individual no more than 7 days apart.

[0700] 14. The method or regimen according to any one of the preceding aspects, wherein said method or regimen maintains the blood hemoglobin level in said individual at greater than 10 g / dL in said individual.

[0701] 15. A method or regimen according to any preceding aspect, wherein the method or regimen maintains or increases blood hemoglobin levels in the individual to at least 10 g / dL at least 13 or 14 days after the individual has received the anti-BMP6 antagonist and the ESA.

[0702] 16. The method or regimen according to aspect 14 or 15, wherein the anti-BMP6 antagonist and the ESA are administered to the individual no more than 1 day apart.

[0703] 17. The method or regimen according to any preceding aspect, wherein the anti-BMP6 antagonist and the ESA are administered to the individual simultaneously.

[0704] 18. The method or regimen according to any preceding aspect, wherein the subject's blood hemoglobin level is prevented from falling to less than 10, 10.5, 11, 11.5, 12, 12.5 or 13 g / dL (e.g. on day 14).

[0705] 19. The method or regimen according to any preceding aspect, wherein the subject's blood hemoglobin is increased to a level of at least 10, 10.5, 11, 11.5, 12, 12.5 or 13 g / dL (eg, on day 14).

[0706] 20. The method or regimen according to any preceding aspect, wherein moderate or severe anemia is prevented in the individual (eg, on day 14).

[0707] 21. A method or regimen according to any preceding aspect, wherein the individual suffers from

[0708] a. Inflammatory diseases or conditions; or

[0709] b. Infection;

[0710] c. Kidney disease;

[0711] d. HIV or undergoing HIV treatment; or

[0712] e. cancer; and

[0713] Anemia is treated or prevented in the individual.

[0714] In one example, the individual suffers from HIV infection, HIV, hepatitis, rheumatoid arthritis, chronic kidney disease, or end-stage renal disease. For example, the infection is a gram-negative bacterial infection. For example, the infection is a gram-positive bacterial infection.

[0715] HIV-infected humans treated with anti-HIV therapy may develop anemia. Therefore, the present invention can be used to treat or prevent anemia in such patients. In one example, the method or regimen can utilize the administration of anti-HIV therapy, such as the administration of <4200 mg / week zidovudine, to treat or prevent anemia in HIV-infected humans.

[0716] Anemia may occur in cancer patients treated with anti-cancer chemotherapy (e.g., immunotherapy, such as by administering immune checkpoint inhibitors, such as anti-CTLA4, anti-PD-L1, anti-TIGIT, anti-ICOS or anti-PD1 antibodies to the individual). Therefore, the present invention can be used to treat or prevent anemia in the patient. In one example, the method or regimen treats or prevents anemia in humans suffering from cancer. In the art, ESAs such as erythropoietin are usually initially administered at a dose of 150 units / kg IV or SC three times a week; or 40,000 units SC once a week are administered to the patient until the chemotherapy course is completed. In one example, the present invention treats or prevents anemia in human cancer patients, wherein the ESA is administered intravenously or subcutaneously to humans at less than 150 units / kg three times a week; or a total weekly dose of less than 450 units / kg is administered to humans; or less than 40,000 units are administered subcutaneously to humans each week.

[0717] ESA therapy is used in the art to reduce the need for red blood cell (RBC) transfusions in patients (e.g., in patients undergoing surgery). Thus, ESA therapy is used, for example, in human patients with perioperative hemoglobin>10 g / dL but ≤13 g / dL, who are at high risk of perioperative blood loss due to surgery such as elective, non-cardiac, non-vascular surgery. ESA is administered once daily at 300 units / kg SC for 15 consecutive days (10 days before surgery, the day of surgery, and 4 days after surgery); alternatively, 600 units / kg SC is administered in 4 doses, 21 days, 14 days, and 7 days before surgery and on the day of surgery. In one example, the present invention treats or prevents anemia in human surgical patients, wherein the ESA is administered to humans as follows: 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 less than 4500 units / kg for a total 15-day dose; or less than 600 units / kg in 3-5 or 4 doses, for example, 21 days, 14 days, and 7 days before surgery and on the day of surgery.

[0718] 22. The method according to aspect 21, wherein moderate or severe anemia is treated or prevented in the individual.

[0719] 23. The method or regimen according to any preceding aspect, wherein the individual is a mammal.

[0720] 24. A combination therapy for use in a method or regimen according to any preceding aspect to treat or prevent anemia in an individual, the combination comprising

[0721] a. Anti-BMP6 antagonist;

[0722] b. ESA; and

[0723] c. Optionally, instructions for use of the method or protocol.

[0724] 25. An anti-BMP6 antagonist for use in a method or regimen according to any preceding aspect to treat or prevent anemia in an individual.

[0725] 26. A combination according to aspect 24 or an antagonist according to aspect 25 for use in the treatment or prevention of moderate or severe anaemia.

[0726] 27. A combination of an antagonist according to any one of aspects 24 to 26 and an anti-inflammatory agent.

[0727] 28. A method, regimen, combination or antagonist according to any preceding aspect, wherein the antagonist comprises an anti-BMP6 antibody binding site, for example, wherein the antagonist is an antibody or an anti-BMP6 capture agent.

[0728] In one example, the capture agent comprises a human BMP6 receptor domain fused to a human antibody Fc region. In one embodiment, the Fc comprises a human gamma-1 or gamma-4 heavy chain constant region.

[0729] 29. A method, regimen, combination or antagonist according to any preceding aspect, wherein the ESA is erythropoietin.

[0730] 30. The method or regimen according to any one of aspects 1 to 23, 28 and 29, wherein an anti-inflammatory agent is administered to the individual.

[0731] In one example, the present invention uses an anti-BMP6 monoclonal antibody (mAb) to mobilize endogenous iron reserves and increase hemoglobin synthesis and optionally erythropoiesis. In one aspect, the present invention can reduce the need for simultaneous and widespread use of intravenous iron or transfusions in ACD patients. Additionally or alternatively, the present invention can reduce the dose of ESA (e.g., EPO) for basic standard of care treatment or make ESA (e.g., EPO) unresponsive patients (or those with low responders) respond to co-administration of ESA with anti-BMP6 antagonists. Additionally or alternatively, the present invention can treat or prevent refractory anemia or anemia in patients who are unresponsive to the ESA standard of care. In patients with uncontrolled hypertension or those suffering from the treatment of ESA (e.g., darbepoetin α, such as or for example, epoetin alfa, such as or ESAs may be contraindicated in patients with pure red cell aplasia (PRCA), a type of anemia caused by leukemia.

[0732] Therefore, in one embodiment of the present invention, the individual (e.g., human) is

[0733] i. Refractory or unresponsive to ESA (e.g., darbepoetin alfa or epoetin alfa);

[0734] ii. suffer from or have developed hypertension (e.g. uncontrolled high blood pressure); or

[0735] iii. Suffering from or having suffered from pure red cell aplasia (e.g. caused by receiving ESA such as darbepoetin alfa or epoetin alfa).

[0736] "Refractory" with respect to drug therapy such as ESA therapy is apparent to one skilled in the art and means, for example, that the individual is resistant to ESA or is less responsive to ESA (i.e., has a less than average response) and is not responding to anemia treatment by the standard of care using ESA.

[0737] ESA is usually used to maintain hemoglobin at a minimum level, which can minimize transfusions and meet the needs of patients. As described above, the present invention can be used for ESA anemia reduction therapy in its various configurations, aspects, embodiments and embodiments, that is, it can use ESA treatment with a lower than standard dose of ESA. This is useful in view of the potential harmful side effects of ESA. Table AD provides relevant information of the aspects.

[0738] Table A: Dosing Information

[0739]

[0740]

[0741] In one example, the individual is a chronic kidney disease (CKD) patient who is not undergoing dialysis. In one example, the individual is a chronic kidney disease (CKD) patient who is undergoing dialysis. In one example, the individual is a chemotherapy patient (e.g., receiving or having received cancer chemotherapy treatment).

[0742] [Table B is as follows]

[0743] Table B: side effect

[0744]

[0745]

[0746]

[0747]

[0748] In one embodiment, the treatment or prevention of the invention reduces the incidence or risk of one or more side effects listed in Table B (e.g., one or more than one "common," "more common," or "very common" side effects) in a subject.

[0749] In one aspect, the present invention provides a method of reducing the side effects of ESA therapy in an individual suffering from anemia or at risk of anemia, the method comprising administering an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA) to the individual, wherein the anemia is treated or prevented. Optionally, the incidence or risk of one or more ESA side effects listed in Table B (e.g., one or more than one "common", "more common" or "very common" side effects) is reduced. In one example, the therapy is anemia treatment. In one example, the therapy is anemia prevention. In one example, the anemia is moderate or severe anemia.

[0750] Table C: US Black Box Warning

[0751]

[0752] In one embodiment, the treatment or prevention of the present invention reduces the incidence or risk of one or more than one side effect listed in Table C, for example, shortened overall survival and / or increased risk of tumor progression or recurrence in an individual, wherein the individual is a patient with breast cancer, non-small cell lung cancer, head and neck cancer, lymphoma and cervical cancer; or a cardiovascular or thromboembolic reaction, such as stroke.

[0753] In one aspect, the present invention provides a method of reducing the side effects of ESA therapy for an individual suffering from anemia or at risk of anemia, the method comprising administering an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA) to the individual, wherein the anemia is treated or prevented. Optionally, the incidence or risk of one or more ESA side effects listed in Table C (e.g., shortening overall survival and / or increasing the risk of tumor progression or recurrence, wherein the individual is a patient with breast cancer, non-small cell lung cancer, head and neck cancer, lymphoma, and cervical cancer; or cardiovascular or thromboembolic reactions, such as stroke) is reduced. In one example, the therapy is anemia treatment. In one example, the therapy is anemia prevention. In one example, the anemia is moderate anemia. In one example, the anemia is moderate to severe anemia. In one example, the anemia is severe anemia. In one example, the anemia in the present invention is anemia derived from myelosuppressive chemotherapy.

[0754] Table D: Estimated starting dose of Aranesp for CKD patients on dialysis based on previous Epoetin alfa dose (in units) (μg / week)

[0755]

[0756] *There are insufficient data to determine the Aranesp conversion dose for pediatric patients receiving Epoetin alfa doses <1,500 units / week.

[0757] Aspects of the present invention provide (i) and (ii)

[0758] (i) A method of reducing the administration of an erythropoiesis stimulating agent (ESA) to an individual suffering from anemia to treat anemia, the method comprising administering an anti-BMP6 antagonist and the ESA, wherein the anemia in the individual is treated.

[0759] (ii) A method of treating or reducing the risk of anemia in an individual suffering from anemia or at risk of anemia, the method comprising administering an anti-BMP6 antagonist and a low dose of an erythropoiesis stimulating agent (ESA) to the individual, wherein the anemia in the individual is treated or the risk of anemia is reduced.

[0760] In examples of said aspects, the ESA is

[0761] a. Epoetin alfa and administered at a weekly dose of less than 1000, 1500, 2500, 5000, 11000, 18000, 34000 or 90000 units, optionally wherein the individual has previously been treated with Epoetin alfa at <1500, 1500 to 2499, 2500 to 4999, 5000 to 10999, 11000 to 17999, 18000 to 33999, 34000 to 89999 or ≥90000 units per week, respectively;

[0762] b. Darbepoetin alfa or and administered at a weekly dose of less than 6.25, 10, 12.5, 20, 25, 40, 60, 100 or 200 micrograms, optionally wherein the individual has previously received 6.25, 10, 12.5, 20, 25, 40, 60, 100 or 200 micrograms per week of darbepoetin alfa or treatment; or

[0763] c. Darbepoetin alfa or and administered at a weekly dose of less than 6.25, 10, 20, 40, 60, 100 or 200 micrograms, optionally wherein the individual has previously received 1500 to 2499, 2500 to 4999, 5000 to 10999, 11000 to 17999, 18000 to 33999, 34000 to 89999 or ≥90,000 units of Epoetin alfa per week, respectively.

[0764] In an example, the ESA is

[0765] (i) Epoetin alfa administered in a weekly dose ranging from 3000 to 30000 units; or

[0766] (ii) Darbepoetin alfa or and is administered in a weekly dose ranging from 15 to 100 micrograms; and

[0767] The individual is a human, such as an adult.

[0768] In one example, the blood hemoglobin is increased to or maintained at a level above 10 g / dL.

[0769] In one example, the individual is an adult. In one example, the individual is a child. In one example, the individual is a human CKD patient undergoing dialysis treatment. In one example, the individual is a human with end-stage renal disease.

[0770] In the method of the present invention, a therapeutically or prophylactically effective amount of an antagonist and an ESA is administered to an individual. In one example, the interval between the administration of an anti-BMP6 antagonist and an ESA to an individual is no more than 10, 14, 21, or 28 days. For example, the interval between the administration of an anti-BMP6 antagonist and an ESA to an individual is no more than 1 or 2 months.

[0771] Examples of erythropoiesis stimulating agents (ESAs) are epoetin alfa, Erythropoietin, Darbepoetin alfa, Epoetin β, Methoxypolyethylene glycol-epoetin beta, and In one embodiment, the ESA of the present invention is any one of the above or a combination of two or more of the above.

[0772] In one example, the ESA comprises or consists of a recombinant erythropoietin, such as selected from the following table. Erythropoietin has various glycosylation patterns, resulting in α, β, δ and ω forms:

[0773] Table E: Exemplary erythropoietins

[0774]

[0775] In one example, the ESA of the present invention is selected from the group consisting of α, β, δ, ζ and ω forms.

[0776] In one example, ESA is a hypoxia-inducible factor-prolyl hydroxylase (HIF-PH) inhibitor, such as roxadustat or FG-4592. HIF is a major regulator of red blood cells (RBC) production in vivo and a potential new mechanism for the treatment of anemia. The new mechanism of action is called hypoxia-inducible factor-prolyl hydroxylase (HIF-PH) inhibitor. HIF-PH inhibitors work by simulating the natural response of the human body to anemia. This allows controlled adaptive stimulation of the erythropoietic system in vivo. This activation of the entire system leads to an increase in red blood cell (RBC) production and an increase in the stability of bone marrow iron supply, which ensures that the iron required for the red blood cell production is appropriately incorporated into hemoglobin. This adaptive simulation is very similar to the natural response caused by people when the altitude rises. At higher altitudes, the low oxygen content circulating in the bloodstream causes the HIF-PH activity of related cells in the kidney and liver to decrease. HIF-PH activity decreases and increases the content of intracellular 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 secretion of erythropoietin (EPO) and subsequent increased RBC production. HIF-PH inhibitors work by blocking the action of prolyl hydroxylases, which promote the decomposition of HIFα protein. As decomposition is inhibited, the content of HIFα protein in the cell increases. The HIF is the main protein mediator that enables the body and all its individual cells to adapt to changes in oxygen levels. HIFα protein is continuously produced and regulated in cells by the activity of the HIF-PH enzyme, which targets HIFα protein for degradation. HIF1α helps cells survive under extremely low oxygen conditions, while HIF2α helps cells and the body adapt to moderate oxygen changes, which occurs with altitude changes from sea level to 7,500 feet. When HIFα is stabilized, it migrates to the nucleus of the cell, where it binds to the protein HIFβ. When combined together, it induces genetic signals to produce EPO and several other proteins. HIF-PH inhibitors can greatly increase HIFα levels in the same way that reducing oxygen by inhibiting the HIF-PH enzyme in the body increases HIFα levels. As HIFα continues to be stabilized (either by staying at a higher altitude or by daily dosing with HIF-PH inhibitors), the levels of hemoglobin and RBCs will rise in order to increase the amount of oxygen circulating in the blood.

[0777] An example of an anti-BMP6 antibody is MAB507, which is commercially available from R&D Systems (monoclonal mouse IgG2B, clone #74219). Other suitable antibodies are disclosed in US8980582, WO2016098079 and US20160176956A1, the disclosures of which (and specifically the sequences of antibodies, variable regions and CDRs therein) are incorporated herein by reference for possible use in the present invention.

[0778] 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 a polypeptide of SEQ ID NO: 2, LCDR2 is a polypeptide of SEQ ID NO: 3, LCDR3 is a polypeptide of SEQ ID NO: 4, HCDR1 is a polypeptide of SEQ ID NO: 5, HCDR2 is a polypeptide of SEQ ID NO: 6 or SEQ ID NO: 7, and HCDR3 is a polypeptide of SEQ ID NO: 8. The SEQ ID NOs in the paragraph are those disclosed in US8980582, and the sequences are expressly incorporated herein by reference for possible use in the present invention and may be included in one or more embodiments of the present invention.

[0779] 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 a polypeptide of SEQ ID NO: 2, LCDR2 is a polypeptide of SEQ ID NO: 3, LCDR3 is a polypeptide of SEQ ID NO: 4, HCDR1 is a polypeptide of SEQ ID NO: 5, HCDR2 is a polypeptide of SEQ ID NO: 6, and HCDR3 is a polypeptide of SEQ ID NO: 8. The SEQ ID NOs in the paragraph are those disclosed in US8980582, and the sequences are expressly incorporated herein by reference for possible use in the present invention and may be included in one or more embodiments of the present invention.

[0780] 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 a polypeptide of SEQ ID NO: 2, LCDR2 is a polypeptide of SEQ ID NO: 3, LCDR3 is a polypeptide of SEQ ID NO: 4, HCDR1 is a polypeptide of SEQ ID NO: 5, HCDR2 is a polypeptide of SEQ ID NO: 7, and HCDR3 is a polypeptide of SEQ ID NO: 8. The SEQ ID NOs in the paragraph are those disclosed in US8980582, and the sequences are expressly incorporated herein by reference for possible use in the present invention and may be included in one or more embodiments of the present invention.

[0781] In one embodiment, the antagonist comprises or consists of an antibody or antigen-binding fragment thereof that binds to human BMP-6 (SEQ ID NO: 1), comprising a LCVR and a HCVR, wherein the LCVR is a polypeptide of SEQ ID NO: 9 and the HCVR is a polypeptide of SEQ ID NO: 10 or SEQ ID NO: 11. In another embodiment, the antagonist comprises or consists of an antibody or antigen-binding fragment thereof that binds to human BMP-6 (SEQ ID NO: 1), comprising a LCVR and a HCVR, wherein the LCVR is a polypeptide of SEQ ID NO: 9 and the HCVR is a polypeptide of SEQ ID NO: 10. In another embodiment, the antagonist comprises or consists of an antibody or antigen-binding fragment thereof that binds to human BMP-6 (SEQ ID NO: 1), comprising a LCVR and a HCVR, wherein the LCVR is a polypeptide of SEQ ID NO: 9 and the HCVR is a polypeptide of SEQ ID NO: 11. The SEQ ID NOs in the paragraph are those disclosed in US8980582, and the sequences are expressly incorporated herein by reference for possible use in the present invention and may be included in one or more embodiments of the present invention.

[0782] In one embodiment, the antagonist comprises or consists of an antibody that binds to human BMP-6 (SEQ ID NO: 1), comprising a LCVR and a HCVR, wherein the LCVR is a polypeptide of SEQ ID NO: 9 and the HCVR is a polypeptide of SEQ ID NO: 10 or SEQ ID NO: 11. In another embodiment, the invention provides an antibody that binds to human BMP-6 (SEQ ID NO: 1), comprising a LCVR and a HCVR, wherein the LCVR is a polypeptide of SEQ ID NO: 9 and the HCVR is a 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 a LCVR and a HCVR, wherein the LCVR is a polypeptide of SEQ ID NO: 9 and the HCVR is a polypeptide of SEQ ID NO: 11. The SEQ ID NOs in the paragraph are those disclosed in US8980582, and the sequences are expressly incorporated herein by reference for possible use in the present invention and for possible inclusion in one or more embodiments of the present invention.

[0783] In one embodiment, the antagonist comprises or consists of an antibody that binds to human BMP-6 (SEQ ID NO: 1), comprising a light chain (LC) and a heavy chain (HC), wherein the LC is a polypeptide of SEQ ID NO: 12, and the HC is a polypeptide of SEQ ID NO: 13 or SEQ ID NO: 14. 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, wherein the LC is a polypeptide of SEQ ID NO: 12, and the HC is a 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, wherein the LC is a polypeptide of SEQ ID NO: 12, and the HC is a polypeptide of SEQ ID NO: 14. The SEQ ID NOs in the paragraph are those disclosed in US8980582, and the sequences are expressly incorporated herein by reference for possible use in the present invention and for possible inclusion in one or more embodiments of the present invention.

[0784] 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, wherein each light chain is a polypeptide of SEQ ID NO: 12, and each heavy chain is a polypeptide of SEQ ID NO: 13. In one embodiment, the antagonist comprises or consists of an antibody that binds to human BMP-6 (SEQ ID NO: 1), comprising two light chains and two heavy chains, wherein each light chain is a polypeptide of SEQ ID NO: 12, and each heavy chain is a polypeptide of SEQ ID NO: 14. The SEQ ID NOs in the paragraph are those disclosed in US8980582, and the sequences are expressly incorporated herein by reference for possible use in the present invention and may be included in one or more technical embodiments herein.

[0785] In one embodiment, the present invention provides a pharmaceutical composition comprising an anti-BMP6 antagonist of the present invention (e.g., an antibody or a BMP6 binding fragment thereof) and an acceptable carrier, diluent or excipient. More specifically, the composition of the present invention further comprises one or more than one additional therapeutic agent, such as an ESA and / or an anti-inflammatory agent. Suitable anti-inflammatory agents may be antibodies or antibody fragments, for example, anti-TNFα antibodies (e.g., adalimumab, Infliximab (infliximab), Golimumab, ); or a capture agent (e.g., etanercept or ); or an anti-TNFR antibody or antibody fragment, or an anti-IL6R antibody (e.g., sarilumab, tocilizumab, or ).

[0786] In one embodiment, the anti-BMP6 antagonist, antibody or fragment is present in an amount of less than about 1×10 -8 M, preferably less than about 1×10 -9 The KD of M binds to BMP6 as determined by common methods known in the art, for example by using a surface plasmon resonance (SPR) biosensor at 37°C.

[0787] "Effective amount" means an amount of an antagonist (e.g., antibody) or ESA of the invention or a pharmaceutical composition of the invention that will elicit the biological or medical response or desired therapeutic effect in an individual, mammal or human being that is being sought by a researcher, physician or other clinician. The effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody and / or ESA to elicit a desired response in the individual. An effective amount is also an amount in which any toxic or detrimental effects are outweighed by the therapeutically beneficial effects.

[0788] The term "treatment" etc. is intended to include slowing down or reversing the progress of a disease such as anemia, moderate anemia, severe anemia or blood hemoglobin reduction. The term also includes alleviating, improving, weakening, eliminating or reducing one or more than one symptom of a disease or condition (such as anemia, moderate anemia, severe anemia or blood hemoglobin reduction), even if the disease or condition is not actually completely eliminated. Individual or patient refers to a mammal with a disease, disease or condition (such as anemia or anemia risk), preferably a human being, which will benefit from BMP-6 activity inhibition. The term "prevention" is, for example, to reduce the risk of a disease or condition such as anemia.

[0789] The ESA, anti-BMP6 antagonist antibody or antigen-binding fragment thereof, a combination or pharmaceutical composition comprising the same, of the present invention may be administered by a parenteral route (e.g., subcutaneous, intravenous, intraperitoneal, intramuscular or transdermal). Administration may be administered to an individual in a single or multiple doses alone or in combination with a pharmaceutically acceptable carrier and / or diluent. The pharmaceutical compositions, combinations or antagonists of the present invention may be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 19th edition, (1995), A. Gennaro et al., Mack Publishing Co.) and may include or combine one or more than one pharmaceutically acceptable carrier, diluent or excipient.

[0790] In one example, the individual suffers from moderate or severe anemia before administering the BMP6 antagonist, and the moderate or severe anemia is treated. In one embodiment, the individual suffers from moderate anemia before administration and the individual has mild anemia or no anemia after treatment. In one embodiment, the individual suffers from severe anemia before administration and the individual has mild, moderate anemia or no anemia after treatment. In one embodiment, the individual has mild anemia or no anemia after treatment, rather than moderate or severe anemia. In another embodiment, the individual does not have anemia after treatment. In one embodiment, the blood hemoglobin content of the individual before administration is less than 9.5g / dL, and the blood hemoglobin content of the individual after treatment is at least 10, 11, 12, 13 or 14g / dL.

[0791] Anemia is usually considered when the hemoglobin concentration is less than 11 g / dL in pregnant women, less than 12 g / dL in nonpregnant women, and less than 13 g / dL in men.

[0792] The severity of anemia is classified according to the following hemoglobin concentration ranges:

[0793] When hemoglobin is 9.5-13.0 g / dL, consider mild anemia

[0794] When hemoglobin is 8.0-9.5 g / dL, consider moderate anemia

[0795] When the hemoglobin concentration is less than 8.0 g / dL, consider severe anemia

[0796] In one example, the hemoglobin level is at or equal to a measurement at sea level.

[0797] In one embodiment, the subject is a human male, such as an adult or an infant. In one embodiment, the subject is a human female, such as an adult or an infant, such as a non-pregnant female or a pregnant female. For example, the human is a dialysis patient. The infant may be a human > 1 month old.

[0798] In one example, the method is a method of eliminating or reducing the need to administer iron or blood transfusions to an individual suffering from anemia, for example, for reducing the dosage or frequency of administration of iron to the individual.

[0799] The present invention may include the simultaneous or sequential administration of anti-BMP6 antagonists and ESA. In one example, the antagonist and ESA administration interval is no more than 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. As illustrated herein, if the administration interval of the antagonist and ESA is no more than 7 days (e.g., no more than one day), it may be effective. In one example, the interval between the administration of anti-BMP6 antagonists and ESA to an individual is no more than 10, 14, 21 or 28 days.

[0800] In one example, ESA is administered 2, 3 or 4 times per week. In one example, ESA is administered 1, 2, 3 or 4 times per month or over an 8-week period. In one example, ESA (e.g., Epoetin alpha) is administered at a total dose of <3000, 2900, 2800, 2700, 2600, <2500, 2500, 2400, 2300, 2200, 2100, <2000, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100 or 1000 units / kg per week. In another example, ESA is administered 1, 2, 3 or 4 times per month or over an 8-week period. In one example, the ESA (eg, darbepoetin alfa) is administered at a total dose of <15, <30, 12, 11, 10, 9, 8, 7, 6, or 5 micrograms / kg per week.

[0801] In one example, the ESA and / or antagonist is administered to the subject intravenously or subcutaneously.

[0802] In one example, the anemia is in a subject receiving or having received zidovudine therapy.

[0803] Optionally, any configuration of the present invention is also one or more of the following:

[0804] (a) To increase or maintain blood iron levels, e.g. to treat or reduce the risk of anemia;

[0805] (b) Treatment of iron deficiency;

[0806] (c) Treat or reduce the risk of anemia of chronic inflammation (ACI);

[0807] (d) Treat or reduce the risk of anemia of chronic disease (ACD);

[0808] (e) treating or reducing the risk of anemia associated with cancer, renal pathology or GvHD;

[0809] (f) increased blood or serum iron levels;

[0810] (g) Increased reticulocyte count;

[0811] (h) Increased red blood cell count;

[0812] (i) increase hemoglobin; and

[0813] (j) increasing the hematocrit in an individual (e.g., a human).

[0814] In one embodiment, the invention is used to modulate (eg, increase) erythropoiesis in an individual.

[0815] In one embodiment, the individual is a human comprising the BMP6 gene SNP rs111588693. This may be associated with an increased tendency to anemia.

[0816] In one example, the anemia is anemia of chronic disease (ACD), such as anemia of cancer, or anemia of chronic kidney disease (CKD). Certain chronic diseases, such as cancer, kidney disease, and autoimmune disorders, can lead to ACD when overactive inflammatory cytokines cause dysregulated iron homeostasis, decreased erythropoiesis, and shortened red blood cell lifespan. Hepcidin has been identified as a key hormone involved in iron homeostasis; high levels of hepcidin are associated with iron-restricted erythropoiesis seen in ACD. BMP-6 has been shown to increase hepcidin expression. In one example, the present invention is used to reduce or maintain reduced hepcidin levels in an individual.

[0817] CKD anemia is anemia of early common complications of CKD patients. Cancer anemia is anemia caused by hematological malignancies and some solid tumors; however, chemotherapy-induced (e.g., immunotherapy-induced) anemia is anemia caused by treatment of cancer patients with chemotherapeutic agents. Anemia in CKD aggravates diabetic neuropathy, cardiovascular disease, and retinopathy, as well as other conditions. Anemia associated with cancer is associated with an increased relative risk of death. Current treatment options for cancer-related anemia are limited to blood transfusions, because erythropoiesis stimulating agents are only applicable to chemotherapy-induced anemia.

[0818] In one example, the individual suffers from a chronic disease, such as cancer (e.g., hematological malignancies or solid tumors), kidney disease, autoimmune disorders, or chemotherapy-induced anemia. In one example, the individual (e.g., human) suffers from CKD, and one or more of diabetic neuropathy, cardiovascular disease, and retinopathy.

[0819] In one example, anemia is hepcidin-related iron-restricted anemia. In one example, anemia is iron-refractory iron deficiency anemia (IRIDA). In one embodiment, IRIDA is caused by a TMPRSS6 gene defect in an individual, for example, wherein IRIDA is caused by a TMPRSS6 gene mutation (e.g., SNP, such as rs855791; rs2543519; rs2235324; or rs1421312).

[0820] In one example, in addition to or instead of treating or preventing anemia, the method is a method of treating or preventing Sjögren's syndrome.

[0821] In one example, the invention is used to increase blood iron levels, serum iron levels, reticulocyte count, red blood cell count, hemoglobin, and / or hematocrit in an individual (eg, a human).

[0822] In one embodiment, the present invention provides the use of anti-BMP6 antagonists and ESA for the manufacture of medicines. In another embodiment, the present invention provides the use of anti-BMP6 antagonists and ESA for the manufacture of medicines for the treatment or prevention of anemia (e.g., moderate to severe anemia). In another embodiment, the present invention provides the use of anti-BMP6 antagonists and ESA for the manufacture of medicines for the treatment of chronic disease anemia. In another embodiment, the present invention provides the use of anti-BMP6 antagonists and ESA for the manufacture of medicines for the treatment of chronic kidney disease anemia. In another embodiment, the present invention provides the use of anti-BMP6 antagonists and ESA for the manufacture of medicines for the treatment of cancer anemia. In one embodiment, the present invention provides the use of anti-BMP6 antagonists and ESA for the manufacture of medicines for the treatment of IRIDA. In another embodiment, the present invention provides the use of anti-BMP6 antagonists and ESA for the manufacture of a medicament for treating IRIDA, wherein IRIDA is caused by a TMPRSS6 gene mutation (e.g., SNP, such as rs855791; rs2543519; rs2235324; or rs1421312). In one embodiment, the present invention provides the use of anti-BMP6 antagonists and ESA for the manufacture of a medicament for treating Sjögren's syndrome.

[0823] Implementation

[0824] The embodiments of the present invention are as follows, and the embodiments (and any unnumbered paragraphs) can be combined with any other configuration, clause, paragraph, example, feature or aspect of the present invention as described herein; the antagonists (e.g., anti-BMP6 antibodies or fragments) or ESAs of the present invention can be provided for (or can be used for) the methods in the following embodiments:

[0825] 1. An anti-bone morphogenetic protein 6 (BMP6) antagonist for use in a method for treating or preventing anemia in an individual, the method comprising administering the anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA) to the individual, wherein the anemia is treated or prevented.

[0826] In one embodiment, the method is for treating anemia in an individual, wherein the anemia is treated.

[0827] In one example, the antagonist comprises or consists of an anti-BMP6 antibody or fragment, such as a human, humanized or chimeric antibody. As an alternative to antibodies or fragments, the present invention encompasses different BMP6 antagonists, such as an anti-BMP6 capture agent or HJV-Fc.

[0828] 2. The antagonist according to embodiment 1, wherein the antagonist comprises or consists of an anti-BMP6 antibody or fragment, the method comprising:

[0829] (a) On the initial day (D 0 ) administering the anti-BMP6 antibody or fragment to the individual; and

[0830] (b) during a period of at least 3 consecutive weeks, the period commencing on D 0 , administering multiple doses of an ESA, wherein throughout the duration of the period, the blood Hb concentration in the individual is reduced compared to D 0 The baseline concentration increased when

[0831] So that:

[0832] (i) throughout the duration of the period, the Hb concentration does not fall below 100% of the baseline Hb concentration; and during the period, the Hb concentration reaches at least 120% of the baseline; and / or

[0833] (ii) the Hb concentration increases by at least 1 g / dl compared to baseline throughout the duration of the period.

[0834] In any embodiment herein, in the example of the entire duration of the period, the Hb concentration is increased by at least 1 g / dl, e.g., at least 1.5, 2, or 2.5 g / dl compared to the baseline. In one example, the Hb concentration does not exceed 11, 11.5, or 12 g / dl in an individual (e.g., an adult male or female human).

[0835] Hb concentration and MCH (see below) can be determined using one or more blood samples obtained from the individual. For example, using blood samples taken at the end of each week of the period (and using blood samples taken at D 0 The baseline is determined by the samples obtained at the time of the test.

[0836] 3. A combination of an amount of an anti-BMP6 antibody or fragment and an amount of an ESA (eg, comprising multiple doses of said ESA) for use in a method for treating anemia, wherein said antibody, fragment and method are according to embodiment 2.

[0837] 4. The combination according to embodiment 3, wherein the method comprises obtaining a single dose from said amount of antibody or fragment, wherein in D 0 administering the single dose to the individual at D 0 Apply weekly.

[0838] In any of the embodiments herein, in one example, in D 0 Administer the first dose of ESA at .

[0839] 5. A combination according to embodiment 3 or 4, wherein the antibody or fragment is comprised of a pharmaceutical composition, wherein the antibody or fragment is mixed with a dose of the ESA to be administered at D 0 administered to the individual.

[0840] 6. A medical kit comprising the combination according to any one of embodiments 3 to 5, a first sterile container comprising said amount of antibody or fragment, and a second sterile container comprising said amount of ESA, and optionally instructions for carrying out said method.

[0841] 7. The antagonist according to embodiment 1, wherein the antagonist comprises or consists of an erythropoietin stimulating agent (ESA) (e.g., comprises multiple doses of the ESA), the method comprising:

[0842] (a) On the initial day (D 0 ) administering an anti-BMP6 antibody or fragment to said individual; and

[0843] (b) during a period of at least 3 consecutive weeks, the period commencing on D 0 , administering multiple doses of said ESA, wherein throughout the duration of said period, the blood hemoglobin (Hb) concentration in said individual is reduced by 10% compared to D 0 The baseline concentration increased when

[0844] So that:

[0845] (i) throughout the duration of the period, the Hb concentration does not fall below 100% of the baseline Hb concentration; and during the period, the Hb concentration reaches at least 120% of the baseline; and / or

[0846] (ii) the Hb concentration increases by at least 1 g / dl compared to baseline throughout the duration of the period.

[0847] 8. An antagonist, combination or kit according to any one of embodiments 2 to 7, wherein

[0848] (iii) the Hb concentration on the last day of the consecutive week period is at least 120% of the Hb concentration on the 7th day immediately before the last day.

[0849] 9. The antagonist, combination or kit according to any one of embodiments 2 to 8, wherein an ESA is administered to said individual within 24 hours of administration of said anti-BMP6 antibody or fragment.

[0850] 10. An antagonist, combination or kit according to any one of embodiments 2 to 9, wherein said period of consecutive cycles consists of a period of 3 or 4 consecutive weeks.

[0851] 11. An antagonist, combination or kit according to any one of embodiments 2 to 10 (eg embodiment 10), wherein the Hb concentration during said period achieves an increase over baseline in the range of 1 to 8 g / dl.

[0852] In any of the embodiments herein, in examples within the time period, the Hb concentration achieves an increase ranging from 1 to 3, 2.5, 2, 1.5 or 1.25 g / dl compared to baseline. For example, the Hb concentration achieves an increase of 1 to 2 g / dl.

[0853] 12. An antagonist, combination or kit according to any one of embodiments 2 to 11, wherein said period consists of 3 or 4 consecutive weeks and at the end of said period the Hb concentration reaches at least 150% of the baseline.

[0854] 13. An antagonist, combination or kit according to any one of embodiments 2 to 12, wherein the period consists of 3 or 4 consecutive weeks, and

[0855] (a) throughout the duration of the period, the Hb concentration does not fall below 110% of the baseline Hb concentration; and during the period, the Hb concentration reaches at least 150% of the baseline; and / or

[0856] (b) The Hb concentration increases by at least 1 g / dl compared to the baseline over the entire duration of the period, and the Hb concentration achieves an increase in the range of 1 to 8 g / dl compared to the baseline during the period. For example, the Hb concentration achieves an increase from 1 g / dl to 2 g / dl.

[0857] 14. An antagonist, combination or kit according to any one of embodiments 2 to 13, wherein during said period, at D 0 The antibody or fragment is administered at the time of administration.

[0858] 15. An antagonist, combination or kit according to embodiment 14, wherein in D 0 The antibody or fragment is administered to the individual in a single dose.

[0859] In any of the embodiments herein, in one example, in D 0 The antibody or fragment is administered to the individual in a single dose, wherein the single dose is administered to the individual in one or more divided doses.

[0860] 16. An antagonist, combination or kit according to any one of embodiments 2 to 15, wherein the initial ESA dose is in the range of D 0 Or no later than 2 days afterward.

[0861] 17. An antagonist, combination or kit according to any one of embodiments 2 to 16, wherein the ESA dose is D 0 Administer on day 4-9 thereafter (eg, day 7).

[0862] 18. An antagonist, combination or kit according to any one of embodiments 2 to 17, wherein the ESA dose is D 0 Administer on day 12-16 thereafter (eg day 14).

[0863] 19. An antagonist, combination or kit according to any one of embodiments 2 to 18, wherein the ESA dose is D 0 Administer on day 19-23 thereafter (eg, day 21).

[0864] In any of the embodiments herein, in one example, the ESA dose is D 0 Thereafter, administration is performed on each of (i) days 4-9 (eg, day 7), (ii) days 12-16 (eg, day 14), and (iii) days 19-23 (eg, day 21).

[0865] 20. The antagonist, combination or kit according to any one of embodiments 2 to 19, wherein the equivalent of 4 ESA doses are administered within said period.

[0866] Here, "equivalent" means that multiple equally divided doses of the ESA can be administered (e.g., on the same day or sequentially), where the divided doses add up to the total dose of the ESA. In one example, the ESA is darbepoetin alfa or and the dosage range is 15 to 100 mcg (micrograms); or 30 to 100 micrograms. In one example, the ESA is epoetin alfa and the dosage range is 3000 to 30000 units (ie, units refer to international units, also known as IU, UI, IE, ME, NE in various languages).

[0867] Generally speaking herein, a dose (of, for example, an antibody, fragment or ESA) may be administered in one aliquoted dose or in multiple aliquoted doses (eg, on the same day, at the same time, within a 30, 1 or 24 hour window).

[0868] 21. The antagonist, combination or kit according to any one of embodiments 2 to 20, wherein the ESA is administered to the individual during each of the first and second weeks following the initial ESA dose.

[0869] 22. The antagonist, combination or kit according to any one of embodiments 2 to 21, wherein the ESA is administered to the individual during each of the first week, the second week and the third week after the initial ESA dose.

[0870] 23. The antagonist, combination or kit according to embodiment 21 or 22, wherein the ESA is administered as a single dose at the end of each of said weeks, optionally wherein said period starts at D 0 Composition of 3 or 4 weeks.

[0871] In an example of any embodiment, the period is from D 0 The 4-week regimen was completed and anemia was cured in the 4th week.

[0872] 24. The antagonist, combination or kit according to any one of embodiments 2 to 23, wherein no more than 4 doses of an ESA and optionally a single dose of said antibody or fragment are administered to said individual within said period.

[0873] 25. The antagonist, combination or kit according to any one of embodiments 2 to 24, wherein during the period (wherein the period is a continuous 4-week period), the total dose of the antibody and the total dose of the ESA are administered to the individual at a ratio of X:Y, wherein X is 10 to 2×10 6 And Y=4.

[0874] In one example, the total weekly dose of the ESA (e.g., where the subject is a human) is 10 or 15 to 80, 100, 200, or 300 mcg (micrograms). For example, the total weekly dose is 10 to 80; 15 to 80; or 30 to 80 micrograms. For example, the ESA comprises or consists of darbepoetin alpha, epoetin alpha, or any other ESA disclosed herein. In one example, each dose (or weekly dose) of the ESA administered to the subject is in the range of 1.5 to 2 micrograms / kg of ESA.

[0875] In certain configurations, the method involves reducing or sparing the administration of ESA. In such cases, for example, the total weekly dose of ESA (e.g., where the individual is a human) is 1 to 20 micrograms, for example, 1 to 15 micrograms. In instances where ESA is sparingly or reduced, each dose (or weekly dose) of ESA administered to the individual is within the range of 0.01 or 0.1 to 0.3 or 1 microgram / kg ESA, for example, 0.01 to 0.3; or 0.1 to 0.3; or 0.01 to 1; or 0.1 to 1 microgram / kg.

[0876] 26. An antagonist, combination or kit according to any one of embodiments 2 to 25, wherein the Hb concentration at the end of the period (e.g., a period of 3 or 4 consecutive weeks) is at least 130% of the Hb concentration in a control anemic patient of the same species who has received administration of an anti-BMP6 antibody or fragment in the same dosing regimen as the individual, with the exception that the patient has not received administration of an ESA during the period.

[0877] 27. An antagonist, combination or kit according to embodiment 26, wherein said Hb concentration at the end of said period is significantly higher than said control at the end of said period as determined by a p value of p<0.0001.

[0878] 28. An antagonist, combination or kit according to any one 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 anemic patient of the same species who has received administration of an anti-BMP6 antibody or fragment in the same dosing regimen as the individual, with the exception that the patient has not received administration of an ESA during the period.

[0879] Mean corpuscular hemoglobin (MCH) is the average mass of hemoglobin per red blood cell in a blood sample.

[0880] 29. An antagonist, combination or kit according to any one of embodiments 2 to 28, wherein the Hb concentration at the end of the period (e.g., a continuous period of 3 or 4 weeks) is at least 120% of the Hb concentration in a control anemic patient of the same species, the control anemic patient having received the ESA administration in the same dosing regimen as the individual, with the exception that the patient has not received anti-BMP6 antibody or fragment administration during the period. Optionally, the control patient has received a control IgG4 antibody that does not specifically bind to BMP6 (e.g., wherein the BMP6 antibody and the control antibody are administered to the individual and the control patient, respectively, at the same dose). Optionally, X is 10 to 2×10 5 , 2×10 4 or 2×10 3 .

[0881] 30. An antagonist, combination or kit according to embodiment 29, wherein said Hb concentration at the end of said period is significantly higher than said control at the end of said period as determined by a p value of p<0.0001.

[0882] 31. An antagonist, combination or kit according to any one of embodiments 2 to 30, wherein the mean corpuscular hemoglobin (MCH) at the end of the period (e.g., a continuous 3 or 4 week period) is at least 119% of the MCH in a control anemic patient of the same species, who has received administration of the ESA in the same dosing regimen as the individual, with the exception that the patient has not received administration of an anti-BMP6 antibody or fragment during the period.

[0883] 32. An antagonist, combination or kit according to embodiment 31, wherein said MCH at the end of said period is significantly higher than said control at the end of said period as determined by a p value of p<0.0001.

[0884] 33. An antagonist, combination or kit according to any one of embodiments 2 to 32, wherein the subject is in D 0 The subject is a patient suffering from anemia of chronic disease (ACD) and optionally wherein the anemia is associated with chronic inflammation (e.g. the subject suffers from arthritis) or a bacterial infection (e.g. a streptococcal infection), or wherein the subject is a chronic kidney disease (CKD) patient.

[0885] 34. An antagonist, combination or kit according to any one of embodiments 2 to 33, wherein at the end of said period, the anemia in said individual is less than D 0 When serious.

[0886] 35. The antagonist according to embodiment 1, wherein the antagonist comprises or consists of an anti-BMP6 antibody or fragment.

[0887] 36. An antagonist, combination or kit according to any one of embodiments 2 to 34, wherein the antibody or fragment competes with a reference antibody for binding to BMP6, wherein the reference antibody is mAb507 (R&D Systems) or an antibody comprising:

[0888] a. a heavy chain, each comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or 2, and a light chain, each comprising or consisting of the amino acid sequence of SEQ ID NO: 3; or

[0889] b. heavy chains, each comprising or consisting of the amino acid sequence of SEQ ID NO: 4, and light chains, each comprising or consisting of the amino acid sequence of SEQ ID NO: 5.

[0890] Competition herein can be determined, for example, by SPR (e.g., at 37°C at pH 7.6 and optionally as a Fab); by ELISA; by fluorescence activated cell sorting (FACS); or in a homogeneous time-resolved fluorescence (HTRF) assay. SPR can be determined using Biacore TM 、Proteon TM Or another standard SPR technique. In one embodiment, the ForteBio Competition is determined by string-layer interferometry (BLI), techniques which will be apparent to those skilled in the art.

[0891] In an alternative, the reference antibody is any anti-BMP6 antibody disclosed in WO2016098079 (the sequences and disclosures concerning said antibodies are incorporated herein for possible use in the present invention).

[0892] 37. An antagonist, combination or kit according to any one of embodiments 2 to 36, wherein the antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 6. The SEQ ID NO: 6 can be used as a peptide itself, as a part of a larger peptide or as a part of a BMP6 protein (e.g. wild-type human BMP6 or recombinantly produced BMP6).

[0893] Additionally or alternatively, the antibody or fragment competes with the reference antibody for binding to another sequence selected from the group consisting of SEQ ID NOs: 7-19. The other sequence can be used in the form of a peptide itself, a portion of a larger peptide (e.g., comprising SEQ ID NO: 6), or a portion of a BMP6 protein (e.g., wild-type human BMP6 or recombinantly produced BMP6, e.g., comprising SEQ ID NO: 6). For example, the antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 7. For example, the antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 8. For example, the antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 9. For example, the antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 10. For example, the antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 11. For example, the antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 12. For example, the antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 13. For example, the antibody or fragment competes with the reference antibody for binding to SEQ ID NO: 14. For example, 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 the paragraphs and embodiments herein are those disclosed in WO2017191437, the disclosure of which is incorporated herein.

[0894] 38. An antagonist, combination or kit according to any one of embodiments 2 to 37, wherein the antibody or fragment competitively inhibits the binding of soluble hemojuvelin (HJV) to BMP6.

[0895] 39. An antagonist, combination or kit according to any one of embodiments 1 to 38, wherein the antibody or fragment does not competitively inhibit the binding of soluble hemojuvelin (HJV) to BMP6 (eg as determined by SPR, HTRF or ELISA).

[0896] 40. An antagonist, combination or kit according to any one of embodiments 2 to 39, wherein the antibody comprises a VH domain encoded by a VDJ region sequence, wherein the VDJ is derived from the recombination of a VH gene segment, a D gene segment and a JH gene segment, wherein the VH is a human germline (i) VH1-3, (ii) VH2-5 or (iii) VH3-15 gene segment.

[0897] 41. An antagonist, combination or kit according to any one of embodiments 2 to 40, wherein the antibody comprises a VL domain encoded by a VJ region sequence, wherein the VJ is derived from the recombination of a VL gene segment and a JL gene segment, wherein the VL is human germline (iv) Vκ3-20, (v) Vλ3-1, (vi) Vκ1-17 or (vii) Vλ1-40.

[0898] 42. An antagonist, combination or kit according to any one of embodiments 2 to 41, wherein the antibody or fragment binds to BMP6 with a stronger affinity (lower KD determined by SPR) than to BMP7; and optionally binds to BMP6 with a stronger affinity than to BMP5.

[0899] Optionally, the antibody or fragment binds to BMP6 with a stronger affinity than to each of BMP2, 4, 5, and 9.

[0900] 43. An antagonist, combination or kit according to any one of embodiments 2 to 42, wherein said antibody or fragment binds to a human BMP6 sequence comprising SEQ ID NO:6.

[0901] 44. An antagonist, combination or kit according to any one of embodiments 2 to 43, wherein the antibody binds to BMP6 with an affinity (KD) of 1 pM to 5 nM, optionally wherein binding is determined by SPR using a Fab of the antibody at 37°C at pH 7.6.

[0902] In one example, the antibody (eg, as a Fab) or fragment binds to BMP6 with an affinity (KD) of

[0903] (a) 2, 3, 4, 5 or 10 pM to 3, 4 or 5 nM;

[0904] (b) 1-10 pM to 5 nM;

[0905] (c) 10 pM to 3, 4 or 5 nM;

[0906] (d) 50 or 80 pM to 200 nM;

[0907] (e) 50 or 80 pM to 150 nM; or

[0908] (f) 50 or 80 pM to 100 nM.

[0909] In one example, the KD is (or is about) 5-15 pM (e.g., 10 pM). In one example, the KD is (or is about) 2-5 nM (e.g., 3 nM). In one example, the KD is (or is about) 100-400 pM (e.g., 140 or 390 pM).

[0910] 45. An antagonist, combination or kit according to any one of embodiments 2 to 44 (eg, embodiment 44), wherein the antibody has a dissociation rate (K off ) is 1×10 -5 to 1×10 -3 S -1 , optionally wherein binding is determined by SPR using a Fab of said antibody at 37°C at pH 7.6.

[0911] In one example, the antibody (e.g., as a Fab) or fragment has a dissociation rate (K) of binding to BMP6. off )for

[0912] (a)1×10 -5 to 5×10 -4 S -1 ;

[0913] (b)1×10 -5 to 6×10 -4 S -1 ;

[0914] (c)1×10 -5 to 7×10 -4 S -1 ;

[0915] (d)1×10 -5 to 8×10 -4 S -1 ;

[0916] (e)2×10 -5 to 1×10 -3 S -1 ;

[0917] (f)2×10 -5 to 5×10 -4 S -1 ;

[0918] (g)2×10 -5 to 6×10 -4 S -1 ;

[0919] (h)2×10 -5 to 7×10 -4 S -1 ;or

[0920] (i)2×10 -5 to 8×10 -4 S -1 .

[0921] In one example, K off is (or about) 5×10 -4 S -1 (For example, when KD is (or is about) 2 nM to 400 pM; when KD is (or is about) 2-5 nM (for example, 3 nM); or when KD is (or is about) 100-400 pM (for example, 140 or 390 pM). In one example, K off is (or about) 3×10 -5 S -1 (For example, when KD is (or is about) 5-15 pM (eg, 10 pM)).

[0922] 46. ​​An antagonist, combination or kit according to any one of embodiments 2 to 45 (eg, embodiments 44 and / or 45), wherein the binding rate (K) of the antibody to BMP6 is on ) is 1×10 5 to 1×10 7 M -1 S -1 , optionally wherein binding is determined by SPR using a Fab of said antibody at 37°C at pH 7.6.

[0923] In one example, the antibody (eg, as a Fab) or fragment has an on-rate (K) for binding to BMP6. on )for

[0924] (a)1×10 5 to 1×10 6 M -1 S -1 ;

[0925] (b)1×10 5 to 2×10 6 M -1S -1 ;

[0926] (c)1×10 5 to 3×10 6 M -1 S -1 ;

[0927] (d)1×10 5 to 4×10 6 M -1 S -1 ;

[0928] (e)1×10 5 to 5×10 6 M -1 S -1 ;

[0929] (f)2×10 5 to 5×10 6 M -1 S -1 ;

[0930] (g)3×10 5 to 5×10 6 M -1 S -1 ;

[0931] (h)4×10 5 to 5×10 6 M -1 S -1 ;

[0932] (i)5×10 5 to 5×10 6 M -1 S -1 ;or

[0933] (j)6×10 5 to 5×10 6 M -1 S -1 .

[0934] In one example, K on is (or is approximately) 1 or 2×10 -5 M -1 S -1 (For example, when KD is 2-5nM (for example, 3nM)). In one example, K on is (or is about) 1-4, 1, 2, 3 or 4×10 -6 M -1 S -1(For example, when KD is (or is about) 5-400 pM (eg, 140 or 390 pM) or 5-15 pM (eg, 10 pM)).

[0935] 47. An antagonist, combination or kit according to any one of embodiments 2 to 46, wherein

[0936] (a) the period consists of 3 or 4 consecutive weeks, and

[0937] (i) throughout the duration of the period, the Hb concentration does not fall below 110% of the baseline Hb concentration; and during the period, the Hb concentration reaches at least 120% of the baseline; and / or

[0938] (ii) an increase in Hb concentration of at least 1 g / dl over baseline over the entire duration of the period, and an increase in Hb concentration over baseline of 1 to 8 g / dl is achieved during the period;

[0939] (b) wherein the dose of the ESA is administered at least twice within two weeks or three weeks prior to said period;

[0940] (c) wherein the antibody or fragment binds to BMP6 with a stronger affinity (lower KD as determined by SPR) than to BMP7; and optionally binds to BMP6 with a stronger affinity than to BMP5 (and optionally binds to BMP6 with a stronger affinity than to each of BMP2, 4, 5 and 9); and

[0941] (d) wherein the antibody or fragment competes with a reference antibody for binding to BMP6, wherein the reference antibody is mAb507 (R&D Systems), or an antibody comprising:

[0942] I. a heavy chain, each comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or 2, and a light chain, each comprising or consisting of the amino acid sequence of SEQ ID NO: 3; or

[0943] II. heavy chains, each comprising or consisting of the amino acid sequence of SEQ ID NO:4, and light chains, each comprising or consisting of the amino acid sequence of SEQ ID NO:5.

[0944] 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.

[0945] In one example (according to the antibodies used in Example 2 below), in part (d), the anti-BMP6 antibody of the 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 invention is a labeled antibody that is pre-incubated with human BMP6 and then combined with an unlabeled reference antibody (according to part I or II), wherein the competition between the antibodies is detected by the assay. In one example, the assay uses AlexaFluor TM In an alternative embodiment, human BMP6 is labeled (e.g., with AlexaFluor TM 647, the test antibody was labeled with biotin to bind Eu3+ cryptate-streptavidin, and the reference antibody was unlabeled).

[0946] Optionally, an anti-BMP6 antibody of the invention (test antibody) competes with a reference antibody for binding to human BMP6 (or to the same epitope of human BMP6 as the reference antibody) in an HTRF assay, wherein the assay uses a fluorophore that directly or indirectly utilizes a donor (such as, for example, a Eu3+ cryptate) or an acceptor fluorophore (such as, for example, an AlexaFluor TM 647) labeled directly or indirectly labeled test antibody and a target BMP6 labeled with a donor or acceptor fluorophore to achieve energy transfer between the donor and the acceptor, thereby generating and detecting a fluorescent signal. In one example, AlexaFluor TM 647 label, competition is detected when the fluorescence signal of the test antibody at 665nM in the presence of the reference antibody is reduced by at least 20% compared to the signal in the absence of the reference antibody. Optionally, the signal reduction at 665nM is at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

[0947] Optionally, the anti-BMP6 antibody (test antibody) is an antibody that competes with a reference antibody for binding to human BMP6 (or binds to the same antigenic determinant of human BMP6 as the reference antibody) in an HTRF assay, wherein the reference antibody comprises a heavy chain each comprising the amino acid sequence of SEQ ID NO: 1 or 2 and a light chain each comprising the amino acid sequence of SEQ ID NO: 3, wherein the assay uses a fluorescent marker using a donor label (such as, for example, Eu3+ cryptate) or an acceptor fluorophore label (such as, for example, AlexaFluor TM 647) A test antibody directly or indirectly labeled and human BMP6 labeled with an acceptor fluorophore or a donor to achieve energy transfer between the donor and the acceptor, wherein the competition between the antibodies is detected by a fluorescence signal of the test antibody in the presence of a reference antibody being reduced by at least 20% compared to the signal in the absence of the reference antibody. For example, the test antibody is labeled with AlexaFluor TM The competition is detected by a fluorescence signal of the test antibody at 665 nM in the presence of the reference antibody compared to a signal in the absence of the reference antibody of at least 20%. Optionally, the signal reduction at 665 nM is at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

[0948] Optionally, the anti-BMP6 antibody (test antibody) also competes with a reference antibody for binding to human BMP6 (or binding to the same antigenic determinant of human BMP6 as the reference antibody) in an HTRF assay, wherein the reference antibody comprises a heavy chain each comprising the amino acid sequence of SEQ ID NO: 4 and a light chain each comprising the amino acid sequence of SEQ ID NO: 5, wherein the assay, for example, uses a fluorescent marker using a donor label (such as, for example, Eu3+ cryptate) or an acceptor fluorophore label (such as, for example, AlexaFluor TM 647) A test antibody directly or indirectly labeled and human BMP6 labeled with an acceptor fluorophore or a donor to achieve energy transfer between the donor and the acceptor, wherein the competition between the antibodies is detected by a fluorescence signal of the test antibody in the presence of a reference antibody being reduced by at least 20% compared to the signal in the absence of the reference antibody. For example, the test antibody is labeled with AlexaFluor TM The competition is detected by a fluorescence signal of the test antibody at 665 nM in the presence of the reference antibody compared to a signal in the absence of the reference antibody of at least 20%. Optionally, the signal reduction at 665 nM is at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

[0949] For example, in part (b), the dose of the ESA is administered 2, 3 or 4 times within 3 weeks prior to the period or during the period.

[0950] In an alternative, the reference antibody is any anti-BMP6 antibody disclosed in WO2016098079 (the sequences and disclosures concerning said antibodies are incorporated herein for possible use in the present invention).

[0951] 48. An antagonist, combination or kit according to any one of embodiments 1 to 47 for use

[0952] a. Treating ACD in individuals;

[0953] b. Treating or preventing moderate or severe anemia in an individual;

[0954] c. treating or preventing anemia in a subject, wherein the subject suffers from an inflammatory disease or condition;

[0955] d. Eliminating or reducing the need to administer iron or blood transfusions to an individual;

[0956] e. treating or preventing anemia in a subject suffering from a microbial infection; or

[0957] f. Reducing the individual's ESA administration.

[0958] 49. A method of treating anemia in an individual, the method comprising:

[0959] (a) On the initial day (D 0 ) administering an anti-BMP6 antibody or fragment to said individual; and

[0960] (b) during a period of at least 3 consecutive weeks, the period commencing on D 0 , administering multiple doses of an erythropoietin stimulating agent (ESA), wherein throughout the duration of said period, the blood hemoglobin (Hb) concentration in said individual is reduced compared to D 0 The baseline concentration increased when

[0961] Such that for the entire duration of the period:

[0962] (i) the Hb concentration does not fall below 100% of the baseline Hb concentration; and during the period, the Hb concentration reaches at least 120% of the baseline; and / or

[0963] (ii) Hb concentration increased by at least 1 g / dl compared to baseline.

[0964] 50. The method according to embodiment 49, wherein the method, antibody fragment or ESA is as described in any one of embodiments 2 to 48.

[0965] 51. The antagonist, combination, kit or method according to any preceding embodiment, wherein the anemia is moderate or severe anemia.

[0966] 52. An anti-bone morphogenetic protein 6 (BMP6) antagonist for use in a method of maintaining a blood hemoglobin (Hb) level of at least 10 g / dL in an individual, the method comprising administering to the individual an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA).

[0967] Any anti-BMP6 antibody or fragment of the present invention can be used as an anti-bone morphogenetic protein 6 (BMP6) antagonist.

[0968] 53. An anti-bone morphogenetic protein 6 (BMP6) antagonist for use in a method of preventing a subject's blood hemoglobin level from falling to less than 10 g / dL, the method comprising administering to the subject an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA).

[0969] 54. An anti-bone morphogenetic protein 6 (BMP6) antagonist for use in a method of raising blood hemoglobin to a level of at least 10 g / dL in an individual suffering from anemia, the method comprising administering to the individual an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA), wherein the anemia is treated.

[0970] 55. An anti-bone morphogenetic protein 6 (BMP6) antagonist for use in a method of treating or preventing anemia in an individual suffering from an inflammatory disease or condition, the method comprising administering an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA) to the individual, wherein the anemia is treated or prevented.

[0971] 56. An anti-bone morphogenetic protein 6 (BMP6) antagonist for use in a method of eliminating or reducing the need for administration of iron or blood transfusion to an individual suffering from anemia, the method comprising administering to the individual an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA), wherein the need is eliminated or reduced.

[0972] 57. An anti-bone morphogenetic protein 6 (BMP6) antagonist for use in a method of treating or preventing anemia in an individual suffering from a microbial infection, the method comprising administering to the individual an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA).

[0973] 58. An anti-bone morphogenetic protein 6 (BMP6) antagonist for use in a method of reducing the administration of an erythropoiesis stimulating agent (ESA) to an individual suffering from anemia to treat anemia, the method comprising administering an anti-BMP6 antagonist and the ESA, wherein the anemia in the individual is treated.

[0974] 59. An anti-bone morphogenetic protein 6 (BMP6) antagonist for use in a method for treating or reducing the risk of anemia in an individual suffering from anemia or at risk of anemia, the method comprising administering an anti-BMP6 antagonist and a low dose of an erythropoiesis stimulating agent (ESA) to the individual, wherein the anemia in the individual is treated or the risk of anemia is reduced.

[0975] 60. The antagonist according to any one...

Claims

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

302.

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

3. The antibody or fragment of claim 1 or 2, wherein the antibody or fragment comprises a VH domain amino acid sequence of SEQ ID NO: 114 paired with a VL domain amino acid sequence of SEQ ID NO:

123.

4. The antibody or fragment according to claim 1 or 2, wherein the antibody or fragment comprises (i) the VH domain amino acid sequence of SEQ ID NO: 114; and The VL domain amino acid sequence of SEQ ID NO: 123; or (ii) the VH domain amino acid sequence of SEQ ID NO: 294; and the VL domain amino acid sequence of SEQ ID NO: 303; Optionally, the antibody or fragment comprises a VH domain amino acid sequence of SEQ ID NO: 294 paired with a VL domain amino acid sequence of SEQ ID NO:

303.

5. The antibody or fragment of claim 1 or 2, comprising a first copy and a second copy of the VH domain.

6. The antibody or fragment according to claim 1 or 2, wherein the VL domain is encoded by a nucleotide sequence derived from the recombination of a human VL gene segment and a JL gene segment, wherein (a) the VL gene segment is selected from IGKV3-20; and / or (b) The JL gene segment is selected from IGKJ1.

7. The antibody or fragment of claim 1 or 2, comprising a first copy and a second copy of the VL domain.

8. The antibody or fragment according to claim 1 or 2, comprising (i) a heavy chain amino acid sequence of SEQ ID NO: 116; and (ii) a light chain sequence of SEQ ID NO:

125.

9. The antibody or fragment according to claim 1 or 2, comprising a heavy chain amino acid sequence of SEQ ID NO: 296; and a light chain sequence of SEQ ID NO:

305.

10. The antibody or fragment according to claim 1 or 2, wherein the antibody or fragment comprises a human constant region, optionally an IgG4 constant region or an IgG1 constant region.

11. The antibody or fragment according to claim 10, wherein the constant region is an IgG4-PE constant region, and optionally the amino acid sequence of the constant region is shown in SEQ ID NO:

454. 12 . A bispecific antibody or fragment comprising the antibody or fragment specifically binding to BMP6 according to claim 1 or 2 .

13. The bispecific antibody or fragment of claim 12, which specifically binds to (i) BMP6 and (ii) human hemojuvelin, transferrin receptor or another BMP receptor.

14. The bispecific antibody or fragment of claim 12, which specifically binds to (i) BMP6 and (ii) BMP2, 4, 7 or 9.

15. The antibody or fragment according to claim 1 or 2, for use in treating or preventing anemia.

16. The antibody or fragment of claim 15, wherein the antibody or fragment is administered to the individual simultaneously or sequentially with an erythropoietin stimulating agent (ESA).

17. A pharmaceutical composition comprising an amount of an antibody or fragment and a pharmaceutically acceptable excipient, diluent or carrier, and an amount of ESA, wherein the antibody or fragment is according to any preceding claim.

18. A kit comprising a first sterile container comprising an amount of the antibody or fragment according to any one of claims 1 to 16, and a second sterile container comprising an amount of ESA.

19. The pharmaceutical composition of claim 17 or the kit of claim 18, wherein the composition or kit comprises multiple doses of the antibody and / or ESA.

20. The antibody or fragment according to claim 1 or 2, or the pharmaceutical composition according to claim 17, or the kit according to claim 18, for use in the following method: (a) preventing a subject's blood hemoglobin level from falling to less than 10 g / dL, the method comprising administering to the subject the antibody or fragment and an erythropoiesis stimulating agent (ESA); (b) raising blood hemoglobin to a level of at least 10 g / dL in an individual suffering from anemia, the method comprising administering to the individual the antibody or fragment and an erythropoiesis stimulating agent (ESA), wherein the anemia is treated; (c) treating or preventing anemia in a subject suffering from an inflammatory disease or condition, the method comprising administering to the subject the antibody or fragment and an erythropoiesis stimulating agent (ESA), wherein the anemia is treated or prevented; (d) eliminating or reducing the need for administration of iron or blood transfusion to an individual suffering from anemia, the method comprising administering to the individual the antibody or fragment and an erythropoiesis stimulating agent (ESA), wherein the need is eliminated or reduced; (e) treating or preventing anemia in an individual suffering from a microbial infection, the method comprising administering to the individual an anti-BMP6 antagonist and an erythropoiesis stimulating agent (ESA); (f) reducing the administration of an erythropoiesis stimulating agent (ESA) to an individual suffering from anemia to treat the anemia, the method comprising administering the antibody or fragment and the ESA, wherein the anemia in the individual is treated; or (g) treating or reducing the risk of anemia in an individual suffering from anemia or at risk of anemia, the method comprising administering to the individual the antibody or fragment and a low dose of an erythropoiesis stimulating agent (ESA), wherein the anemia in the individual is treated or the risk of anemia is reduced.

21. The pharmaceutical composition according to claim 17 or the kit according to claim 18, wherein the ESA is a. Epoetin alfa and administered at a weekly dose of less than 1000, 1500, 2500, 5000, 11000, 18000, 34000 or 90000 units, optionally wherein the individual has previously received < 1500, 1500 to 2499, 2500 to 4999, 5000 to 10999, 11000 to 17999, 18000 to 33999, 34000 to 89999 or ≥ 90000 units of Epoetin alfa per week, respectively; b. darbepoetin alfa and administered at a weekly dose of less than 6.25, 10, 12.5, 20, 25, 40, 60, 100 or 200 micrograms, optionally wherein the individual has previously been treated with 6.25, 10, 12.5, 20, 25, 40, 60, 100 or 200 micrograms of darbepoetin alfa per week, respectively; or c. darbepoetin alfa and administered at a weekly dose of less than 6.25, 10, 20, 40, 60, 100 or 200 micrograms, optionally wherein the individual has previously received 1500 to 2499, 2500 to 4999, 5000 to 10999, 11000 to 17999, 18000 to 33999, 34000 to 89999 or ≥ 90,000 units of epoetin alfa per week, respectively.

22. The antibody or fragment of claim 1 or 2 or the pharmaceutical composition of claim 17 or the kit of claim 18 for use in maintaining or increasing blood hemoglobin levels in the individual to at least 10 g / dL for at least 13 or 14 days after the individual has received the antibody or fragment and an ESA.

23. Use of the antibody or fragment of any one of claims 1 to 16, 20 and 22, or the pharmaceutical composition of any one of claims 17 and 19 to 22, or the kit of any one of claims 18 to 22 for the manufacture of a medicament for treating or preventing anemia.

24. The use according to claim 23, further comprising administering to the individual another therapeutic agent, optionally wherein the other therapeutic agent is selected from the group consisting of: (a) Intravenous iron; (b) ESA; (c) ActRIIa inhibitors; (d) ActRIIb inhibitors; (e) IL-6 or IL-6 receptor inhibitor; (f) TNF-α or TNF-α receptor inhibitors; (g) HJV inhibitors; (h) matriptase-2 (MTP2) agonist; (i) HIF-PH inhibitors; (j) transferrin receptor 2 (TFR2) inhibitors; (k) HFE inhibitors; (l) NRf2 inhibitors; (m) transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptor inhibitor; (n) Activin receptor inhibitors; (o) GDF11 inhibitors; and (p) Myostatin inhibitors.

25. The use according to claim 24, wherein (i) the ESA is EPO; (ii) the IL-6 or IL-6 receptor inhibitor is an anti-IL-6 or IL-6 receptor antibody; (iii) the TNF-α or TNF-α receptor inhibitor is an anti-TNF-α or TNF-α receptor antibody; (iv) the HJV inhibitor is an anti-HJV antibody; (v) the MTP2 agonist is a MTP2 agonist antibody; and / or (vi) The activin receptor inhibitor is an activin receptor Fc fusion.

26. A pharmaceutical composition comprising an antibody or fragment according to any one of claims 1 to 16, and a pharmaceutically acceptable excipient, diluent or carrier, and optionally in combination with another therapeutic agent as defined in claim 24 or 25.

27. Use of the pharmaceutical composition according to claim 26 in the manufacture of a medicament for treating or preventing anemia.

28. A kit comprising the pharmaceutical composition of claim 26 in combination with a label or instructions for use.

29. The kit of claim 28, wherein the label or instructions include a marketing authorization number.

30. The kit of claim 28 or 29, wherein the kit comprises an injection device comprising the antibody or fragment.

31. A nucleic acid, (a) encoding a VH domain and a VL domain of an antibody or fragment according to any one of claims 1 to 12; (b) encoding (i) the VH domain amino acid sequence of SEQ ID NO: 114; and (ii) the VL domain amino acid sequence of SEQ ID NO: 123; (c) having (i) a nucleotide sequence encoding a VH domain, wherein the nucleotide sequence encoding the VH domain is the sequence of SEQ ID NO: 115; and (ii) a nucleotide sequence encoding a VL domain, wherein the nucleotide sequence encoding the VL domain is the sequence of SEQ ID NO: 124; (d) encoding the heavy and light chains of the antibody or fragment according to any one of claims 1 to 13; or (e) encoding (ii) the heavy chain amino acid sequence of SEQ ID NO: 116; and (iii) the light chain amino acid sequence of SEQ ID NO:

125.

32. A vector comprising the nucleic acid according to claim 31.

33. A host cell comprising the nucleic acid according to claim 31 or the vector according to claim 32.

34. The host cell of claim 33, wherein the host cell is a CHO or HEK293 or Cos cell.

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